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Details for Patent: 7,022,500

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Details for Patent: 7,022,500

Title:Humanized immunoglobulins
Abstract: Novel methods for producing, and compositions of, humanized immunoglobulins having one or more complementarity determining regions (CDR's) and possible additional amino acids from a donor immunoglobulin and a framework region from an accepting human immunoglobulin are provided. Each humanized immunoglobulin chain will usually comprise, in addition to the CDR's, amino acids from the donor immunoglobulin framework that are, e.g., capable of interacting with the CDR's to effect binding affinity, such as one or more amino acids which are immediately adjacent to a CDR in the donor immunoglobulin or those within about about 3.ANG. as predicted by molecular modeling. The heavy and light chains may each be designed by using any one or all of various position criteria. When combined into an intact antibody, the humanized immunoglobulins of the present invention will be substantially non-immunogenic in humans and retain substantially the same affinity as the donor immunoglobulin to the antigen, such as a protein or other compound containing an epitope.
Inventor(s): Queen; Cary L. (Los Altos, CA), Selick; Harold E. (Belmont, CA)
Assignee: Protein Design Labs, Inc. (Fremont, CA)
Filing Date:Nov 22, 2000
Application Number:09/718,998
Claims:1. A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant within about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises at least three amino acids from the donor immunoglobulin heavy chain framework outside the Kabat and Chothia CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, wherein each of these at least three donor amino acids: (I) is immediately adjacent to one of the CDRs, or (II) is capable of interacting with the CDRs, or (III) is typical at its position for human immunoglobulin sequences, and the replaced amino acid is rare at its position for human immunoglobulin sequences.

2. A humanized immunoglobulin according to claim 1, wherein said humanized immunoglobulin binds to the antigen with an affinity constant of at least 10.sup.8 M.sup.-1.

3. A humanized immunoglobulin having a complementarity determining region from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant within about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises at least three amino acids from the donor immunoglobulin heavy chain framework outside the Kabat and Chothia CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, wherein each of these at least three donor amino acids: (I) is immediately adjacent to one of the CDRs, or (II) is capable of interacting with the CDRs.

4. A humanized immunoglobulin according to claim 3, wherein said humanized immunoglobulin binds to the antigen with an affinity constant of at least 10.sup.8 M.sup.-1.

5. A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant within about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises at least three amino acids from the donor immunoglobulin heavy chain framework outside the Kabat and Chothia CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids: is capable of interacting with the CDRs.

6. A humanized immunoglobulin according to any one of claims 1 through 5, wherein said humanized immunoglobulin is an antibody tetramer, Fab, or (Fab').sub.2.

7. A humanized immunoglobulin according to any one of claims 1 through 5, which is substantially pure.

8. A pharmaceutical composition comprising a humanized immunoglobulin according to claim 7 in a pharmaceutically acceptable carrier.

9. A humanized immunoglobulin comprising a complementarity determining region from a donor immunoglobulin and heavy and light chain acceptor immunoglobulin variable region frameworks, which humanized immunoglobin in specifically binds to an antigen with an affinity constant within about four-fold that of the donor immunoglobulin, wherein the heavy and light chain acceptor immunoglobin variable region frameworks are consensus frameworks from many human antibodies, and wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin variable region framework outside the CDRs that replace the corresponding amino acids in the one or both of the heavy and light chain acceptor immunoglobulin variable region frameworks, wherein each of these said donor amino acids is capable of interacting with the CDRs.

10. A humanized immunoglobulin according to claim 9, wherein said humanized immunoglobulin binds to the antigen with an affinity constant of at least 10.sup.8 M.sup.-1.

11. A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant within about four-fold that of the donor immunoglobulin, wherein the acceptor immunoglobulin heavy chain variable region framework is a consensus of human immunoglobulin heavy chain variable region frameworks, and wherein said humanized immunoglobulin heavy chain comprises amino acids from the donor immunoglobulin framework outside the CDRs that replace the corresponding amino acids in the acceptor immunoglobulin framework, wherein each of these said donor amino acids is capable of interacting with the CDRs.

