Super humanized antibodies

• US 20050261480A1
• Filed: 02/08/2005
• Published: 11/24/2005
• Est. Priority Date: 07/12/2001
• Status: Active Grant

• Current Assignee: Arrowsmith Technology Licensing LLC
• Original Assignee: Arrowsmith Technology Licensing LLC
• Inventors: Foote, Jefferson
• Granted Patent: US 7,732,578 B2
• Time in Patent Office: Days
• US Class Current: 530/387.100
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/2818 against CD28 or CD152
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 16/464 Igs containing CDR-residues from one specie grafted between FR-residues from another
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/54 F(ab')2
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/567 Framework region [FR]
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

Disclosed herein are methods for humanizing antibodies based on selecting variable region framework sequences from human antibody genes by comparing canonical CDR structure types for CDR sequences of the variable region of a non-human antibody to canonical CDR structure types for corresponding CDRs from a library of human antibody sequences, preferably germline antibody gene segments. Human antibody variable regions having similar canonical CDR structure types to the non-human CDRs form a subset of member human antibody sequences from which to select human framework sequences. The subset members may be further ranked by amino acid similarity between the human and the non-human CDR sequences. Top ranking human sequences are selected to provide the framework sequences for constructing a chimeric antibody that functionally replaces human CDR sequences with the non-human CDR counterparts using the selected subset member human frameworks, thereby providing a humanized antibody of high affinity and low immunogenicity without need for comparing framework sequences between the non-human and human antibodies. Chimeric antibodies made according to the method are also disclosed.

Claims

1-46. -46. (canceled)

47. A method of designing a molecule that binds an antigen, comprising,

determining a canonical CDR structure type for at least two CDRs in a variable region of a subject antibody that binds the antigen;

selecting an object variable region from an object antibody based on whether the object variable region has the same canonical CDR structure types at corresponding locations in the object variable region as the at least two canonical CDR structure types of the subject variable region; and

designing a molecule that binds the same antigen as the subject antibody comprising a chimeric variable region having the at least two CDRs from the subject variable region grafted into a framework sequence of the selected object variable region at the corresponding locations, where the framework sequence of the chimeric variable region differs from the framework sequence of the selected object variable region by no more than 10 amino acid residues.

48. The method of claim 47 wherein designing the chimeric variable region includes grafting each of three CDRs from the subject variable region into corresponding locations in the object framework sequence.

49. The method of claim 47 wherein the framework sequence of the chimeric variable region differs from the framework sequence of the object variable region by no more than 5 amino acid residues.

50. The method of claim 47 wherein the framework sequence of the chimeric variable region differs from the framework sequence of the object variable region by no more than 2 amino acid residues.

51. The method of claim 47 wherein selecting the object variable region includes obtaining a set of candidate object variable regions having the same canonical CDR structure types as the subject CDRs and ranking the set of candidate object variable regions by comparing position by position similarity of amino acid residues of the subject CDRs to the corresponding object CDRs according to a ranking criterion.

52. The method of claim 51 wherein the object variable region is selected from the top 25% of the ranked candidate variable regions.

53. The method of claim 51 wherein the ranking criterion includes a score of amino acid identity between the subject and object CDR sequences at corresponding residue positions of at least one CDR.

54. The method of claim 51 wherein the ranking criterion includes a score of amino acid identity between the subject and object CDR sequences at corresponding residue positions of at least two CDRs.

55. The method of claim 51 wherein the ranking criterion includes a score of amino acid identity between the subject and object CDR sequences at corresponding residue positions in each of three CDRs.

56. The method of claim 54 wherein the ranking criterion further includes a score of amino acid homology between the subject and object CDRs at corresponding residue positions of at least one CDR.

57. The method of claim 54 wherein the ranking criterion further includes a score of amino acid homology between the subject and object CDRs at corresponding residue positions of at least two CDRs.

58. The method of claim 54 wherein the ranking criterion further includes a score of amino acid homology between the subject and object CDRs at corresponding residue positions for each of three CDRs.

59. The method of claim 47 wherein the CDRs are Kabat defined CDRs.

60. The method of claim 47 wherein the CDRs are Chothia defined CDR loops.

61. The method of claim 47 wherein designing the chimeric variable region further includes substituting at least one amino acid residue of a subject CDR sequence with a different amino acid, with the proviso that no more than 4 residues are substituted in any of the subject'"'"'s light chain CDR1, light chain CDR2, light chain CDR 3, heavy chain CDR1, or heavy chain CDR3 and no more than 10 amino acids are substituted in the subject heavy chain CDR2.

