CYSTEINE ENGINEERED ANTIBODIES AND CONJUGATES
First Claim
1. A cysteine engineered antibody comprising one or more free cysteine amino acids having a thiol reactivity value in the range of 0.6 to 1.0,wherein the cysteine engineered antibody is prepared by a process comprising replacing one or more amino acid residues of a parent antibody by cysteine.
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Abstract
Antibodies are engineered by replacing one or more amino acids of a parent antibody with non cross-linked, highly reactive cysteine amino acids. Antibody fragments may also be engineered with one or more cysteine amino acids to form cysteine engineered antibody fragments (ThioFab). Methods of design, preparation, screening, and selection of the cysteine engineered antibodies are provided. Cysteine engineered antibodies (Ab), optionally with an albumin-binding peptide (ABP) sequence, are conjugated with one or more drug moieties (D) through a linker (L) to form cysteine engineered antibody-drug conjugates having Formula I:
Ab-(L-D)p I
where p is 1 to 4. Diagnostic and therapeutic uses for cysteine engineered antibody drug compounds and compositions are disclosed.
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Citations
126 Claims
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1. A cysteine engineered antibody comprising one or more free cysteine amino acids having a thiol reactivity value in the range of 0.6 to 1.0,
wherein the cysteine engineered antibody is prepared by a process comprising replacing one or more amino acid residues of a parent antibody by cysteine.
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47. A method of screening cysteine engineered antibodies for thiol reactivity comprising:
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(a) introducing one or more cysteine amino acids into a parent antibody in order to generate a cysteine engineered antibody; (b) reacting the cysteine engineered antibody with a thiol-reactive affinity reagent to generate an affinity labelled, cysteine engineered antibody; and (c) measuring the binding of the affinity labelled, cysteine engineered antibody to a capture media. - View Dependent Claims (48, 49, 50, 51, 52, 53, 54, 55, 56)
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57. An antibody-drug conjugate compound comprising a cysteine engineered antibody (Ab), and a drug moiety (D) wherein the cysteine engineered antibody is attached through one or more free cysteine amino acids by a linker moiety (L) to D;
- the compound having Formula I;
Ab—
(L-D)p
Iwhere p is 1, 2, 3, or 4; and
wherein the cysteine engineered antibody is prepared by a process comprising replacing one or more amino acid residues of a parent antibody by one or more free cysteine amino acids.- View Dependent Claims (58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117)
where; A is a Stretcher unit covalently attached to a cysteine thiol of the cysteine engineered antibody (Ab);
a is 0 or 1;each W is independently an Amino Acid unit; w is an integer ranging from 0 to 12; Y is a Spacer unit covalently attached to the drug moiety; and y is 0, 1 or 2.
- the compound having Formula I;
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67. The anibody-drug conjugate compound of claim 66 having the formula:
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68. The antibody-drug conjugate compound of claim 67 wherein Ww is valine-citrulline.
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69. The antibody-drug conjugate compound of claim 67 wherein R17 is (CH2)5 or (CH2)2.
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70. The antibody-drug conjugate compound of claim 66 having the formula:
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71. The antibody-drug conjugate compound of claim 70 wherein R17 is (CH2)5 or (CH2)2.
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72. The antibody-drug conjugate compound of claim 66 having the formula:
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73. The antibody-drug conjugate compound of claim 57 wherein L is SMCC.
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74. The antibody-drug conjugate compound of claim 57 wherein L is BMPEO.
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75. The antibody-drug conjugate compound of claim 57 wherein the drug moiety D is selected from a microtubulin inhibitor, a mitosis inhibitor, a topoisomerase inhibitor, and a DNA intercalator.
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76. The antibody-drug conjugate compound of claim 57 wherein the drug moiety D is selected from a maytansinoid, an auristatin, a dolastatin, and a calicheamicin.
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77. The antibody-drug conjugate compound of claim 57 wherein D is MMAE, having the structure:
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78. The antibody-drug conjugate compound of claim 57 wherein D is MMAF, having the structure:
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79. The antibody-drug conjugate compound of claim 57 wherein D is DM1, having the structure:
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80. The antibody-drug conjugate compound of claim 57 wherein the parent antibody is a fusion protein comprising the albumin-binding peptide (ABP).
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81. The antibody-drug conjugate compound of claim 57 wherein the parent antibody is selected from a monoclonal antibody, a bispecific antibody, a chimeric antibody, a human antibody, and a humanized antibody.
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82. The antibody-drug conjugate compound of claim 57 wherein the parent antibody is huMAb4D5-8 (trastuzumab).
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83. The antibody-drug conjugate compound of claim 57 wherein the parent antibody is an anti-ErbB2 antibody.
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84. The antibody-drug conjugate compound of claim 57 wherein the parent antibody is an anti-EphB2R antibody.
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85. The antibody-drug conjugate compound of claim 57 wherein the parent antibody is an anti-CD22 antibody.
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86. The antibody-drug conjugate compound of claim 57 wherein the parent antibody is an anti-MUC16 antibody.
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87. The antibody-drug conjugate compound of claim 57 wherein the parent antibody is an intact antibody selected from IgA, IgD, IgE, IgG, and IgM.
