EP1495323A2 - Procede de clivage et de deglycosylation d'anticorps pour promouvoir une liaison de ligand - Google Patents

Procede de clivage et de deglycosylation d'anticorps pour promouvoir une liaison de ligand

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Publication number
EP1495323A2
EP1495323A2 EP03718249A EP03718249A EP1495323A2 EP 1495323 A2 EP1495323 A2 EP 1495323A2 EP 03718249 A EP03718249 A EP 03718249A EP 03718249 A EP03718249 A EP 03718249A EP 1495323 A2 EP1495323 A2 EP 1495323A2
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European Patent Office
Prior art keywords
antibody
fragment
carbohydrate
binding
hcg
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EP03718249A
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German (de)
English (en)
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Lisa S. Kelly, Ph.D.
David J. Puett
Michael J. Pierce
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University of Georgia Research Foundation Inc UGARF
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University of Georgia Research Foundation Inc UGARF
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Publication of EP1495323A2 publication Critical patent/EP1495323A2/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
    • G01N2333/42—Lectins, e.g. concanavalin, phytohaemagglutinin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins

Definitions

  • the present invention details methods to detect glycoproteins in samples to identify a disease state using binding assays and methods for increasing the efficiency of interaction of an antibody with a ligand by cleaving the antibody and orienting it on a matrix.
  • glycosylation characterized by O-linked oligosaccharides, which are attached to serine or threonine residues
  • glycosylation characterized by N- linked oligosaccharides which are attached to asparagine residues in an Asn-X- Ser/Thr sequence, where X can be any amino acid except proline.
  • N- acetylneuramic acid (also known as sialic acid) is usually the terminal residue of both N-linked and O-linked oligosaccharides.
  • Variables such as protein structure and cell type influence the number and nature of the carbohydrate units within the chains at different glycosylation sites. Glycosylation isomers are also common at the same site even within a given cell type.
  • the levels of glycosylation are a reflection of the levels and activities of different glycosyltransferases and glycosidases responsible for the intracellular construction of oligosaccharides.
  • glycosylation of the glycoproteins produced by diseased cells is markedly altered.
  • An increase in glycosylation is a common feature in the transformation to malignancy of certain cancers and has been related to the metastatic potential of tumor cell lines.
  • Altered glycosylation of membrane glycoproteins and glycolipids is observed in mammalian cells transformed with diverse tumor viruses, carcinogens, or transfection with certain oncogenes.
  • there is a quantitative increase in a particular substituent e.g., sialylation (Dall'Olio F. Protein glycosylation in cancer biology: an overview. Clin Mol Pathol 49:M126-M135 (1996)).
  • oligosaccharide structure in the tumor which is normally only found in fetal tissue; for instance, certain Lewis histo- blood group antigens have been detected in adenocarcinomas.
  • Qualitative differences in oligosaccharides may also be observed in certain transformed cells.
  • BHK fibroblasts transformed with polyoma virus or with Rous sarcoma virus display more highly branched complex N-linked oligosaccharides than do the corresponding normal cells.
  • the increase in the ⁇ -(l,6) branching of the cell surface-bound oligosaccharides has been associated, at least in some cases, with capacity for metastasis.
  • Increased levels of ⁇ -1,6 branching over the level in normal tissue has been observed for some human breast tumor tissues.
  • hCG human chorionic gonadotropin
  • hCG is a 38 kDa heterodimeric glycoprotein hormone that is approximately 30% oligosaccharide by weight.
  • hCG human luteinizing
  • FSH follicle stimulating
  • TSH thyroid-stimulating
  • hCG is structurally a member of the superfamily of 'cystine-loop' growth factors, along with transforming growth factor ⁇ (TGF ⁇ ), nerve growth factor (NGF), and platelet-derived growth factor ⁇ (PDGF ⁇ ), that are characterized by distinctive folds stabilized by disulfide bonds (Lapthorn et al., Nature, 369, 455-461 (1994)).
  • TGF ⁇ transforming growth factor ⁇
  • NEF nerve growth factor
  • PDGF ⁇ platelet-derived growth factor ⁇
  • Trophoblastic cells during pregnancy produce hCG, where it plays a central role in maintaining pregnancy by rescuing the corpus luteum.
  • hCG occurs in a number of molecular forms in serum and urine, including intact hCG, free subunits, and the ' ⁇ -core fragment'.
  • hCG hCG
  • ⁇ -core fragment the major form in the urine and is believed to be derived from proteolysis in the kidneys.
  • the intact hCG heterodimer binds to an ovarian hLH/hCG receptor.
  • expression of this receptor was initially considered to be restricted to gonadal tissues, recent studies have indicated low levels of expression on a variety of normal and neoplastic tissues, including those derived from breast and prostate (Lojun et al., Biol. Reprod., 57, 1202-1210 (1997); Dirnhofer et al., Prostate, 35, 212-220 (1998)).
  • hCG human immunoglobulin C
  • Lectins are proteins with the ability to bind specific
  • lectins in diagnostic assay because of their capacity to recognize the specific carbohydrate epitopes that can be associated with disease states.
  • the principal technique used to analyze carbohydrate modification of proteins in native samples is by developing "glycosylation profiles" using lectin binding assays. These assays are usually performed by separating a cell or tissue extract and probing this using one or more conjugated lectin. The levels of lectin binding are thereafter compared between samples to identify protein bands with altered levels of glycosylation.
  • P.C.T Publication No. WO 89/04490 Sudmar et al.
  • Canfield (WO 87/00289, published Jan. 15, 1987) proposes a method of detecting soluble desialylated glycoproteins (particularly hCG, thyroglobulin, CEA and CA19-9) in biological fluids. His assay preferably uses a solid phase lectin and a labeled antibody, but he recognizes that the roles of the lectin and the antibody may be reversed.
  • ft have a high affinity for the antigen, preferably at least 10 liters/mole, and more preferably, at least 10 9 liters/mole. David does not recognize that lectins may replace antibodies in a sandwich assay, and indeed teaches against such a substitution in view of the relatively low affinity of lectins for their targets.
  • Samuel et al. discloses a heterologous sandwich immunoassay for human TF antigen using a monoclonal antibody to capture the antigen from a sample of bodily fluid and a labeled lectin (peanut agglutinin) as a probe to confirm that TF antigen was captured.
  • Holtzhauer et al. discloses a sandwich immunoassay for detecting the presence of fucosylated ⁇ -fetoprotein, using a specific antibody in conjunction with specific lectins (selected from the group of Ulex-europaeus agglutinin, Lotus-tetragonolobus agglutinin, and Anguilla-anguilla agglutinin).
  • specific lectins selected from the group of Ulex-europaeus agglutinin, Lotus-tetragonolobus agglutinin, and Anguilla-anguilla agglutinin.
  • covalent lectin-antibody conjugates in immunoassays is described in Chu, U.S. Patent No. 4,371,515 (1983); Chu, U.S. Pat. No. 4,259,747; and Guesdon et al., J. Immunol. Meth, 39:1-13 (1980).
