U.S. patent application number 15/597624 was filed with the patent office on 2017-12-07 for anti-cmet antibody drug conjugates and methods for their use.
The applicant listed for this patent is ABBVIE BIOTHERAPEUTICS INC.. Invention is credited to DANIEL E. AFAR, CHRISTIAN B. ALLAN, MARK G. ANDERSON, LOUIE NAUMOVSKI, EDWARD B. REILLY, JIEYI WANG.
Application Number | 20170348429 15/597624 |
Document ID | / |
Family ID | 59014737 |
Filed Date | 2017-12-07 |
United States Patent
Application |
20170348429 |
Kind Code |
A1 |
REILLY; EDWARD B. ; et
al. |
December 7, 2017 |
ANTI-cMET ANTIBODY DRUG CONJUGATES AND METHODS FOR THEIR USE
Abstract
The present disclosure provides antibody drug conjugates that
bind human cMET, their methods of making, and their uses to treat
patients having cancer.
Inventors: |
REILLY; EDWARD B.;
(LIBERTYVILLE, IL) ; NAUMOVSKI; LOUIE; (LOS ALTOS,
CA) ; ALLAN; CHRISTIAN B.; (SAN MATEO, CA) ;
WANG; JIEYI; (BELMONT, CA) ; ANDERSON; MARK G.;
(GRAYSLAKE, IL) ; AFAR; DANIEL E.; (LOS ALTOS
HILLS, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ABBVIE BIOTHERAPEUTICS INC. |
REDWOOD CITY |
CA |
US |
|
|
Family ID: |
59014737 |
Appl. No.: |
15/597624 |
Filed: |
May 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62337796 |
May 17, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6803 20170801;
A61P 35/00 20180101; A61K 31/517 20130101; A61K 47/6811 20170801;
A61K 45/06 20130101; A61K 47/6849 20170801; A61K 31/517 20130101;
A61K 2300/00 20130101 |
International
Class: |
A61K 47/68 20060101
A61K047/68; A61K 31/517 20060101 A61K031/517 |
Claims
1. A method of treating a solid tumor cancer that overexpresses
cMet, comprising administering to a human subject having said
cancer an anti-cMet antibody drug conjugate ("ADC") in an amount
and for a period of time sufficient to provide a therapeutic
benefit.
2. The method of claim 1 in which the cMet overexpressing cancer is
of a cancer type wherein cMet is overexpressed in at least about
10% of a patient population having the cancer type.
3. The method of claim 1 in which a biopsy of the cMet
overexpressing tumor tissue from the subject has an IHC score of 2+
and/or an H-score from 150 to 224, when measured according to the
cMet ABBV-ADC staining protocol.
4. The method of claim 1 in which a biopsy of the cMet
overexpressing tumor tissue from the subject has an IHC score of 3+
and/or an H-score greater than 225, when measured according to the
cMet ABBV-ADC staining protocol.
5. The method according to any one of claims 1-4 in which the cMet
overexpressing cancer is non-small cell lung cancer ("NSCLC").
6. The method of claim 5, in which the NSCLC is a non-squamous
NSCLC.
7. The method of claim 5 in which the NSCLC is squamous NSCLC.
8. The method of claim 6 in which the non-squamous NSCLC is an
adeonocarcinoma.
9. The method of claim 1 in which the cancer is colorectal cancer
("CRC").
10. The method of claim 1 in which the cancer is head & neck
("H&N") cancer.
11. The method of claim 1 in which the cancer is pancreatic
cancer.
12. The method of claim 1 in which the cMet overexpressing cancer
is resistant to prior treatment with targeted and/or non-targeted
chemotherapy.
13. The method of claim 1 in which the cMet overexpressing cancer
is resistant to prior treatment with an anti-cMet antibody.
14. The method of claim 1 in which the anti-cMet ADC is
administered as monotherapy.
15. The method of claim 1 in which the anti-cMet ADC is
administered adjunctive to an additional anticancer agent, where
the additional agent is administered according to its FDA-approved
dosing regimen.
16. The method of claim 15 in which the additional anticancer agent
is an inhibitor of epidermal growth factor receptor ("EGFR").
17. The method of claim 16 in which the additional anticancer agent
is erlotinib.
18. The method of claim 15 in which the additional anticancer agent
is an inhibitor of PD1.
19. The method of claim 18 in which the inhibitor of PD1 is an
anti-PD1 antibody.
20. The method of claim 19 in which the anti-PD1 antibody is
nivolumab.
21. The method of any one of claims 1-20 in which the anti-cMet ADC
is administered in an amount ranging from about 0.15 mg/kg to about
3.3 mg/kg once every three weeks.
22. The method of claim 21 in which the anti-cMet ADC is
administered in an amount of about 2.7 mg/kg.
23. The method of any one of claims 1-20 in which the anti-cMet ADC
is administered in an amount ranging from about 0.15 mg/kg to about
3.3 mg/kg once every two weeks.
24. The method of claim 23 in which the anti-cMet ADC is
administered in an amount of about 1.6 mg/kg once every two
weeks.
25. The method of claim 23 in which the anti-cMet ADC is
administered in an amount of about 1.9 mg/kg once every two
weeks.
26. The method of any one of claims 1-25 in which the anti-cMet ADC
comprises an anti-cMet antibody linked to a cytostatic and/or
cytotoxic agent by way of a linker.
27. The method of claim 26 in which the anti-cMet antibody is a
full-length antibody.
28. The method of claim 26 in which the anti-cMet antibody
comprises a V.sub.H chain comprising three CDRs, namely V.sub.H CDR
#1 (SEQ ID NO:112), V.sub.H CDR #2 (SEQ ID NO:113) and V.sub.H CDR
#3 (SEQ ID NO: 114); a V.sub.L chain comprising three CDRs, namely
V.sub.L CDR #1 (SEQ ID NO: 115), V.sub.L CDR #2 (SEQ ID NO: 116)
and V.sub.L CDR #3 (SEQ ID NO: 117); and a modified hinge region of
SEQ ID NO: 170.
29. The method of claim 26 in which the anti-cMet antibody
comprises a V.sub.H chain of SEQ ID NO: 78; a V.sub.L chain of SEQ
ID NO: 79; and a modified hinge region of SEQ ID NO: 170.
30. The method of claim 29 in which the anti-cMet antibody is an
IgG1.
31. The method of claim 26 in which the anti-cMet antibody
comprises a heavy chain of SEQ ID NO: 86 and a light chain of SEQ
ID NO: 87.
32. The method of claim 31 in which the anti-cMet antibody is
ABBV399.
33. The method of claim 26 in which the anti-cMet antibody
comprises a heavy chain of SEQ ID NO: 171 and a light chain of SEQ
ID NO: 172.
34. The method of claim 33 in which the anti-cMet antibody is
ABT-700 (S238C)-PBD.
35. The method of claim 26 in which the anti-cMet antibody
comprises the six CDRs of the antibody STI-D0602/STI-0602.
36. The method of claim 26 in which the anti-cMet antibody
comprises a V.sub.H chain of STI-D0602/STI-0602 and a V.sub.L chain
of STI-D0602/STI-0602.
37. The method of claim 26 in which the linker comprises a segment
according to one or more of structural formulae (IVa), (IVb), (IVc)
and (IVd): ##STR00042## or a salt thereof, in which: peptide
represents a peptide (illustrated C.fwdarw.N and not showing the
carboxy and amino "termini") cleavable by Cathepsin B; T represents
a polymer comprising one or more ethylene glycol units or an
alkylene chain, or combinations thereof; R.sup.a is selected from
hydrogen, alkyl, sulfonate and methyl sulfonate; p is an integer
ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1;
represents the point of attachment of the linker to the cytotoxic
and/or cytostatic agent; and * represents the point of attachment
to the remainder of the linker.
38. The method of claim 37 in which peptide is selected from the
group consisting of Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala;
Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser;
Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; and Val-Ala and salts
thereof.