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88. The antibody-drug conjugate compound of claim 87 wherein the IgG is selected from subclasses:
- IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
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89. The antibody-drug conjugate compound of claim 57 wherein the parent antibody is an antibody fragment.
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90. The antibody-drug conjugate compound of claim 89 wherein the antibody fragment is a Fab fragment.
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91. The antibody-drug conjugate compound of claim 90 wherein the Fab fragment is hu4D5Fabv8.
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92. The antibody-drug conjugate compound of claim 91 wherein the one or more of the amino acid residues of hu4D5Fabv8 replaced by cysteine are selected from L-V15, L-A43, L-V110, L-A144, L-S168, H-A40, H-A88, H-S119, H-A121, H-S122, H-A175, and H-S179.
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95. A pharmaceutical composition comprising the antibody-drug conjugate compound of claim 57 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier or excipient.
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96. The pharmaceutical composition of claim 95 further comprising a therapeutically effective amount of an additional chemotherapeutic agent
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97. A method for killing or inhibiting the proliferation of tumor cells or cancer cells comprising treating tumor cells or cancer cells with an amount of the antibody-drug conjugate compound of claim 57, or a pharmaceutically acceptable salt or solvate thereof, being effective to kill or inhibit the proliferation of the tumor cells or cancer cells.
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98. A method of treating cancer comprising administering to a patient a formulation of an antibody-drug conjugate compound of claim 57 and a pharmaceutically acceptable diluent, vehicle, carrier or excipient.
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99. The method of claim 98 further comprising administering an effective amount of an additional chemotherapeutic agent.
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100. The method of claim 98 wherein the antibody-drug conjugate compound binds to a receptor encoded by an ErbB gene.
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101. The method of claim 98 wherein the amount of antibody-drug conjugate compound administered to the patient is in the range of about 0.1 to about 10 mgfkg of patient weight.
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102. The method of claim 98 wherein said antibody-drug conjugate compound is administered by infusion.
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103. The method of claim 98 wherein said antibody-drug conjugate compound is formulated in a unit dosage injectable form.
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104. The method of claim 98 further comprising the administration of a second antibody which binds ErbB2 and blocks antibody activation of an ErbB receptor.
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105. The method of claim 98 further comprising the administration of a cytotoxic agent or chemotherapeutic agent.
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106. A method of inhibiting cellular proliferation comprising:
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(a) exposing mammalian cells in a cell culture medium to the antibody-drug conjugate compound of claim 57, and (b) measuring a cytotoxic or cytostatic activity of the antibody-drug conjugate compound, whereby proliferation of the cells is inhibited
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107. A method of inhibiting the growth of tumor cells that overexpress a growth factor receptor selected from a HER2 receptor and an EGF receptor comprising administering to a patient an antibody-drug conjugate compound of claim 57 which binds specifically to said growth factor receptor, and a chemotherapeutic agent wherein said antibody-drug conjugate and said chemotherapeutic agent are each administered in amounts effective to inhibit growth of tumor cells in the patient.
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108. The method of claim 107 wherein said antibody-drug conjugate compound sensitizes the tumor cells to said chemotherapeutic agent.
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109. A method for the treatment of a human patient susceptible to, or diagnosed with, a disorder characterized by overexpression of ErbB2 receptor, comprising administering an effective amount of a combination of an antibody-drug conjugate compound of claim 57 and a chemotherapeutic agent.
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110. A method for the treatment of cancer in a mammal, wherein the cancer is characterized by the overexpression of an ErbB receptor and does not respond, or responds poorly, to treatment with an unconjugated anti-ErbB antibody, comprising administering to the mammal a therapeutically effective amount of an antibody-drug conjugate compound of claim 57.
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111. The method of claim 110 wherein the mammal is human.
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112. The method of claim 110 wherein the ErbB receptor is selected from the group consisting of ErbB1 (EGFR), ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4).
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113. The method of claim 110 wherein the antibody of the antibody-drug conjugate compound is an anti-ErbB2 antibody.
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114. The method of claim 110 wherein the cancer is selected from the group consisting of breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, colorectal, thyroid, pancreatic, prostate and bladder cancer.
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115. The method of claim 114 wherein the cancer is breast cancer and the breast cancer overexpresses ErbB2 at a 2+ level or more.
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116. A method of treating an autoimmune disease in a patient comprising administration of a formulation of an antibody-drug conjugate compound of claim 57 and a pharmaceutically acceptable diluent, vehicle, carrier or excipient.
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117. An article of manufacture comprising
an antibody-drug conjugate compound of claim 57, a container, and a package insert or label indicating that the compound can be used to treat cancer.
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93. An antibody-drug conjugate compound selected from the structures:
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94. An antibody-drug conjugate compound having the structure:
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118. A method for making an antibody drug conjugate compound comprising a cysteine engineered antibody (Ab), and a drug moiety (D) wherein the cysteine engineered antibody is attached through the one or more engineered cysteine amino acids by a linker moiety (L) to D;
- the compound having Formula I;
Ab—
(L-D)p
Iwhere p is 1, 2, 3, or 4;
the method comprising replacing one or more amino acid residues of a parent antibody by cysteine to prepare the cysteine engineered antibody.- View Dependent Claims (119, 120, 121, 122, 123, 124, 125, 126)
- the compound having Formula I;
Specification