  • antibodies contain 3-12% carbohydrates at conserved N- glycosylation sites located in the constant regions of their heavy chains. Although glycosylation of this site seems to have little effect on antigen binding (Donadel, G., Calabro, A., Sigounas, G., Hascall, V. C, Notkins, A. L. and Harindranath, N. (1994) Glycobiology 4, 491-496), this glycan moiety provides a high level of background in assays which are designed to detect glycans. These assays do not account for the glycosylation of the antibodies themselves, lending unknown and variable background to each invention and limiting the benefit that can be gained by using them.
  • Antibodies are comprised of four subunits: two light chains and two heavy chains, which associate via disulfide bonds and non-covalent interactions. Together the four subunits form a Y-shaped molecule, with the top of the "Y” being made of both heavy and light chains while the stem of the "Y” is a heavy chain dimer. Each chain is organized into a constant region, which differs only minimally between antibodies, and a variable region which comprises the antigen binding sites and is found at the amino-terminal of each chain, at the top of the "Y". The sequence of the variable region determines the affinity of antibodies for specific antigens, which can differ dramatically. In addition, the method of production of an antibody drastically affects the utility of the antibody as a diagnostic tool. For example, the affinity of monoclonal antibodies is often lower than that of corresponding polyclonal antibodies (see for e.g. Chappey O.N., Pharm Res. 9(11): 1375-9 (1992)).
  • Antibody fragments are effective in binding antigen.
  • Many studies have shown that antibody fragments produced by recombinant means (i.e. by producing isolated fragments from recombinant DNA) can be used to detect antigens in samples and can be utilized to detect antigens by linking the fragment to a matrix (described in: Oelschlaeger, et al. Analytical Biochemistry. 309(1):27 (2002); Ramirez N., et al. Transgenic Research. ll(l):61-4 (2002); Hock B. , et al. Biosensors & Bioelectronics. 17(3):239-49 (2002); Foy BD., et al. Journal of Immunological Methods.
  • Antibody fragmentation by enzymes has long been known in the art.
  • the enzymes known include pepsin, papain, and ficin, which each release an Fc region (which comprises the constant region of the immunoglobulin and is in some cases fragmented), and either two F(ab) fragments (antigen binding) or one F(ab) 2 fragment comprised of the two individual F(ab) fragments and a short "hinge region" at its carboxy-terminal.
  • Fc region which comprises the constant region of the immunoglobulin and is in some cases fragmented
  • Fc region which comprises the constant region of the immunoglobulin and is in some cases fragmented
  • Fc region which comprises the constant region of the immunoglobulin and is in some cases fragmented
  • Fc region which comprises the constant region of the immunoglobulin and is in some cases fragmented
  • Fc region which comprises the constant region of the immunoglobulin and is in some cases fragmented
  • Fc region which comprises the constant region of the immunoglobulin
  • U.S. Patent No. 5,622,872 discloses antibody fragments bound to an electric sensor, wherein binding of an analyte can change the electrical, optical, or structural properties of a polymeric layer. This tool provides for a sensitive measurement, but only fluorometric changes are analyzed. There is therefore still no suggestion in the prior art that one can combine these teachings to limit lectin binding and enhance antibody recognition by cleaving known antibodies to yield antibody fragments and orient them on a matrix to allow enhanced recognition of analyte.
  • Another object of the present invention is to provide methods for identifying glycoproteins that can act as disease markers because they possess glycosylation patterns that differ from the glycoproteins from non-diseased cells.
  • a further object of the present invention is to provide a diagnostic tool for detecting diseases from patient samples.
  • a specific object of the present invention to provide novel methods for the diagnosis and prognosis of cancers associated with elevated levels of human chorionic gonadotropin, or altered glycosylation patterns of hCG.
  • Yet another object of the present invention is to provide methods for identifying glycoproteins that can act as disease markers because they are differentially expressed in diseased versus non-diseased cells.
  • a further object of the present invention is to develop therapeutic devices which can target a cytotoxic agent to a diseased cell by virtue of the enhanced detection and reduced background afforded by a combination of lectin and antibody fragment recognition.
  • a capturing agent is used to recover a protein from a biological sample and a probe is used to recognize the carbohydrate moieties on the protein.
  • the glycosylation pattern of a protein is analyzed by contacting a biological fluid with an antibody specific for the protein, forming a bound antibody-protein complex; contacting the bound protein with a carbohydrate probe; and detecting association between the bound protein and the carbohydrate probe.
  • This aspect of the invention is most applicable to highly expressed proteins such as tumoral hCG.
  • the capturing agent is an antibody specific for hCG and the probe is specific for the unique glycosylation pattern on tumoral hCG.
  • the increased sensitivity of this method relies in part on methods to improve the binding capacity on a matrix and reduce carbohydrate load of antibodies by cleaving these antibodies using an enzyme and linking the cleaved antibodies to a substrate, prior to analyte binding.
  • one embodiment provides a method to improve the binding capacity of antibodies on a matrix by cleaving the antibodies with a protease and linking them in an oriented manner to a solid matrix. Because the antibodies are in this way aligned with their variable regions equally available for antigen binding, this method allows enhanced interaction with antigen and increased antibody load on the matrix.
  • the invention utilizes antibodies prepared through traditional means, thereby expanding the utility of the invention beyond that of recombinant antibody fragments.
  • Another embodiment of this invention provides a higher affinity method to detect specific glycoproteins comprising: providing an antibody; cleaving said antibody using a protease to yield a cleaved antigen recognition fragment; linking said cleaved antigen recognition fragment to a solid matrix; contacting said cleaved antigen recognition fragment with an antigen, yielding a bound antigen; contacting said bound antigen with a carbohydrate-binding molecule; and recognizing the contacted carbohydrate-binding molecule.
  • This method achieves a higher apparent affinity of the antibody for the antigen by orienting the antibody binding fragments (F(ab)) in a directional manner, as well as decreasing nonspecific background binding of the carbohydrate-binding molecule when the cleavage renders the antibody free of carbohydrates, preferably N-linked oligosaccharides.
  • One aspect of this invention applies to the diagnosis of disease in tissue, urine, or serum samples.
  • the invention allows the analysis of glycoproteins found in diseased versus normal states.
  • the inventors unexpectedly discovered that a sandwich-type assay that relies upon the unique glycosylation patterns of tumoral hCG can be used to distinguish tumoral hCG from pregnancy- associated hCG and other non-tumoral hCG.
  • the carbohydrate probe in this instance preferably is specific for the presence of enhanced fucosylation and/or triantennary branching, or decreased terminal mannose, and most preferably is the Aleuria aurantia lectin, the Galanthus nivalis agglutinin, Sambucis nigra agglutinin, or Phaseolus vulgaris erythroagglutinin.
  • the sandwich type- assay is the preferred vehicle for practicing the instant invention, it will be appreciated by workers of skill in the art that other assays that employ this dual targeting scheme can also be constructed.