39. The method of claim 38 in which the peptide is Val-Cit.
40. The method of claim 26 in which the anti-cMet ADC has an
average drug-to-antibody ratio ("DAR") in the range of 0-10.
41. The method of claim 26 in which the anti-cMet ADC has an
average drug-to-antibody ratio ("DAR") in the range of 1-4.
42. The method of claim 41 in which the anti-cMet ADC has a DAR in
the range of 2-4.
43. The method of claim 41 in which the anti-cMet ADC has a DAR of
about 3.1.
44. The method of claim 41 in which the anti-cMet ADC has an about
1:1 ratio of E2 and E4 ADC.
45. The method of claim 41 in which the anti-cMet ADC has a DAR of
3.0.
46. The method of claim 26 in which the cytostatic and/or cytotoxic
agent is a microtubule inhibitor.
47. The method of claim 46 in which the microtubule inhibitor is an
auristatin.
48. The method of claim 47 in which the auristatin is MMAE or
MMAF.
49. The method of claim 47 in which the auristatin is MMAE.
50. The method of claim 26 in which the anti-cMet ADC is a compound
according to structural formula (I): [D-L-XY-].sub.n-Ab (I) or a
salt thereof, in which: D is the cytotoxic and/or cytostatic agent;
L is the linker; Ab is the anti-cMet antibody; XY represents a
covalent linkage linking linker L to antibody Ab; and n has a value
ranging from 2 to 8.
51. The method of claim 50 in which n has a value of 2, 3 or 4.
52. The method of claim 50 in which XY is a linkage formed with an
amino group on anti-cMet antibody Ab.
53. The method of claim 50 in which XY is an amide or a
thiourea.
54. The method of claim 50 in which XY is a linkage formed with a
sulfhydryl group on anti-cMet antibody Ab.
55. The method of claim 50 in which XY is a thioether.
56. The method of claim 50 in which the compound according to
structural formula (I) has the structure of formula (IIa):
##STR00043##
57. The method of claim 56 in which anti-cMet antibody is
ABT-700.
58. The method of claim 50 in which the compound of structural
formula (I) has the following structure: ##STR00044##
59. The method of claim 58 in which anti-cMet antibody is
ABT-700.
60. The method of claim 50 in which the compound according to
structural formula (I) has the structure of formula (IIb):
##STR00045##
61. The method of claim 60 in which anti-cMet antibody is
ABT-700.
62. The method of claim 50 in which the compound according to
structural formula (I) has the following structure:
##STR00046##
63. The method of claim 62 in which anti-cMet antibody b is
ABT-700.
64. A method of treating a human patient diagnosed with non-small
cell lung cancer ("NSCLC") comprising administering to the patient
an anti-cMet antibody drug conjugate ("ADC") in an amount and for a
period of time sufficient to provide therapeutic benefit.
65. The method of claim 64 in which the NSCLC tumor tissue has an
immunohistochemistry ("IHC") H-score of greater than or equal to
150 when measured according to the cMet ABBV-ADC staining protocol
or an IHC score of 2+.
66. The method of claim 64 in which the NSCLC tumor tissue has an
immunohistochemistry ("IHC") H-score of greater than 225 when
measured according to the cMet ABBV-ADC staining protocol or an IHC
score of 3+.
67. The method of claim 64 in which the NSCLC tumor tissue has an
IHC score of 2+ and/or an H-score from 150 to 224, when measured
according to the cMet ABBV-ADC staining protocol.
68. The method according to any one of claims 64, 65, and 66 in
which the NSCLC is a non-squamous cell carcinoma.
69. The method according to any one of of claims 64, 65, and 67 in
which the NSCLC is a squamous cell carcinoma.
70. The method according to claim 64 in which the anti-cMet ADC is
administered as monotherapy.
71. The method according to claim 64 in which the anti-cMet ADC is
administered adjunctive to an additional anticancer agent, where
the additional agent is administered according to its FDA-approved
dosing regimen.
72. The method of claim 71 in which the additional anticancer agent
is an inhibitor of epidermal growth factor receptor ("EGFR").
73. The method of claim 72 in which the additional anticancer agent
is erlotinib, administered once daily.
74. The method of claim 71 in which the additional anticancer agent
is an inhibitor of PD1.
75. The method of claim 74 in which the inhibitor of PD1 is an
anti-PD1 antibody.
76. The method of claim 75 in which the anti-PD1 antibody is
nivolumab.
77. The method of claim 64 in which the anti-cMet ADC is
administered in an amount ranging from about 0.15 mg/kg to about
3.3 mg/kg, once every 3 weeks.
78. The method of claim 77 in which the anti-cMet ADC is
administered in an amount of about 2.7 mg/kg once every 3
weeks.
79. The method of claim 64 in which the anti-cMet ADC is
administered in an amount ranging from about 0.15 mg/kg to about
3.3 mg/kg, once every 2 weeks.
80. The method of claim 79 in which the anti-cMet ADC is
administered in an amount of about 1.6 mg/kg, once every 2
weeks.
81. The method of claim 64 in which the anti-cMet ADC is
administered in an amount of about 1.9 mg/kg, once every 2
weeks.
82. The method of any one of claims 64 through 81 in which the
anti-cMet ADC comprises an anti-cMet antibody linked to a
cytostatic and/or cytotoxic agent by way of a linker.
83. The method of claim 82 in which the anti-cMet antibody is a
full-length antibody.
84. The method of claim 82 in which the anti-cMet antibody
comprises a V.sub.H chain comprising three CDRs, namely V.sub.H CDR
#1 (SEQ ID NO:112), V.sub.H CDR #2 (SEQ ID NO:113) and V.sub.H CDR
#3 (SEQ ID NO: 114); a V.sub.L chain comprising three CDRs, namely
V.sub.L CDR #1 (SEQ ID NO: 115), V.sub.L CDR #2 (SEQ ID NO: 116)
and V.sub.L CDR #3 (SEQ ID NO: 117); and a modified hinge region of
SEQ ID NO: 170.
85. The method of claim 84 in which the anti-cMet antibody
comprises a V.sub.H chain of SEQ ID NO: 78; a V.sub.L chain of SEQ
ID NO: 79; and a modified hinge region of SEQ ID NO: 170.
86. The method of claim 85 in which the anti-cMet antibody is an
IgG1.
87. The method of claim 84 in which the anti-cMet antibody
comprises a heavy chain of SEQ ID NO: 86 and a light chain of SEQ
ID NO: 87.
88. The method of claim 84 in which the anti-cMet antibody
comprises a heavy chain of SEQ ID NO: 171 and a light chain of SEQ
ID NO: 172.
89. The method of claim 82 in which the anti-cMet antibody
comprises comprises the six CDRs of the antibody
STI-D0602/STI-0602.
90. The method of claim 89 in which the anti-cMet antibody is an
IgG1.
91. The method of claim 82 in which the anti-cMet antibody
comprises a V.sub.H chain of STI-D0602/STI-0602 and a V.sub.L chain
of STI-D0602/STI-0602.
92. The method of claim 91 in which the anti-cMet antibody is an
IgG1.
93. The method of claim 82 in which the linker is cleavable by a
lysosomal enzyme.
94. The method of claim 93 in which the lysosomal enzyme is
Cathepsin B.
95. The method of claim 94 in which the linker comprises a segment
according to one or more of structural formulae (IVa), (IVb), (IVc)
and (IVd): ##STR00047## or a salt thereof, in which: peptide
represents a peptide (illustrated C.fwdarw.N and not showing the
carboxy and amino "termini") cleavable by Cathepsin B; T represents
a polymer comprising one or more ethylene glycol units or an
alkylene chain, or combinations thereof; R.sup.a is selected from
hydrogen, alkyl, sulfonate and methyl sulfonate; p is an integer
ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1;
represents the point of attachment of the linker to the cytotoxic
and/or cytostatic agent; and * represents the point of attachment
to the remainder of the linker.
96. The method of claim 95 in which peptide is selected from the
group consisting of Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala;
Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser;
Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; and Val-Ala and salts
thereof.
