JP2009531289A6 - Method for modifying and labeling proteins with oligosaccharides - Google Patents

Abstract

The present invention relates generally to methods for conferring functions on proteins, particularly antibodies, at the position of oligosaccharide linkages, methods for humanizing antibodies by modifying sugar chains, and novel antibodies linked to modified oligosaccharides. . The present invention further relates to kits used to prepare the antibodies of the present invention.

Description

  This patent application has priority to US Provisional Patent Application No. 60 / 772,221 filed on February 10, 2006 and US Provisional Patent Application No. 60 / 804,640 filed on June 13, 2006. All of which are incorporated by reference in their entirety as if set forth herein.

The present invention generally relates to a method for labeling proteins in novel oligosaccharide linkages, a method for humanizing antibodies by modifying sugar chains, and novel antibodies linked to modified oligosaccharides.

BACKGROUND Isolated or synthesized proteins and antibodies (eg, IgG) are used in therapeutic, diagnostic, and research applications. By labeling the antibody with a detectable label (eg, a fluorophore), the antibody can be used specifically for the detection of target biomolecules or cells. A substance that binds to the antibody (eg biotin) may be bound as a tag, so that they specifically bind to the target biomolecule or cell and further use a substance that binds to the tagged antibody (eg Purification of streptavidin) biomolecules or cells can be performed. The antibody may typically be labeled with a cysteine or lysine residue (which may be present on the Fab), or an antibody binding site. By adding a tag or label to this site, the binding properties of the antibody can be impaired or at least altered. Furthermore, it is very difficult to quantify the number of labeled molecules bound to each antibody.

  Therapeutic monoclonal antibodies (Mabs) are currently used as essential drugs to combat cancer, rheumatoid arthritis, macular degeneration, and other diseases or symptoms. However, antibodies produced in non-human cell lines have antigenic characteristics and can be recognized as foreign by the human immune system, thereby limiting the half-life and effectiveness of the antibody. Introducing human IgG sequences into transgenic mice leads to a reduction in immunogenicity problems, but does not completely eliminate them. In addition to the protein sequence, the nature of the oligosaccharides that bind to IgG also has a significant impact on recognition by the immune system. Since glycosylation is specific to cell type, IgG produced in different host cells has different oligosaccharide patterns, which can affect biological function. Even when cells (eg, human embryonic stem cells) are grown on the mouse feeder layer while exchanging serum derived from animals, the cells take up sialic acid, which is an immunogen derived from non-human, and further Acid appears on the cell surface (Non-patent Document 1). Therapeutic antibody manufacturers tried to circumvent these challenges by using defucosylated oligosaccharides to produce less antigenic IgG, but defucosylated antibodies are equivalent to humanized antibodies. In addition, the problem of immunogenicity and still having a half-life different from that of natural human antibodies has not been solved.

  There is metabolic oligosaccharide engineering, which refers to making minor modifications to monosaccharide residues in cellular glycans. Researchers use metabolic engineering to disrupt glycan biosynthesis, chemically modify the cell surface, scrutinize metabolic flow inside the cell, and identify specific glycoprotein subtypes from the proteome. It identified (refer nonpatent literature 2).

Martin, M.M. J. et al. , Et al., Nature Medicine, 2005, 11: 228-232. Dube, D.D. H. , And Bertozzi, C.I. R. , Current Opinion in Chemical Biology, 2003, 7: 616-625.

  From the above, there is a need for an antibody having a tag or label at a site other than the binding site, and an antibody that can be easily labeled by a simple and efficient chemical reaction. There is also a need for antibodies that have post-translational modifications similar to human antibodies.

SUMMARY OF THE INVENTION The present invention generally relates to novel methods for remodeling and labeling proteins and antibodies in oligosaccharide linkages, methods for humanizing antibodies by modifying glycosylation, and antibodies or antibodies that bind to modified oligosaccharides. It relates to protein. Note that the antibody or protein (eg, IgG) is labeled using in vitro or in vivo methods.

  In some in vitro embodiments, the oligosaccharide present in the first antibody is cleaved and another second oligosaccharide is attached to the cleavage site on the first antibody. This second oligosaccharide may be cleaved from the second antibody, for example. By using this method, for example, oligosaccharides obtained from human antibodies may be conjugated to antibodies produced in non-human cell lines. By using this method, a secondary label may be bound to the antibody at the position of the oligosaccharide.

  In some in vivo embodiments, non-natural sugars with chemical handles are taken up by antibody producing cells and the oligosaccharide is incorporated onto the antibody. After the antibody is isolated, a secondary label may be attached at the position of the chemical handle.

  By labeling the antibody at the site of the glycan residue of the Fc portion of the antibody, possible disruption of epitope binding is avoided instead of the conventional labeling of cysteine or lysine residues in the binding region of the antibody. Furthermore, the number of glycan residues present in IgG is generally known. In contrast, each epitope-specific IgG has a different number of lysine or cysteine residues in the peptide sequence, and each can be labeled differently based on the structure of the IgG. Therefore, it is usually difficult to determine how much label is present on each IgG. By using the method of the present invention, it is expected that almost all of the glycan residues will be labeled, so that quantitative labeling and detection using antibodies, eg, by fluorescence activated cell sorting (FACS), etc. Is possible.

  In other methods of the invention, the linking of human oligosaccharides to non-human IgG actually results in the same glycoforms as those in humans, the only differences being the presence of extra galactose near the sugar binding site and the ring Only the presence of a ring structure derived from the addition.

  In addition to the humanization of therapeutic antibodies, the method of the present invention can have many applications. For example, glycosylation patterns can change in certain diseases or symptoms (eg, rheumatoid arthritis and pregnancy). The ability to mix and adapt oligosaccharides allows researchers to investigate human diseases associated with altered glycosylation in animal models.

One embodiment of the present invention includes the following steps:
Attaching a modified sugar comprising a chemical handle to a GlcNAc residue on the first protein;
Mixing a reporter molecule, carrier molecule or solid support capable of reacting the first protein with the chemical handle;
And the reporter molecule, carrier molecule, or solid support is bound to the protein at the position of the chemical handle, thereby forming a sugar-modified protein.

  In another embodiment, the first protein is an antibody. In a further embodiment, the antibody is IgG.

  In another embodiment, the binding step is performed in a solution that is substantially free of proteases.

  In another embodiment, the method includes cleaving the oligosaccharide present on the first protein at the position of GlcNAc-GlcNAc binding to obtain a protein comprising a GlcNAc residue prior to the binding step. In a further embodiment, the oligosaccharide is cleaved by endoglycosidase H cleavage at the position of GlcNAc-GlcNAc binding. In another embodiment, the oligosaccharide is cleaved by endoglycosidase M cleavage at the position of the GlcNAc-GlcNAc bond.

  In another embodiment, the reporter molecule, carrier molecule, or solid support is attached to the GlcNAc using a galactosyltransferase variant. In another embodiment, the variant is a Y289L variant.

  In another embodiment, the modified sugar is an azide modified sugar and the reporter molecule, carrier molecule, or solid support is labeled with alkyne or activated alkyne. More specifically, the azide modified sugar is UDP-GalNAz.

In another embodiment, the method includes the following steps prior to the combining step:
Cleaving the oligosaccharide present on the second protein at the position of GlcNAc-GlcNAc binding to obtain an oligosaccharide having a GlcNAc residue;
Attaching the oligosaccharide to a reporter molecule, solid support or carrier molecule capable of reacting with the chemical handle.

  In another embodiment, an oligosaccharide having a GlcNAc residue is pretreated with ammonium bicarbonate and the oligosaccharide is coupled to an alkyne using a succinimidyl ester. In other embodiments, the second protein is synthesized in a different cell line or different type of cell than the first protein. In another embodiment, the second protein is synthesized in human cells.

  In other embodiments, the reporter molecule is selected from the group consisting of a fluorescent dye, an enzyme, a radiolabel, a metal chelator, or a detectable substrate. In other embodiments, the carrier molecule is selected from the group consisting of therapeutic agents, DNA, proteins, peptides, and sugars.

  In other embodiments, the sugar-modified protein is more or less antigenic compared to the first protein before it is modified. In other embodiments, the first protein is from a non-human source. In other embodiments, the sugar-modified protein is a humanized protein.

  In another embodiment, the cleavage of the first protein is carried out in the presence of OH-alkyne, and the endoglycosidase M binds the OH-alkyne to a cleaved GlcNAc residue and the modified The oligosaccharide is labeled with an azide residue.

  Another aspect of the present invention relates to the provision of a method for labeling a protein by labeling an oligosaccharide that binds to the protein. The method includes incubating the protein-producing cell in the presence of a non-natural sugar, the non-natural sugar having a chemical handle.

  Another aspect of the invention relates to the provision of an antibody comprising a labeled oligosaccharide.

  Regardless of their location, the above embodiments are provided as any one specific description of embodiments of the present invention.

  Other objects, features and advantages of the present invention will become apparent from the following detailed description. Preferred embodiments of the present invention are shown below, and those skilled in the art can easily conceive various modifications and changes within the scope of the present invention from those detailed descriptions. It should be understood that the examples have been described for illustrative purposes only.

Detailed Description of the Invention Introduction:
Various glycosidase enzymes (such as endo-H) can cleave N-linked oligosaccharides, and other enzymes (such as Gal-T) can convert selected oligosaccharides to acceptor molecules containing -OH groups. Can be transferred. The most efficient acceptor molecules are N-acetylglucosamine and glucose. Alternatively, a selected sugar can be introduced to the acceptor (eg, protein) by metabolic labeling. Described herein are the use of this enzyme or metabolic system for several applications, including modification of proteins with oligosaccharides (eg, therapeutic antibodies), or conjugation of proteins to solid substrates with oligosaccharides. In some applications, transfer of oligosaccharides to proteins with complementary chemical handles for solid support surfaces coated with appropriate acceptor molecules may be performed. In addition, the sugar may be transferred to a detection molecule (eg, a fluorescent or fluorogenic compound or protein that streamlines detection, purification and evaluation by mass spectrometry). In addition to the appropriate acceptor OH group, transfer to large molecules with affinity peptide tags, active chemical moiiety (eg azide, alkyne) or biotin is also possible.

  One suitable use of this method is the humanization of non-human antibodies by replacing non-human N-linked oligosaccharides with human N-linked oligosaccharides derived from human antibodies. In this case, the non-human antibody is modified with a series of hetero-human oligosaccharides. Alternatively, the same type of purified or synthesized human-type oligosaccharide (coupled to a protein or peptide substrate) can be used as a donor conferring specificity to the antibody acceptor. Such properties include control of serum half-life, targeting specific cells or tissues (eg, Fc acceptors) or antibody stability modification. The donor may have a functional moiety (eg, metal chelator, fluorescent molecule, antigen, oligonucleotide, biotin compound, or cell ligand).

Definition:
Before describing the present invention in detail, it should be understood that the present invention is not limited to a specific composition or process and may be varied as appropriate. Where the singular terms "a", "an", "the" are used in this specification and the appended claims, the plural forms unless the context clearly dictates otherwise. Is also included. That is, for example, the term “ligand” includes a plurality of ligands, and the term “antibody” includes a plurality of antibodies.

  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 this invention belongs. Terms used in this specification are defined as follows.

(Table 1) List of abbreviations

  As used herein, the term “acceptor” refers to a substituent capable of forming a bond with an oligosaccharide or monosaccharide containing a donor by an endoglycosidase catalyst. Preferably, the acceptor unit is a GlcNAc residue added to the antibody. Preferably, the acceptor unit is a GlcNAc that is targeted by Endo-M or Endo-A and is linked to a further sugar moiety prior to contact with Endo-M or End-A.

  As used herein, the term “donor” refers to a substituent capable of forming a bond with an oligosaccharide or monosaccharide acceptor by an endoglycosidase catalyst. Preferably, the donor unit is a GlcNAc residue added to asparagine. Preferably, the donor unit is a GlcNAc that is targeted to Endo-M or End-A and binds to an additional GlcNAc moiety prior to contact with Endo-M or End-A. A suitable donor has the structure Asn-GlcNAc-X, where X is an oligosaccharide or monosaccharide that is transferred to a target protein containing an acceptor.

  As used herein, the term “activated alkyne” refers to a chemical group that selectively reacts with an alkyne modifying group on another molecule to form a covalent bond between the alkyne modifying group and the alkyne reactive group. Point to. An example of an alkyne reactive group is azide. A “reactive alkyne” may also be a molecule having a chemical group that selectively reacts with an alkyne group.

As used herein, the term “affinity” refers to the degree of binding interaction between two molecules, eg, an antibody and an antigen, or a positively charged group and a negatively charged group. In the case of a bivalent molecule such as an antibody, affinity is typically defined as the binding force between the antigen and one binding region (eg, between the antigen and one Fab fragment). The binding force between both binding domains and the antigen is referred to as “avidity”. “High affinity” in the present invention means that a ligand binds to an antibody with an affinity constant (K _a ) greater than 10 ⁴ M ⁻¹ (usually 10 ⁵ to 10 ¹¹ M ⁻¹ ). Sex figures are measured by inhibition ELISA, or equivalent techniques (eg, Scatchard plot or K _d / dissociation constant (inversely proportional to K _a )).

  As used herein, the term “reactive alkyne” refers to a chemical group that selectively reacts with an alkyne modifying group on another molecule to form a covalent bond between the alkyne modifying group and the alkyne reactive group. Refers to that. An example of a reactive alkyne group is azide. A “reactive alkyne” may also be a molecule having a chemical group that selectively reacts with an alkyne group.

  The term “antibody” as used herein refers to an immunoglobulin molecule or a portion thereof having immunological activity (ie, an antigen-binding portion). Examples of immunologically active portions of immunoglobulin molecules include immunoglobulin molecules or fragments thereof, which are sufficient to retain an F (ab) site, and an oligosaccharide binding site (eg, an asparagine-GlcNAc binding site). Contains an Fc site. The antibody may optionally be a polyclonal, monoclonal, recombinant (eg, chimeric or humanized) fully human, non-human (eg, mouse), or single chain antibody. . The antibody may have an effector function, can fix the complement, and may be coupled with a toxin or imaging agent as required. The antibody is, for example, IgG.