  • the dual targeting feature of the instant invention substantially reduces the possibility of a false positive signal because a positive reactivity in this assay depends on the ability of the antigen to meet the epitope requirements of two different anti-hCG reagents, (e.g., a capture antibody and a carbohydrate probe).
  • Human choriogonadotropin Human choriogonadotropin, sometimes abbreviated "hCG,” is defined as the intact heterodimer consisting of an ⁇ - and a ⁇ -subunit.
  • Free ⁇ subunit The ⁇ subunit of hCG refers to the non-combined ⁇ - subunit consisting of a 23 kD polypeptide containing 145 amino acid residues.
  • the ⁇ -subunit contains 2 N-glycosidic moieties attached to asparagine residues 13 and 30, a carboxyl terminal extension peptide (CTP), and four O-glycosidic linked oligosaccharides bound to serine residues 121, 127, 132, and 138.
  • CTP carboxyl terminal extension peptide
  • Free ⁇ subunit The ⁇ subunit of hCG refers to the non-combined ⁇ - subunit consisting of a glycosylated 14 kD polypeptide containing 92 amino acid residues and two N-linked complex branched oligosaccharide moieties bound to asparagine residues 52 and 78. It is common to hCG, LH, FSH and TSH.
  • Core fragment of hCG ⁇ refers to a 10 kD two-chain polypeptide lacking the amino acid sequences 1-5, 41-54 and 93-145. The two chains are held together by disulfide bonds.
  • the term may be abbreviated hCG ⁇ cf.
  • Aleuria aurantia lectin The Aleuria aurantia lectin is a dimer of two identical subunits of about 36,000 daltons each with an isoelectric point of about pH 9, isolated from Aleuria aurantia mushrooms. Unlike Ulex europaeus and Lotus tetragonolobus lectins which prefer ( ⁇ -1,2) linked fucose residues, Aleuria aurantia lectin binds preferentially to fucose linked ( ⁇ -1,6) to N- acetylglucosamine or to fucose linked ( ⁇ -1,3) to N-acetyllactosamine related structures.
  • Antibody load (on a matrix): In this application this is defined as the number of antibody molecules that can be accommodated on a given area of a binding matrix.
  • Phaseolus vulgaris erythroagglutinin is a member of the Phaseolus vulgaris agglutinin family of lectins, each of which consists of four subunits, isolated from Phaseolus vulgaris (red kidney bean) seeds.
  • the Phaseolus vulgaris erythroagglutinin appears to be
  • E subunit for erythroagglutinin
  • GNA Galanthus nivalis agglutinin
  • SNA Sambucis nisra agglutinin
  • Tumoral hCG refers to hCG derived from cancer cells that express abnormal levels of hCG or abnormally glycosylated hCG when compared to hCG excreted as a consequence of pregnancy, or baseline hCG expression from non-cancerous cells.
  • Cancer is meant to include pre-malignant conditions, malignant cancer, metastatic cancer, and non-metastasized cancer.
  • Such cancers include trophoblastic cancers such as choriocarcinoma, hydatidiform mole, and embryonal carcinoma of the testis, as well as non-trophoblastic cancers such as uterine cervical cancer, ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, breast cancer, bladder cancer, hepatic cancer, and gastric cancer.
  • the invention has particular utility in diagnosing or treating diseases associated with altered levels of hCG, such as cancers that secrete hCG or its subunits.
  • the invention provides methods to enhance the efficacy of binding between a known antibody and an antigen, thus increasing the
  • the antibodies of the invention can be any known antibodies, from any class.
  • the antibodies can include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies. Methods of preparing polyclonal or monoclonal antibodies are well known to those skilled in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1988)).
  • An antibody useful in the invention, or antigen binding fragment of such an antibody is characterized by having specific binding activity for a ligand or sample epitope.
  • Specific binding activity of an antibody for a ligand can be readily determined by one skilled in the art, for example, by comparing the binding activity of an antibody to a particular ligand versus a control ligand that differs from the particular ligand.
  • Both naturally occurring as well as non-naturally occurring antibodies may be utilized in this invention, including, for example, chimeric, bifunctional and humanized antibodies.
  • Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al. (Science 246:1275-1281 (1989); Kang et al., Proc. Natl. Acad. Sci. USA, 88:4363-4366 (1991)).
  • the advantage of using such a combinatorial antibody library is that antibodies do not have to be individually generated for each ligand.
  • These and other methods of making functional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341 :544-546 (1989); Hilyard et al., 1992, Protein Engineering: A practical approach; Borrabeck, 1995, Antibody Engineering, 2d ed.).
  • the antibody recognizes an epitope on hCG.
  • /7 hCG can be prepared by using hCG as an immunogen to challenge a mammal, e.g. a mouse, rabbit, rat or goat.
  • the antibody can be raised against the entire hCG molecule, the hCG ⁇ subunit, the hCG ⁇ subunit, hCG ⁇ cf, or any immunogenic determinant thereof.
  • the antibody is most preferably specific for the ⁇ -subunit of the human chorionic gonadotropin molecule.
  • the animal is challenged over a substantial period (e.g. 5 months). See generally, Haavik et al., Glycobiology 2:217-224 (1992).
  • mice monoclonal antibodies For mouse monoclonal antibodies, standard fusion partners may be used to immortalize spleen cells from the challenged animal. See, e.g., Kohler and Milstein, Nature 256:495-497 (1977). The resulting anti-hCG monoclonal antibodies or polyclonal antibodies may be used in the various immunological procedures described below.
  • the antibody is preferably treated to remove any sugars on the antibody that might inadvertently compete for binding by the carbohydrate probe.
  • Techniques for cleaving carbohydrates from glycoproteins can include treatment with a deglycosylating enzyme such as N- glycanase.
  • the antibody is cleaved by a protease before contacting it with an antigen.
  • the protease can be any protease that releases a fragment at least containing the variable antibody recognition region of the antibody. Suitable protease enzymes include, but are not limited to: papain, chymopapain, bromelain, ficin, tryspin, etc. Further proteases that may be used in the present invention can be found at http://merops.sanger.ac.uk/index.html.
  • the thiol protease enzymes are activated by reaction with cysteine or a low molecular weight sulfhydryl reducing agent such as dithiothreitol, mercaptoethanol, mercaptoethylamine, etc.
  • cysteine or a low molecular weight sulfhydryl reducing agent such as dithiothreitol, mercaptoethanol, mercaptoethylamine, etc.
  • an antibody is reacted with an amount of an activated thiol protease enzyme equivalent (on a weight basis) to about 1-20% of the antibody composition.
  • the reaction mixture may be buffered using a buffer such as phosphate, citrate or acetate buffer at a pH from about 5.0-7.0, preferably pH 5.0-6.0.
  • the reaction mixture is incubated at a temperature from about 4°C-37°C, generally about room temperature (about 25°C).
  • the reaction is preferably performed in the presence of a chelating agent such as, for example, ethylenediaminetetraacetic acid (EDTA) at about 1-3 mM, which enhances the stability of the thiol protease enzyme.
