OLIGOSACCHARIDE MODIFICATION AND LABELING OF PROTEINS

• US 20110207171A1
• Filed: 01/14/2011
• Published: 08/25/2011
• Est. Priority Date: 02/10/2006
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: CORRY, Schuyler B., Agnew, Brian, GEE, Kyle R.
• Granted Patent: US 8,785,212 B2
• Time in Patent Office: Days
• US Class Current: 435/52
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: C12Y 302/01096 Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (3.2.1.96)
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/534 with radioactive label
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/583 with non-fluorescent dye label
• CPC Class Codes: Y10T 436/17 Nitrogen containing

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.

Claims

1-20. -20. (canceled)

21. A method of labeling a protein by labeling an oligosaccharide attached to said protein, comprising incubating an protein-producing cell in the presence of an unnatural sugar to provide a labeled protein, wherein said unnatural sugar comprises a chemical handle.

22. An antibody comprising a labeled oligosaccharide.

23. 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.

24. The method of claim 23, wherein the group comprises an oligosaccharide group.

25. The method of claim 24, wherein the modified protein comprises a modified recombinant protein or a modified antibody.

26. The method of claim 25, wherein the antibody comprises an IgG.

27. The method of claim 25, wherein the antibody comprises a monoclonal antibody, recombinant antibody, or a fragment thereof which is immunologically active.

28. The method of claim 23, wherein the protein-producing cell comprises a hybridoma cell or a cell producing a recombinant protein.

29. The method of claim 23, wherein the protein-producing cell is a murine cell, a plant cell, a goat cell, a rabbit cell, a yeast cell, or a chinese hamster ovary cell.

30. The method of claim 23, wherein the unnatural substance comprises an unnatural sugar.

31. The method of claim 30, wherein the unnatural sugar comprises GalNAz, ManNAz, GlcNAz, Ac₄GalNAz, Ac₄ManNAz, or Ac₄GlcNAz.

32. The method of claim 23, wherein the chemical handle comprises an alkyne reactive group.

33. The method of claim 32, wherein the alkyne reactive group comprises an azide group.

34. The method of claim 23, wherein the chemical handle comprises an azide reactive group.

35. The method of claim 34, wherein the azide reactive group comprises an alkyne, phosphine or a cyclooctyne group.

36. The method of claim 25, 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.

37. The method of claim 36, wherein the step of attaching results in the formation of a covalent bond.

38. The method of claim 36, wherein the label comprises a reporter molecule, carrier molecule or solid support.

39. The method of claim 38, wherein the reporter molecule comprises a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme, or a radioisotope.

40. The method of claim 38, wherein the carrier molecule comprises 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.

41. The method of claim 38, wherein the solid support comprises 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.

42. The method of claim 36, wherein the label comprises a therapeutic moiety.

43. The method of claim 42, wherein the therapeutic moiety comprises taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, a glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, or analogs or homologs thereof;

or, an antimetabolite, an alkylating agent, an anthracycline, an antibiotic, an anti-mitotic agent, an antimetabolite, an alkylating agent, an anthracycline, an antibiotic, an anti-mitotic agent, abrin, ricin A, pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor, γ

-interferon, α

-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, interleukin-1, interleukin-2, interleukin-6, granulocyte macrophage colony stimulating factor, or granulocyte colony stimulating factor.

44. The method of claim 25, further comprising the step of isolating the modified antibody.

45. The method of claim 44, 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.

46. The method of claim 45, wherein the step of attaching results in the formation of a covalent bond.

47. The method of claim 45, wherein the label comprises a reporter molecule that comprises a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme or a radioisotope.

48. The method of claim 45, wherein the label comprises a carrier molecule that comprises 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.

49. The method of claim 45, wherein the label comprises a solid support that comprises 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, a 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.

50. The method of claim 45, wherein the label comprises a therapeutic moiety.

51. The method of claim 50, wherein the therapeutic moiety comprises taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, a glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, or analogs or homologs thereof;

or, an antimetabolite, an alkylating agent, an anthracycline, an antibiotic, an anti-mitotic agent, abrin, ricin A, pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor, γ

-interferon, α

-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, interleukin-1, interleukin-2, interleukin-6, granulocyte macrophage colony stimulating factor, or granulocyte colony stimulating factor.

52. A modified antibody made by the method of claim 25.

53. A labeled antibody made by the method of claim 36.

54. An isolated modified antibody made by the method of claim 44.

55. A labeled antibody made by the method of claim 45.

56. A kit comprising the modified antibody made by the method of claim 25 and a label that is reactive with the chemical handle of the antibody.

57. The kit of claim 56, wherein the label comprises a reporter molecule.

58. The kit of claim 56, further comprising a copper catalyst.

59. A kit comprising the modified antibody made by the method of claim 44 and a label that is reactive with the chemical handle of the antibody.

60. The kit of claim 59, wherein the label comprises a reporter molecule.

61. The kit of claim 59, further comprising a copper catalyst.

62. 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.

63. The kit of claim 62, wherein the unnatural substance comprises an unnatural sugar.

64. The kit of claim 63, wherein the unnatural sugar comprises GalNAz, ManNAz, GlcNAz, Ac₄GalNAz, Ac₄ManNAz, or Ac₄GlcNAz.

65. The kit of claim 62, 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.

66. The kit of claim 62, wherein the 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.

67. The kit of claim 62, 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.

68. The kit of claim 62, further comprising a copper catalyst.