The term “antibody fragment” as used herein refers to an antibody fragment that has major selective binding in a complete antibody. Specific fragments are known in the art (eg, Fab, Fab ′ and F (ab ′) ₂ (obtained by incubation with various proteases, pepsin or papain)) and lack the Fc fragment of a complete antibody. Or referred to as “half-molecule” fragments, which are obtained by reductively cleaving disulfide bonds that bind the heavy chain portion of a complete antibody. In addition, as such a fragment, an isolated fragment composed of a light chain variable region, an “Fv” fragment composed of a heavy chain and a light chain variable region, and a light chain and a heavy chain variable region are connected by a peptide linker. A recombinant single chain polypeptide molecule may be mentioned. Examples of other binding fragments include (i) Fd fragment consisting of VH and CH1 regions, (ii) dAb fragment (Ward, et al., Nature 341,544 (1989)) (consisting of VH region), (iii) single Isolated CDR regions, and (iv) single chain Fv molecules (scFv) (above). In addition, recombinant fragments can be used to generate any fragment that retains antigen recognition properties.

  As used herein, the term “antigen” refers to a molecule that induces or is capable of inducing the formation of an antibody, or a molecule to which an antibody selectively binds, including but not limited to biological material. As mentioned. Antigens are also referred to as “immunogens”. A target binding antibody selectively binds an antigen (used interchangeably herein with the term “target”).

  As used herein, the term “anti-region antibody” means an antibody obtained by immunizing an animal with a selected site (a fragment of an external antibody), and only that fragment is an immunogen. It is used as. Examples of the antibody region include an Fc site, a hinge region, and a Fab site. Anti-regional antibodies include monoclonal and polyclonal antibodies. As used herein, the term “anti-region fragment” means a monovalent fragment obtained by enzymatic treatment from the anti-region antibody of the present invention.

  As used herein, the term “aqueous solution” means a solution whose main component is water that retains the solution properties of water. Even if the aqueous solution contains a solvent other than water, typically water is the main solvent.

  As used herein, the term “azido-reactive” refers to a chemical group that selectively reacts with an azide modifying group on another molecule to form a covalent bond between the azide modifying group and the azide reactive group. Means that. Examples of azide reactive groups include alkynes and phosphines (eg triarylphosphine). “Azide-reactive” may also be a molecule that contains a chemical group that selectively reacts with an azide group.

  The term “buffer” as used herein refers to a system for minimizing changes in the acidity or basicity of a solution with respect to addition or deletion of chemicals.

  The term “carrier molecule” as used herein refers to a biological or non-biological component that is covalently linked to a compound of the invention. Such components include, but are not limited to, amino acids, peptides, proteins, polysaccharides, nucleosides, nucleotides, oligonucleotides, nucleic acids, haptens, psoralens, drugs, hormones, lipids, lipid assemblies, synthetic polymers, polymeric microparticles, biological cells, viruses And combinations thereof.

  As used herein, the term “chemical handle” refers to a specific functional group (eg, azide, alkyne, activated alkyne, phosphite, phosphine, etc.). The chemical handle is separate from the reactive group (defined below), the chemical handle is not present in naturally occurring biomolecules, and for biomolecules (eg, native cellular components) Refers to a chemical group that is chemically inert. However, when reacted with an azide or alkyne reactive group, the reaction is performed efficiently under biological reaction conditions (eg, conditions in cell culture in the absence of excess heat or strong reactants). Is called.

  As used herein, the term “click chemistry” refers to a reaction in which 1,2,4-triazole is formed by a Huisgen or 1,3-dipolar cycloaddition between an azide and a terminal alkyne. Point to. Such chemical reactions include, but are not limited to, by using a simple heteroatom organic reactant, it is possible to carry out a highly reliable, selective, stereospecific and exothermic reaction.

  As used herein, the term “cycloaddition” refers to a net reduction in bond diversity by combining two or more π-electron systems (eg, an unsaturated molecule or an unsaturated portion of the same molecule). It means a chemical reaction that forms a cyclic product. In the cycloaddition, a new π bond is formed by π electrons. The product of the cycloaddition is referred to as “adduct” or “cycloadduct”. Different types of cycloaddition are known in the art and include, but are not limited to, [3 + 2] cycloaddition and Diels-Alder reactions. [3 + 2] cycloaddition (also called 1,3-dipolar cycloaddition) occurs between 1,3-dipole and dipolar affinity substances and is typical during the formation of 5-membered heterocycles Used. The term “[3 + 2] cycloaddition” is also described in Bertozzi et al. Am. Chem. Soc. , 2004, 126: 15046-15047, including "copper free" [3 + 2] cycloadditions between azide and cyclooctyne derivatives and difluorocyclooctyne.

  As used herein, the term “detectable response” refers to the generation or change of a signal that can be detected directly or indirectly by observation or use of an instrument. Typically, the detectable reaction is the generation of a signal, characterized in that the fluorophore emits intrinsic fluorescence and does not immediately cause a change in signal upon binding to a metal ion or biological compound. . Alternatively, the detectable reaction may be a change in absorbance or fluorescence wavelength distribution pattern or intensity, or an optical response that results in light scattering, fluorescence duration, fluorescence polarization change, or a combination of the above parameters. . Other detectable reactions include, for example, chemiluminescence, phosphorescence, radiation from radioisotopes, magnetic force and electron density.

  As used herein, the term “detectably significant” refers to a signal whose physical properties can be identified or separated by observation or use of instruments. For example, fluorophores can be easily identified by spectral properties, fluorescence intensity, lifetime, polarization, or light decay rate due to other fluorophores in the sample and any additional material present.

  As used herein, the term “directly detectable” refers to the presence of a material, or the signal generated from the material does not require chemical modification or the presence of further substances, observation, instrumentation, or film. This refers to the state that can be easily detected.

As used herein, the term “fluorophore” refers to a composition that fluoresces inherently or a fluorogenic composition that exhibits a change in fluorescence upon binding to a biological compound or metal ion. Substituents that change the solubility, spectral properties, or physical properties of the fluorophore may be included in the fluorophore. Many fluorophores are known to those skilled in the art and include xanthenes such as coumarin, cyanine, benzofuran, quinoline, quinazolinone, indole, benzazole, borapolyazaindacene, fluorescein, rhodamine and rhodol, and RICHARD P. et al. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (9 th edition, CD-ROM, September 2002) , but like other fluorophores described, without limitation.

  The term “sugar modified” refers to a particle, such as a protein or an antibody, that has an altered number or configuration of attached sugar molecules. A preferred sugar-modified antibody is a therapeutic antibody derived from a non-human that has been humanized by modification of the sugar chain.

  As used herein, the term “glycoprotein” refers to naturally glycosylated proteins and proteins that have been enzymatically modified in vivo or in vitro to have sugar residues.

  As used herein, the term “humanized” refers to a protein (eg, antibody) that is less immunogenic or less likely to elicit an immune response in a human compared to non-humanized. Refers to the modification. Preferably, non-human proteins and antibodies are “humanized” by modification to be similar or identical to those expressed in humans.

  As used herein, the term “kit” means a set of related components, typically one or more compounds or compositions packaged.

  As used herein, the term “label” is directly or indirectly detectable (eg, with respect to its spectral properties, conformation, or activity) that binds to a target or compound and is used in the methods of the invention. Refers to chemical moieties or proteins, examples include reporter molecules, solid supports, carrier molecules, and the like. In the present invention, the label is used as a generic term for a reporter molecule, a solid support, or a carrier molecule. The label may be directly detectable (fluorophore) or indirectly detectable (hapten or enzyme). Such labels include, but are not limited to, radioisotopes for labels that can be measured with a radiation counter, dyes that can be visually observed or measured with a spectrophotometer, dyes or other dye sources, and spin labels that can be measured with a spin label analyzer. As well as fluorescent labels (fluorophores) (the output signal is generated by excitation of the appropriate molecular adduct, which can be visualized by excitation with light absorbed by the dye, or for example a standard fluorometer or Measurement by an imaging system). The label may be a chemiluminescent substrate, in which case the output signal is generated by a chemical modification of the signal compound, a metal-containing material or an enzyme, in which case an enzyme-dependent secondary signal (eg colored from a colorless substrate) Formation of material). The term label may also be a “tag” or hapten that can selectively bind to a conjugated molecule, which is characterized in that it generates a detectable signal when added subsequently with a substrate. . For example, using biotin as a tag, binding to the tag using horseradish peroxidase (HRP) avidin or streptavidin conjugate, and then a colorimetric substrate (eg tetramethylbenzidine (TMB)), or fluorescence A substrate (eg, Amplex Red reagent (Molecular Probe)) can be used to detect the presence of HRP. Many labels are known to those skilled in the art, for example, RICHARD P. et al. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORENCECENT PROBES AND RESEARCH PRODUCTS (10th edition, CD-ROM, September 2005), as well as for chemiluminescent substances, fluorophores, haptens, enzymes and colorimetric substrates that react with them However, it is not limited to these.

  The term “linker” or “L” of the present invention refers to a single covalent bond or a series of stable groups consisting of 1-30 non-hydrogen atoms selected from the group consisting of C, N, O, S and P. It means a covalent bond. Exemplary linker members include moieties such as —C (O) NH—, —C (O) O—, —NH—, —S—, —O—. A “cleavable linker” refers to a linker having one or more cleavable groups that can be cleaved as a result of a reaction or reaction conditions. The term “cleavable group” refers to a portion of a conjugate (eg, a reporter molecule, carrier molecule or solid support) from the conjugate residue by cleaving the bond between the conjugate residue and the release moiety. Refers to a group capable of releasing the body. Such cleavage may be chemical cleavage or enzymatic cleavage. Exemplary groups that are enzymatically cleavable include natural amino acid or peptide sequences having natural amino acids at their termini.

  In addition to enzymatically cleavable groups, one or more groups that are cleaved by reactions with substances other than enzymes are also included within the scope of the present invention. Typical non-enzymatic cleavage materials include, but are not limited to, acids, bases, light (eg, nitrobenzyl derivatives, phenacyl groups, benzoin esters) and heat. Many cleavable groups are known in the prior art. For example, Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983), Joshi et al., J. Biol. Biol. Chem. 265: 14518-14525 (1990), Zarling et al. Immunol. , 124: 913-920 (1980), Bouizar et al., Eur. J. et al. Biochem. 155: 141-147 (1986), Park et al., J. MoI. Biol. Chem. 261: 205-210 (1986), Browning et al., J. MoI. Immunol. 143: 1859-1867 (1989). In addition, universal cleavable bifunctional (homo- and heterobifunctional) spacer arms are commercially available.

  A typical cleavable group (ester) refers to a compound that can be cleaved by a chemical (eg, sodium hydroxide) to give a carboxylic acid-containing fragment and a hydroxyl-containing product.

  The terms “protein” and “polypeptide” are used herein in a general sense and encompass polymers of amino acid residues of any length. The term “peptide” is used herein as a term that refers to a polypeptide having less than 100 amino acid residues (typically less than 10 amino acid residues). The term encompasses not only natural amino acid polymers, but also amino acid polymers in which one or more amino acid residues are artificial analogs of the corresponding natural amino acids.

  As used herein, the term “purified” refers generally to a preparation of glycoprotein in a cell mixture or environment in which the protein or complex is endogenously present, such as serum protein or cell lysate. It refers to substantially free of contaminant proteins present in association with glycoproteins.

  As used herein, the term “reactive group” refers to a group that can react with other chemical groups to form a covalent bond (ie, cause a covalent reaction under appropriate reaction conditions), generally Means a group representing a binding site with another substance. In the present invention, a reactive group is a moiety that is generally present in a biological system and refers to a chemical group that reacts under normal biological conditions. And an activated alkyne moiety. The reactive group is, for example, a carboxylic acid or succinimidyl ester moiety and can react with a functional group present in a different compound to form a covalent bond. Reactive groups generally include nucleophiles, electrophiles and photoexcitable groups.

  The term “reporter molecule” refers to any moiety that can bind to a modified post-translationally modified protein of the present invention and that can be detected directly or indirectly. Reporter molecules include, but are not limited to, chromophores, fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, particles, haptens, enzymes, and radioisotopes. Suitable reporter molecules include fluorophores, fluorescent proteins, haptens and enzymes.

  As used herein, the term “sample” refers to any material containing an analyte of interest or a modified post-translationally modified protein of the present invention. The analyte may include a reactive group such as a group that can conjugate the compound of the invention to the analyte. Samples may also contain substances that are usually mixed with the target, such as diluents, buffers, surfactants, and impurities, debris, and the like. Examples include urine, serum, plasma, whole blood, saliva, tears, cerebrospinal fluid, nipple secretions, and the like. In addition, it may be a solid, gel, or sol substance such as mucus, body tissue, cells dissolved in a liquid material (eg, buffer, extract, solvent, etc.). Typically, samples are living cells, biological fluids containing endogenous host cell proteins, nucleic acid polymers, nucleotides, oligonucleotides, peptides and buffer solutions. The sample may be present in an aqueous solution, may be a viable cell culture, or may be immobilized on a solid or semi-solid surface such as a polyacrylamide gel, blot membrane or microarray.

  As used herein, the term “solid support” is substantially insoluble in a selected solvent system or can be normally sequestered (eg, precipitated) from a given solvent system in which it is dissolved. ) Refers to the material. The term solid support includes semi-solid supports. A solid support useful in the practice of the present invention may have a group activated or capable of being activated to bind a given molecular species to the solid support. The solid support may exist in various forms (chips, wafers, wells, etc.), and individual or multiple compounds of the invention may be bound to, for example, polymer beads or particles.

  As used herein, the term “Staudinger binding reaction” refers to a chemical reaction developed by Saxon and Bertozzi (E. Saxon and C. Bertozzi, Science, 2000, 287: 2007-2010), It refers to a modified reaction of the classic Staudinger reaction. The classical Staudinger reaction is a chemical reaction in which a combination of phosphine or phosphite and an azide yields an aza-ylide intermediate that yields phosphine oxide and amine upon hydrolysis. The Staudinger reaction is a mild method for reducing an azide to an amine, and triphenylphosphine is usually used as the reducing agent. In the Staudinger ligation reaction, an electrophilic trap (usually a methyl ester) is appropriately placed on a triarylphosphine (usually ortho to the phosphorus atom) and reacted with an azide to prepare an aza-ylide intermediate. Further, it is prepared in an aqueous solvent to obtain a compound having an amide group and a phosphine oxide functional group. Although the Staudinger ligation reaction has such a name because the two initiating molecules bind (contact / covalently bond), in the classic Staudinger reaction, the two products do not covalently bond after hydrolysis.