  • a chelating agent such as, for example, ethylenediaminetetraacetic acid (EDTA) at about 1-3 mM, which enhances the stability of the thiol protease enzyme.
  • EDTA ethylenediaminetetraacetic acid
  • the thiol protease enzyme is preactivated by incubation with cysteine or a sulfhydryl reducing agent such as dithiothreitol, mercaptoethanol, mercaptoethylamine, etc.
  • a thiol protease enzyme may be activated by dissolving the enzyme at about 1 mg/ml in 50 mM cysteine-containing buffer and incubating the reaction mixture for about 1 hour at room temperature.
  • the cysteine can be removed by gel filtration chromatography ("desalting") and then the eluted activated enzyme can be used as described to digest an antibody composition.
  • the enzyme can be immobilized by attachment to a substrate such as agarose, Sepharose. Sephadex, polyacrylamide, or agarose-acrylamide beads, before activation.
  • a substrate such as agarose, Sepharose. Sephadex, polyacrylamide, or agarose-acrylamide beads
  • agarose beads activated by reaction with carbonyldiimidazole commercially available as "Reacti Gel” Pierce Chemical Corporation
  • the antibody fragment can be separated by gently reducing interchain disulfide bonds with such reagents as mercaptoethanolamine.
  • antibody fragment refers to any derivative of an antibody which is less than full-length. Preferably, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments.
  • the antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide
  • the fragment may also optionally be a multimolecular complex.
  • Single-chain Fvs are antibody fragments consisting of only the variable light chain and variable heavy chain covalently connected to one another by a polypeptide linker. Either the variable light chain or the variable heavy chain may be the amino-terminal domain.
  • the polypeptide linker may be of variable length and composition so long as the two variable domains are bridged without serious steric interference.
  • the antigen binding domains can be separated from other cleaved products by a variety of methods.
  • the Fc domain is removed by incubation with a protein A or G (Staphylococcus aureus) linked to a support that can be separated (these are well known in the art and many versions are commercially available).
  • An aspect of this invention is to link the antibody binding fragments to a solid support in an oriented manner. This can be accomplished by a variety of methods.
  • the antibody fragment is linked to a solid support via a thiol linkage based on a cysteine residue in the antibody "hinge" region.
  • the antibody can be linked to a solid support via a carboxy-terminal amino acid other than a cysteine via, for example, an amide linkage.
  • the antibody fragment can be linked to a linking agent, such as streptavadin, and thereafter linked to a biotinylated surface by interaction of the streptavidin and biotin.
  • Solid supports which may be employed include, but are not limited to, those made of cellulosic materials such as paper, cellulose, and cellulose derivatives such as cellulose acetate and nitrocellulose; silica; fiberglass; inorganic materials such as deactivated aluminum, diatomaceous earth, or other inorganic finely divided material dispersed uniformly in a porous polymer matrix made of polymers such as vinyl chloride-propylene copolymer and vinyl chloride- vinyl acetate copolymer; cotton; nylon; porous gels such as silica gel, agarose,
  • polystyrene particles such as polyacrylamide; magnetic particles; microtiter plates; polystyrene tubes; protein-binding membranes; Sephadex (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.); Trisacryl (Pointet-Girard); silicon particles; polystyrene particles; vinyl chloride particles; latex particles; and porous fibrous matrices.
  • hCG is separated from a complex solution (such as human urine) for analysis of its glycan moieties. Isolation of hCG may be performed by numerous methods which are well-known to those of the skill in the art.
  • the hCG may be isolated and purified by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, and hydroxyapatite chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the protein. Finally, high performance liquid chromatography can be employed for final purification steps.
  • a sample suspected of containing human chorionic gonadotropin is subjected to immunoprecipitation or to immunoaffinity purification using an antibody which recognizes human chorionic gonadotropin.
  • Isolated human chorionic gonadotropin then is subjected to gel electrophoresis, such as, for example, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to an appropriate membrane, such as a polyvinylidene fluoride (PVDF) membrane.
  • PVDF polyvinylidene fluoride
  • Appropriate bands then are excised from the membrane, and treated with an exoglycosidase or with a combination of an exoglycosidase and a neuraminidase to release the oligosaccharides from the human chorionic gonadotropin.
  • the oligosaccharides then are labeled and analyzed through separation of the oligosaccharides on a gel. The gel then is analyzed for the presence of oligosaccharides with enhanced fiicosylation and/or triantennary branching.
  • a carbohydrate probe such as Aleuria aurantia lectin, Galanthus nivalis agglutinin, Sambucis nigra agglutinin, or Phaseolus vulgaris erythroagglutinin is incubated with a predetermined amount of a first portion of a sample suspected of containing human chorionic gonadotropin. After incubation, the sample is contacted with an antibody supported on a solid support.
  • the supported antibody which may be a monoclonal or polyclonal antibody, is cleaved by a protease to remove the carbohydrate moiety on the Fc region.
  • the antibody or antibody fragment recognizes an epitope of human chorionic gonadotropin which, if an oligosaccharide that binds the carbohydrate binding agent is present on the hCG, is proximal to such oligosaccharide, whereby binding of the hCG antigen to the supported antibody is inhibited by the binding of the carbohydrate binding agent.
  • hCG having linked oligosaccharides that bind the carbohydrate binding agent preferably Aleuria aurantia lectin, Galanthus nivalis agglutinin, Sambucis nigra agglutinin, or Phaseolus vulgaris erythroagglutinin
  • carbohydrate binding agent preferably Aleuria aurantia lectin, Galanthus nivalis agglutinin, Sambucis nigra agglutinin, or Phaseolus vulgaris erythroagglutinin
  • any hCG which binds to the supported antibody is hCG which does not bind the carbohydrate binding agent and thus is less modified by these specific oligosaccharides. Bound hCG then is separated from free hCG. The sample then is contacted with a labeled antibody which recognizes an epitope of human chorionic gonadotropin. Thus, the labeled antibody binds to human chorionic gonadotropin which has been bound by the supported antibody. Bound labeled antibody then is separated from free labeled antibody. The amount of bound, labeled antibody is then determined.
  • the antibody or antibody fragment reactive to hCG which is supported on a solid support is contacted with a second portion of the sample suspected of containing hCG.
  • the second portion contains an amount of sample identical to that of the first portion.
  • the labeled antibody to hCG is added, and the amount of bound labeled antibody is determined. If the amounts of bound labeled antibody in the first assay and the second assay are the same, then no hCG which binds the carbohydrate binding agent used is present in the sample. If the amount of bound labeled antibody in the second assay is greater than that in the first assay, then hCG which binds the carbohydrate binding agent used is present in the sample.
  • the glycosylation of antigens is recognized by a method comprising: providing an antibody having an affinity for an antigen; cleaving said antibody using a protease to yield a cleaved antigen recognition fragment; linking said cleaved antigen recognition fragment to a solid matrix; contacting said cleaved antigen recognition fragment with an antigen, yielding a bound antigen; contacting said bound antigen with a carbohydrate-binding molecule; and recognizing the contacted carbohydrate- binding molecule.