12. A humanized immunoglobulin according to claim 11, wherein said humanized immunoglobulin binds to the antigen with an affinity constant of at least 10.sup.8 M.sup.-1.

13. A humanized immunoglobulin according to any one of claims 9 through 12, wherein said humanized immunoglobulin is an antibody tetramer, Fab, or (Fab').sub.2.

14. A humanized immunoglobulin according to any one of claims 9 through 12, which is substantially pure.

15. A pharmaceutical composition comprising a humanized immunoglobulin according to claim 14 in a pharmaceutically acceptable carrier.

16. A method of producing a humanized immunoglobulin that specifically binds to an antigen with an affinity constant within about four-fold that of the donor immunoglobulin, comprising the steps of (1) selecting an acceptor immunoglobulin heavy chain variable region framework whose sequence is a consensus sequence of human heavy chain variable region framework sequences; (2) synthesizing a DNA segment encoding a humanized immunoglobulin heavy chain variable region, comprising complementarity determining regions (CDRs) from a donor immunoglobulin heavy chain variable region and the selected acceptor immunoglobulin heavy chain variable region framework; (3) introducing the DNA segment encoding the humanized immunoglobulin heavy chain variable region and a DNA segment encoding a humanized immunoglobulin light chain variable region into a cell; and (4) expressing the DNA segments in the cell to produce the humanized immunoglobulin.

17. The method of claim 16, further comprising the step of substituting an amino acid in the acceptor immunoglobulin heavy chain variable region framework outside the CDRs with the corresponding amino acid from the donor immunoglobulin heavy chain variable region, wherein the amino acid is capable of interacting with the CDRS.

18. The method of claim 16, further comprising the step of purifying the humanized immunoglobulin.

19. The method of claims 16 or 17, wherein said humanized immunoglobulin binds to the antigen with an affinity constant of at least 10.sup.8 M.sup.-1.

20. A method of producing a humanized immunoglobulin that specifically binds to an antigen with an affinity constant within about four-fold that of the donor immunoglobulin, the method comprising: providing a cell containing DNA segments encoding humanized light and heavy chain variable regions; and expressing the DNA segments in the cell to produce the humanized immunoglobulin; wherein the cell containing the DNA segments was produced by: (1) selecting an acceptor immunoglobulin heavy chain variable region framework whose sequence is a consensus sequence of human immunoglobulin heavy chain variable region framework sequences; (2) synthesizing a DNA segment encoding a humanized immunoglobulin heavy chain variable region, comprising a complementarity determining region (CDR) from a donor immunoglobulin heavy chain variable region and the selected acceptor immunoglobulin heavy chain variable region framework and further comprising amino acids from the donor immunoglobulin heavy chain framework outside the CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain variable region framework, at positions in the immunoglobulins where the amino acids are capable of interacting with the CDRs; (3) introducing the DNA segment encoding the humanized immunoglobulin heavy chain variable region and a DNA segment encoding a humanized immunoglobulin light chain variable region into a cell.

21. The method of claim 20, further comprising the step of purifying the humanized immunoglobulin.

22. The method of claims 20 or 21, wherein said humanized immunoglobulin binds to the antigen with an affinity constant of at least 10.sup.8 M.sup.-1.

23. The method of claims 20 or 21, wherein said humanized immunoglobulin is an antibody tetramer, Fab, or (Fab').sub.2.

24. A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant within about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises an amino acid from the donor immunoglobulin framework outside the CDRs that replaces the corresponding amino acid in the acceptor immunoglobulin frameworks, at a position selected from the group consisting of L9, LIO, L13, L36, L41, L42, L48, L49, L63, L70, L71, L79, L87, L106, H37, H48, H49, H66, H67, H68, H72, H79, H81, H82b, H86, H87, H89, H91, H94, H103, H104, H105 and H107, utilizing the Kabat nunbering system where the donor amino acid: (I) is adjacent to a CDR in the donor immunoglobulin sequence, or (II) is capable of interacting with the CDRs, or (III) is typical at its position for human immunoglobulin sequences, and the replaced amino acid is rare at its position for human immunoglobulin sequences.

25. A humanized immunoglobulin according to claim 24, having three CDRs from the heavy chain of the donor immunoglobulin and three CDRs from the light chain of the donor immunoglobulin.