97. The method of claim 96 in which the peptide is Val-Cit.
98. The method of claim 82 in which the anti-cMet ADC has an
average drug-to-antibody ratio ("DAR") in the range of 0-10.
99. The method of claim 82 in which the anti-cMet ADC has an
average drug-to-antibody ratio ("DAR") in the range of 1-4.
100. The method of claim 99 in which the anti-cMet ADC has a DAR in
the range of 2-4.
101. The method of claim 99 in which the anti-cMet ADC has a DAR of
about 3.1.
102. The method of claim 99 in which the anti-cMet ADC has an about
1:1 ratio of E2 and E4 ADC.
103. The method of claim 99 in which the anti-cMet ADC has a DAR of
3.0.
104. The method according to any one of claims 82 through 103 in
which the cytostatic and/or cytotoxic agent is a microtubule
inhibitor.
105. The method of claim 104 in which the microtubule inhibitor is
an auristatin.
106. The method of claim 105 in which the auristatin is MMAE or
MMAF.
107. The method of claim 106 in which the auristatin is MMAE.
108. The method according to claim 82 in which the anti-cMet ADC is
a compound according to structural formula (I): [D-L-XY-].sub.n-Ab
(I) or a salt thereof, in which: D is the cytotoxic and/or
cytostatic agent; L is the linker; Ab is the anti-cMet antibody; XY
represents a covalent linkage linking linker L to antibody Ab; and
n has a value ranging from 2 to 8.
109. The method of claim 108 in which n has a value of 2, 3 or
4.
110. The method of claim 108 in which XY is a linkage formed with
an amino group on anti-cMet antibody Ab.
111. The method of claim 108 in which XY is an amide or a
thiourea.
112. The method of claim 108 in which XY is a linkage formed with a
sulfhydryl group on anti-cMet antibody Ab.
113. The method of claim 108 in which XY is a thioether.
114. The method of claim 108 in which the compound according to
structural formula (I) has the structure of formula (IIa):
##STR00048##
115. The method of claim 114 in which anti-cMet antibody is
ABT-700.
116. The method of claim 108 in which the compound of structural
formula (I) has the following structure: ##STR00049##
117. The method of claim 116 in which anti-cMet antibody is
ABT-700.
118. The method of claim 108 in which the compound according to
structural formula (I) has the structure of formula (IIb):
##STR00050##
119. The method of claim 118 in which anti-cMet antibody is
ABT-700.
120. The method of claim 108 in which the compound according to
structural formula (I) has the following structure:
##STR00051##
121. The method of claim 120 in which anti-cMet antibody is
ABT-700.
122. A method of treating a human subject having a NSCLC tumor with
an IHC score of at least 2+ or an H score of 150 or greater in at
least one tumor biopsy from the subject, comprising administering
to the subject an anti-cMet ADC in an amount of about 2.7 mg/kg
once every two weeks or once every 3 weeks, in which the anti-cMet
ADC is a compound according to the following structure:
##STR00052## or a pharmaceutically acceptable salt thereof, in
which n has a value ranging from 2-4 and Ab is a full-length
anti-cMet antibody.
123. The method of claim 122 in which the anti-cMet antibody is
ABT-700.
124. The method of claim 123 in which the anti-cMet ADC is
administered as monotherapy.
125. The method of claim 122 in which the anti-cMetADC is
administered adjunctive to an additional anticancer agent.
126. The method of claim 125 in which the additional anticancer
agent is erlotinib.
127. The method of claim 125 in which the additional anticancer
agent is Nivolumab.
128. The method of claim 50 in which the drug is a
pyrrolobenzodiazepine (PBD), preferably PBD
((S)-2-(4-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-
-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propo-
xy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one); SG2000
(SJG-136;
(11aS,11a'S)-8,8'-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-
-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one)) (or
SGD-1882).
129. The method of claim 128 in which the drug is a
pyrrolobenzodiazepine (PBD), preferably SGD-1882.
130. The method according to claim 50, in which the compound of
formula I has the following structure: ##STR00053## in which Ab is
the antibody and n is 2.
131. The method according to claim 130 in which the antibody
comprises an heavy chain of SEQ ID NO: 171 and a light chain of SEQ
ID NO:172.
132. The method according to claim 50 in which the cMet ADC is the
compound of formula ##STR00054## in which the antibody comprises an
heavy chain of SEQ ID NO: 171 and a light chain of SEQ ID
NO:172.
133. A method of treating a human subject having a NSCLC
adenocarcinoma, comprising administering to the subject ABBV-399
once every 3 weeks in an amount of about 2.7 mg/kg, in which the
adenocarcinoma has an H-score of at least 225.
134. A method of treating a human subject having a NSCLC
adenocarcinoma, comprising administering to the subject ABBV-399
once every 3 weeks in an amount of about 2.7 mg/kg, in which the
adenocarcinoma has an IHC score of 3+.
135. The method according to any one of claims 133 and 134, in
which ABBV-399 is administered adjunctive to erlotinib, in which
the the erlotinib is administered once daily at 150 mg.
136. A method of treating a human subject having a NSCLC squamous
cell carcinoma, comprising administering to the subject ABBV-399
once every 2 weeks in an amount of about 1.6 mg/kg or 1.9 mg/kg, in
which the squamous cell carcinoma has an H-score of from 150 to
224.
137. A method of treating a human subject having a NSCLC squamous
cell carcinoma, comprising administering to the subject ABBV-399
once every 2 weeks in an amount of about 1.6 or 1.9 mg/kg, in which
the squamous cell carcinoma has an IHC score of 2+.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
Provisional Application No. 62/337,796, filed May 17, 2016, the
contents of which are incorporated herein by reference in their
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 17, 2017, is named 12252_0206-00000_SL.TXT and is 96,370
bytes in size.
1. FIELD
[0003] This application pertains to, among other things, anti-cMet
antibody drug conjugates ("ADCs"), compositions including the ADCs,
methods of making the ADCs, methods of selecting specific patient
populations for cancer treatment with a anti-cMet ADC, and methods
of using the ADCs to treat cancers.
2. BACKGROUND
[0004] Oncogenic protein kinases such as cMet represent a class of
biologically important targets for cancer intervention. cMet, a
well characterized receptor tyrosine kinase encoded by the MET
proto-oncogene, is the cell surface receptor for hepatocyte growth
factor (HGF; Gherardi E, Birchmeier W, Birchmeier C et al.
Targeting MET in cancer: rationale and progress. Nat Rev Can. 2012;
12:89-103). cMet overexpression occurs in approximately 30%-50% of
solid tumors including non-small cell lung cancer (NSCLC),
colorectal cancer (CRC), and advanced gastroesophageal cancer
(AGEC) (Spigel D R, Ervin T J, Ramlau R A, et al. Randomized Phase
II trial of onartuzumab in combination with erlotinib in patients
with advanced non-small-cell lung cancer. J Clin Oncol. 2013;
31(32):41054114; Resnick M B, Routhier J, Konkin T et al. Epidermal
growth factor receptor, cMET, B-catenin, and p53 expression as
prognostic indicators in stage II colon cancer: a tissue microarray
study. Clin Can Res. 2004; 10:3069-3075; Lee H E, Kim M A, Lee H S,
et al. MET in gastric carcinomas: comparison between protein
express and gene copy number and impact on outcome. Br J Can. 2012;
107(2):325-333).
[0005] Overexpression of cMet has been associated with poor patient
outcome. Thus, there remains a need for cancer therapeutics that
target solid tumor cancers characterized by overexpression of
cMet.
3. SUMMARY
[0006] The therapies described herein target solid tumor cancers in
which cMet is overexpressed in at least 10% of the patient
population having the cancer. cMet (cellular mesenchymal-epithelial
transition factor) is a cell-surface receptor tyrosine kinase that
transduces signals from the extracellular matrix into the cytoplasm
by binding to hepatocyte growth factor/HGF ligand. This cell
surface receptor is expressed in epithelial cells of many organs,
including the liver, pancreas, prostate, kidney, muscle and bone
marrow, during both embryogenesis and adulthood. cMet regulates
many physiological processes including cell proliferation and
survival, migration and scattering (cell-cell repulsion), tissue
morphogenesis, organ regeneration, and tissue remodeling. In cancer
and other pathological processes, cMet is often aberrantly
activated via mutation, amplification, or protein
overexpression.