62. The method of claim 47 wherein the act of designing the chimeric molecule further includes substituting at least one but no more than 10 amino acid residues of the selected framework sequence with a different amino acid residue.

63. The method of claim 47 wherein each of three subject CDRs is a light chain CDR, and if one of the three subject CDR sequences is of a canonical structure type absent from the object light chain variable region, then the act of selecting further includes selecting an object variable region with a CDR of a different canonical structure type than the subject CDR type that is absent, with the proviso that the different object canonical structure type has a length no more than two amino acid residues smaller or larger than the subject canonical structure type that is absent.

64. The method of claim 63 wherein if the absent CDR canonical structure type is of canonical type 1, then the act includes selecting an object variable region with a canonical type 2 CDR at the corresponding location, or if the subject CDR sequences is of canonical type 5 then the act includes selecting an object variable region with a canonical type 4 or 3 at the corresponding location.

65. The method of claim 47 wherein the subject variable region is a mouse variable region.

66. The method of claim 47 wherein the object variable region is a human variable region.

67. The method of claim 47 wherein the subject variable region is a mouse variable region and the object variable region is a human variable region.

68. The method of claim 47 wherein the object variable region sequence is selected from the group consisting of human V_k, V_λ

, V_H, J_H, J_k and J_λ

sequences.

69. The method of claim 47 wherein the act of designing the molecule that binds the antigen includes designing chimeric variable regions for each of the light chain and the heavy chain.

70. The method of claim 69 wherein the chimeric variable region includes the frameworks from human V_k, and V_H sequences.

71. The method of claim 69 wherein the chimeric variable light chains and chimeric heavy chains are designed to form a molecule selected from the group consisting of a Fab fragment, a (Fab)′

₂ molecule, and a single chain Fv molecule.

72. The method of claim 69 wherein the chimeric variable light chains and chimeric variable heavy chains are further designed to include an antibody constant region domain for the object antibody to form a complete antibody.

73. The method of claim 47 wherein the selected object variable region is encoded by a human germline variable region gene.

74. The method of claim 47 wherein the selected object variable region is from a mature human antibody.

75. The method of claim 47 through 74 further comprising preparing a nucleic acid encoding said chimeric variable region.

76. A host capable of expressing a nucleic acid made according to claim 75.

77. The method of claim 47 further including the act of constructing a set of molecules with different object variable regions, determining a dissociation constant between the antigen and different members of the set of molecules, and selecting a molecule having a dissociation constant of at least 10⁶ M⁻

1, at least 10⁷ M<sup>−

1</sup> or at least 10⁸ M<sup>−

1.</sup>

78. A molecule designed according to the method of claim 47 characterized in that said molecule binds the antigen, and has a chimeric variable region containing at the at least two CDRs from the subject variable region grafted into a framework sequence of the object variable region.

79. A humanized antibody variable region assembled into a molecule that binds an antigen, comprising

a chimeric antibody variable region containing at least two non-human CDR sequences grafted into a human variable region framework sequence, the chimeric variable region being characterized by having no more than 10 amino acid residues that differ from a framework sequence of a candidate human antibody variable region having at least two human CDR sequences with the same canonical structure type as the non-human CDR sequences for at least two corresponding CDR positions.

80. The humanized variable region of claim 79 wherein the non-human variable region is a mouse variable region.

81. The humanized variable region of claim 79 wherein the human variable region sequence is selected from the group consisting of V_k, V_λ

, V_H, J_H, J_k and J_λ

sequences.

82. The humanized variable region of claim 79 wherein the assembled molecule includes chimeric variable region sequences for each of a variable light chain and a variable heavy chain.

83. The humanized variable region of claim 82 wherein the chimeric variable light chains and chimeric variable heavy chains are assembled to form a molecule selected from the group consisting of a Fab fragment, a (Fab)₂ molecule, and a single chain Fv molecule.

84. The humanized variable region of claim 82 wherein the chimeric molecule further includes a human antibody constant region domain to form a complete antibody.

85. The humanized variable region of claim 79 wherein the candidate human variable region is encoded by a human germline variable region gene.

86. The humanized variable region of claim 79 wherein the candidate human variable region is a sequence from a human mature antibody.

87. The humanized variable region of claim 79 wherein the assembled molecule has a dissociation constant for its antigen of at least at least 10⁶ M⁻

1, at least 10⁷ M<sup>−

1</sup> or at least 10⁸ M<sup>−

1</sup>.

88. The humanized variable region of claim 79 wherein the assembled molecule does not elicit an immune response when administered to a human.

89. The humanized variable region of claim 79 wherein the assembled molecule binds a human CD28 antigen.