Antibodies directed to the deletion mutants of epidermal growth factor receptor and uses thereof
The present invention relates to novel antibodies, particularly antibodies directed against deletion mutants of epidermal growth factor receptor and particularly to the type III deletion mutant, EGFRvIII. The invention also relates to human monoclonal antibodies directed against deletion mutants of epidermal growth factor receptor and particularly to EGFRvIII. Diagnostic and therapeutic formulations of such antibodies, and immunoconjugates thereof, are also provided.
1. An isolated human monoclonal antibody that specifically binds to EGFRvIII, wherein EGFRvIII comprises a peptide that consists of the sequence LEEKKGNYVVTDHC (SEQ ID NO: 56), wherein said antibody is conjugated to a toxin selected from the group consisting of: Maytansinoid (DM-1), saporin, Auristatin E toxin (AEFP), monomethyl auristatin E (MMAE), and Auristatin E, wherein said isolated antibody comprises:
a heavy chain variable region amino acid sequence selected from the group consisting of:
SEQ ID NO: 142, and
a variant of SEQ ID NO: 142 that has a point mutation selected from the group consisting of: His35Asn, LeulO4Thr, Ala105Thr, Val50Gly, Val50Ala, Leu104Ile, Trp52Glu, Tyr59Arg, Leu104Tyr, Val50His, His35Ser, His35Val, His35Ala, His35Gly, His35Thr, Tyr59Ser, Leu104Glu, Leu104Gln, Tyr59His, Val50Asn, Tyr59Asn, Ala105Lys, Trp52Lys, Trp52Arg, Tyr59Thr, Leu104Ser, Leu104Asn, and Leu104Asp; and
a light chain variable region amino acid sequence selected from the group consisting of: SEQ ID NO: 144, and
a variant of SEQ ID NO: 144 that has a point mutation selected from the group consisting of: Leu99Glu, Leu99Asn, Leu99His, Leu99Thr, Gln98Glu, Asn35Gly, Asn35Arg, Leu99Tyr, Ala96Thr, Ala96Ser, Pro100Trp, Ser32Ile, His31Glu, Leu99Ser, Pro100Asp, Pro100Gln, Ser32Val, Ser32Pro, Pro100Gly, Pro100Ala, Ser32Ala, Ser32Thr, Pro100Ser, Gln98Thr, Pro100Met, Ser32Met, Ser32Gly, Pro100Asn, and Tyr37Phe, wherein if the heavy chain variable region is the variant of SEQ ID NO: 142 then the light chain variable region is SEQ ID NO: 144, and wherein if the light chain variable region is the variant of SEQ ID NO: 144, then the heavy chain variable region is SEQ ID NO: 142.
2. The antibody of claim 1 that specifically binds to an epitope of EGFRvIII, wherein the epitope consists of the sequence LEEKKGNYVVTDHC (SEQ ID NO:56).
3. The antibody of claim 1 wherein the antibody specifically recognizes a glycine residue at position 6 of an epitope consisting of the sequence LEEKKGNYVVTDHC (SEQ ID NO:56).
4. The antibody of claim 1 , wherein the antibody further binds to the epitope consisting of the sequence EEKKGNYVVT (SEQ ID NO:57).
5. A composition comprising:
the antibody of claim 1 ;
and a pharmaceutically acceptable carrier or diluent.
6. The antibody of claim 1 , wherein the antibody has a K D of less than 1.3×10 −9 M.
7. The antibody of claim 1 , wherein said antibody has a K D of less than 500 pM.
8. The antibody of claim 1 , wherein said antibody is capable of being internalized.
9. The antibody of claim 1 , wherein the residues in the amino acid sequence L E E K K G N Y V V T D H C (SEQ ID NO: 56) that are involved in binding with the antibody are selected from the group consisting of EEK, KKNYV, LEK, EKNY, and EEKGN.
10. An isolated human monoclonal antibody that specifically binds to EGFRvIII, wherein EGFRvIII comprises an epitope consisting of the amino acid sequence EEKKGNY (SEQ ID NO: 131), wherein the antibody comprises a heavy chain variable region in SEQ ID NO: 142 and a light chain variable region in SEQ ID NO: 144, and wherein said antibody is conjugated to a toxin selected from the group consisting of: Maytansinoid (DM-1), saporin, Auristatin E toxin (AEFP), monomethyl auristatin E (MMAE), and Auristatin E.