  As used herein, the terms “the integrity of the structure of [biomolecule] is not impaired” or “the integrity of the structure of [biomolecule] is maintained” are: 1) gel electrophoresis And, when analyzed by detection (for example, staining), the intensity of the band or spot derived from the labeled biomolecule is not decreased by 20% or more, preferably 10% or more (derived from the labeled biomolecule to be analyzed) Compared to the corresponding band or spot from the same amount of electrophoresed unlabeled biomolecule), or 2) the band or spot from the labeled biomolecule when analyzed by gel electrophoresis Is significantly more obscured than the corresponding band or spot from the same amount of electrophoresed unlabeled biomolecule. Here, “remarkably obscured” (synonymous with “remarkably diffused”) means that the detectable band or spot is at least more than 5%, preferably 10% greater than the corresponding unlabeled biomolecule. It means that it appears in the gel with an area of more than%, more preferably more than 20%. Other reproducible test methods for structural integrity of labeled biomolecules include, but are not limited to, detection of released amino acids or peptides or mass spectrometry.

  An antibody that is “synthesized” in a cell refers to an antibody that is naturally produced and isolated in the cell or that has been synthesized and further isolated in a recombinant manner in the cell.

  As used herein, the term “about” means within 10% (ie ± 10%), within 5% (ie ± 5%), within 2.5% (ie ± 2.5%) of the subsequent parameter value. ), Or a term used to include a numerical value within 1% (that is, ± 1%), and in some cases, it may be a parameter having no fluctuation range. That is, a length of “about 20 nucleotides” may be 19 or 21 nucleotides long (ie 5% variation), or in some embodiments 20 nucleotides long (ie no variation). There can be. In the present invention, when the term “one” is used, one or more subsequent elements may be included (for example, in a protein microarray, one protein includes one protein sequence or a plurality of proteins. sell). The term “or” is not limited to the one or term it refers to. For example, the structure “A or B” can mean A alone, B alone, or both A and B.

  Usually, metabolic and enzymatic labeling of biomolecules with azide moieties, alkyne moieties or phosphine and azide reactive moieties, and reactive alkyne moieties or phosphine reactive moieties to facilitate understanding of the present invention. The chemical labeling of the moiety is first detailed. Subsequently, some examples are shown that show that such labeled biomolecules can be detected, separated and / or analyzed. An exemplary method is shown below.

The method of the present invention:
Preparation of antibodies and antibody-producing cells:
Antibodies used for cleavage and substitution of the oligosaccharides of the present invention can be prepared using any means known to those skilled in the art. General information regarding procedures for antibody production and labeling can be found, for example, in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chap. 14 (1988). Cell lines expressing antibodies for metabolic labeling in vivo can be prepared using any means known to those skilled in the art. For therapeutic applications, when administered frequently to patients, chimeric antibodies, humanized antibodies, and fully human antibodies are preferred. Chimeric and humanized monoclonal antibodies (including portions derived from human and non-human) can be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be prepared by known recombinant DNA techniques and may be performed, for example, using methods described in the following literature: Robinson et al., PCT / US86 / No. 02269, Akira, et al., European Patent Application Publication No. 184,187, Taniguchi, M .; , European Patent Application Publication No. 171,496, Morrison et al., European Patent Application Publication No. 173,494, Ncubrgcr et al., International Publication No. 86/01533, Cabilly et al., US Pat. No. 4,816,567, Cabilly et al. European Patent Application Publication No. 125,023, Better et al., Science 240: 1041-1043 (1988), Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443 (1987), Liu et al., J. Biol. Immunol 139: 3521-3526 (1987), Sun et al., Proc. Natl. Acad. Sci. USA 84: 214-218 (1987), Nishimura et al., Canc. Res. 47: 999-1005 (1987), Wood et al., Nature 314: 446-449 (1985), and Shaw et al., J. Biol. Natl. Cancer Inst. 80: 1553-1559 (1988), Morrison, S .; L. , Science 229: 1202-1207 (1985), Oi et al., BioTechniques 4: 214 (1986), Winter US Pat. No. 5,225,539, Jones et al., Nature 321: 552-525 (1986), Verhoeyan et al., Science. 239: 1534, and Beidler et al., J. MoI. Immunol. 141: 4053-4060 (1988).

  Transgenic mice that are unable to express endogenous immunoglobulin heavy and light chain genes but are capable of expressing human heavy and light chain genes can be used to prepare human antibodies according to the present invention. (Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 (1995) and U.S. Pat. Nos. 5,625,126, 5,633,425, 5,569,825, No. 5,661,016 and 5,545,806). In addition, Invitrogen (Carlsbad, Calif.), Abgenix, Inc. Companies such as (Fremont, California) and Medarex (Princeton, NJ) outsource human antibodies that react with specific antigens using techniques similar to those described above. Human antibodies that recognize selected epitopes can also be prepared using a technique called “guide selection”. In this method, selected non-human monoclonal antibodies (eg, mouse antibodies) are used to guide select human antibodies that recognize the same epitope. This technique is described, for example, in Jespers et al., Bio / Technology 12: 899-903 (1994). It is described in.

Glycoprotein:
The glycoprotein component of the present invention may be any glycoprotein, such as hormones, enzymes, antibodies, virus receptors, viral surface glycoproteins, parasitic glycoproteins, parasitic receptors, T cell receptors, MHC molecules , Immunomodulator, tumor antigen, mucin, inhibitor, growth factor, trophic factor, lymphokine, cytokine, toxoid, nerve growth hormone, blood coagulation factor, adhesion molecule, multidrug resistance protein, adenylate cyclase, bone morphogenetic protein and lectin Etc.

  In addition, glycoproteins include hormones and cytokines. Examples of hormones include follicle stimulating hormone, human chorionic gonadotropin, luteinizing hormone, thyroid stimulating hormone, and sheep, bovine, porcine, mouse and rat equivalents of these hormones. Examples of cytokine glycoproteins include α-interferon, lymphotoxin and interleukin-2. Furthermore, tumor-associated antigens of glycoproteins such as fetal cancer antigen (CEA), human mucin, her-2 / neu and prostate specific antigen (PSA) [RA Henderson and OJ Finn, Advances in Immunology. 62, pp. 217-56 (1996)]).

  Alternatively, the glycoprotein component may be selected from personal care glycoproteins, facial glycoproteins, veterinary glycoproteins, food glycoproteins, feed glycoproteins, diagnostic glycoproteins, sugars used in chemical reactions Examples include proteins, industrial glycoproteins, detergent glycoproteins (including surfactant glycoproteins and contaminant-free glycoproteins). Such glycoproteins include enzymes such as hydrolases, transferases, isomerases, lyases, ligases, transferases and oxidoreductases. Examples of hydrolases include lipase, cholinesterase, alkaline phosphatase, β-amylase, deoxyribonuclease, glucoamylase A and B, α-galactosidase I and II, β-fructofuranosidase, β-glucuronidase, N-acetyl-β- Examples include glucosaminidase, hyaluronidase, oxytocinase, kallikrein, bromelain, enterokinase, proteinases a, b and c, pepsinogen and pepsin. Examples of oxidoreductases include glucose oxidase, peroxidase and chloroperoxidase. Examples of transferases include γ-glutamyl transpeptidase and ribonuclease.

  Additional glycoproteins encompassed for the present invention include cross-linked glycoproteins (for example, as described in US Pat. No. 6,359,118, the contents of which are incorporated).

In vitro modification of IgG oligosaccharides Oligosaccharides bind to asparagine residues on immunoglobulin gamma (IgG) antibodies by N-acetylglucosamine disaccharide linkages (GlcNac-GlcNac). Generally, each IgG has two sites for the oligosaccharide to bind to GlcNac-GlcNac near the IgG neck. Endoglycosidase treatment (eg, endoglycosidase H (endoH)) cleaves between two GlcNac sugars. The cut end of each sugar is called the “reducing end”. The oligosaccharide having the reducing end GlcNAc is obtained by cleaving the oligosaccharide from the first IgG and collecting the cleaved oligosaccharide. Next, the second IgG is treated with an endoglycosidase (eg, Endo H). Next, the second IgG (having GlcNAc having a reducing end) is recovered. Next, the reducing end of the GlcNAc residue bound to the first oligosaccharide and the reducing end of the GlcNAc residue oligosaccharide bound to the second IgG are treated, and the first oligosaccharide is treated with the second oligosaccharide. Bind to IgG.

  One method for binding the first oligosaccharide is, for example, a method of chemically conjugating the first oligosaccharide to alkyne. For example, the reducing end can be treated with ammonium bicarbonate to convert it to an amine, and the alkyne can be attached via a succinimidyl ester. An azido modified sugar is then conjugated to the reducing end of a GlcNAc residue on a second IgG such as UDP-GalNAz. This transfer reaction can be performed using, for example, a mutant galactosyltransferase. The azide labeled IgG is then mixed with the alkyne labeled oligosaccharide. The alkyne and azide moieties are subjected to a cycloaddition reaction, whereby oligosaccharides derived from the first IgG and oligosaccharides on the second IgG are linked.

Oligosaccharide removal from IgG Oligosaccharides bind to IgG via asparagine residues on the Fc portion of the antibody (FIG. 1B). At the amino acid position, the two GlcNAc sugars are each β (1-4) linked. The enzyme endo-H cleaves this bond, so that one GlcNAc residue remains bound to asparagine on IgG, while the other GlcNac remains bound to the remaining oligosaccharide. GlcNAc that binds to an oligosaccharide has a reactive reducing end that can be selectively modified without altering other sugar residues.

Azide Binding to IgG GlcNAc The enzyme galactosyltransferase typically transfers galactose from UDP-galactose to a terminal GlcNAc residue. Kidekel et al. (J. Am. Chem. Soc. 2003, 125: 16162-16163, Hsieh-Wilson, L., et al., U.S. Patent Application Publication No. 20050130235, published June 16, 2005, U.S. Patent Application No. 10/990. , No. 767) transfer an acetone-containing galactose substrate to a GlcNAc residue using a mutant enzyme (Y289L mutant). An azide-containing galactose substrate (UDP-GalNAz) can be synthesized by transfer to the GlcNAc site by a mutant galactosyltransferase.

Binding of alkyne to cleaved oligosaccharide reducing end The reducing end of a sugar is a reactive site that can be selectively modified. Some sugars (eg sucrose) do not have a reducing end and are more stable than sugars with a free reducing end. Cleavage of the GlcNAc-GlcNAc bond by the enzyme endo-H exposes the reducing end of the oligosaccharide GlcNAc.

  Using the method of Tamura et al. (Analytical Biochem. 1994, 216: 335-344), the reducing end of an N-linked oligosaccharide is reacted with ammonium bicarbonate to convert it to an amine, thereby obtaining an oligosaccharide-glycosylamine. This reaction can be carried out using methods known to those skilled in the art, for example, using a 10 ml screwed glass vial in the presence of about 10 μmol of water in the presence of about 1 g ammonium bicarbonate / about 0.5 ml water. This can be done by mixing the oligosaccharides and incubating the mixture at about 50 ° C. for about 24 hours. The resulting oligosaccharide-glycosylamine may be conjugated with a fluorophore, for example by reacting with an acylating agent, or conjugated with an N-glycyl linker, thereby allowing other biological or A biophysical probe can be received.

For example, Boc protected tyrosine succinimidyl ester can be used to couple tyrosine to an amine. Boc protection is performed to avoid polymerization of the tyrosine succinimidyl ester. An alkyne-succinimidyl ester is thereby obtained and can be used for the conjugation reaction. When bound to tyrosine residues, oligosaccharides may be quantified at A280 _nm .

Glycoprotein labeling using a [3 + 2] cycloaddition reaction:
The azide and terminal alkyne undergo a cycloaddition reaction in the presence of a copper catalyst. This cycloaddition reaction can be performed using, for example, the method of Sharpless et al. (US Patent Application Publication No. 200502224427 (published October 6, 2005, PCT / US03 / 17311, Lewis WG, et al., Angelwandte Chemie-Int). '1 Ed. 41 (6): 1053, method reviewed in Kolb, HC, et al., Angew. Chem. Inst. Ed. 2001, 40: 2004-2021), the reaction yields a high yield, and It is possible to change the reagents to react without causing side reactions of heteroatom bonds (not carbon-carbon bonds) and prepare a library of chemicals in the presence of metal catalysts and reducing agents. For example, Cu (II) is contained in the reaction system and a reducing agent ( Example, if ascorbic acid, metallic copper, quinone, hydroquinone, vitamin _{K 1,} glutathione, ^cysteine, Fe 2+, in the presence of ^{Co 2+),} may apply a potential. As a further suitable reducing agent is, Al, Be, Examples thereof include metals selected from the group consisting of Co, Cr, Fe, Mg, Mn, Ni, and Zn Examples of other metals that can catalyze the cycloaddition reaction include Au, Ag, Hg, Cd, Zr, and Ru. Fe, Co, Pt, Pd, Ni, Rh and W. One skilled in the art can determine the appropriate metal depending on the use of the ligand of interest.

In vitro modification of IgG using endo-M or endo-A Endo-M (or endo-A) is an endoglycosidase with glycosyltransferase activity. Similar to Endo H, the enzyme cleaves the disaccharide GlcNAc-GlcNAc between the two residues. Endo M has further enzymatic activity, i.e., the enzyme cleaves oligosaccharides with GlcNAc residues and then attaches the reducing end of GlcNAc residues to OH groups. That is, another method of the invention relates to providing a method for in vitro labeling of alkynes to cleaved oligosaccharides. In one embodiment, IgG with an oligosaccharide having a GlcNAc-GlcNAc linkage is incubated with OH-alkyne in the presence of endo-M.

  One aspect of the present invention relates to the provision of methods for preparing sugar-modified antibodies comprising at least one monosaccharide or oligosaccharide. The method comprises the steps of contacting an antibody, endo-M or endo-A, and a donor having a monosaccharide or oligosaccharide to prepare a contact solution, wherein the antibody has an acceptor, and the contact solution Incubating for a sufficient time to form a covalent bond between the acceptor and the monosaccharide or oligosaccharide, and obtaining a sugar-modified antibody.

  In a further embodiment, the antibody is derived from non-human. More specifically, the antibody is derived from mouse cells, plant cells, goat cells, rabbit cells, yeast or CHO cells. In other embodiments, the sugar-modified antibody is humanized.