  • the carbohydrate binding agent comprises one or more lectins.
  • Lectins are herein defined as a sugar-binding protein of non-immune origin.
  • the structural information obtained from a large number of mammalian lectins has led to their classification into several families which exhibit a number of variable properties, including ion dependence and solubility (see for example, Drickamer,K., J. Biol. Chem., 1988, 263: 9557; Drickamer, K., Curr. Opin. Struc. Biol., 1993, 3: 393; Drickamer, K., Biochemical Society Transactions, 1993, 21 : 456).
  • certain lectins bind DNA molecules, depending on conditions (see Vieira-Breitwieser O, 20 m Annual International Lectin Meeting Abstracts, T25, 2002), therefore the conditions of the
  • the lectins that can be used in this application include those derived from the group consisting of animal derived, galactose-binding lectins (termed Galectins); Ca-dependent (C-type) animal lectins including sialyl-LewisX recognizing selectins and mannose-specific collectins; glycosaminoglycan binding annexins; plant-derived lectins including concanavalin A, and ricin, and invertebrate lectins such as tachylectins or Xenopus oocyte lectins.
  • Lectins also include: Arachis Hypogaea Agglutinin (PNA), Bauhinia Purpurea Agglutinin (BPA),Bendeirea Simplicifolia Agglutinin (BSA), Canavalia Ensoformis Agglutinin (CON A), Dolichos Biflorus Agglutini (DBA), Glycine Max (SBA), Lens Culinaris (LcH), Limulus Polyhemus (LPA), Lotus Tetragonolobus (Lotus A), Phaseolus Vulgaris (L-PHA), Phaseolus Limensis (LBA I), Phaseolus Vulgaris (H-PHA), Pisum Sativum (PEA), Phytolacca Americana (Pokeweed), Ricinus Communis (RCA I), Ricinus Consis (RCA II), Sophora Japonica (SJA), Triticum Vulgaris (WGA), Ulex Europeus (UEA I), Ulex Europaeus (UEA II
  • the choice of lectin will dictate the subpopulation of glycoproteins that are analyzed in each iteration.
  • lectins that are known to recognize carbohydrate moieties that are altered in disease states are used.
  • the lectins used include 1-6 oligosaccharide binding leukoagglutinin (L-PHA), Concanavalin A (ConA), which has affinity for oligomannosyl saccharides found in N-glycans, galectins including Galectin LEC- 6, specific for LacN Ac-containing glycans, Aleuria aurantia lectin (AAL) with broad specificity for L-Fuc-containing oligosaccharides, Peanut agglutinin (PNA), which is specific for Gall-3GalNAc), found widely in O-gl yeans, and Helix pomatia lectin (HP A), which binds N-acetylgalactosamine, Sambucis nig
  • conjugated lectins In addition to conjugated lectins, other non-lectin sugar binding proteins including sugar-specific enzymes and transport proteins can be used in this method.
  • Mono- or poly-clonal antibodies specific to one or more carbohydrate moieties may be used. These can include antibodies that are commercially available, such as anti-blood Group A Isotypes; anti-blood Group B Isotype; anti-
  • a blood Group H Isotypes; anti-Le Isotypes; anti-GM3 Isotypes; anti-E-selectin Isotypes; anti-MUCl Isotypes- anti-Extended sialyl-Lewis Isotypes anti-Gb3
  • hCG is immobilized and the bound hCG then is contacted with a compound or binding molecule that probes for enhanced fiicosylation and/or triantennary branching, or decreased terminal mannose, and most preferably Aleuria aurantia lectin, Galanthus nivalis agglutinin, Sambucis nigra agglutinin, or Phaseolus vulgaris erythroagglutinin.
  • a compound or binding molecule that probes for enhanced fiicosylation and/or triantennary branching, or decreased terminal mannose, and most preferably Aleuria aurantia lectin, Galanthus nivalis agglutinin, Sambucis nigra agglutinin, or Phaseolus vulgaris erythroagglutinin.
  • hCG antigen is isolated from a biological sample derived from a patient and any assay described herein is followed.
  • the biological sample may be derived from serum, plasma, prostatic fluid, semen, urine, spinal fluid, or other suitable biological fluid.
  • the assay of the present invention is particularly applicable for detection and quantitation of hCG antigen in various body fluids, such as serum, pleural effusions, and urine, of carcinoma patients.
  • the biological sample is derived from urine. This is particularly important clinically because the assay provides early detection of cancer, as altered hCG expression and glycosylation are associated with certain pre-malignant changes of tissues. Similarly, it is possible to detect increased levels of hCG antigens in carcinoma patients before cancer tissues reaches a critical mass which is detectable by other non-invasive methods. In these cases the assay is particularly valuable for its early diagnosis of cancer.
  • the interaction of the antibody fragments of the invention with the antigens and carbohydrate recognition agents can be distinguished by a variety of means.
  • the antibody fragments are linked to a solid matrix and antigen binding is recognized by a perturbation of that matrix. This can occur through, in one example, surface plasmon resonance (SPR).
  • SPR is a phenomenon which occurs when light is reflected off thin metal films. A fraction of the light energy incident at a sharply defined angle can interact with the delocalized electrons in the metal film (plasmon) thus reducing the reflected light intensity.
  • the precise angle of incidence at which this occurs is determined by a number of factors, preferably the refractive index close to the backside of the metal film, to which target molecules are immobilized and addressed by ligands in a mobile phase running along a flow cell. If binding occurs to the immobilized target the local refractive index changes, leading to a change in SPR angle, which can be monitored in real-time by detecting changes in the intensity of the reflected light, producing a sensorgram. The rates of change of the SPR signal can be analyzed to yield apparent rate constants for the association and dissociation phases of the reaction. The ratio of these values gives the apparent equilibrium constant (apparent affinity).
  • the size of the change in SPR signal is directly proportional to the mass being immobilized and can thus be interpreted crudely in terms of the stoichiometry of the interaction. Signals are easily obtained from sub-microgram quantities of material. It will also be appreciated that the invention also can be practiced using surface plasmon resonance in which binding of the lectin to the carbohydrate moiety of the antigen is detected by variations in the local refractive index.
  • the carbohydrate binding agent may be conjugated to a detectable marker or label.
  • detectable markers or labels include, but are not limited to, fluorescent labels, radioisotopes, such as, for example, isotopes of iodine, cobalt, or tritium, spin labels, chemiluminescent materials, enzymes, absorbing dyes, biotin, or colored particles.
  • the presence of the analyte contacted with the binding molecule, and therefore having linked oligosaccharides that bind to the carbohydrate binding molecule, can be determined by detecting this label.
  • the presence of the label on the bound antigen may be determined by means known to
  • the bound analyte includes linked oligosaccharide, the levels of which can be determined by means known to those skilled in the art, for instance by fluorescence detection or by chemiluminescence.