11. The antibody of claim 1 , wherein the toxin is selected from the group consisting of: Maytansinoid (DM-1) and saporin.
12. The antibody of claim 1 , wherein the toxin is selected from the group consisting of: Auristatin E toxin (AEFP), monomethyl auristatin E (MMAE), and Auristatin E.
13. The antibody of claim 10 , wherein the toxin is selected from the group consisting of: Auristatin E toxin (AEFP), monomethyl auristatin E (MMAE), and Auristatin E.
14. The antibody of claim 10 , wherein the toxin is selected from the group consisting of: Maytansinoid (DM-1) and saporin.
15. The antibody of claim 1 , wherein said antibody specifically binds SEQ ID NO: 56.
16. The antibody of claim 15 , wherein nonspecific binding of the antibody to the wild type EGFR peptide (SEQ ID NO: 134) is less than 10% of that of the specific binding of the antibody to EGFRvIII (SEQ ID NO: 135).
17. The antibody of claim 1 , wherein the antibody inhibits the binding of Epidermal Growth Factor (EGF) to Epidermal Growth Factor Receptor vIII (EGFRvIII).
18. An isolated human monoclonal antibody that binds to EGFRvIII and comprises a light chain variable region and a heavy chain variable region,
wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 2, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 19, and wherein said antibody is conjugated to a toxin selected from the group consisting of: Maytansinoid (DM-1), saporin, Auristatin E toxin (AEFP), monomethyl auristatin E (MMAE), and Auristatin E.
19. The antibody of claim 18 , wherein said antibody has a K D lower than 500 pM.
20. The antibody of claim 18 , wherein the antibody selectively binds to EGFRvIII with a K D lower than 1.3×10 −9 M.
21. The antibody of claim 18 , wherein the antibody inhibits the binding of Epidennal Growth Factor (EGF) to Epidermal Growth Factor Receptor vIII (EGFRvIII).
22. The antibody of claim 18 , wherein said antibody is capable of being internalized.
23. The antibody of claim 18 , wherein the residues that the antibody interacts with in the sequence L E E K K G N Y V V T D H C (SEQ ID NO: 56) are selected from the group consisting of EEK, KKNYV, LEK, EKNY, and EEKGN.
24. The antibody of claim 18 , wherein the antibody binds to the peptide consisting of the sequence EEKKGNYVVT (SEQ ID NO: 57).
25. The antibody of claim 18 , wherein said antibody specifically binds SEQ ID NO: 56.
26. The antibody of claim 25 , wherein nonspecific binding of the antibody to the wild type EGFR peptide (SEQ ID NO: 134) is less than 10% of that of the specific binding of the antibody to EGFRvIII (SEQ ID NO: 135).
27. The antibody of claim 18 , wherein the toxin is selected from the group consisting of: Auristatin E toxin (AEFP), monomethyl auristatin E (MMAE), and Auristatin E.
28. The antibody of claim 18 , wherein the toxin is selected from the group consisting of: Maytansinoid (DM-1) and saporin.
29. The antibody of claim 18 that specifically binds to an epitope of EGFRvIII, wherein the epitope consists of the sequence LEEKKGNYVVTDHC (SEQ ID NO: 56).
30. The antibody of claim 18 wherein the antibody specifically recognizes a glycine residue at position 6 of the epitope consisting of the sequence LEEKKGNYVVTDHC (SEQ ID NO: 56).
31. An isolated human monoclonal antibody that binds to EGFRvIII and that comprises a light chain variable region and a heavy chain variable region,
wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 25, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10, and wherein said antibody is conjugated to a toxin selected from the group consisting of: Maytansinoid (DM-1), saporin, Auristatin E toxin (AEFP), monomethyl auristatin E (MMAE), and Auristatin E.
32. The antibody of claim 31 , wherein the antibody has a K D lower than l.3×10 −9 M.
33. The antibody of claim 32 , wherein said antibody has a K D lower than 500 pM.
34. The antibody of claim 31 , wherein the antibody inhibits the binding of Epidermal Growth Factor (EGF) to Epidermal Growth Factor Receptor vIII (EGFRvIII).