  In another embodiment, the acceptor is N-acetylglucosamine (hereinafter GlcNAc). In other embodiments, the antibody contacts endo-M. In other embodiments, the antibody contacts Endo-A. In other embodiments, the donor has the sequence: Asn-GlcNAc-X, where X is an oligosaccharide or monosaccharide. More specifically, X is -GlcNAc- (oligosaccharide or monosaccharide).

  In other embodiments, alkynyl or azide functional groups are present on the monosaccharide or oligosaccharide. In other embodiments, a chemical handle is present on the monosaccharide or oligosaccharide.

  In other embodiments, the reporter molecule is present in a monosaccharide or oligosaccharide. More specifically, the reporter molecule is a fluorescent dye, enzyme, radiolabel, metal chelator or detectable substrate.

  In other embodiments, the monosaccharide or oligosaccharide contains a carrier molecule, solid support, receptor, ligand, peptide, protein, nucleic acid polymer, polysaccharide, antigen, or hapten.

  In another embodiment, the step of purifying the sugar-modified antibody is included in the preparation step. More specifically, after purification, the sugar-modified antibody is mixed with an excipient, diluent or pharmacologically acceptable salt. More specifically, the sugar-modified antibody is administered to the patient after mixing with an excipient, diluent or pharmacologically acceptable salt.

  In other embodiments, the sugar-modified antibody is a therapeutic antibody. More particularly, the therapeutic antibody is selected from the group consisting of trastuzumab, cetuximab, bevacizumab, alemtuzumab, gemtuzumab, ibritumomab, rituximab, and panitumumab.

  In other embodiments, the presence of the mono- or oligosaccharide affects the serum half-life of the sugar-modified antibody, a specific cell or tissue is the target of the sugar-modified antibody, or the stability of the sugar-modified antibody Affects sex.

  In other embodiments, the contacting step is performed in an aqueous solution. In other embodiments, the contacting step is performed in an organic solution. In other embodiments, the contacting step is performed in a protic solution. In other embodiments, the contacting step is performed in a polar solution. In other embodiments, the contacting step is performed in a solvent that is not a ketone or furan.

  In other embodiments, the sugar-modified antibody comprises at least one oligosaccharide.

  In other embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

Another aspect of the invention relates to providing a method for covalently attaching a monosaccharide or oligosaccharide to an antibody. The method is
Contacting an antibody with endo-M or endo-A and a donor having a monosaccharide or oligosaccharide to prepare a contact solution, the antibody having an acceptor;
Incubating the contact solution for a sufficient time to form a covalent bond between the acceptor and the monosaccharide or oligosaccharide.

Another aspect of the invention relates to providing a method for humanizing a therapeutic antibody. The method is
Contacting the therapeutic antibody with endo-M or endo-A and a donor having a monosaccharide or oligosaccharide to prepare a contact solution, the antibody having an acceptor;
Incubating the contact solution for a sufficient time to form a covalent bond between the acceptor and the monosaccharide or oligosaccharide, and obtaining a humanized therapeutic antibody.

  In other embodiments, the therapeutic antibody is a monoclonal antibody.

  In other embodiments, the therapeutic antibody is selected from the group consisting of trastuzumab, cetuximab, bevacizumab, alemtuzumab, gemtuzumab, ibritumomab, rituximab and panitumumab.

Another aspect of the invention relates to the provision of a kit for the preparation of a detectable sugar-modified antibody. The kit is
(A) End-M or End-A;
(B) a donor containing oligosaccharides or monosaccharides;
(C) Includes instructions for preparing detectable sugar-modified antibodies.

  In other embodiments, the oligosaccharide or monosaccharide comprises a reporter group, azide group or alkynyl group.

  In other embodiments, the reporter molecule is a fluorescent dye, enzyme, radiolabel, metal chelator, or detectable substrate.

Another aspect of the invention relates to providing a composition comprising:
(A) an antibody containing an acceptor;
(B) End-M or End-A;
(C) a donor comprising an oligosaccharide or monosaccharide;
(D) Solvent.

Metabolic labeling of antibodies in antibody-producing cells Using in vivo metabolic labeling methods, labeling antibodies in antibody-producing cells (eg, hybridoma cells and other cells that produce antibodies or recombinant antibodies) it can. Antibody-producing cells can take up non-natural sugar substrates (eg, non-natural sugar substrates with reactive compounds / affinity handles). Chemical handles include, but are not limited to, for example, azide, triarylphosphine, or alkyne residues, eg, groups that can participate in “click” type chemical reactions. In a specific embodiment, it is preferred that the unnatural sugar has a modification that is sufficiently small for efficiency of uptake into cells. The substrate is incorporated into an N-linked glycan or an O-linked glycan bound to an antibody by an intracellular metabolic mechanism. After the in vivo labeled antibody is released from the cells and separated, the labeled antibody may be directly labeled using a detection / affinity / immobilization compound that is reactive with the chemical handle. These compounds include, but are not limited to, fluorophores, solid support resins, microarray slides, or affinity tags.

Metabolic labeling of antibodies Cell surface glycoproteins can be modified by incubating antibody-producing cells in the presence of unnatural sugars (see, eg, US Pat. No. 6,936,701 (Bertozzi et al.)). Can be used to incubate cells in the presence of unnatural sugars). For example, the cells are incubated with unnatural sugar (about 20 mM) for about 72 hours.

Synthesis of Non-Natural Sugar Substrates Non-natural sugar substrates that can incorporate reactive chemical handles can be synthesized and used in click chemistry reactions. Using azide / alkyne cycloaddition reactions, affinity probes (biotin), dyes, polymers (eg, poly (ethylene glycol) or polydextran), or other monosaccharides (eg, glucose, galactose, fucose, O-GlcNAc, appropriate A mannose-derived sugar having a simple chemical handle). In specific embodiments, these handles include, for example, azide, triarylphosphine, or alkyne residues. The chemical handle may also be a Staudinger-reactive azide group (see Saxon, E., et al., J. Am. Chem. Soc., 2002, 124 (50): 14893-14902). The Staudinger reaction involves a reaction between a trivalent phosphorus compound and an organic azide (Staudinger et al., Hclv. Chim. Acta 1919, 2: 635) and is used in many applications (Gololobov et al., Tetrahedron 1980). , 37, 437), (Gololobov et al., Tetrahedron 1992, 48, 1353). The phosphine may have an adjacent acyl group (eg, ester, thioester, or N-acylimidazole) (ie, phosphinoether, phosphinothioether, phosphinoimidazole), thereby trapping the aza-ylide intermediate; Forms a stable amide bond upon hydrolysis. The phosphine may typically be a diaryl phosphine or a triaryl phosphine in the sense of stabilizing the phosphine.

  For example, non-natural sugar substrates that can be incorporated in vivo in the present invention include, but are not limited to, GalNAz, ManNaz and GlcNAz. Non-natural sugar substrates include, for example, sugar substrates containing small sugar groups, which are taken up by cellular mechanisms and are less likely to be recognized as foreign substances.

Labeling of cleaved oligosaccharides and in vivo IgG labeling Various labels or tags (reporter molecules, solid support and carrier molecules) are linked or conjugated to the in vitro cleaved oligosaccharides of the invention, which are further according to the invention. It may be bound to cleaved IgG. These labels or tags may be linked to in vivo sugar-modified IgG isolated from the cells after metabolic labeling and labeled or tagged, the label or tag incorporated between metabolic labels It may be coupled with a chemical / affinity handle on the non-natural sugar.

  The label or tag may be, for example, a therapeutic moiety (eg, cytotoxin, therapeutic agent or radioactive metal ion). The cytotoxin or cytotoxin agent can be any agent that is detrimental to cells. For example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mitramycin, actinomycin D, 1- Examples thereof include dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof. Therapeutic agents include antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (mechloretamine, thiotepachlorambucil, melphalan, carmustine (BCNU) and Lomustine (CCNU), cyclic phosphate amide, busulfan, dibromomannitol, strcptozotocin, mitomycin C and cis-dichlorodiamineplatinum (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly Daunomycin) and doxorubicin), antibiotics (eg dactinomycin (formerly actinomycin), bleomycin, mitramycin and anthramycin (AMC)), side by side Antimitotic agents (e.g. vincristine and vinblastine) include, but are not limited to.

  Antibody conjugates may be used to modulate a given biological response. For example, DNA or protein or polypeptide having a desired biological activity may be provided as a drug moiety. Examples of such proteins include toxins (eg, abrin, castor venom A, Pseudomonas exotoxin or diphtheria toxin), polypeptides (eg, tumor necrosis factor, γ-interferon, α-interferon, nerve growth factor, platelet-derived growth factor, tissue plus) Minogen activator) or modulators of biological responses (eg lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL- 6 ") granulocyte-macrophage colony-stimulating factor (" GM-CSF "), granulocyte colony-stimulating factor (" G-CSF ") or other growth factors). Alternatively, an antibody heteroconjugate may be prepared by conjugating an antibody with a second antibody, as described, for example, in US Pat. No. 4,676,980 by Segal.

  The label or tag can also be a detectable label, eg, for diagnostic or research purposes, or it can bind other biomolecules or reagents to antibodies (eg, biotin / avidin binding or microarray chips, beads, or plates). It may be a tag that binds an antibody). Examples of such labels or tags include fluorescent dyes (eg fluorescein (FITC), Oregon Green 488 dye, Marina Blue dye, Pacific Blue dye), and Texas Red-X dye, Alexa Fluor dye (Invitrogen, Carlsbad, CA). ), Radioisotope-containing compounds, light scattering compounds (eg containing gold or silver), dyes, photogenerating compounds (eg luciferase), haptens (eg biotin, desthiobiotin, DSB-X biotin and dinitrophenol (DNP)) Enzymes (eg horseradish peroxidase (HRP), alkaline phosphatase and [β] -lactamase), phycobilin proteins (eg R-phycoerythrin (R-PE), allophycocyanin (AP)), Include, but are not limited to, particles (eg, Qdots, gold, ferrofluid, dextran and microspheres).

Reporter molecule:
The reporter molecule of the present invention may be any directly or indirectly detectable reporter molecule known to those skilled in the art that can bind to the modified glycoprotein of the present invention. Such reporter molecules include, but are not limited to, chromophores, fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, particles, haptens, enzymes, and radioisotopes. Suitable reporter molecules include fluorophores, fluorescent proteins, haptens, and enzymes.

  The fluorophores of the present invention can be any chemical group that exhibits an absorption maximum of greater than 280 nm and maintains its spectral properties when covalently bound to a labeling reagent. Fluorophores of the present invention include, but are not limited to: pyrene (including all corresponding derivatives disclosed in US Pat. No. 5,132,432), anthracene, naphthalene, acridine, Stilbene, indole or benzindole, oxazole or benzoxazole, thiazole or benzothiazole, 4-amino-7-nitrobenz-2-oxa-1, 3-diazole (NBD), cyanine (US patent application Ser. No. 09 / 968,401) And all corresponding compounds described in US patent application Ser. No. 09 / 969,853), carbocyanine (US patent application Ser. Nos. 09 / 557,275, 09 / 969,853, and 09). / 968,401, U.S. Pat. Nos. 4,981,977, 5,268,4 No. 6, No. 5,569,587, No. 5,569,766, No. 5,486,616, No. 5,627,027, No. 5,808,044, No. 5,877,310, 6,002,003, 6,004,536, 6,008,373, 6,043,025, 6,127,134 No. 6,130,094, No. 6,133,445, and WO02 / 26891, WO97 / 40104, WO99 / 51702, WO01 / 21624, EP1065250A1), carbostyril, porphyrin, salicylic acid, anthranilate , Azulene, perylene, pyridine, quinoline, borapolyazaindacene (US Pat. Nos. 4,774,339, 5,187,288, 5,248,7) 2, any of the corresponding compounds disclosed in US Pat. Nos. 5,274,113 and 5,433,896), xanthene (US Pat. Nos. 6,162,931, 6,130). , 101, 6,229,055, 6,339,392, 5,451,343, and all corresponding disclosures in US patent application Ser. No. 09 / 922,333. Compound), oxazine (including all corresponding compounds disclosed in US Pat. No. 4,714,763) or benzoxazine, carbazine (including all corresponding compounds disclosed in US Pat. No. 4,810,636). Compounds), phenalenone, coumarin (US Pat. Nos. 5,696,157, 5,459,276, 5,501,980, and 5,8). Including the corresponding compounds disclosed in US Pat. No. 30,912), benzofurans (including corresponding compounds disclosed in US Pat. Nos. 4,603,209 and 4,849,362), benzphenalenone (including Including any corresponding compounds disclosed in US Pat. No. 4,812,409), and derivatives thereof. In the present invention, oxazines include resorufin (including all corresponding compounds disclosed in US Pat. No. 5,242,805), aminooxazinones, diaminooxazines and their benzo-substituted analogs.

  When the fluorophore is xanthene, the fluorophore is optionally fluorescein, rhodol (US Pat. Nos. 5,227,487 and 5,442,045, all corresponding compounds also disclosed). Or rhodamine (including all corresponding compounds disclosed in US Pat. Nos. 5,798,276, 5,846,737, US 09 / 129,015). Also good. In the present invention, the fluorescein may be benzofluorescein or dibenzofluorescein, seminaphthofluorescein or naphthofluorescein. Similarly, in the present invention, rhodol may be seminaphtho rhodofluor (including all corresponding compounds disclosed in US Pat. No. 4,945,171). Alternatively, the fluorophore may be xanthene having a bond through a single covalent bond present at the 9-position of xanthene. Suitable xanthenes include 3H-xanthen-6-ol-3-one derivatives having a bond at the 9-position, 6-amino-3H-xanthen-3-one derivatives having a bond at the 9-position, or 9-position. And a 6-amino-3H-xanthene-3-imine derivative having a bond.

  Suitable fluorophores of the present invention include xanthene (rhodol, rhodamine, fluorescein and its derivatives), coumarin, cyanine, pyrene, oxazine, and borapolyazaindacene. Sulfonated xanthene, fluorinated xanthene, sulfonated coumarin, fluorinated coumarin, and sulfonated cyanine are optimal. The choice of fluorophore to be bound to the labeling reagent determines the absorption spectrum and fluorescence emission characteristics of the labeling reagent and the immunolabeling complex. The physical properties of fluorophore labels include spectral properties (absorption, emission and stalk shift), fluorescence intensity, duration, polarity, and photobleaching rate, all of which are used to distinguish fluorophores from each other. can do.

  Typically, the fluorophore has one or more aromatic or aromatic heterocycles and various substituents (including but not limited to halogen, nitro, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl). , Cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring structures, benzo or other substituents typically present in known fluorophores, etc.).