[0007] Solid tumor cancers in which cMet is overexpressed in at
least 10% of the patient population include lung cancer, colorectal
cancer, head and neck cancer, pancreatic cancer, gastric cancer,
glioblastoma, ovarian, breast, prostate, cervical, and esophageal
cancer. Data presented herein demonstrate, for the first time, that
antibody drug conjugates ("ADCs") that specifically target cMet
overexpression have demonstrated anti-tumor activity in patients
diagnosed with non-small cell lung cancer. Data demonstrating in
vivo anti-tumor efficacy of anti-cMet ADCs administered as
monotherapy or combination are provided in Examples 10-14 and 16,
and FIGS. 8-12C and FIGS. 14-18.
[0008] cMet overexpression can be defined by an
immunohistorychemistry (IHC) H-score of greater than or equal to
150 when measured according to the assay of Example 17. Briefly,
IHC staining protocol for cMet overexpression has been developed
using the Ventana cMet CONFIRM (SP44) kit. Tissue samples are
stained with the Ventana antibody and then scored by determining
the percentages of target tissue cells staining at various
intensity levels of low to high. FIG. 20 depicts representative
H-scores using the assay described in Example 17.
[0009] Alternatively, cMet overexpressing tumor tissue using an IHC
score from 0 to 3+ as described in Example 17. FIG. 19 and FIG. 21
depict representative IHC scores using the assay described in
Example 17.
[0010] The anti-cMet ADCs may be administered as single therapeutic
agents (monotherapy) or adjunctively with or to other anti-cancer
treatments and/or therapeutic agents, typically but not necessarily
those used to treat the type of cancers being treated. Indeed, data
presented herein demonstrate that tumors that exhibit resistance to
other targeted or non-targeted chemotherapies retain sensitivity to
anti-cMet ADCs (see, e.g., Example 14 and FIGS. 12A-12C).
Accordingly, the anti-cMet ADCs described herein provide
significant benefits over current targeted and non-targeted
approaches toward the treatment of solid tumor cancers that
overexpress cMet. Adjunctive therapies and/or therapeutic agents
typically will be used at their approved dose, route of
administration, and frequency of administration, but may be used at
lower dosages and/or less frequently. When administered as
monotherapy, the anti-cMet ADC will typically be administered on a
schedule that provides therapeutic benefit. It is contemplated that
anti-cMet ADCs administered once a week, once every two weeks, once
every three weeks, once every four weeks, once every five weeks,
once every six weeks, once every seven weeks or once every eight
weeks will provide therapeutic benefit, although more or less
frequent administration may be beneficial. When administered
adjunctive to or with another therapy and/or agent, the anti-cMet
ADC may be administered before, after or concurrently with the
other therapy or agent.
[0011] The anti-cMet ADCs may be administered via a variety of
routes or modes of administration, including but not limited to,
intravenous infusion and/or injection and subcutaneous injection.
The amount administered will depend upon the route of
administration, the dosing schedule, the type of cancer being
treated, the stage of the cancer being treated, and other
parameters such as the age and weight of the patient, as is well
known in the art. Specific exemplary dosing schedules expected to
provide therapeutic benefit are provided in the Detailed
Description. Generally, an amount of anti-cMet ADC in the range of
about 0.005 to 15 mg/kg when administered intravenously on a weekly
basis from once weekly to and including once every eight weeks is
expected to provide therapeutic benefit.
[0012] Accordingly, in one aspect, the present disclosure provides
ADCs that specifically bind cMet ("anti-cMet ADCs"). The anti-cMet
ADCs comprise cytotoxic and/or cytostatic agents linked by way of
linkers to an antigen binding moiety that specifically binds cMet.
In some embodiments, the antigen binding moiety is an antibody
and/or an antigen binding fragment.
[0013] Antibodies and/or binding fragments composing the anti-cMet
ADCs generally comprise a heavy chain comprising a variable region
(V.sub.H) having three complementarity determining regions ("CDRs")
referred to herein (in N.fwdarw.C order) as V.sub.H CDR#1, V.sub.H
CDR#2, and V.sub.H CDR#3, and a light chain comprising a variable
region (V.sub.L) having three complementarity determining regions
referred to herein (in N.fwdarw.C order) as V.sub.L CDR#1, V.sub.L
CDR#2, and V.sub.L CDR#3. The amino acid sequences of exemplary
CDRs, as well as the amino acid sequence of the V.sub.H and V.sub.L
regions of the heavy and light chains of exemplary anti-cMet
antibodies and/or binding fragments that can compose the anti-cMet
ADCs are provided herein. Specific embodiments of anti-cMet ADCs
include, but are not limited to, ABT-700 and STI-0602.
[0014] For therapeutic uses, it may be desirable to utilize
anti-cMet ADCs that bind cMet with an affinity of at least 100 nM.
Accordingly, in some embodiments, the anti-cMet ADCs comprise an
anti-cMet and/or anti-cMet binding fragment that binds cMet with an
affinity of at least about 100 nM, or even higher, for example, at
least about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM,
20 nM, 15 nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1
nM, 0.01 nM, or greater. Affinity of anti-cMet antibodies and/or
binding fragments can be determined using techniques well known in
the art or described herein, such as for example, ELISA, isothermal
titration calorimetry (ITC), surface plasmon resonance, flow
cytometry, or fluorescent polarization assay. In some embodiments,
the affinity refers to apparent affinity EC.sub.50 values, measured
according to Example 5. In one embodiment, the antibody has an
apparent affinity EC.sub.50 value from lower than about 10
nanomol/L, preferably from about 1 picomol/L to 10 nanomol/L,
preferably about 0.3 nanomol/L, as determined according to Example
5.
[0015] Antibodies may be in the form of full-length antibodies,
bispecific antibodies, dual variable domain antibodies, multiple
chain or single chain antibodies, surrobodies (including surrogate
light chain construct), single domain antibodies, camelized
antibodies, scFv-Fc antibodies, and the like. They may be of, or
derived from, any isotype, including, for example, IgA (e.g.,
IgA.sub.1 or IgA.sub.2), IgD, IgE, IgG (e.g., IgG.sub.1, IgG.sub.2,
IgG.sub.3 or IgG.sub.4), IgM, or IgY. In some embodiments, the
anti-cMet antibody is an IgG (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3
or IgG.sub.4). Antibodies may be of human or non-human origin.
Examples of non-human origin include, but are not limited to,
mammalian origin (e.g., simians, rodents, goats, and rabbits) or
avian origin (e.g., chickens). In specific embodiments, antibodies
composing the anti-cMet ADCs are suitable for administration to
humans, such as, for example, humanized antibodies and/or fully
human antibodies.
[0016] Antigen binding fragments composing the anti-cMet ADCs may
include any fragment of an antibody capable of specifically binding
cMet. Specific examples of antibody binding fragments that may be
included in the anti-cMet ADCs include, but are not limited to,
Fab, Fab', (Fab').sub.2, Fv and scFv.
[0017] Antibodies and/or binding fragments composing the anti-cMet
ADCs may include modifications and/or mutations that alter the
properties of the antibodies and/or fragments, such as those that
increase half-life, increase or decrease ADCC, etc., as is known in
the art.
[0018] The cytotoxic and/or cytostatic agents composing the
anti-cMet ADCs may be any agents known to inhibit the growth and/or
replication of, and/or kill cells. Numerous agents having cytotoxic
and/or cytostatic properties are known in the literature.
Non-limiting examples of classes of cytotoxic and/or cytostatic
agents include, by way of example and not limitation, cell cycle
modulators, apoptosis regulators, kinase inhibitors, protein
synthesis inhibitors, alkylating agents, DNA cross-linking agents,
intercalating agents, mitochondria inhibitors, nuclear export
inhibitors, topoisomerase I inhibitors, topoisomerase II
inhibitors, RNA/DNA antimetabolites and antimitotic agents.