35. The antibody of claim 31 , wherein said antibody is capable of being internalized.
36. The antibody of claim 31 , wherein the residues that the antibody interacts with in the sequence L E E K K G N Y V V T D H C (SEQ ID NO: 56) are selected from the group consisting of EEK, KKNYV, LEK, EKNY, and EEKGN.
37. The antibody of claim 31 , wherein the antibody binds to the peptide consisting of the sequence EEKKGNYVVT (SEQ ID NO: 57).
38. The antibody of claim 31 wherein said antibody specifically binds SEQ ID NO: 56.
39. The antibody of claim 38 , wherein nonspecific binding of the antibody to the wild type EGFR peptide (SEQ ID NO: 134) is less than 10% of that of the specific binding of the antibody to EGFRvIII (SEQ ID NO: 135).
40. The antibody of claim 31 , wherein the toxin is selected from the group consisting of: Auristatin E toxin (AEFP), monomethyl auristatin E (MMAE), and Auristatin E.
41. The antibody of claim 31 , wherein the toxin is selected from the group consisting of: Maytansinoid (DM-1) and saporin.
42. The antibody of claim 31 that specifically binds to an epitope of EGFRvIII, wherein the epitope consists of the sequence LEEKKGNYVVTDHC (SEQ ID NO: 56).
43. The antibody of claim 31 wherein the antibody specifically recognizes a glycine residue at position 6 of an epitope consisting of the sequence LEEKKGNYVVTDHC (SEQ ID NO: 56) of EGFRvIII.
44. An isolated human monoclonal antibody that binds to EGFRvIII and that comprises a light chain variable region and a heavy chain variable region,
wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 23, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7, and wherein said antibody is conjugated to a toxin selected from the group consisting of: Maytansinoid (DM-1), saporin, Auristatin E toxin (AEFP), monomethyl auristatin E (MMAE), and Auristatin E.
45. The antibody of claim 44 , wherein the residues in the sequence L E E K K G N Y V V T D H C (SEQ ID NO: 56) involved in binding with the antibody are selected from the group consisting of EEK, KKNYV, LEK, EKNY, and EEKGN.
46. The antibody of claim 44 , wherein the antibody has a K D lower than 1.3 nM.
47. The antibody of claim 44 wherein the antibody has a K D lower than 500 pM.
48. The antibody of claim 44 , wherein said antibody specifically binds SEQ ID NO: 56.
49. The antibody of claim 48 , wherein nonspecific binding of the antibody to the wild type EGFR peptide (SEQ ID NO: 134) is less than 10% of that of the specific binding of the antibody to EGFRVIII (SEQ ID NO: 135).
50. The antibody of claim 44 , wherein the antibody inhibits the binding of Epidermal Growth Factor (EGF) to Epidermal Growth Factor Receptor vIII (EGFRvIII).
51. The antibody of claim 44 , wherein said antibody is capable of being internalized.
52. The antibody of claim 44 , wherein the antibody binds to the peptide consisting of the sequence EEKKGNYVVT (SEQ ID NO: 57).
53. The antibody of claim 44 , wherein the toxin is selected from the group consisting of: Auristatin E toxin (AEFP), monomethyl auristatin E (MMAE), and Auristatin E.
54. The antibody of claim 44 , wherein the toxin is selected from the group consisting of: Maytansinoid (DM-1) and saporin.
55. The antibody of claim 44 , that specifically binds to an epitope of EGFRvIII, wherein the epitope consists of the sequence LEEKKGNYVVTDHC (SEQ ID NO: 56).
56. An isolated human monoclonal antibody that binds to EGFRvIII and that comprises a light chain variable region and a heavy chain variable region,
wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9, and wherein said antibody is conjugated to a toxin selected from the group consisting of: Maytansinoid (DM-1), saporin, Auristatin E toxin (AEFP), monomethyl auristatin E (MMAE), and Auristatin E.
57. The antibody of claim 56 , wherein the residues in the sequence L E E K K G N Y V V T D H C (SEQ ID NO: 56) involved in binding with the antibody are selected from the group consisting of EEK, KKNYV, LEK, EKNY, and EEKGN.