  In one aspect of the invention, the fluorophore has an absorption maximum greater than 480 nm. In particularly useful embodiments, the fluorophore has an absorption spectrum at or near 488 nm to 514 nm (especially suitable for excitation by an argon-ion laser excitation source) or near 546 nm (especially suitable for excitation by a mercury arc lamp). .

  Many fluorophores can also function as chromophores, so that the fluorophores described above are also preferred chromophores of the present invention.

  In addition to the fluorophore, enzymes are also used as labels for detection reagents. Enzyme labels are desirable labels because they can amplify a detectable signal and improve test sensitivity. The enzyme itself does not produce a detectable reaction when contacted with an appropriate substrate, but has the function of destroying the substrate, whereby the converted substrate produces a fluorescent or colorimetric or luminescent signal. Since one enzyme on the labeling reagent can bind to multiple substrates that change to a detectable signal, the enzyme can amplify the detectable signal. This is advantageous when the amount of target present in the sample is small, or when there is no fluorophore that gives a signal similar or stronger than the enzyme. However, fluorophores are most preferred because they do not require additional assay procedures, thereby reducing the overall time required to perform the assay. It is preferred to select an enzyme substrate that produces a suitable measurable product (eg colorimetric, fluorescent or chemiluminescent). Such substrates are widely used, many of which are described in the above MOLECULAR PROBES HANDBOOK.

  A suitable colorimetric or fluorescent generating substrate and enzyme combination includes an oxidoreductase (eg, horseradish peroxidase) and a substrate (eg, 3,3′-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC). ) (Resulting in characteristic colors (brown and red, respectively)) Other colorimetric oxidoreductase substrates that produce detectable products include, but are not limited to: Not: 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3 ′, 5,5′-tetramethylbenzidine (TMB), o-Dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol. However, homovanillic acid or 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazine and reduced benzothiazine (Amplex® Red reagent and its derivatives (US Pat. No. 4,384,042), Amplex UltraRcd and its derivatives (WO050504504)), and reduced dihydroxanthene (including dihydrofluorescein (US Pat. No. 6,162,931)), dihydrorhodamine (including dihydrorhodamine 123), etc. Tyramide (US Pat. No. 5,196). , 306, 5,583,001, and 5,731,158) (peroxidase substrates) are unique as peroxidase substrates, ie, may be endogenously detectable prior to enzyme activity. But tyramide It is unique because it is “fixed in place” by the action of peroxidase in a process called signal amplification (TSA), and these substrates are used to target and label samples such as cells, tissues, or arrays. Then they are extensively detected by microscopy, flow cytometry, optical scanning and fluorometric analysis.

  In other suitable colorimetric (optionally fluorescent) substrate and enzyme combinations, a phosphatase enzyme such as acid phosphatase, alkaline phosphatase or a recombinant of such phosphatase and a colorimetric substrate (eg 5-bromo-6-chloro) In combination with 3-indolyl phosphate (BCIP), 6-chloro-3-indolyl phosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenyl phosphate, or o-nitrophenyl phosphate) Or 4-methylumbelliferyl phosphate, 6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, US Pat. No. 5,830,912), fluorescein diphosphate, phosphoric acid 3 -O-methylfluorescein, phosphorus Resorufin, 9H- (1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (phosphate DDAO) or ELF97, ELF39, or related phosphates (US Pat. No. 5,316,906) And a combination with a fluorescent substrate such as No. 5,443,986).

  Glycosidases (especially β-galactosidase, β-glucuronidase and β-glucosidase) are further suitable enzymes. Suitable colorimetric substrates include 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) and similar indolyl galactosides, glucosides and glucuronides, o-nitrophenyl β-D. -Galactopyranoside (ONPG) and p-nitrophenyl β-D-galactopyranoside and the like are included, but are not limited thereto. Suitable fluorescent substrates include resorufin β-D-galactopyranoside, fluorescein digalactoside (FDG), fluorescein diglucuronide and their structural isomers (US Pat. Nos. 5,208,148 and 5,242). 805, 5,362,628, 5,576,424, and 5,773,236), 4-methylumbelliferyl β-D-galactopyranoside, carboxyumbellide And ferryl β-D-galactopyranoside and fluorinated coumarin β-D-galactopyranoside (US Pat. No. 5,830,912).

  Additional enzymes include, but are not limited to, hydrolases such as cholinesterase and peptidase, oxidases and reductases such as glucose oxidase and cytochrome oxidase, for which suitable substrates are known.

  Enzymes that produce chemiluminescence and their appropriate substrates are suitable in some assays. Examples include, but are not limited to, natural and recombinant luciferases and aequorin. In addition, chemiluminescent substrates for phosphatases, glycosidases and oxidases containing stable dioxetanes, luminols, isoluminols and acridinium esters are also useful.

  In addition to enzymes, haptens such as biotin are also suitable labels. Biotin is useful because it has the function of amplifying a detectable signal in an enzyme system, and it can also function as a tag, so it can also be used for affinity chromatography. For detection purposes, biotin-affinity enzyme conjugates such as avidin-HRP are used. A peroxidase substrate is then added to produce a detectable signal.

  Haptens also include hormones, natural and synthetic drugs, contaminants, allergens, influencing factors, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, intermediates, nucleotides, and the like.

  Fluorescent proteins can also be used as labeling substances for the labeling reagents of the present invention. Examples of fluorescent proteins include green fluorescent protein (GFP) and phycobilin protein and derivatives thereof. Fluorescent proteins (particularly phycobilin proteins) are particularly useful in the preparation of labeling reagents with tandem dye labels. These tandem dyes contain fluorescent proteins and fluorophores so that the emission spectrum is far away from the wavelength of the fluorescent protein's absorption spectrum and a greater Stoke shift can be obtained. This is especially advantageous when detecting target substances present at low levels in a sample (the emitted fluorescence is maximally optimized, in other words the emitted light is mostly reabsorbed by the fluorescent protein. Not) When this works, the fluorescent protein and fluorophore function as an energy transfer pair (the fluorescent protein emits light and the fluorophore absorbs the wavelength of the light), and the fluorophore is a wavelength that can only be obtained by the fluorescent protein. And radiate from the fluorescent protein. Particularly useful combinations include the phycobilin proteins disclosed in US Pat. Nos. 4,520,110, 4,859,582, 5,055,556, and US Pat. No. 5,798,276. Or the sulfonated cyanine fluorophores disclosed in US patent application Ser. Nos. 09/968/401 and 09/969/853, or US Pat. No. 6,130,101. And sulfonated xanthene derivatives disclosed in US Pat. No. 4,542,104, and combinations thereof. Alternatively, the fluorophore may function as an energy donor and the fluorescent protein may function as an energy acceptor.

  In an exemplary embodiment, a glycoprotein is provided that is covalently conjugated to a carrier molecule. This includes, but is not limited to, any azide modified glycoprotein and any carrier molecule disclosed herein.

  In one embodiment, a first composition is provided comprising a glycoprotein of the invention, a first reporter molecule and a carrier molecule. In other embodiments, a second glycoprotein is provided that contains the first composition in combination with a second conjugate. The second conjugate includes a carrier molecule or solid support (below) covalently bound to a second reporter molecule. The first and second reporter molecules have different structures and preferably have different emission spectra. More preferably, the first and second reporter molecules are selected so that their fluorescence emission does not essentially overlap. In another embodiment, the reporter molecules have different excitation spectra or the reporter molecules are excited by the same laser.

Carrier molecule:
The carrier molecule (or solid support) of the conjugate in the second composition may be the same or different molecule. The discussion herein relating to the properties of various carrier molecules is generally applicable to this embodiment of the invention as well as other embodiments.

  In the present invention, various carrier molecules can be used. Typical carrier molecules include antigens, steroids, vitamins, drugs, haptens, metabolites, poisons, environmental pollutants, amino acids, peptides, proteins, nucleic acids, nucleic acid polymers, carbohydrates, lipids, and polymers.

  In certain specific embodiments, the carrier molecule is an amino acid, peptide, protein, polysaccharide, nucleoside, nucleotide, oligonucleotide, nucleic acid, hapten, psoralen, drug, hormone, lipid, lipid assembly, synthetic polymer, polymeric microparticle, biological Including cells, viruses and combinations thereof. In other exemplary embodiments, the carrier molecule is selected from haptens, nucleotides, oligonucleotides, nucleic acid polymers, proteins, peptides or polysaccharides. In preferred embodiments, the carrier molecule is an amino acid, peptide, protein, polysaccharide, nucleoside, nucleotide, oligonucleotide, nucleic acid, hapten, psoralen, drug, hormone, lipid, lipid conjugate, tyramine, synthetic polymer, polymeric microparticle, biological A cell, cellular component, ion chelating moiety, enzyme substrate, or virus. Examples of other preferred carrier molecules include antibodies or fragments thereof, antigens, avidin or streptavidin, biotin, dextran, IgG binding proteins, fluorescent proteins, agarose and non-biological microparticles.

In a specific embodiment, the enzyme substrate is selected from amino acids, peptides, sugars, alcohols, alkanonic acids, 4-guanidinobenzoic acid, nucleic acids, lipids, sulfates, phosphates, —CH ₂ OCO alkyl groups and combinations thereof. That is, the enzyme substrate is an enzyme selected from the group consisting of peptidase, phosphatase, glycosidase, dealkylase, esterase, guanidinobenzotase, sulfatase, lipase, peroxidase, histone deacetylase, endoglycoceramidase, exonuclease, reductase and endonuclease. Disconnected by.

In other exemplary embodiments, the carrier molecule is an amino acid (including those protected or substituted by phosphates, carbohydrates or C ₁ -C ₂₂ carboxylic acids) or a polymer of amino acids (eg, a peptide or protein). In a related embodiment, the carrier molecule comprises at least 5 amino acids (preferably 5-36 amino acids). Exemplary peptides include but are not limited to neuropeptides, cytokines, toxins, protease substrates and protein kinase substrates. Other typical peptides can function as organelle-localized peptides (ie, peptides that have the function of localizing conjugated compounds in specific cellular structures through cellular transport mechanisms). Suitable protein carrier molecules include enzymes, antibodies, lectins, glycoproteins, histones, albumin, lipoproteins, avidin, streptavidin, protein A, protein G, phycobilin proteins and other fluorescent proteins (hormones, toxins and growth factors) Is mentioned. Typically, the protein carrier molecule is an antibody, antibody fragment, avidin, streptavidin, toxin, lectin or growth factor. Typical haptens include biotin, digoxigenin, and fluorophores.

  In other exemplary embodiments, the carrier molecule comprises a nucleobase, nucleoside, nucleotide or nucleic acid polymer, optionally with a further linker or spacer (eg, an alkynyl bond (US Patent) for attachment to a fluorophore or other ligand. 5,047,519), aminoallyl bonds (US Pat. No. 4,711,955) or other bonds). In other exemplary embodiments, the nucleotide carrier molecule is a nucleoside or deoxynucleoside or dideoxynucleoside.

  Exemplary nucleic acid polymer carrier molecules may be single-stranded or multi-stranded, natural or synthetic DNA or RNA, or DNA / RNA hybrids (such as morpholine-derived phosphates (AntiVirals, Corvallis OR)). Non-natural linkers may be incorporated), or peptide nucleic acids (eg, N- (2-aminoethyl) glycine units) (nucleic acids containing less than 50 nucleotides, preferably containing less than 25 nucleotides), and the like.

  In other exemplary embodiments, the carrier molecule is a carbohydrate (usually a polysaccharide (eg dextran, ficoll, heparin, glycogen, amylopectin, mannan, inulin, starch, agarose and cellulose)) or a polyol (usually a polymer (eg poly ( Ethylene glycol))). In a related embodiment, dextran, agarose or ficoll is used as the polysaccharide carrier molecule.

  In other exemplary embodiments, the carrier molecule contains a lipid (typically 6-25 carbon atoms), including glycolipids, phospholipids and sphingolipids. Alternatively, the carrier molecule contains a lipid sac (eg, a liposome) or a lipoprotein (see below). Several lipophilic substituents are useful for facilitating transport of dye conjugates to cells or cellular organelles.

  Alternatively, the carrier molecule can be a cell, cell line, cellular fragment or cell-associated particle (virus particle, bacterial particle), viral component, biological cell (eg, animal cell, plant cell, bacterium, or yeast) or cell organelle. is there. Examples of cellular organelles that are useful as carrier molecules include lysosomes, endosomes, cytoplasm, nuclei, histones, filaments, Golgi apparatus, endoplasmic reticulum and vacuoles.

  In other exemplary embodiments, the carrier molecule is non-covalently associated with the organic or inorganic material. Using exemplary embodiments of carrier molecules with lipophilic substituents allows for the targeting of lipid assemblies, such as biological membranes or liposomes, by non-covalent uptake of dye compounds in the membrane, for example, Probes for membrane structure analysis or for incorporation into liposomes, lipoproteins, films, plastics, lipophilic microspheres or similar materials.

  In a specific embodiment, the carrier molecule comprises a specific binding pair member, and in particular, the compound of the invention is conjugated with a specific binding pair member to form a binding pair. And Alternatively, the presence of a labeled specific binding pair member indicates the presence of a complementary member of that specific binding pair, and each specific binding pair member is otherwise identified on its surface or in a cavity. It has a region for specifically binding or complementary binding to a substance having the following three-dimensional structure and polarity. In this case, the dye compound of the present invention functions as a reporter molecule for a specific binding pair. A typical binding pair is shown in Table 2.

^＊ＩｇＧは免疫グロブリンである。
^†ｃＤＮＡ及びｃＲＮＡは、ハイブリッド形成に使用する相補鎖である。 Table 2 Representative specific binding pairs

^* IgG is an immunoglobulin.
^† cDNA and cRNA are complementary strands used for hybridization.

In specific embodiments, the carrier molecule is an antibody fragment (eg, anti-Fc, anti-Fc isotype, anti-J chain, anti-κ light chain, anti-λ light chain or single chain fragment variable protein), or non-antibody peptide or protein (eg, Non-limiting examples include soluble Fc receptors, protein G, protein A, protein L, lectin, or fragments thereof). In one embodiment, the carrier molecule is an Fc portion of a target binding antibody or an Fab fragment specific for an isotype of the Fc portion of a target binding antibody (US Patent Application No. 10 / 118,204). Monovalent Fab fragments are typically produced as murine monoclonal antibodies or may be prepared as polyclonal antibodies using various animals, including but not limited to rabbits or goats. These fragments may be derived from any isotype (eg, mouse IgM, IgG ₁ , IgG _2a , IgG _2b or IgG ₃ ).