[0019] In a specific embodiment, a cytotoxic and/or cytostatic
agent composing an anti-cMet ADC is a cell-permeating antimitotic
agent, such as, for example, an auristatin. Specific examples of
cell-permeating auristatins include, but are not limited to,
dolastatin-10 and monomethyl auristatin E ("MMAE"). In another
specific embodiment, a cytotoxic and/or cytostatic agent composing
an anti-cMet ADC is a cell-permeating DNA cross-linking agent, such
as a cell-permeating minor groove-binding DNA cross-linking agent.
Specific examples of cell-permeating DNA minor groove-binding
agents include, but are not limited to, pyrrolobenzodiazepines
("PBD") and PBD dimers.
[0020] The linkers linking the cytotoxic and/or cytostatic agents
to the antigen binding moiety of an anti-cMet ADC may be long,
short, flexible, rigid, hydrophilic or hydrophobic in nature, or
may comprise segments that have different characteristics, such as
segments of flexibility, segments of rigidity, etc. The linker may
be chemically stable to extracellular environments, for example,
chemically stable in the blood stream, or may include linkages that
are not stable and release the cytotoxic and/or cytostatic agents
in the extracellular milieu. In some embodiments, the linkers
include linkages that are designed to release the cytotoxic and/or
cytostatic agents upon internalization of the anti-cMet ADC within
the cell. In some specific embodiments, the linkers includes
linkages designed to cleave and/or immolate or otherwise breakdown
specifically or non-specifically inside cells. A wide variety of
linkers useful for linking drugs to antigen binding moieties such
as antibodies in the context of ADCs are known in the art. Any of
these linkers, as well as other linkers, may be used to link the
cytotoxic and/or cytostatic agents to the antigen binding moiety of
the anti-cMet ADCs described herein.
[0021] The number of cytotoxic and/or cytostatic agents linked to
the antigen binding moiety of an anti-cMet ADC can vary (called the
"drug-to-antibody ratio," or "DAR"), and will be limited only by
the number of available attachments sites on the antigen binding
moiety and the number of agents linked to a single linker.
Typically, a linker will link a single cytotoxic and/or cytostatic
agent to the antigen binding moiety of an anti-cMet ADC. In
embodiments of anti-cMet ADCs which include more than a single
cytotoxic and/or cytostatic agent, each agent may be the same or
different. As long as the anti-cMet ADC does not exhibit
unacceptable levels of aggregation under the conditions of use
and/or storage, anti-cMet ADCs with DARs of twenty, or even higher,
are contemplated. In some embodiments, the anti-cMet ADCs described
herein may have a DAR in the range of about 1-10, 1-8, 1-6, or 1-4.
In certain specific embodiments, the anti-cMet ADCs may have a DAR
of 2, 3, or 4. In other specific embodiments, the anti-cMet ADCs
may have an average DAR of 3.1.
4. BRIEF DESCRIPTION OF THE FIGURES
[0022] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings) will be provided by the Office
upon request and payment of the necessary fee.
[0023] FIGS. 1A-1E show the amino acid sequences of several cMet
antibodies.
[0024] FIGS. 2A-2B: illustrate ABBV-399 Process 1.
[0025] FIGS. 3A-3B illustrate ABBV-399 Process 2.
[0026] FIGS. 4A-4D depict ABBV-399 cytotoxicity in cMet expressing
cell lines.
[0027] FIG. 5 provides proliferation inhibition results with
ABBV-399 and ABT-700 PBD.
[0028] FIGS. 6A-6B show in vitro activity of ABT-700 PBD in
colorectal cancer cell lines.
[0029] FIG. 7 shows in vitro activity of ABT-700 PBD in brain
cancer cell lines.
[0030] FIG. 8 shows ABT-700 PBD activity in SW48 xenografts.
[0031] FIGS. 9A-9C show the activity of ABT-700 PBD and ABBV-399 in
NSCLC patient xenografts.
[0032] FIGS. 10A-10B show the activity of ABBV-399 in NSCLC patient
xenografts using Kaplan-Meier plots.
[0033] FIGS. 11A-11B compare the activity of ABT-700 versus
ABBV-399 in human tumor xenografts; FIG. 11C shows the activity of
ABBV-339 alone or in combination with FOLFIRI.
[0034] FIGS. 12A-12C depict the activity of ABBV-399 in human
xenograft models refractory to ABT-700.
[0035] FIG. 13 provides the ABBV-399 dose escalation scheme for the
monotherapy phase I trial.
[0036] FIG. 14 provides a waterfall plot showing best percent
change in target lesions.
[0037] FIG. 15 provides a waterfall plot showing best percent
change in target lesions/cMet levels with ABBV-399 monotherapy.
[0038] FIG. 16 shows the number of weeks before clinical
progression in 16 patients treated with ABBV-399.
[0039] FIG. 17 is a waterfall plot showing best percent change in
target lesions ABBV-399 combination with erlotinib.
[0040] FIG. 18 shows the number of weeks before clinical
progression in 6 patients treated with ABBV-399 and erlotinib.
[0041] FIG. 19 illustrates the Ventana's SP44 scoring guide.
[0042] FIG. 20 illustrates patient selection based on cMet
overexpression.
[0043] FIG. 21 provides exemplary IHC scores using the method of
Example 17.
5. DETAILED DESCRIPTION
5.1. Abbreviations
[0044] The antibodies, binding fragments, ADCs and polynucleotides
described herein are, in many embodiments, described by way of
their respective polypeptide or polynucleotide sequences. Unless
indicated otherwise, polypeptide sequences are provided in
N.fwdarw.C orientation; polynucleotide sequences in 5'.fwdarw.3'
orientation. For polypeptide sequences, the conventional three or
one-letter abbreviations for the genetically encoded amino acids
may be used, as noted in TABLE 1, below.
TABLE-US-00001 TABLE 1 Encoded Amino Acid Abbreviations Three
Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine Ala
A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys
C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H
Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M
Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T
Tryptophan Trp W Tyrosine Tyr Y Valine Val V
[0045] Certain sequences are defined by structural formulae
specifying amino acid residues belonging to certain classes (e.g.,
aliphatic, hydrophobic, etc.). The various classes to which the
genetically encoded amino acids belong as used herein are noted in
TABLE 2, below. Some amino acids may belong to more than one class.
Cysteine, which contains a sulfhydryl group, and proline, which is
conformationally constrained, are not assigned classes.
TABLE-US-00002 TABLE 2 Encoded Amino Acid Classes Class Amino Acids
Aliphatic A, I, L, V Aromatic F, Y, W Non-Polar M, A, I, L, V Polar
N, Q, S, T Basic H, K, R Acidic D, E Small A, G
5.2. Definitions
[0046] Unless otherwise defined herein, scientific and technical
terms used in connection with the present disclosure shall have the
meanings that are commonly understood by those of ordinary skill in
the art.
5.3. Antibody Drug Conjugates that Bind to cMet and cMet
Overexpression Assay
[0047] The present disclosure concerns antibody drug conjugates
that specifically bind human cMet, compositions comprising the
ADCs, anti-cMet antibodies and/or binding fragments that can
comprise the ADCs, polynucleotides encoding anti-cMet antibodies
and/or binding fragments that comprise the ADCs, host cells capable
of producing the antibodies and/or binding fragments, methods and
compositions useful for making the antibodies, binding fragments
and ADCs, and various methods of using the ADCs in cancer
treatment.
[0048] Data provided herein demonstrate, for the first time, that
antibody drug conjugates ("ADCs") specifically targeting cMet
exhibit potent antitumor effects, both alone and in combination
with other targeted and non-targeted antitumor therapies, against
solid tumors in which cMet is overexpressed, particularly those
with an IHC-score of 2+ and 3+ when measured by
immunohistochemistry with the SP44 antibody. Data demonstrating in
vivo anti-tumor efficacy of ABBV-399 administered as monotherapy
are provided in the Examples.