26. A humanized immunoglobulin according to claim 24, wherein said humanized immunoglobulin binds to the antigen with an affinity constant of at least 10.sup.8 M.sup.-1.

27. A humanized immunoglobulin according to claim 24, wherein the amino acid at position L28 has been replaced.

28. A humanized immunoglobulin according to claim 24, wherein the amino acid at position L10 has been replaced.

29. A humanized immunoglobulin according to claim 24, wherein the amino acid at position LI 3 has been replaced.

30. A humanized immunoglobulin according to claim 24, wherein the amino acid at position L36 has been replaced.

31. A humanized immunoglobulin according to claim 24, wherein the amino acid at position L41 has been replaced.

32. A humanized immunoglobulin according to claim 24, wherein the amino acid at position L42 has been replaced.

33. A humanized immunoglobulin according to claim 24, wherein the amino acid at position L48 has been replaced.

34. A humanized immunoglobulin according to claim 24, wherein the amino acid at position L49 has been replaced.

35. A humanized immunoglobulin according to claim 24, wherein the amino acid at position L63 has been replaced.

36. A humanized immunoglobulin according to claim 24, wherein the amino acid at position L70 has been replaced.

37. A humanized immunoglobulin according to claim 24, wherein the amino acid at position L71 has been replaced.

38. A humanized immunoglobulin according to claim 24, wherein the amino acid at position L79 has been replaced.

39. A humanized immunoglobulin according to claim 24, wherein the amino acid at position L87 has been replaced.

40. A humanized immunoglobulin according to claim 24, wherein the amino acid at position L106 has been replaced.

41. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H137 has been replaced.

42. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H48 has been replaced.

43. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H49 has been replaced.

44. A humanized immunoglobin according to claim 24, wherein the amino acid at position H66 has been replaced.

45. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H67 has been replaced.

46. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H68 has been replaced.

47. A humanized-immunoglobulin according to claim 24, wherein the amino acid at position H72 has been replaced.

48. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H79 has been replaced.

49. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H81 has been replaced.

50. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H82b has been replaced.

51. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H86 has been replaced.

52. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H87 has been replaced.

53. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H89 has been replaced.

54. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H91 has been replaced 166.

55. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H94 has been replaced.

56. A humanized immunoglobulin according to claim 24, wherein the amino acid at position HI 03 has been replaced.

57. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H104 has been replaced.

58. A humanized immunoglobulin according to claim 24, wherein the amino acid at position 11105 has been replaced.

59. A humanized immunoglobulin according to claim 24, wherein the amino acid at position H107 has been replaced.

60. A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant within about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises an amino acid from the donor immunoglobulin framework outside the CDRs that replaces the corresponding amino acid in the acceptor immunoglobulin framework, at a position where the donor amino acid: is adjacent to a CDR in the donor immunoglobulin sequence; wherein said position is selected from the group consisting of H49, H66, 1H94, and H103, utilizing the Kabat numbering system.

61. A humanized immunoglobulin according to claim 60 having three CDRs from the heavy chain of the donor immunoglobulin and three CDRs from the light chain of the door immunoglobulin.

62. A humanized immunoglobulin according to claim 60, wherein said humanized immunoglobulin binds to the antigen with an affinity constant of at least 10.sup.8 M.sup.-1.

63. A humanized immunoglobulin according to claim 60, wherein the amino acid at position H49 has been replaced.

64. A humanized immunoglobulin according to claim 60, wherein the amino acid at position H66 has been replaced.

65. A humanized immunoglobulin according to claim 60, wherein the amino acid at position H94 has been replaced.

66. A humanized immunoglobulin according to claim 60, wherein the amino acid at position H103 has been replaced.

67. A humanized immunoglobulin having complementarity determining regions (CDRS) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant within about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises an amino acid from the donor immunoglobulin light chain framework outside the CDRs that replaces the corresponding amino acid in the acceptor immunoglobulin light chain framework, at a position where the donor amino acid; is capable of interacting with the CDRs.

68. A humanized immunoglobulin according to claim 67 wherein said position is selected from the group consisting of L36, L48, L49, L70, L71 and L87, utilizing the Kabat numbering system.