  Alternatively, non-antibody proteins or peptides (eg, protein G or other suitable protein) may be used alone or in combination with albumin. Suitable albumins include human and bovine serum albumin or ovalbumin. Proteins A, G, and L are defined as those proteins or derivatives thereof known to those skilled in the art, which include at least one binding region with IgG (ie, proteins that have an affinity for IgG). These proteins may be modified but need not be conjugated with a reactive label, as with other carrier molecules of the invention.

  In another embodiment, the carrier molecule is an intact intact antibody. Antibody is a term that refers to a soluble substance or molecule that is secreted or produced in an animal in response to an antigen and that has the property of specifically binding to the antigen that induced its production. Antibodies are also glycoproteins themselves and can therefore be used as antigens or immunogens for the preparation of anti-species antibodies. Antibodies (also known as immunoglobulins) are classified into five different classes (IgG, IgA, IgM, IgD and IgE). The basic IgG immunoglobulin structure is composed of two identical light chain polypeptides and two identical heavy chain polypeptides joined by disulfide bonds.

Monovalent antigen-binding fragments can be isolated by treating IgG with the enzyme papain, which is described herein as a Fab fragment. Also, larger fragments are produced when IgG is treated with pepsin (another proteolytic enzyme) (F (ab ′) ₂ ). This fragment can be further cleaved in half by treatment with a mild reducing buffer, resulting in a monovalent Fab ′ fragment. The Fab ′ fragment is slightly larger than the Fab and contains one or more free sulfhydryl groups derived from the hinge region (it is not present in the small Fab fragment). The term “antibody fragment” is defined herein as the Fab ′, F (ab ′) ₂ and Fab portions of an antibody. It is a known technique to obtain antibody fragments by treating antibody molecules with pepsin and papain (Gorevic et al., Methods of Enzyol., 116: 3 (1985)).

  The monovalent Fab fragment of the present invention may be prepared from any murine monoclonal or polyclonal antibody, in which case various animals are immunized with an external antibody or fragment thereof (US Pat. No. 4,196,265). Discloses a method for preparing a monoclonal antibody). Typically, the secondary antibody is derived from a polyclonal antibody produced in a rabbit or goat, but of course the anti-species antibody is prepared using any animal known to those skilled in the art used for the preparation of polyclonal antibodies. May be. The term “primary antibody” refers to an antibody that binds directly to an antigen, as opposed to a “secondary antibody” that binds to a portion of the primary antibody. Monoclonal antibodies are equivalent to, and in some cases, preferred, polyclonal antibodies, in which case ligand-bound antibodies are typically produced from murine hybridoma cell lines prepared using methods known to those skilled in the art. It is preferably compatible with the monoclonal antibody to be prepared.

  In one embodiment, the antibody is an antibody prepared using only the Fc site of an external antibody as an immunogen. Basically, animals are immunized with only Fc region fragments of external antibodies (eg, from mice). Next, blood is collected to prepare a polyclonal antibody and incubated with an enzyme, pepsin or papain to prepare a monovalent fragment. The fragment is further affinity purified using a column containing only whole immunoglobulin protein of the immunized animal, or Fc fragment only.

  In an exemplary embodiment, the azido glycoprotein is covalently bound to the solid support. This includes, but is not limited to, any azido glycoprotein described above and any solid support disclosed herein.

  In one embodiment, a first composition is provided comprising a compound of the invention, a first reporter molecule and a solid support. In other embodiments, a second composition is provided that contains the first composition in combination with a second conjugate. The second conjugate includes the solid support or carrier molecule described above that is covalently linked to a second reporter molecule. The first and second reporter molecules have different structures and preferably have different emission spectra. More preferably, the first and second reporter molecules are selected so that their fluorescence emission does not essentially overlap. In another embodiment, the reporter molecule may have a different excitation spectrum, or the reporter molecule may be excited with the same laser.

  The solid support (or carrier molecule) of the conjugate in the second composition may be the same or different molecules. The discussion herein relating to the properties of various carrier molecules is generally applicable to this embodiment of the invention as well as other embodiments.

Solid Support In the present invention, various solid supports can be used. A solid support suitable for use in the present invention typically has a substituent that is substantially insoluble in the liquid phase and capable of reacting with a chemical handle. In preferred embodiments, the solid support comprises an alkyne or an activated alkyne and may react with a modified sugar or vice versa. The solid support of the present invention is not limited to a specific type of support, and may be a semi-solid support. Rather, many prior art supports known to those skilled in the art are available. That is, useful solid supports include solid and semi-solid matrices (eg, aerogels and hydrogels, resins, beads, biochips (such as biochips with a thin film coat), microfluidic chips, silicon chips, multiwell plates (also (Also called microtiter plates or microplates), membranes, conductive and non-conductive metals, glasses (such as microscope slides), and magnetic supports, etc. More specific examples of useful solid supports , Silica gel, polymer film, particle, derivatized plastic thin film, glass bead, cotton, plastic bead, alumina gel, polysaccharide (eg Sepharose), poly (acrylate), polystyrene, poly (acrylamide), polyol, agarose, agar ,cellulose, Kisstran, starch, ficoll, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, polyvinyl chloride, polypropylene, polyethylene (poly (ethylene glycol), etc.), nylon, latex beads, magnetic beads, paramagnetic beads, Examples include superparamagnetic beads and starch.

  In some embodiments, the solid support is a hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amide, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide group, etc. ( (It is not limited to these.) You may have a reactive functional group for making the compound which concerns on this invention couple | bond. Any of the above reactive groups can be effectively used as a reactive functional group on the solid support according to the present specification.

  A suitable solid support can be selected as appropriate based on the desired end use and suitability for various synthetic procedures. For example, when an amide bond is desired to bind the compound of the present invention to a solid support, a resin that is usually useful in peptide synthesis is used, such as polystyrene (PAM resin, Bachcm, Peninsula Laboratories). ), POLYHIPE (trademark) resin (Aminotech (Canada)), polyamide resin (Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGel (trademark), Rapp Polymere, Tubingen, Germany), poly Dimethyl-acrylamide resin (Milligen / Biosearch (California)) or PEGA beads (Polymer Laboratories) s Co., Ltd.) and the like.

kit:
Kits comprising components for use in in vivo metabolic labeling of proteins and antibodies are also provided. Such a kit may include, for example, a non-natural sugar labeled with a chemical / affinity handle (eg, azide, triarylphosphine or alkyne). Such a kit may further comprise reagents for labeling or purifying metabolically labeled antibodies isolated from cells, for example. For example, the kit may include non-natural oligosaccharides (eg, GalNAz, ManNAz or GlcNAz). The kit may further comprise a fluorophore label or an affinity tag (reactive with the chemical / affinity handle on the non-natural oligosaccharide). Those skilled in the art will envision various labels or tags (eg, but not limited to those listed herein) that can be used, for example, to label or tag the modified antibody. For example, the label or tag can be conjugated to an alkyne so that a click-type chemical reaction can be performed by contacting the azide on the metabolically labeled antibody.

  The kit of the present invention may package one or more components separately in any number of containers, packets, tubes, vials, microtiter plates, etc., or the components may vary in such containers. You may mix in combination. In the kit of the invention, for example, non-natural oligosaccharides (including chemical / affinity handles) may be provided in a separate container from the fluorophore label or affinity tag.

  The kits of the invention may include instructions for performing one or more methods described herein, and / or a description of one or more compositions or reagents described herein. May include a letter. The instructions and / or instructions may be printed and may be included as a kit insert. Alternatively, instruction manuals and / or instructions for the kit may be provided via the Internet.

Specific embodiments of the present invention:
For example, in the method of the present invention, a terminal sugar residue on a glycoprotein or antibody can be directly labeled by using the Gal-T enzyme described in the present specification. Alternatively, the sugar group is cleaved by, for example, endo-H, and then the sugar containing the chemical handle is made into a protein or antibody using, for example, endo-M or endo-A, both of which can catalyze sugar cleavage and transfer Add. In other embodiments, a sugar containing a chemical handle is attached to a glycoprotein or antibody by metabolic labeling. “Sugar” refers to monosaccharide or oligosaccharide.

One embodiment of the present invention relates to a method for preparing a sugar-modified protein. The method is described in detail below.
Cleaving an oligosaccharide present on the first protein at a GlcNAc-GlcNAc bond to obtain a protein comprising a GlcNAc residue having a reducing end;
Attaching a modified sugar having a chemical handle to the reducing end of the GlcNAc residue;
Mixing the first protein with a modified oligosaccharide having a label capable of reacting with the chemical handle;
The modified oligosaccharide binds to the protein at the site of the chemical handle, thereby forming a sugar modified protein.

  In another embodiment, the protein is an antibody. More particularly, the antibody is IgG. In another embodiment, the oligosaccharide is cleaved by endoglycosidase H at the position of the GlcNAc-GlcNAc bond. In another embodiment, the modified sugar is bound to the reducing end by catalysis of a mutant galactosyltransferase. In another embodiment, the variant is a Y289L variant. In another embodiment, the modified sugar is an azide modified sugar and the modified oligosaccharide is alkyne labeled. In another embodiment, the azide-modified sugar is UDP-GalNAz.

In another embodiment, the modified oligosaccharide is
Cleaving the oligosaccharide present on the second protein at the position of the GlcNAc-GlcNAc bond to obtain an oligosaccharide having a GlcNAc residue having a reducing end;
It is obtained by labeling the oligosaccharide with a label capable of reacting with the chemical handle.

  In another embodiment, the reducing end of the cleaved oligosaccharide is treated with ammonium bicarbonate, and the treated oligosaccharide is coupled to an alkyne using a succinimidyl ester. In another embodiment, the second protein is synthesized in a different cell line or different type of cell than the first protein. In another embodiment, the second protein is synthesized in human cells.

  In another embodiment, the modified oligosaccharide further comprises a second label. In another embodiment, the second label is selected from the group consisting of a therapeutically active moiety, a therapeutic agent, a radioactive metal ion, DNA, protein, peptide, sugar, detectable label, biotin and avidin.

  Another aspect of the invention relates to the provision of an oligosaccharide modified protein obtained using the method of any one of the above embodiments.

  Another aspect of the invention relates to the provision of oligosaccharide cleaved proteins obtained using a method comprising treating a protein with endoglycosidase H. In another embodiment, the protein is an antibody. More particularly, the antibody is IgG.

  Another aspect of the invention relates to the provision of antibodies containing labeled oligosaccharides. In another embodiment, the label is selected from the group consisting of a moiety having therapeutic activity, a therapeutic agent, a radioactive metal ion, DNA, protein, peptide, sugar, detectable label, biotin and avidin.

  In other embodiments, the oligosaccharide is cleaved by endoglycosidase M at the position of the GlcNAc-GlcNAc bond.

In another embodiment, the cleavage of the first antibody is performed in the presence of OH-alkyne,
The endoglycosidase M binds the OH-alkyne to the reducing end of the cleaved GlcNAc residue;
The modified oligosaccharide is labeled with an azide residue.

  In another embodiment, the oligosaccharide is cleaved from the second antibody at the position of GlcNAc-GlcNAc binding, catalyzed by endoglycosidase M.

In other embodiments,
The cleavage of the second antibody is performed in the presence of OH-alkyne, and the endoglycosidase M binds the OH-alkyne to the reducing end of the cleaved GlcNAc residue, thereby modifying the alkyne. Obtained oligosaccharides,
The chemical handle on the modified sugar on the first antibody is an azide.

  Another aspect of the present invention relates to the provision of an oligosaccharide cleaved antibody obtained using a method comprising treating an antibody with endoglycosidase M. In other embodiments, the antibody is IgG.

  Another aspect of the invention relates to the provision of a method for labeling antibodies. The method is performed by labeling an oligosaccharide that binds to the antibody and includes incubating the antibody-producing cells in the presence of a non-natural sugar, the non-natural sugar comprising a chemical handle.

  In other embodiments, the antibody-producing cell is a recombinant cell. In another embodiment, the antibody-producing cell is a hybridoma. In another embodiment, the antibody is IgG. In another embodiment, the chemical handle is selected from the group consisting of azide, triarylphosphine or alkyne. In another embodiment, the unnatural sugar is selected from the group consisting of GalNAz, ManNaz, and GlcNAz.

  Another embodiment includes isolating the labeled antibody from the antibody-producing cells.

  Other embodiments further comprise binding a second label to the isolated antibody, the step comprising reacting the isolated antibody with a second label that is reactive with the chemical handle. Including contacting with.

  In another embodiment, the chemical handle is an azide and the second label has an alkyne or is modified to have an alkyne. In another embodiment, the second label is selected from the group consisting of a therapeutically active moiety, a therapeutic agent, a radioactive metal ion, DNA, protein, peptide, sugar, detectable label, biotin and avidin.

  Another aspect of the invention relates to the provision of labeled antibodies obtained using the methods of any of the embodiments described herein.

  Another aspect of the invention relates to providing a kit comprising a non-natural sugar labeled with a chemical handle and a second label that binds to the non-natural sugar at the position of the chemical handle.

  In other embodiments, the kit further comprises instructions for in vivo labeling of the antibody in the antibody producing cell. In another embodiment, the chemical handle is selected from the group consisting of azide, triarylphosphine and alkyne. In another embodiment, the unnatural sugar is selected from the group consisting of GalNAz, ManNAz, and GlcNAz. In another embodiment, the chemical handle is an azide and the secondary label comprises an alkyne.

  The following examples illustrate the invention and describe those methods of the invention in detail to those skilled in the art and describe specific embodiments of the invention. The following examples are merely for the purpose of providing specific methodologies useful in understanding and practicing the present invention and should not be construed as limiting the invention to the embodiments. . The reagents used in the examples are either commercially available or can be prepared using commercially available instruments, methods or reagents known in the art. The above embodiments illustrate various aspects of the present invention and method embodiments of the present invention. The disclosures of all references in the examples are incorporated herein by reference. Such examples do not exclude many different embodiments of the present invention, nor do they exclude the selection of suitable reagents beyond the scope described above. That is, for convenience of understanding, the above-described present invention will be described as some specific embodiments with reference to the drawings and examples. However, those skilled in the art will appreciate that many changes and modifications can be made to the technical idea or the present invention. It will be readily understood that implementations can be made without departing from the scope of the appended claims.