[0049] For purposes of this application, including the claims, the
particular assay used in the study described herein is referred to
as the "cMet ABBV-ADC staining protocol." This protocol is
described in detail in Example 17 and the results are expressed in
terms of H-score and may also be expressed in terms of IHC score or
other scoring system well known in the art.
[0050] The H-score approach provides optimal data resolution for
determining variation in intensity and tumor percentage of staining
within and among tumor types. It also provides a good tool for
determining thresholds for positive staining. In this method, the
percentage of cells (0-100) within a tumor with staining
intensities ranging from 0-3+ are provided. This protocol results
in staining of the cMet protein both in the cytoplasm and in the
cell surface/membrane. The staining intensity for each cell in a
fixed field (typically, 100 cells) of the processed tumor biopsy is
determined, and an individual value is attributed to each cell as
follows, depending on the cell surface/membrane staining:
[0051] 0=no staining
[0052] 1+=weak staining
[0053] 2+=moderate staining
[0054] 3+=strong staining
[0055] To obtain an H-score, the percentage of tumor cells are
multiplied by each intensity and added together. The maximum
H-score is 300 if 100% of tumor cells label with 3+ intensity. The
H-score is calculated as follows:
H-score=[1.times.(% cells 1+)+2.times.(% cells 2+)+3.times.(% cells
3+)]
[0056] This protocol results both in cytoplasmic and membrane cMet
staining. For the H-score calculations referred to herein, membrane
staining was used. The final tumor H-score (0-300) score gives more
relative weight to higher-intensity membrane staining (3+
cell>2+ cell>1+ cell). FIG. 20 shows exemplary staining
results for various tumor H-scores (15, 90, 180, and 290) obtained
with the "cMet ABBV-ADC staining protocol."
[0057] Each tumor can also be given an IHC score of IHC 0, IHC 1+,
IHC 2+, or IHC 3+. While both the IHC and H scores involve 0, 1+,
2+, and 3+ values they are not to be confused. For the H-score, 0,
1+, 2+, and 3+ values refer to the intensity of staining of an
individual cell. For the IHC score, 0, 1+, 2+, and 3+ values refer
to the overall staining of a particular area of the tumor sample.
FIG. 21 shows exemplary staining results for various tumor
IHC0/1+/2+/3+ scores obtained with the "cMet ABBV-ADC staining
protocol."
[0058] For the purposes on this disclosure, and following the
protocol described herein, if none of the cells in a fixed field
are stained, the value attributed to the tumor is IHC 0. If the
overall level of staining in a fixed field is low, the value
attributed is IHC 1+. If most of the cells in a fixed field exhibit
moderate staining, the value attributed is IHC 2+. If most of the
cells in a fixed field exhibit strong staining, the value
attributed is IHC 3+.
[0059] In another embodiment, and for the purposes on this
disclosure, and following the protocol described herein, if none of
the cells in a fixed field are stained, the value attributed to the
tumor is IHC 0. If the overall level of staining in a fixed field
is low, the value attributed is IHC 1+. If at least 15% of the
cells in a fixed field exhibit moderate staining, the value
attributed is IHC 2+. If at least 15% of the cells in a fixed field
exhibit strong staining, the value attributed is IHC 3+.
[0060] For purposes of this disclosure, an H-score between 150 and
224 is equivalent to an IHC score of 2+ and an H-score of 225 and
above is equivalent to an IHC score of 3+.
[0061] Accordingly, in one aspect, the present disclosure provides
ADCs that specifically bind cMet ("anti-cMet ADCs"). The anti-cMet
ADCs comprise cytotoxic and/or cytostatic agents linked by way of
linkers to an antigen binding moiety that specifically binds cMet.
In the case of ABBV-399, the antigen binding moiety (ABT-700) binds
cMet at IPT domain 1 of human cMet. In other anti-cMet ADCs, the
antigen binding moiety may be any moiety capable of specifically
binding cMet. In some embodiments, the antigen binding moiety is an
antibody and/or an antibody binding fragment.
[0062] In a specific embodiment, a cytotoxic and/or cytostatic
agent composing an anti-cMet ADC is a cell-permeating antimitotic
agent, such as, for example, an auristatin. Specific examples of
cell-permeating auristatins include, but are not limited to,
dolastatin-10 and monomethyl auristatin E ("MMAE"). In another
specific embodiment, a cytotoxic and/or cytostatic agent composing
an anti-cMet ADC is a cell-permeating DNA cross-linking agent, such
as a cell-permeating minor groove-binding DNA cross-linking agent.
Specific examples of cell-permeating DNA minor groove-binding
agents include, but are not limited to, pyrrolobenzodiazepines
("PBD") and PBD dimers.
[0063] As will be appreciated by skilled artisans, antibodies
and/or binding fragments are "modular" in nature. Throughout the
disclosure, various specific embodiments of the various "modules"
composing the antibodies and/or binding fragments are described. As
specific non-limiting examples, various specific embodiments of
V.sub.H CDRs, V.sub.H chains, V.sub.L CDRs and V.sub.L chains are
described. It is intended that all of the specific embodiments may
be combined with each other as though each specific combination
were explicitly described individually.
[0064] The ADCs disclosed herein are also "modular" in nature.
Throughout the disclosure, various specific embodiments of the
"modules" composing the ADCs are described. As non-limiting
examples, specific embodiments of antibodies, linkers, and
cytotoxic and/or cytostatic agents that may compose the ADCs are
described. It is intended that all of the specific embodiments
described may be combined with each other as though each specific
combination were explicitly described individually.
[0065] It will also be appreciated by skilled artisans that the
various ADCs described herein may be in the form of salts, and in
some specific embodiments, pharmaceutically acceptable salts. The
ADCs of the disclosure that possess a sufficiently acidic, a
sufficiently basic, or both functional groups, can react with any
of a number of inorganic bases, and inorganic and organic acids, to
form a salt. Alternatively, compounds that are inherently charged,
such as those with a quaternary nitrogen, can form a salt with an
appropriate counter ion, e.g., a halide such as a bromide,
chloride, or fluoride.
[0066] Acids commonly employed to form acid addition salts are
inorganic acids such as hydrochloric acid, hydrobromic acid,
hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and
organic acids such as p-toluenesulfonic acid, methanesulfonic acid,
oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic
acid, citric acid, etc. Base addition salts include those derived
from inorganic bases, such as ammonium and alkali or alkaline earth
metal hydroxides, carbonates, bicarbonates, and the like.
5.4. Antibodies to cMet
[0067] In specific exemplary embodiments, the antigen binding
moiety is an antibody or an antigen binding fragment.
[0068] As used herein, the term "antibody" (Ab) refers to an
immunoglobulin molecule that specifically binds to, or is
immunologically reactive with, a particular antigen--here, cMet.
Antibodies comprise complementarity determining regions (CDRs),
also known as hypervariable regions, in both the light chain and
heavy chain variable domains. The more highly conserved portions of
the variable domains are called the framework (FR). As is known in
the art, the amino acid position/boundary delineating a
hypervariable region of an antibody can vary, depending on the
context and the various definitions known in the art. Some
positions within a variable domain may be viewed as hybrid
hypervariable positions in that these positions can be deemed to be
within a hypervariable region under one set of criteria, while
being deemed to be outside a hypervariable region under a different
set of criteria. One or more of these positions can also be found
in extended hypervariable regions. The variable domains of native
heavy and light chains each comprise four FR regions, largely by
adopting a .beta.-sheet configuration, connected by three CDRs,
which form loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The CDRs in each chain are held together in
close proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen binding site of
antibodies. See Kabat et al., Sequences of Proteins of
Immunological Interest (National Institute of Health, Bethesda, Md.
1987). As used herein, numbering of immunoglobulin amino acid
residues is done according to the immunoglobulin amino acid residue
numbering system of Kabat et al. unless otherwise indicated.