69. A humanized immunoglobulin according to claim 67 or 68 having three CDRs from the heavy chain of the donor immunoglobulin and three CDRs from the light chain of the donor immunoglobulin.

70. A humanized immunoglobulin according to claim 67 or 68, wherein said humanized immunoglobulin binds to the antigen with an affinity constant of at least 10.sup.8 M.sup.-1.

71. A humanized immunoglobulin according to claim 67, wherein the amino acid at position L36 has been replaced.

72. A humanized immunoglobulin according to claim 67, wherein the amino acid at position L48 has been replaced.

73. A humanized immunoglobulin according to claim 67, wherein the amino acid at position L49 has been replaced.

74. A humanized immunoglobulin according to claim 67, wherein the amino acid at position L70 has been replaced.

75. A humanized immunoglobulin according to claim 67, wherein the amino acid at position L71 has been replaced.

76. A humanized immunoglobulin according to claim 67, wherein the amino acid at position L87 has been replaced.

77. A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant within about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises an amino acid from the donor immunoglobulin heavy chain framework outside the CDRs that replaces the corresponding amino acid in the acceptor immunoglobulin heavy chain framework, at a position where the donor amino acid: is capable of interacting with the CDRs; wherein said position is selected from the group consisting of H37, H48, H67, H71 and H72, utilizing the Kabat numbering system.

78. A humanized immunoglobulin according to claim 77 having three CDRs from the heavy chain of the donor immunoglobulin and three CDRs from the light chain of the donor immunoglobulin.

79. A humanized immunoglobulin according to claim 77, wherein said humanized immunoglobulin binds to the antigen with an affinity constant of at least 10.sup.8 M.sup.-1.

80. A humanized immunoglobulin according to claim 77, wherein the amino acid at position H37 has been replaced.

81. A humanized immunoglobulin according to claim 77, wherein the amino acid at position H48 has been replaced.

82. A humanized immunoglobulin according to claim 77, wherein the amino acid at position H67 has been replaced.

83. A humanized immunoglobulin according to claim 77, wherein the amino acid at position H71 has been replaced.

84. A humanized immunoglobulin according to claim 77, wherein the amino acid at position H72 has been replaced.

85. A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant within about four-fold that of the donor immunoglobulin, wherein the acceptor immunoglobulin heavy chain variable region framework is a consensus sequence of human immunoglobulin heavy chain variable region frameworks, and wherein said humanized immunoglobulin comprises an amino acid from the donor immunoglobulin framework outside the CDR that replaces the corresponding amino acid in the acceptor immunoglobulin framework, at a position in the immunoglobulins where the donor amino acid: (I) is adjacent to a CDR in the donor immunoglobulin sequence, or (II) is capable of interacting with the CDRs.

86. A humanized immunoglobulin according to claim 85, wherein said position is selected from the group of T16, L48, L49, L70, L71, L87, H37, H48, H49, H66, H67, H71, H72, H94, and H103, utilizing the Kabat numbering system.

87. A humanized immunoglobulin according to claim 85 or 86 having three CDRs from the heavy chain of the donor immunoglobulin and three CDRs from the light chain of the donor immunoglobulin.

88. A humanized immunoglobulin according to claim 85 or 86 wherein said humanized immunoglobulin binds to the antigen with an affinity constant of at least 10.sup.8 M.sup.-1.

89. A humanized immunoglobulin according to claim 85, wherein the amino acid at position H37 has been replaced.

90. A humanized immunoglobulin according to claim 85, wherein the amino acid at position H49 has been replaced.

91. A humanized immunoglobulin according to claim 85, wherein the amino acid at position H67 has been replaced.

92. A humanized immunoglobulin according to claim 85, wherein the amino acid at position H71 has been replaced.

93. A humanized immunoglobulin according to claim 85, wherein the amino acid at position H94 has been replaced.

94. A humanized immunoglobulin according to any one of claims 24, 60, 67, 68, 77, 85 and 86 which is substantially pure.

95. A pharmaceutical composition comprising a humanized immunoglobulin according to claim 94 in a pharmaceutically acceptable carrier.
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