58. The antibody of claim 56 , wherein the antibody has a K D lower than 1.3 nM.
59. The antibody of claim 56 , wherein the antibody has a K D lower than 500 pM.
60. The antibody of claim 56 , wherein said antibody specifically binds SEQ ID NO: 56.
61. The antibody of claim 60 , wherein nonspecific binding of the antibody to the wild type EGFR peptide (SEQ ID NO: 134) is less than 10% of that of the specific binding of the antibody to EGFRVIII (SEQ ID NO: 135).
62. The antibody of claim 56 , wherein the antibody inhibits the binding of Epidermal Growth Factor (EGF) to Epidermal Growth Factor Receptor vIII (EGFRvIII).
63. The antibody of claim 56 , wherein said antibody is capable of being internalized.
64. The antibody of claim 56 , wherein the antibody binds to the peptide consisting of the sequence EEKKGNYVVT (SEQ ID NO: 57).
65. The antibody of claim 56 , wherein the toxin is selected from the group consisting of: Auristatin E toxin (AEFP), monomethyl auristatin E (MMAE), and Auristatin E.
66. The antibody of claim 56 , wherein the toxin is selected from the group consisting of: Maytansinoid (DM-1) and saporin.
67. The antibody of claim 56 that specifically binds to an epitope of EGFRvIII, wherein the epitope consists of the sequence LEEKKGNYVVTDHC (SEQ ID NO: 56).
68. The antibody of claim 56 wherein the antibody specifically recognizes a glycine residue at position 6 of the epitope consisting of the sequence LEEKKGNYVVTDHC (SEQ ID NO: 56) of EGFRvIII.
69. A kit comprising:
a container,
a composition contained therein, and
a package insert or label indicating that the composition can be used to treat cancer characterized by the expression of EGFRvIII, wherein the composition comprises the antibody of claim 1 .
70. The kit of claim 69 , wherein the cancer is a lung carcinoma, breast carcinoma, head & neck cancer, prostate carcinoma or glioblastoma.
71. An isolated human monoclonal antibody that binds to EGFRvIII and that comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 144, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 142, and wherein said antibody is conjugated to a toxin selected from the group consisting of: Maytansinoid (DM-1), saporin, Auristatin E toxin (AEFP), monomethyl auristatin E (MMAE), and Auristatin E.
72. A composition comprising:
the antibody of claim 18 ; and
a pharmaceutically acceptable carrier or diluent.
73. A composition comprising:
the antibody of claim 31 ; and
a pharmaceutically acceptable carrier or diluent.
74. A composition comprising:
the antibody of claim 44 ; and
a pharmaceutically acceptable carrier or diluent.
75. A composition comprising:
the antibody of claim 56 ; and
a pharmaceutically acceptable carrier or diluent.
76. A kit comprising:
a container,
a composition contained therein, and
a package insert or label indicating that the composition can be used to treat cancer characterized by the expression of EGFRvIII, wherein the composition comprises the antibody of claim 18 .
77. The kit of claim 76 , wherein the cancer is a lung carcinoma, breast carcinoma, head & neck cancer, prostate carcinoma or glioblastoma.
78. A kit comprising:
a container,
a composition contained therein, and
a package insert or label indicating that the composition can be used to treat cancer characterized by the expression of EGFRvIII, wherein the composition comprises the antibody of claim 31 .
79. The kit of claim 78 , wherein the cancer is a lung carcinoma, breast carcinoma, head & neck cancer, prostate carcinoma or glioblastoma.
80. A kit comprising:
a container,
a composition contained therein, and
a package insert or label indicating that the composition can be used to treat cancer characterized by the expression of EGFRvIII, wherein the composition comprises the antibody of claim 44 .
81. The kit of claim 80 , wherein the cancer is a lung carcinoma, breast carcinoma, head & neck cancer, prostate carcinoma or glioblastoma.
82. A kit comprising:
a container,
a composition contained therein, and
a package insert or label indicating that the composition can be used to treat cancer characterized by the expression of EGFRvIII, wherein the composition comprises the antibody of claim 56 .
83. The kit of claim 82 , wherein the cancer is a lung carcinoma, breast carcinoma, head & neck cancer, prostate carcinoma or glioblastoma.