[0069] Antibodies and/or binding fragments composing the anti-cMet
ADCs generally comprise a heavy chain comprising a variable region
(V.sub.H) having three complementarity determining regions ("CDRs")
referred to herein (in N.fwdarw.C order) as V.sub.H CDR#1, V.sub.H
CDR#2, and V.sub.H CDR#3, and a light chain comprising a variable
region (V.sub.L) having three complementarity determining regions
referred to herein (in N.fwdarw.C order) as V.sub.L CDR#1, V.sub.L
CDR#2, and V.sub.L CDR#3. The amino acid sequences of exemplary
CDRs, as well as the amino acid sequence of the V.sub.H and V.sub.L
regions of the heavy and light chains of exemplary anti-cMet
antibodies and/or binding fragments that can be included in antigen
binding moieties composing the anti-cMet ADCs are provided herein.
Specific embodiments of anti-cMet ADCs include, but are not limited
to, those that comprise antibodies and/or binding fragments that
include these exemplary CDRs and/or V.sub.H and/or V.sub.L
sequences, as well as antibodies and/or binding fragments that
compete for binding cMet with such antibodies and/or binding
fragments.
[0070] Antibodies may be in the form of full-length antibodies,
bispecific antibodies, dual variable domain antibodies, multiple
chain or single chain antibodies, surrobodies (including surrogate
light chain construct), single domain antibodies, camelized
antibodies, scFv-Fc antibodies, and the like. They may be of, or
derived from, any isotype, including, for example, IgA (e.g.,
IgA.sub.1 or IgA.sub.2), IgD, IgE, IgG (e.g., IgG.sub.1, IgG.sub.2,
IgG.sub.3 or IgG.sub.4), IgM, or IgY. In some embodiments, the
anti-cMet antibody is an IgG (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3
or IgG.sub.4). Antibodies may be of human or non-human origin.
Examples of non-human origin include, but are not limited to,
mammalian origin (e.g., simians, rodents, goats, and rabbits) or
avian origin (e.g., chickens). In specific embodiments, antibodies
composing the anti-cMet ADCs are suitable for administration to
humans, such as, for example, humanized antibodies and/or fully
human antibodies.
[0071] Antibodies composing anti-cMet ADCs may be polyclonal,
monoclonal, genetically engineered, and/or otherwise modified in
nature, including but not limited to, chimeric antibodies,
humanized antibodies, human antibodies, primatized antibodies,
single chain antibodies, bispecific antibodies, dual-variable
domain antibodies, etc. In various embodiments, the antibodies
comprise all or a portion of a constant region of an antibody. In
some embodiments, the constant region is an isotype selected from:
IgA (e.g., IgA.sub.1 or IgA.sub.2), IgD, IgE, IgG (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3 or IgG.sub.4), IgM, and IgY. In specific
embodiments, antibodies composing an anti-cMet ADC comprise an
IgG.sub.1 constant region isotype.
[0072] The term "monoclonal antibody" as used herein is not limited
to antibodies produced through hybridoma technology. A monoclonal
antibody is derived from a single clone, including any eukaryotic,
prokaryotic, or phage clone, by any means available or known in the
art. Monoclonal antibodies useful with the present disclosure can
be prepared using a wide variety of techniques known in the art
including the use of hybridoma, recombinant, and phage display
technologies, or a combination thereof. In many uses of the present
disclosure, including in vivo use of ADCs including anti-cMet
antibodies in humans, chimeric, primatized, humanized, or human
antibodies can suitably be used.
[0073] The term "chimeric" antibody as used herein refers to an
antibody having variable sequences derived from a non-human
immunoglobulin, such as a rat or a mouse antibody, and human
immunoglobulin constant regions, typically chosen from a human
immunoglobulin template. Methods for producing chimeric antibodies
are known in the art. See, e.g., Morrison, 1985, Science
229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies
et al., 1985, J. Immunol. Methods 125:191-202; U.S. Pat. Nos.
5,807,715; 4,816,567; and 4,816,397, which are incorporated herein
by reference in their entireties.
[0074] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins that contain minimal sequences derived
from non-human immunoglobulin. In general, a humanized antibody
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin sequence. The humanized antibody can also comprise
at least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin consensus sequence.
Methods of antibody humanization are known in the art. See, e.g.,
Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos. 5,530,101;
5,585,089; 5,693,761; 5,693,762; and U.S. Pat. No. 6,180,370 to
Queen et al.; EP239400; PCT publication WO 91/09967; U.S. Pat. No.
5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol.,
28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska
et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Pat. No.
5,565,332, all of which are hereby incorporated by reference in
their entireties.
[0075] "Human antibodies" are antibodies having the amino acid
sequence of a human immunoglobulin and include antibodies isolated
from human immunoglobulin libraries or from animals transgenic for
one or more human immunoglobulin and that do not express endogenous
immunoglobulins. Human antibodies can be made by a variety of
methods known in the art including phage display methods using
antibody libraries derived from human immunoglobulin sequences. See
U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO
98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO
96/33735; and WO 91/10741, each of which is incorporated herein by
reference in its entirety. Human antibodies can also be produced
using transgenic mice which are incapable of expressing functional
endogenous immunoglobulins but which can express human
immunoglobulin genes. See, e.g., PCT publications WO 98/24893; WO
92/01047; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;
5,885,793; 5,916,771; and 5,939,598, which are incorporated by
reference herein in their entireties. In addition, companies such
as Medarex (Princeton, N.J.), Astellas Pharma (Deerfield, Ill.),
Amgen (Thousand Oaks, Calif.) and Regeneron (Tarrytown, N.Y.) can
be engaged to provide human antibodies directed against a selected
antigen using technology similar to that described above. Fully
human antibodies that recognize a selected epitope can be generated
using a technique referred to as "guided selection." In this
approach, a selected non-human monoclonal antibody, e.g., a mouse
antibody, is used to guide the selection of a completely human
antibody recognizing the same epitope (see, Jespers et al., 1988,
Biotechnology 12:899-903).
[0076] "Primatized antibodies" comprise monkey variable regions and
human constant regions. Methods for producing primatized antibodies
are known in the art. See, e.g., U.S. Pat. Nos. 5,658,570;
5,681,722; and 5,693,780, which are incorporated herein by
reference in their entireties.
[0077] Anti-cMet ADCs may comprise full-length (intact) antibody
molecules, as well as antigen binding fragments that are capable of
specifically binding cMet. Examples of antibody binding fragments
include by way of example and not limitation, Fab, Fab',
F(ab').sub.2, Fv fragments, single chain Fv fragments and single
domain fragments.
[0078] A Fab fragment contains the constant domain of the light
chain and the first constant domain (CH.sub.2) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxyl terminus of the heavy chain CH.sub.2
domain including one or more cysteines from the antibody hinge
region. F(ab') fragments are produced by cleavage of the disulfide
bond at the hinge cysteines of the F(ab').sub.2 pepsin digestion
product. Additional chemical couplings of antibody fragments are
known to those of ordinary skill in the art. Fab and F(ab').sub.2
fragments lack the Fc fragment of intact antibody, clear more
rapidly from the circulation of animals, and may have less
non-specific tissue binding than an intact antibody (see, e.g.,
Wahl et al., 1983, J. Nucl. Med. 24:316).
[0079] An "Fv" fragment is the minimum fragment of an antibody that
contains a complete target recognition and binding site. This
region consists of a dimer of one heavy and one light chain
variable domain in a tight, non-covalent association
(V.sub.H-V.sub.L dimer). It is in this configuration that the three
CDRs of each variable domain interact to define an antigen binding
site on the surface of the V.sub.H-V.sub.Ldimer. Often, the six
CDRs confer antigen binding specificity upon the antibody. However,
in some instances even a single variable domain (or half of an Fv
comprising only three CDRs specific for a target) may have the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0080] "Single-chain Fv" or "scFv" antibody binding fragments
comprise the V.sub.H and V.sub.L domains of an antibody, where
these domains are present in a single polypeptide chain. Generally,
the Fv polypeptide further comprises a polypeptide linker between
the V.sub.H and V.sub.L domains which enables the scFv to form the
desired structure for antigen binding.