  • kits or package for detecting hCG linked to an oligosaccharide that binds carbohydrates having enhanced fiicosylation and/or triantennary branching, and/or decreased terminal mannose residues such as Aleuria aurantia lectin, Sambucis nigra agglutinin, Galanthus nivalis agglutinin, or Phaseolus vulgaris erythroagglutinin.
  • the kit includes (a) a solid support upon which is supported a binder, in particular, a cleaved antibody, which recognizes an epitope of human chorionic gonadotropin; and (b) a binding molecule or probe such as Aleuria aurantia lectin, Sambucis nigra agglutinin, Galanthus nivalis agglutinin, or Phaseolus vulgaris erythroagglutinin having a label such as those hereinabove described.
  • a binder in particular, a cleaved antibody, which recognizes an epitope of human chorionic gonadotropin
  • a binding molecule or probe such as Aleuria aurantia lectin, Sambucis nigra agglutinin, Galanthus nivalis agglutinin, or Phaseolus vulgaris erythroagglutinin having a label such as those hereinabove described.
  • the kit includes a pair of solid supports. Each support includes a binder, in particular, an antibody, which recognizes an epitope of human chorionic gonadotropin.
  • the epitope is located proximal to an oligosaccharide which binds Aleuria aurantia lectin, Sambucis nigra agglutinin, or Phaseolus vulgaris erythroagglutinin, if such an oligosaccharide is present on the human chorionic gonadotropin.
  • the kit also includes a probe for carbohydrates that have enhanced fiicosylation and/or triantennary branching, or decreased terminal mannose, such as Aleuria aurantia lectin, Sambucis nigra agglutinin, Galanthus nivalis agglutinin, or Phaseolus vulgaris erythroagglutinin, and a labeled antibody which recognizes an epitope of human chorionic gonadotropin.
  • a probe for carbohydrates that have enhanced fiicosylation and/or triantennary branching, or decreased terminal mannose, such as Aleuria aurantia lectin, Sambucis nigra agglutinin, Galanthus nivalis agglutinin, or Phaseolus vulgaris erythroagglutinin, and a labeled antibody which recognizes an epitope of human chorionic gonadotropin.
  • the binding profiles of samples from patients suspected of harboring a disease may be compared with binding profiles from control samples. These profiles can be developed by either comparing control samples in assays conducted at the same time as those conducted on the suspected samples, or may be developed prior to these assays. Thus, a comparison can readily be made to a benchmark set of values in a clinical setting.
  • the binding profiles are the level of binding of specified lectins to arrays of samples contacted with immobilized antibody or antibody fragments described herein.
  • Control samples can include both those that are known to harbor a disease or those that are known to not harbor a disease.
  • the assay of the present invention is useful as a prognostic test. Therapy can easily be monitored using the test of the present invention. Correlation of hCG antigen expression with tumor burden and tumor progression gives the capability of monitoring the response of the tumor to anti-cancer treatment.
  • a further embodiment of the invention is a method of detecting cancer by contacting a biological sample with a cleaved antibody, preferably an immobilized antibody fragment immunoreactive against hCG as described herein, and detecting the carbohydrate moieties on the hCG derived from the sample by probing the immobilized hCG with one or more lectins, preferably those selected from the group consisting of Aleuria aurantia lectin, Sambucis nigra agglutinin, Phaseolus vulgaris erythroagglutinin, or Galanthus nivalis agglutinin.
  • the same techniques can be applied to detection of pregnancy in biological samples.
  • the invention can be utilized to allow more specific targeting of a therapeutic means, such as a chemotherapeutic molecule, to a diseased cell.
  • a therapeutic means such as a chemotherapeutic molecule
  • the preferred embodiment of this invention comprises a therapeutic means linked to cleaved antigen recognition fragments and carbohydrate recognition agents specific for diseased cells. The combination of these recognition agents can allow more specific and effective targeting of the therapeutic means to diseased cells.
  • Suitable therapeutic means can, in non-limiting examples, include: Cisplatin, dacarbazine, cyclophosphamide, mechlorethamine, cytarabine, streptozocin, carboplatin, Mitomycin, carmustine (BCNU), Mitoxantrone, Pentostatin, dactinomycin, Procarbazine, "-rubicins” such as: daunorubicin - idarubicin- doxorubicin, bleomycin, Methotrexate, chlorambucil, Taxol, plicamycin, Etoposide, thioguanine, Fluorouracil, vinblastine, Hydroxyurea, vincristine, Melphalan, Anastrozole, Bicalutamide, Bromocriptine, Buserelin, Busulfan, Capecitabine, Cyproterone acetate, Demeclocycline, Desmopressin, Dexamethasone, Fluoxymesterone, Flu
  • control sample is a biological sample derived from cells under "normal physiological condition" means conditions that are typical inside a living organism or a cell. It will be recognized that the concentration of various salts depends on the organ, organism, cell, or cellular compartment used as a reference.
  • F(ab) and F(ab') are synonymous.
  • F(ab) 2 fragments are considered to comprise, in part, two F(ab) fragments.
  • Example 1 Antibody preparation for ELISA-based and Biacore assays
  • Example 2 Controls to check lectin binding to antibodies and fragments
  • Antibodies or F(ab)2 were immobilized to microplate wells at 2.5 ⁇ g/ml for 2 h at 37°C and then blocked overnight in blocking buffer (1% bovine serum albumin in phosphate-buffered saline (PBS) with 0.2% Tween) at 4°C. The plate was then probed with an array of biotinylated lectins (40 ⁇ g/ml) diluted in blocking buffer, followed by streptavidin conjugated to horseradish peroxidase
  • JAR cell culture and hCG acquisition JAR choriocarcinoma cells were acquired from ATCC and cultured according to their guidelines. Briefly, cells were grown in 75 mm ⁇ flasks in RPMI media supplemented with 4.5 g/L of glucose, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 10% fetal bovine serum, and IX penicillin-streptomycin-Fungizone. After 48 h of standard culture, the medium was replaced with serum-free medium for 48 h before collection in a 50 ml centrifuge tube. Any suspended cells or cellular debris were first removed by low-speed centrifugation. The clarified medium was concentrated 10-fold in a 10 kD centrifuge filtration device, and the free hCG present was measured with a radioimmunoassay.
  • Lectin binding on hCG derived from pregnancy and malignancy The concentration of pregnancy-derived hCG was verified with the RIA. JAR cell culture media and pregnancy urine-derived hCG were diluted to the same concentration of hCG in PBS and incubated on a microwell plate (7.5 ng/well) after coating with the F(ab)2 as above. Lectin binding was quantified as described
  • CM5 chip containing four flow channels comprised of a carboxymethyl dextran matrix on a gold substrate.
  • the monoclonal antibody or protease-derived antibody fragment as described above was bound through reactive amines to a test channel.
  • the matrix was activated with 50 mM N-hydroxysuccinimide (NHS) and 200 mM ethyl-N (dimethyaminopropyl) carbodimide (EDC) flowing at 5 ⁇ l/min for 7 min.