Example <Example 1>:
HPLC analysis sugar chain removal reaction of monoclonal and polyclonal antibodies with or without oligosaccharide cleavage by endo-H glycosidase:
Add 90 μL reduced and alkylated antibody (1 mg / ml) to 40 μL 0.5 M sodium citrate (pH 5.5) and 10 μL endo H _f (1,000,000 U / mL, New England BioLabs) did. Sealed and incubated at 37 ° C. for 48 hours. The sample was centrifuged to remove the precipitate and then injected directly onto the HPLC. Reversed phase HPLC was performed at 75 ° C. with a Waters 600E LC system, an Agilent Zorbax 300SB-CN column (4.6 × 150 mm, 3.5 μm), at a flow rate of 0.8 mL / min. The composition of the mobile phase was solvent A: water containing 0.1% TFA, and solvent B: 80% n-propanol, 10% acetonitrile, 10% 0.1% TFA. (Conditions were as described in Rehder et al., J. Chrom. A, 1102 (2006) 164-175). Separations were performed using a 20-40% B linear gradient over 30 minutes. The results are shown in FIGS.

<Example 2>:
Enzyme labeling of chicken anti-goat antibody with and without oligosaccharide cleavage by endo-H glycosidase Chicken anti-goat IgG was washed with 50 mM MES (pH 6.5) buffer on a VivaSpin 5000 MWCO filter column. An aliquot of 1 mg / mL washed IgG was incubated for 24 hours at 37 ° C. with 50 mM MES (pH 6.5) containing 47.5 U / μL Endo H (New England BioLabs) for glycosylation. did. Cleaned (not deglycosylated), or 0 in the end _{H f} treated IgG further using Click-iT O-GlcNAc enzymatic labeling kit nondenaturing conditions (50 mM of MES (pH 6.5). 5 mg / ml IgG, 120 mM NaCl, 11 mM MnCl ₂ , 0.1 mM ZnCl ₂ , 50 μM UDP-GalNAz, 5 U / mL Antarctic phosphatase, and 0.65 mg / mL GalT enzyme overnight at 4 ° C. Azide-labeled by incubation). The sample was passed through a P10 size resin packed in a 0.5 mL spin column and purified into 50 mM Tris (pH 8). The collected fraction containing the antibody is labeled with a TAMRA Click-iT ™ detection kit (C33370), the sample is passed through a P10 size resin packed in a 0.5 mL spin column, and purified again into 50 mM Tris (pH 8). did. Approximately 250 ng was analyzed on a 4-12% bis-Tris gel using MOPS buffer. Gels were imaged with a BioRadFX imager using a 532 nm laser and a 555 nm long pass emission filter (FIG. 3A). Gels were post-stained using SYPRO® Ruby protein gel stain and imaged using a 488 nm laser and a 555 nm long pass emission filter (FIG. 3B).

<Example 3>:
Metabolic labeling of antibodies with azido sugars Mouse M96 hybridoma cells were treated with azido sugar analogs, Ac ₄ GalNAz, Ac ₄ ManNAz, or Ac ₄ GlcNAz for 5 days. Cell supernatant (including azide-labeled monoclonal antibody) was collected and protein was precipitated with chloroform / methanol. The pellet pellet was resolubilized with 50 μL of 1% SDS, 100 mM Tris (pH 8), labeled with the TAMRA Click-iT ™ detection kit (C33370), and 1 μg of 4-12 using MOPS buffer. % Bis-Tris gel. The gel was imaged with a BioRadFX imager using a 532 nm laser and a 555 nm long pass emission filter (FIG. 4A). Gels were post-stained with SYPRO® Ruby protein gel stain and imaged using a 488 nm laser and a 555 nm long pass emission filter (FIG. 4B).

<Example 4>:
Metabolic labeling and “click” detection of glycoprotein subclass Jurkat cells were treated with 40 μM Ac ₄ ManNAz or Ac ₄ GalNAz for 3 days (A), or 250 μM Ac ₄ GlcNAz overnight (B). The collected cells were sonicated in 50 mM Tris buffer (with protease and phosphatase inhibitor, pH 8.0) and the lysate was subjected to high speed centrifugation (100 K × g). Membrane pellet proteins from ManN Az- and GalNAz-treated cells and supernatant cell lysates from GlcNAc-treated cells were precipitated with chloroform / methanol, lysed with detergent, 1 mM CuSO4 and 5 mM ascorbine Labeling with a fluorescent alkyne probe in the presence of acid (1). 10 μg of labeled and precipitated protein was subjected to electrophoresis on 1-D NuPAGE® Novcx® 4-12% gel (Invitrocn). Imaged with a Fuji FLA-3000 scanner (Fuji) using 532 nm excitation (FIG. 5A). The gel was then post-stained with SYPRO® Ruby staining (Invitrogen) and imaged with excitation at 473 nm (FIG. 5B). The control lane represents an extract of untreated cells (however, treated with a fluorescent probe) (FIG. 5).

<Example 5>:
Separation of Ac ₄ GlcNAz-treated Jurkat cells soluble protein by two-dimensional gels Jurkat cells were cultured overnight with 250 μM Ac ₄ GlcNAz or DMSO carrier (untreated control). Soluble lysate protein was prepared as in Example 4 using sonication and ultracentrifugation and labeled with a fluorescent alkyne probe for 1 hour. 40 μg of labeled protein is precipitated and redissolved with 7M urea, 2M thiourea, 65 mM DTT, 2% CHAPS, 1% Zwittergent 3-10, 1% pH 3-10 carrier ampholite, Separation in the first dimension with IEF strips of pH 3-10 and in the second dimension with 4-12% Bis-Tris gel with MOPS buffer. Gels excited at 532 nm were post-stained and imaged with SYPRO® Ruby staining (Invitrogen), further excited at 473 nm, and re-imaged using a Fuji FLA-3000 scanner (Fuji). See FIG.

<Example 6>:
Detection of 40 and 50 kD azide-labeled model proteins in gel 25 μmol each of 40 and 50 kD model proteins with one N-terminal azide were incorporated into 100 μg of Jurkat cell lysate (upper panel) and not ( (Lower panel). The protein was labeled with a fluorescent alkyne probe, serially diluted as indicated, and electrophoresed on a NuPAGE® Novex® 4-12% gel. Excitation at 532 nm and images were obtained with a FLA-3000 scanner (left panel). The gel was then stained with SYPRO® Ruby stain, excited at 473 nm, and imaged (right panel). The detection sensitivity of the labeled protein was less than 10 femtomole. See FIG.

<Example 7>:
The labeling efficiency of the 40 and 50 kd azide-labeled model proteins was unchanged in the complex protein extract. Azide-labeled 100 ng (25 pmol) or 10 ng (2.5 pmol) of 40 Kd and 50 Kd proteins, respectively, 100, 50, 25 Or labeled with a fluorescent alkyne probe in the background of 0 μg control Jurkat lysate (left panel) (after labeling, 100 μg of control lysate is added to “0 lysate” to recover the labeled protein by precipitation. Promoted). Gels were post-stained with SYPRO® Ruby total protein stain. See FIG.

<Example 8>:
Enzymatic labeling and detection of α-crystallin O-GlcNAc α-crystallin O-GlcNAc was enzymatically azide (UDP-GalNAz) labeled using a modified b-GalT1 enzyme. The protein was then reacted with a fluorescent alkyne probe as described above. The protein was electrophoresed on a 1-D NuPAGE® Novex® 4-12% gel at the indicated dilution (only 2-10% α-crystallin was O-GlcNAc modified, Therefore, the detection sensitivity of the O-GlcNAc moiety was in the medium-low femtomolar range (10-45 fmol). See FIG.

<Example 9>:
Comparison of a-O-GlcNAc monoclonal antibody CTD110.6 with GalT1 enzyme label In A, α-crystallin O-GlcNAc was enzymatically labeled with a modified GalT1 enzyme and then reacted with a biotin-alkyne probe. Proteins were electrophoresed on 1-D NuPAGE® Novex® 4-12% Vistris gels at the indicated dilutions and blotted onto PVDF membranes. The PVDF membrane was then incubated with streptavidin-HRP and the protein was detected using ECL Plus ™ (GE Biosystems). Lane 2 (NE) shows a control without addition of 8 pmoL of enzyme (in A, the detection sensitivity of O-GlcNAc by Western blot is in the low femtomolar range (3-10 fmol)). In B, untreated α-crystallin was electrophoresed on a 1-D gel and blotted as described above. PVDF membranes were processed using O-GlcNAc Western blot detection kit (Pierce) according to manufacturer's instructions. The kit utilized a CTD110.6α-O-GlcNAc monoclonal antibody. Lane 2 is a lane containing 5 ng of a positive control (O-GlcNAc-modified BSA) attached to the kit. No α-crystallin was detected using the antibody detection system. See FIG.

<Example 10>:
Multiplex detection of O-GlcNAc protein, phosphoprotein and total protein in the same two-dimensional gel:
Soluble fractions were extracted from Jurkat cells fed with Ac ₄ GlcNAz and labeled with UV-excitable alkyne dye for 2 hours. Proteins precipitated with chloroform / methanol as described above were electrophoresed on a two-dimensional gel. The gel was rinsed with water and imaged using a Lumi-Imager ™ (Roche) with UV irradiation and 600 / bp release. The gel is then stained with Pro-Q® diamondrin protein staining, imaged with a FLA-3000 laser imager with 532 nm excitation / 580 LP emission, and SYPRO® Ruby total protein staining. And imaged again with excitation at 473 nm and long pass emission at 580 nm according to the manufacturer's instructions. See FIG.

<Example 11>:
Multiple Western Blot Detection of O-GlcNAc Modified Protein and Cofilin 25 μg of Jurkat cell soluble protein was subjected to two-dimensional gel electrophoresis as described above and further blotted onto a PVDF membrane. The PVDF membrane was incubated with an α-cofilin polyclonal antibody and further detected by ECL Plus ™ detection (manufactured by GE Biosystems) using a GAR-HRP secondary antibody. After imaging, the blot membrane was incubated with streptavidin AP, and O-GlcNAc protein was detected using WesternBreeze® chemiluminescence detection kit (Invitrogen). See FIG.

<Example 12>:
In the control and inhibitor treated cultured cell extract, a differential detection Jurkat cells O-GlcNAc modified proteins were incubated overnight at _Ac 4 GlcNAz, were analyzed for the presence of PUGNAc process. PUGNAc is a commonly used inhibitor of O-GlcNAcase. Soluble Jurkat lysate was labeled with fluorescent alkyne. Lane 1) cells treated with 50 μM PUGNAc and 4 mM glucosamine for 3 hours prior to recovery, lane 2) untreated, lanes 3 to 5) cells cultured overnight in 250 μM Ac ₄ GlcNAz, lanes 6 to 8) 250 μM Ac ₄ Cells treated with 50 μM PUGNAc and further 250 μM Ac ₄ GlcNAz and proteins treated with PUGNAc (lanes 6-8) were cultured overnight in GlcNAz for 3 hours before recovery, and untreated controls (lanes 3-5). Showed a significant increase in O-GlcNAc staining. See FIG.

<Example 13>:
Linkage of glycoproteins in gels Fluorescent alkyne compounds used for linking in gels are shown in FIGS. In addition, two strong fluorogenic alkynes are shown in E and F. The TAMRA-alkyne compound (shown in the upper left frame) is used for in-gel staining tests, using reactive azido-succinimidyl esters in vitro, or feeding feed cells with azide modified sugars in vivo. The azide group was incorporated into the protein.

  Cell lysates were obtained from Jurkat cells incorporating azido modified sugars.

Azide-alkyne reaction conditions for in-gel detection or Western blot detection:

  All ingredients were mixed, finally ascorbic acid was added, the solution was prepared to a final solution of 200 μL, and gently vortexed. 10 μL of 100 mM BCS was added (solution turns orange in the presence of CuI), vortexed gently, then argon was added. The solution was mixed on the rotor for 1 hour at room temperature.

After the reaction, the protein was precipitated using the following procedure. Add 600 μL of MeOH to the reaction mixture and vortex for 20 seconds (freeze for 30 minutes if protein content is low), then add 200 μL of chloroform, vortex for 20 seconds, then add 450 μL of H ₂ O And vortexed for 20 seconds. The solution was centrifuged at 18K xg for 5 minutes. The upper layer was removed, 450 μL of MeOH was added, vortexed for 20 seconds, and centrifuged at 18 K × g for 5 minutes. The supernatant was removed. Finally, 600 μL of MeOH was added, vortexed, and temporarily sonicated to disperse the pellet and centrifuged for 5 minutes at 18 K × g.

To dilute sodium ascorbate to 100 mM, dried sodium ascorbate (5 mg) was mixed with 250 μL H ₂ O. FIG. 19 shows the results of electrophoresis of a protein labeled with a TAMRA-alkyne compound according to the attached protocol. Lanes 2, 3 and 4 on the left side of the gel show cell extracts incorporating azide-modified sugars, and lane 1 shows cells not subjected to azido sugar feed (control). On the right side, the results when a control azide-labeled protein (ovalbumin and myoglobin) (+) or an unlabeled control (−) was applied at different concentrations are shown. As a result, it was shown that detection of an azide modified protein in a gel was possible very efficiently and selectively.

<Example 14>:
Azide ovalbumin and azido-myoglobin were used at 2.5 μg each, and spiked into 80 μg of unlabeled Jurkat lysate. The lysate was then labeled with TAMRA alkyne for 2 hours. The composition of the reaction solution was 50 mM Tris (pH 8), 25% propylene glycol, 1 mM CuSO ₄ , 5 mM sodium ascorbate, 20 μM TAMRA alkyne. Reactions with and without the chelator (10 mM TPEN, EDTA, either batocuproin disulfonic acid (BCS) or neocuproin) were performed. A control reaction was performed without CuSO ₄ . After labeling, the samples were precipitated and approximately 30 μg of each sample was redissolved in 7 mM urea / 2 mM thiourea / 65 mM DTT / 2% CHAPS / to obtain a two-dimensional gel (pH 4-7, IEF strip, 4-12% Bistris gel with MOPS buffer). The TAMRA signal was imaged using a Fuji FLA3000 with 532 nm excitation, 580 long pass emission, and the gel was post-stained with SYPRO® Ruby total protein gel stain (FIG. 14A). As a result, the resolution of protein separation was improved by the addition of a chelator, and the best results were obtained particularly with batocuproine disulfonic acid (BCS) and Cu (I) chelators. See total protein staining (Figure 14B).

