Oligosaccharide modification and labeling of proteins

Abstract

The present invention generally relates to methods of functionalizing proteins, particularly antibodies, at oligosaccharide linkages, methods of humanizing antibodies by modifying glycosylation, as well as to novel antibodies linked to modified oligosaccharides. The invention further relates to kits that may be used to produce the antibodies of the invention.

1 .- 21 . (canceled) 
     
     
         22 . An antibody comprising a labeled oligosaccharide. 
     
     
         23 . A modified antibody made by a method of metabolically modifying a protein by modifying a group attached to the protein, comprising incubating a protein-producing cell in the presence of an unnatural substance to provide the modified protein, wherein the unnatural substance comprises a chemical handle, wherein the modified protein comprises a modified antibody. 
     
     
         24 . A labeled antibody made by the method of  claim 23 , further comprising the step of attaching a label to the modified antibody by contacting the modified antibody with a label that is reactive with the chemical handle. 
     
     
         25 . An isolated modified antibody made by the method of  claim 23 , further comprising the step of isolating the modified antibody. 
     
     
         26 . A labeled antibody made by the method of  claim 23 , further comprising the step of attaching a label to the isolated modified antibody by contacting the isolated modified antibody with a label that is reactive with the chemical handle. 
     
     
         27 . A kit comprising the modified antibody according to  claim 23  and a label that is reactive with the chemical handle of the antibody. 
     
     
         28 . The kit of  claim 27 , wherein the label comprises a reporter molecule. 
     
     
         29 . The kit of  claim 27 , further comprising a copper catalyst. 
     
     
         30 . A kit comprising the modified antibody made by the method of  claim 24  and a label that is reactive with the chemical handle of the antibody. 
     
     
         31 . The kit of  claim 30 , wherein the label comprises a reporter molecule. 
     
     
         32 . The kit of  claim 30 , further comprising a copper catalyst. 
     
     
         33 . A kit comprising (a) an unnatural substance comprising a chemical handle comprising an azide reactive or alkyne reactive group, and (b) a label comprising a reporter molecule, carrier molecule, or a solid support that will attach to the unnatural substance at the chemical handle. 
     
     
         34 . The kit of  claim 33 , wherein the unnatural substance comprises an unnatural sugar. 
     
     
         35 . The kit of  claim 34 , wherein the unnatural sugar comprises GalNAz, ManNAz, GlcNAz, Ac 4 GalNAz, Ac 4 ManNAz, or Ac 4 GlcNAz. 
     
     
         36 . The kit of  claim 33 , wherein the label comprises a reporter molecule comprising a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme, or a radioisotope. 
     
     
         37 . The kit of  claim 33 , wherein the label comprises a carrier molecule comprising an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a tyramine, a synthetic polymer, a polymeric microparticle, a biological cell, cellular components, an ion chelating moiety, an enzymatic substrate, a virus, an antibody or fragment thereof, an antigen, an avidin, a streptavidin, a biotin, a dextran, an IgG binding protein, a fluorescent protein, agarose, or a non-biological microparticle. 
     
     
         38 . The kit of  claim 33 , wherein the label comprises a solid support comprising an aerogel, a hydrogel, a resin, a bead, a biochip, a microfluidic chip, a silicon chip, a multi-well plate, a membrane, a conducting metal, a nonconducting metal, a glass, a magnetic support, a silica gel, a polymeric membrane, a particle, a derivatized plastic film, a glass bead, cotton, a plastic bead, an alumina gel, a polysaccharide, a poly(acrylate), a polystyrene, a poly(acrylamide), a polyol, an agarose, an agar, a cellulose, a dextran, a starch, a FICOLL, a heparin, a glycogen, an amylopectin, a mannan, an inulin, a nitrocellulose, a diazocellulose, a polyvinylchloride, a polypropylene, a polyethylene, a nylon, a latex bead, a magnetic bead, a paramagnetic bead, a superparamagnetic bead, or a starch. 
     
     
         39 . The kit of  claim 33 , further comprising a copper catalyst.