  • the antibody 50 ⁇ g/ml in 10 mM sodium acetate buffer, pH 5.0
  • was passed over the chip at 5 ⁇ l/min until 10,000 RU are reached on the instrument (10-15 min).
  • the surface was deactivated with 1 M ethanolamine, pH 8.5 (5 ⁇ l/min for 7 min).
  • a deactivated but non-derivitized reference channel served as
  • hCG standard or tumor cell-secreted
  • running buffer (10 mM HEPES, pH 7.4, 0.15 M NaCl, 2 mM CaCl 2 , 2 mM MgCl2, 0.005% Tween 20)
  • lectins (5 ⁇ l/min for 2 min) at a saturating concentration of 500 ⁇ g/ml were injected onto the chip.
  • Streptavidin can also be coated to the chip via amine coupling, followed by capture of amine-coupled biotinylated antibody fragment.
  • the surface is pulsed with either 50 mM HCl at 10 ⁇ l/min for 1 min or 10 mM glycine, pH 2.0, at 50 ⁇ l/min for 12 sec, depending upon the antibody used. Binding of any component is calculated as the difference in the resonance units 10 sec before the injection and 5 sec before the injection is complete.
  • the ELISA-based and Biacore protocols enable a distinction of normal glycosylation from abnormal glycosylation of hCG using a unique sandwich assay involving hCG protein capture via an antibody or antibody fragment, followed by lectin binding.
  • Phaseolus vulgaris erythroagglutin (specificity for Gal (l-4)GlcNac (l-2)Man)
  • Example 6 Cell culture and hCG acquisition
  • Cancerous cells were acquired from ATCC and cultured according to their guidelines. Briefly, cells were grown in 75 mm flasks in the recommended media supplemented by IX penicillin-streptomycin-Fungizone. After 48 h of standard culture, the medium was replaced with serum-free medium for 48 h before collection in a 50 ml centrifuge tube. Any suspended cells or cellular debris were first removed with a 5 min spin at 2000 rpm before the clarified media was concentrated roughly 10-fold in a 10 kD centrifuge filtration device. Intact hCG (Coat-a-Count IRMA, Diagnostics Products Corporation) and total ⁇ -subunit (RIA - ICN Pharmaceuticals) present were measured in 3 experiments each. Pregnancy- derived hCG was purchased from Sigma, and its concentration was also verified with IRMA and RIA.
  • Monoclonal antibodies which recognize separate epitopes on hCG ⁇ can be used.
  • an anti-fluorescein isothiocyanate (FITC) antibody chosen for its lack of reactivity with human serum, can be purchased from Sigma.
  • the antibodies must be treated to remove the one site of N-linked glycosylation on each heavy chain or undesired binding will introduce high background on subsequent incubation with lectins.
  • Each MoAb can be enzymatically digested to sever the carbohydrate-containing F c portion.
  • a first antibody can be incubated with 100 units of immobilized ficin (Sigma)/ mg MoAb in 0.1 M citrate buffer, pH 6.0, 5 mM EDTA ([ethylenedinitrilo]tetraacetic acid), and 1 mM cysteine for 2h at 37°C with shaking.
  • a second antibody can be digested with immobilized papain, pre-activated with 10 mM cysteine for 30 min at 37°C in 0.1 M acetate buffer, pH 5.5, 3 mM EDTA.
  • the reaction can be centrifuged over a 0.22 ⁇ m centrifuge filter (Millipore) to remove the cysteine, and the enzyme-linked beads can be
  • each reaction volume can be passed over a 0.22 ⁇ m filter to remove the agarose-bound enzymes.
  • the antibody fragments can be dialyzed across a 10 kD membrane into PBS.
  • the F(ab') 2 fragments can be gently reduced to F(ab') by incubation with 5- 10 mM mercaptoethanolamine for 1 h at 37°C, and dialyzed into immobilization buffer (10 mM sodium acetate, pH 4.0-4.5, 5 mM EDTA). Fragmentation and reduction of the antibodies can be verified on a 10% polyacrylamide gel stained with silver.
  • F(ab') fragments can be immobilized to a CM5 chip (Biacore) through their free hinge thiol groups using a maleimide coupling protocol provided by Biacore.
  • Either anti-FITC, a single antibody, or a mixture of antibodies together, can be diluted to a total concentration of 50 ⁇ g F(ab')/ ml of immobilization buffer.
  • flow over the chip can be set at 5 ⁇ l/min with running buffer (10 mM HEPES (N-[2-hydroxyethyl] piperazine-N'-[2- ethanesulfonic acid]), pH 7.5, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20).
  • the chip can be activated with lO ⁇ l of 200 mM EDC (l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride)/ 50 mM NHS (N- hydroxysuccinimide), followed by 20 ⁇ l of ethylenediamine, pH 8.5.
  • EDC l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride
  • NHS N- hydroxysuccinimide
  • the hetero- bifunctional cross-linking agent sulfo-MBS 40 ⁇ l of 50 mM m- maleimidobenzoyl-N-hydroxysulfo-succinimide ester, Pierce Chemical Company
  • a concentration series of pregnancy-derived hCG can be separately injected in HBS over the coupled reference/experimental channels and the binding response recorded on surfaces derivitized with one monoclonal antibody (or fragment) or derivitized with multiple monoclonal antibodies (or fragments) that recognize separate epitopes on hCG.
  • the channels can be regenerated to the antibody fragment between hCG injections with a 30s pulse of 10 mM glycine, pH 2.0 at 30 ⁇ l/min flow rate.
  • Avidity effects manifested thorough decreased dissociation of hCG from multiple monoclonals over single antibodies, increase the apparent affinity for hCG up to 8-fold.
  • Example 10 Lectin binding on hCG ⁇ derived from pregnancy and malignancy
  • Urine samples from patients with pregnancy, complete hyaditiform mole, choriocarcinoma or germ cell tumors can be diluted into HBS to give a binding response of approximately 150 RU when injected over a F(ab') surface of approximately 12,000 RU. Flow rates for all injections but the regeneration pulse can be 5 ⁇ l/ min.
  • the hormone-containing samples can be injected for 5 min, followed by a 4 min injection of Galanthus nivalis agglutinin, Phaseolus vulgaris erythroagglutinin or Sambucis nigra agglutinin (250 ⁇ g/ ml with 100 ⁇ g/ ml bovine serum albumin in HBS).
  • the sensor chip can be regenerated between samples with 15 ⁇ l of 10 mM glycine, pH 2.0 flowing at 30 ⁇ l /min. Deflections on the sensorgrams caused by residual lectin binding to the antibody fragments or injection noise can be removed by subtracting the response of a blank injection of running buffer followed by the lectin from each experimental sensorgram using BIAevaluation software from BIAcore. Similarly, deflections due to mass transfer effects, air bubbles, and other random events can be removed by subtracting the sensorgram simultaneously generated on a non-derivitized reference channel from each experimental sensorgram. All experiments were repeated three times.