  A second experiment was performed with similar samples and click labeling conditions. However, in the chelator treatment, the addition of 5 mM TPEN, BCS or neocuproin was added at the beginning of the reaction. After labeling, the sample was precipitated and redissolved in LDS buffer + 5 mM TCEP to prepare a serial 2-fold dilution series. Dilutions were loaded onto 4-12% Bistris gels using MOPS running buffer (lane 1 is 250 ng ovalbumin and myoglobin, respectively). FIG. 15A shows that the addition of a chelator reduces the background of the TAMRA signal image without compromising sensitivity. FIG. 15B shows that in post-staining with Sypro® Ruby total protein gel stain, the band resolution is very good in the sample with chelator.

  In the chelator treatment, the effect of the chelator was tested using the same click labeling conditions except that 7 mM, 5 mM or 2 mM BCS, or 7 mM, 5 mM or 2 mM neocuproin was added. The lane marked with an asterisk in FIG. 16 shows the results of the reaction when CuSO 4 and BCS are added to the reaction solution and vortexed before adding sodium ascorbate. In all other reactions, CuSO4 and sodium ascorbate were added and vortexed before adding BCS. Analysis of the gel showed that it was essential to add and mix sodium ascorbate and CuSO4 to the reaction tube before adding the chelator. If the chelator and CuSO4 were added and vortexed before adding sodium ascorbate, azide-alkyne labeling did not proceed, suggesting that the chelator inhibits Cu (II) to Cu (I) reduction.

<Example 15>:
Enzymatic labeling of antibodies using Click chemistry The goat IgG antibody was reduced, alkylated, further divided into two separate aliquots and deglycosylated using endo-H enzyme. The deglycosylated antibody (two separate preparations) was further added in a 150 μL reaction using 33 ng / μL GalT1 (Y289L) enzyme and 500 μM UDP-GalNAz (0.5 μg / μL goat antibody). , GalNAz-labeled. The reaction was incubated overnight at 4 ° C. 4-500 ng goat antibody (see gel shown, control without GalNAz or azide label) was loaded into each lane of a 4-12% Bistris gel. Electrophoresis was performed at 200 v for ~ 50 minutes using MES buffer. Gels were stained with TAMRA-alkyne and imaged with a Fuji imager with emission at 532 nm (excitation) and 580 nm. The gel was post-stained with SYPRO Ruby using an overnight protocol. See FIG.

  US patent applications filed on February 12, 2007 (US Provisional Patent Applications Nos. 60 / 772,221 and 60 / 804,640) filed on February 12, 2007, relating to agent case numbers IVGN745 and IVGN745.1 ) Is incorporated herein by reference.

  The entire contents of each patent, patent application, publication, and document incorporated herein is hereby incorporated by reference, including all tables, drawings and figures. All publications and patents are hereby incorporated by reference to the same extent as if each publication or patent was specifically and individually incorporated herein. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art and makes any approval regarding the contents or dates of these publications or documents. Not a thing.

  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 this invention belongs. Any similar method and apparatus, or equivalents thereof, other than the methods, apparatus and materials described herein can be used in the practice or testing of the present invention.

  The above aspects can be modified as appropriate without departing from the scope, technical idea and basic aspects of the present invention. Although the present invention has been described in considerable detail with respect to one or more specific embodiments, those skilled in the art will recognize that the embodiments specifically disclosed herein can be varied as appropriate. These modifications and improvements are also within the scope and spirit of the present invention. One skilled in the art will recognize that the present invention can be modified as appropriate to solve the problem and obtain the results and the effects described (including inherent effects). The examples provided herein are exemplary only, representative of particular embodiments, and are not intended to limit the scope of the invention.

  The invention best illustrated herein may be practiced in the absence of any component or components not specifically disclosed herein. In other words, the terms and expressions used are explanatory terms, are not limiting, and the features equivalent to the disclosed and described features, or a part thereof, are not exclusive, and various modifications are possible. Is understood to be possible within the scope of the invention. Embodiments of the invention are described in the claims.

Shown is a HPLC comparison of untreated (lower panel) and endo- _Hf treated (upper panel) goat anti-rabbit polyclonal antibodies. The untreated antibody eluted the light chain at 20.2 minutes and the glycosylated heavy chain eluted at 26.8 minutes. For the endo- _Hf treated antibody, the fully deglycosylated heavy chain eluted at 31.2 minutes. Untreated (bottom panel) and end _{H f} treatment (upper panel) were G2A monoclonal antibodies shows a comparison by HPLC analysis. The untreated antibody eluted the light chain at 21.5 minutes and the glycosylated heavy chain eluted at 29.1 minutes. For endo- _Hf- treated antibodies, the light chain elutes at 21.1 minutes and for glycosylated or partially deglycosylated heavy chains at 27.8 and 28.7 minutes, fully deglycosylated The heavy chain eluted at 30.1 minutes. FIG. 3A shows labeling with TAMRA Click-iT ™. Labeling of chicken anti-goat heavy chain (Endry) in End H-treated IgG and untreated IgG (Undegly) are shown. C: Control chicken anti-goat IgG starting material, Degly: endo-H treatment, O-GlcNAc enzymatic labeling and Click iT detection after chicken anti-goat IgG, Undegly: O-GlcNAc enzymatic labeling and Click iT detection Later chicken anti-goat IgG. FIG. 3B shows the same post-stained gel with SYPRO Ruby gel staining (showing the total protein pattern) as described in FIG. 3A. DMSO carrier (control), 20 μM Ac ₄ GalNAz, 40 μM Ac ₄ GalNAz, 20 μM Ac ₄ ManNAz, 40 μM Ac ₄ ManNAz or 30 μM Ac ₄ GlcNAz were introduced into M96 cells, and then the azido sugar was clicked. Show the result FIG. 4B shows the total protein pattern when the same gel described in FIG. 4A is post-stained by SYPRO Ruby gel staining. Diagram (C) showing metabolic labeling of glycoprotein subclasses A) and B) and their “click” detection and procedure after separation in a gel. Fig. 3 shows the results of detecting the click azide sugar Click iT of the Jurkat cell soluble protein introduced with Ac ₄ GlcNAz or DMSO carrier (untreated control), and further subjecting it to separation by two-dimensional polyacrylamide gel electrophoresis . The total protein pattern is shown when the same gel is post-stained by SYPRO Ruby gel stain. The results of detection of azide-labeled model proteins (40 and 50 kD) in the gel (first labeled with a fluorescent alkyne tag and further separated in the gel) are shown. The labeling efficiency of the azide-labeled 40 and 50 kd proteins is shown to be unchanged in the extract obtained by protein synthesis. The detection results after GalTl enzyme labeling and detection of α-crystallin O-GlcNAcA) (schematic) and B) gel separation are shown. The result of a comparison with the detection by GalTl enzyme labeling and Click-iT and the detection of (alpha) -crystallin O-GlcNAc by O-GlcNAc monoclonal antibody CTD 110.6 is shown. The detection of O-GlcNAc modified protein, phosphoprotein and total protein in the same two-dimensional electrophoresis gel is shown. The detection by Western blotting of O-GlcNAc modified protein and cofilin on the same membrane is shown. The differential detection of the cell extract treated with O-G1cNAc modified protein (control) and O-GlcNAcase inhibitory gene (PUGNAC) is shown. Gel showing the results of separating labeled proteins using a click reaction with different chelators. Azidoovalbumin and azidomyoglobin 2.5 μg each incorporated into 80 μg of unlabeled Jurkat lysate were labeled with TAMRA alkyne for 2 hours. The composition of the reaction solution was 50 mM Tris (pH 8), 25% propylene glycol, 1 mM CuSO ₄ , 5 mM sodium ascorbate, 20 μM TAMRA alkyne. The reaction was carried out using the presence or absence of a chelator (10 mM TPEN [upper left gel], EDTA [upper right gel], bathocuproine disulfonic acid (BCS) [center left gel] or neocuproin [center right gel]). Control reactions were performed without CuSO ₄ [lower left gel] or without chelator [lower right gel]. After labeling, the sample was precipitated and redissolved with 7 mM urea / 2 mM thiourea / 65 mM DTT / 2% CHAPS /, and about 30 μg of two-dimensional gel electrophoresis (pH 4-7 IEF strip, 4-12% bis- (Tris gel, MOPS buffer). The TAMRA signal was imaged with excitation at 532 nm, 580 long-pass emission with Fuji FLA3000 (14A), and the gel was post-stained by gel staining with Sypro® Ruby (14B). The result of having isolate | separated the protein labeled in the gel using the click reaction by a different chelator is shown. Sample and click labeling conditions are the same as in FIG. 14 (except that the chelator treatment involves the addition of 5 mM TPEN, BCS or neocuproin at the beginning of the reaction). After labeling, a sample of LDS buffer + 5 mM TCEP was precipitated and dissolved again to prepare a serial 2-fold dilution series. Dilutions were loaded onto 4-12% bis-Tris gels containing MOPS running buffer (250 ng ovalbumin and lane 1 myoglobin each). FIG. 15A shows that the addition of a chelator reduces the background of the image due to the TAMRA signal without compromising sensitivity. FIG. 15B is the result of post-staining by Sypro® Ruby total protein gel staining and shows that the band resolution is very good for the sample containing the chelator. The sample and click labeling conditions are the same as in FIG. 14 (except that the chelator treatment includes the addition of 7 mM, 5 mM or 2 mM BCS, or neocuproin). The lane marked with an asterisk (A) shows the result of the reaction when CuSO ₄ and BCS are added to the reaction solution and vortexed before adding sodium ascorbate. In all other reactions, CuSO ₄ and sodium ascorbate were added and vortexed before adding BCS. Analysis of the gel showed that it was necessary to add sodium ascorbate and CuSO ₄ to the reaction tube and mix before adding the chelator. If the chelator and CuSO ₄ are added and vortexed before adding sodium ascorbate, azide-alkyne labeling is not performed, suggesting that addition of the chelator inhibits the reduction of Cu II to Cu I. . Shows enzymatic labeling of antibodies using click chemistry: A) Lane 1: Goat antibody (GAb) only. Lane 2: GAb with GalTl enzyme (no UDP-GalNAz control). Lane 3: GAb with enzyme and UDP-GalNAz. Lane 4: azide-labeled ovalbumin and myoglobin control protein. Lane 5: unlabeled molecular weight marker. B) Total protein pattern when a dilution series of azide-labeled goat antibody (from above) is prepared, electrophoresed, and further post-stained with SYPRO Ruby gel stain. The structures of four existing alkyne fluorophores (A to D) and two strong alkyne fluorophores (E, F) are shown. Biomolecules can be labeled by using them according to the method of the present invention. A) TAMRA-alkyne, B) Alexa 488-alkyne, C) Alexa 633-alkyne, D) Alexa 532-alkyne, E) strong fluorogenic alkyne, F) strong fluorogenic alkyne. The result of the dyeing | staining of the gel using the TAMRA-alkyne compound shown to FIG. 12A is shown. Lanes 2, 3 and 4 on the left side of the gel represent the results of cell extracts labeled with azide modified sugars. Lane 1 represents control, unlabeled cells. On the right side, control azide-labeled proteins (ovalbumin and myoglobin) (+) or unlabeled control (−) (with concentration change) are shown. As a result, it has been shown that very efficient and selective labeling of azide-modified proteins in gel is possible.

Claims (22)

Attaching a chemical handle comprising a modified sugar to a GlcNAc residue on the first protein;
A method for preparing a sugar-modified protein comprising the step of mixing the first protein with a reporter molecule, carrier molecule, or solid support capable of reacting with the chemical handle,
The method wherein the reporter molecule, carrier molecule, or solid support is attached to a protein at the chemical handle, thereby forming a sugar-modified protein.   The method of claim 1, wherein the first protein is an antibody.   The method according to claim 1, further comprising the step of cleaving an oligosaccharide existing on the first protein at the position of GlcNAc-GlcNAc binding to obtain a protein containing a GlcNAc residue before the binding step.   4. The method of claim 3, wherein the oligosaccharide is cleaved by endoglycosidase H cleavage at the position of GlcNAc-GlcNAc binding.   The method of claim 1, wherein the modified sugar is coupled to the GlcNAc using a galactosyltransferase variant.   6. The method of claim 5, wherein the variant is a Y289L variant.   The method of claim 1, wherein the modified sugar is an azide-modified sugar and the reporter molecule, carrier molecule, or solid support is labeled with an alkyne or activated alkyne.   8. The method of claim 7, wherein the azido modified sugar is UDP-GalNAz. The method of claim 1 further comprising the following steps:
Cleaving the oligosaccharide present on the second protein at the position of GlcNAc-GlcNAc binding to obtain an oligosaccharide having a GlcNAc residue;
Attaching the oligosaccharide to a reporter molecule, solid support or carrier molecule capable of reacting with the chemical handle.   10. The method of claim 9, wherein the oligosaccharide having a GlcNAc residue is treated with ammonium bicarbonate and the oligosaccharide is coupled to the alkyne by a succinimidyl ester.   10. The method of claim 9, wherein the second protein is synthesized in a different cell line or different type of cell than the first protein.   The method of claim 9, wherein the second protein is synthesized in human cells.   The method of claim 1, wherein the modified oligosaccharide comprises a reporter molecule selected from the group consisting of a fluorescent dye, an enzyme, a radiolabel, a metal chelator, or a detectable substrate.   The method of claim 1, wherein the modified oligosaccharide comprises a carrier molecule selected from the group consisting of a therapeutic agent, DNA, protein, peptide, and sugar.   The method of claim 1, wherein the sugar-modified protein is more or less antigenic compared to the first protein before being modified.   The method of claim 1, wherein the first protein is from a non-human source.   17. The method of claim 16, wherein the sugar modified protein is humanized.   4. The method of claim 3, wherein the oligosaccharide is cleaved by endoglycosidase M cleavage at the position of GlcNAc-GlcNAc binding.   19. The method of claim 18, wherein the oligosaccharide is cleaved by endoglycosidase M cleavage at the position of GlcNAc-GlcNAc binding, after which the oligosaccharide is transferred to the first protein.   The method of claim 1, wherein the binding step is performed in a solution that is substantially free of proteases.   A method of labeling a protein by labeling an oligosaccharide bound to a protein comprising incubating a protein-producing cell in the presence of a non-natural sugar, wherein the non-natural sugar includes a chemical handle. Method.   An antibody comprising a labeled oligosaccharide.

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