  • Galanthus nivalis agglutinin has shown more binding to diseased samples; Phaseolus vulgaris erythroagglutinin has shown more binding to pregnancy samples. This trend has continued when pregnancy and choriocarcinoma samples were diluted over a 16-fold range. A ratio above two of Phaseolus vulgaris erythroagglutinin binding to Galanthus nivalis agglutinin binding can distinguish diseased samples from pregnancy samples.
  • Example 11 Lectin binding to hCG ⁇ from other malignant cell lines
  • BeWo choriocarcinoma cells were obtained from the ATCC and grown according to their instructions in 75mm flasks. When the cultures reached 50% confluency, the flasks were washed 3 times with PBS, and the media was replaced with serum-free media for 48 h. The conditioned media was collected and concentrated 10-fold over lOkD centrifuge filters. Hormone levels were measured with a commercial ⁇ -radioimmunoassay. Commercial hCG purified from pregnancy urine (Sigma) was diluted in cell media to the same concentration as hCG from the BeWo cells. Both were then diluted into HBS to 1.0 ⁇ g/ml. Lectin probing and channel regeneration were as described above. The lectins Phaseolus vulgaris erythroagglutinin and Sambucis nigra agglutinin bound more extensively to choriocarcinoma cell-derived hCG than pregnancy-derived hCG.
  • IgG Fifty micrograms of IgG can be digested to F(ab')2 with 1 mg of immobilized ficin (Sigma) in 0.1 M citrate buffer, pH 6.0, 1 mM cysteine, 5 mM EDTA. After digestion, the reaction volume can be passed over a 0.22 ⁇ m centrifuge filter to remove the agarose-bound enzyme. The fragmented antibody can be dialyzed across a 10 kD membrane into PBS and gently reduced by incubation with 5 mM mercaptoethanolamine at 37° C for 1 h. The resulting F(ab') fragments can be dialyzed across a 10 kD membrane into immobilization buffer (10 mM sodium acetate, pH 4.0). Fragmentation of the antibody can be verified on a 10% polyacrylamide gel stained with silver.
  • immobilized ficin Sigma
  • F(ab') fragment can be immobilized to a CM5 chip (BIAcore) through its free hinge thiol groups using a maleimide coupling protocol provided by BIAcore. Any remaining active sites can be blocked with 50 ⁇ l of 100 mM cysteine in 10
  • Flow rates for all injections but the regeneration pulse can be 5 ⁇ l/ min.
  • the hormone-containing samples can be injected for 5 min, followed by a 4 min injection of the lectin (250 ⁇ g/ ml with 100 ⁇ g/ ml bovine serum albumin in HBS).
  • the sensor chip can be regenerated between samples with 15 ⁇ l of 10 mM glycine, pH 2.0 flowing at 30 ⁇ l /min. Deflections on the sensorgrams caused by residual lectin binding to the antibody fragments or injection noise can be removed by subtracting the response of a blank injection of running buffer followed by the lectin from each experimental sensorgram using BIAevaluation software from BIAcore. Similarly, deflections due to mass transfer effects, air bubbles, and other random events were removed by subtracting the sensorgram simultaneously generated on a non-derivitized reference channel from each experimental sensorgram.
  • IgG and F(ab') can be compared in their capacity to be linked to BIAcore chip surfaces with amine capture.
  • IgG can be digested with 1 mg of immobilized ficin (Sigma)/ 50 ⁇ g IgG in 0.1 M citrate buffer, pH 6.0, 1 mM cysteine, 5 mM EDTA. After digestion, the reaction volume can be passed over a 0.22 ⁇ m centrifuge filter to remove the agarose-bound enzyme. The fragmented antibody can be dialyzed across a 10 kD membrane into PBS and gently reduced by incubation with 5 mM mercaptoethanolamine at 37° C for 1 h.
  • the resulting F(ab') fragments can be dialyzed across a 10 kD membrane into immobilization buffer (10 mM sodium acetate, pH 4.0).
  • the F(ab') fragment can be immobilized to a CM5 chip (BIAcore) through its free hinge thiol groups using a maleimide coupling protocol provided by BIAcore.
  • IgG can be immobilized to a BIAcore chip using amine coupling. Passing hCG over these surfaces at 10 ng/ml at 5 ⁇ l/min for 5 min leads to a 3 -fold greater deflection on the cleaved antibody surface than that on intact IgG.
  • the cleaved antibody surface has higher sensitivity given the same amount (approximately 10,000 RU) of immobilized capture molecules.

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Abstract

L'invention concerne des procédés pour détecter des glycoprotéines dans des échantillons pour identifier un état pathologique. En particulier, un essai de sandwich amélioré est décrit, lequel essai faisant appel à la fois à un anticorps et à un réseau de lectines. L'invention concerne des procédés permettant d'accroître l'efficacité d'interaction d'un anticorps et d'un ligand par le biais du clivage de l'anticorps pour retirer la fraction d'hydrate de carbone, et pour orienter le fragment clivé sur une matrice pour améliorer la reconnaissance d'un ligand. L'invention concerne encore des procédés de différenciation d'échantillon dérivés de sujets présentant une grossesse ou une maladie.
EP03718249A 2002-04-05 2003-04-07 Procede de clivage et de deglycosylation d'anticorps pour promouvoir une liaison de ligand Withdrawn EP1495323A2 (fr)

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GB2404734A (en) * 2003-08-05 2005-02-09 Secr Defence A method of screening a sample for abnormally glycosylated and/or expressed proteins
US7838634B2 (en) * 2005-04-15 2010-11-23 Van Andel Research Institute Methods for measuring glycan levels of proteins
US8632983B2 (en) 2005-04-15 2014-01-21 Van Andel Research Institute Biomarkers for pancreatic cancer and diagnostic methods
US7879799B2 (en) 2006-08-10 2011-02-01 Institute For Systems Biology Methods for characterizing glycoproteins and generating antibodies for same
EP2037274A1 (fr) * 2007-09-11 2009-03-18 GALAB Technologies GmbH Investigation de glycane à base de lectine
PL3023777T3 (pl) 2013-09-09 2018-04-30 Shimadzu Corporation Sposób otrzymywania fragmentów peptydowych, zestaw do otrzymywania fragmentów peptydowych przeznaczony do stosowania w nim i sposób analizy
CN104678103A (zh) * 2014-08-05 2015-06-03 首都医科大学附属北京佑安医院 检测血清糖蛋白岩藻糖指数的化学发光蛋白芯片、试剂盒及检测方法
CN107407666A (zh) * 2015-03-09 2017-11-28 株式会社岛津制作所 使用质谱分析的单克隆抗体的检测方法
CN115932065A (zh) * 2015-03-10 2023-04-07 株式会社岛津制作所 用于检测单克隆抗体的样品调制用试剂盒
US10753936B2 (en) 2016-07-22 2020-08-25 Van Andel Research Institute Method of detecting the level of a glycan
CA3230920A1 (fr) * 2021-09-10 2023-03-16 Jason J. SUN Agents d'agglutination, dispositifs et procedes pour dosages sur sang total

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO03087821A2 *

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