[0081] Antibodies and/or binding fragments composing the anti-cMet
ADCs may include modifications and/or mutations that alter the
properties of the antibodies and/or fragments, such as those that
increase half-life, increase or decrease ADCC, etc., as is known in
the art.
[0082] "Single domain antibodies" are composed of a single V.sub.H
or V.sub.L domains which exhibit sufficient affinity to cMet. In a
specific embodiment, the single domain antibody is a camelized
antibody (See, e.g., Riechmann, 1999, Journal of Immunological
Methods 231:25-38).
[0083] Antibodies composing the anti-cMet ADCs may also be
bispecific antibodies. Bispecific antibodies comprised of
monoclonal, often human or humanized, antibodies that have binding
specificities for two different epitopes on the same or different
antigens. In the present disclosure, one of the binding
specificities can be directed towards cMet, the other can be for
any other antigen, e.g., for a cell-surface protein, receptor,
receptor subunit, tissue-specific antigen, virally derived protein,
virally encoded envelope protein, bacterially derived protein, or
bacterial surface protein, etc.
[0084] Antibodies composing anti-cMet ADCs may be derivatized.
Derivatized antibodies are typically modified by glycosylation,
acetylation, pegylation, phosphorylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein. Any of numerous chemical
modifications may be carried out by known techniques, including,
but not limited to, specific chemical cleavage, acetylation,
formylation, metabolic synthesis of tunicamycin, etc. Additionally,
the derivative may contain one or more non-natural amino acids,
e.g., using ambrx technology. See, e.g., Wolfson, 2006, Chem. Biol.
13(10):1011-2.
[0085] Antibodies or binding fragments composing anti-cMet ADCs may
be antibodies or fragments whose sequences have been modified to
alter at least one constant region-mediated biological effector
function. For example, in some embodiments, an anti-cMet antibody
may be modified to reduce at least one constant region-mediated
biological effector function relative to the unmodified antibody,
e.g., reduced binding to the Fc receptor (Fc.gamma.R). Fc.gamma.R
binding may be reduced by mutating the immunoglobulin constant
region segment of the antibody at particular regions necessary for
Fc.gamma.R interactions (See, e.g., Canfield and Morrison, 1991, J.
Exp. Med. 173:1483-1491; and Lund et al., 1991, J. Immunol.
147:2657-2662). Reducing Fc.gamma.R binding may also reduce other
effector functions which rely on Fc.gamma.R interactions, such as
opsonization, phagocytosis and antigen-dependent cellular
cytotoxicity ("ADCC").
[0086] Antibodies included in anti-cMet ADCs may have low levels
of, or lack, fucose. Antibodies lacking fucose have been correlated
with enhanced ADCC activity, especially at low doses of antibody.
See Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa
et al., 2003, J. Biol. Chem. 278:3466-73. Methods of preparing
fucose-less antibodies include growth in rat myeloma YB2/0 cells
(ATCC CRL 1662). YB2/0 cells express low levels of FUT8 mRNA, which
encodes .alpha.-1,6-fucosyltransferase, an enzyme necessary for
fucosylation of polypeptides.
[0087] Antibodies or binding fragments composing anti-cMet ADCs may
include modifications that increase or decrease their binding
affinities to the neonatal Fc receptor, FcRn, for example, by
mutating the immunoglobulin constant region segment at particular
regions involved in FcRn interactions (see, e.g., WO 2005/123780).
In particular embodiments, an anti-cMet antibody of the IgG class
is mutated such that at least one of amino acid residues 250, 314,
and 428 of the heavy chain constant region is substituted alone, or
in any combinations thereof, such as at positions 250 and 428, or
at positions 250 and 314, or at positions 314 and 428, or at
positions 250, 314, and 428, with substitution at positions 250 and
428 being a specific combination. For position 250, the
substituting amino acid residue may be any amino acid residue other
than threonine, including, but not limited to, alanine, cysteine,
aspartic acid, glutamic acid, phenylalanine, glycine, histidine,
isoleucine, lysine, leucine, methionine, asparagine, proline,
glutamine, arginine, serine, valine, tryptophan, or tyrosine. For
position 314, the substituting amino acid residue may be any amino
acid residue other than leucine, including, but not limited to,
alanine, cysteine, aspartic acid, glutamic acid, phenylalanine,
glycine, histidine, isoleucine, lysine, methionine, asparagine,
proline, glutamine, arginine, serine, threonine, valine,
tryptophan, or tyrosine. For position 428, the substituting amino
acid residues may be any amino acid residue other than methionine,
including, but not limited to, alanine, cysteine, aspartic acid,
glutamic acid, phenylalanine, glycine, histidine, isoleucine,
lysine, leucine, asparagine, proline, glutamine, arginine, serine,
threonine, valine, tryptophan, or tyrosine. Specific combinations
of suitable amino acid substitutions are identified in TABLE 1 of
U.S. Pat. No. 7,217,797, which is incorporated herein by reference.
Such mutations increase binding to FcRn, which protects the
antibody from degradation and increases its half-life.
[0088] An anti-cMet antibody and/or binding fragment may have one
or more amino acids inserted into one or more of its hypervariable
regions, for example as described in Jung & Pluckthun, 1997,
Protein Engineering 10:9, 959-966; Yazaki et al., 2004, Protein
Eng. Des Sel. 17(5):481-9; and U.S. Pat. App. No. 2007/0280931.
[0089] Anti-cMet antibodies and/or binding fragments with high
affinity for cMet may be desirable for therapeutic uses.
Accordingly, the present disclosure contemplates ADCs comprising
anti-cMet antibodies and/or binding fragments having a high binding
affinity to cMet. In specific embodiments, the antibodies and/or
binding fragments bind cMet with an affinity of at least about 100
nM, but may exhibit higher affinity, for example, at least about 90
nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM,
10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM, 0.01 nM,
or even higher. In some embodiments, the antibodies bind cMet with
an affinity in the range of about 1 pM to about 100 nM, or an
affinity ranging between any of the foregoing values.
[0090] Affinity of antibodies and/or binding fragments for cMet can
be determined using techniques well known in the art or described
herein, such as for example, but not by way of limitation, ELISA,
isothermal titration calorimetry (ITC), surface plasmon resonance,
flow cytometry or fluorescent polarization assays. In one
embodiment, affinity refers to apparent affinity EC50 values
measured according to Example 5.
[0091] In the context of this disclosure, anti-cMet antibodies can
serve at least two different purposes. In some embodiments, the
anti-cMet antibodies are used for diagnostic purposes, assisting in
and guiding patient selection. For example, these anti-cMet
antibodies can be used for immunohistochemistry assays of tumor
biopsies obtained from the patients to be treated or under
treatment. One of ordinary skill in the art is familiar with the
techniques for selecting a particular antibody for diagnostic
purposes to assay for the levels of cMet protein expression in
tumor biopsies. Typically, the samples are scored under one or more
scoring guides, including IHC scores of 0/1+/2+/3+ or H-scores. The
disclosure details one example of such a diagnostic assay that is
commercially available from Ventana. The Ventana antibody SP44, and
antibodies with similar properties can be made or acquired from
other vendors and the protocol adjusted so that the method has the
same or better diagnostic power as the Ventana assay. In addition,
anti-cMet antibodies other than SP44 can also be used for this
purpose. One of ordinary skill in the art would know how to
properly adjust the protocol to a new antibody in order to obtain a
diagnostic test for cMet expression levels. Companion diagnostics
exist for a variety of other FDA approved cancer treatments and are
within the level of ordinary skill. The FDA maintains a list of
FDA-approved companion diagnostic tests at, for example,
www.fda.gov/.
[0092] Examples of anti-cMet antibodies that can be used include,
for example, the diagnostic antibodies disclosed in U.S. Pat. No.
8,673,302 (224D10 and 221C9) and U.S. Pat. No. 9,120,852 (227D3 and
205A5). The disclosures of each of these patents are fully
incorporated herein by reference, including the amino acid
sequences for the CDRs, heavy chains (full and variable regions),
and light chains (full and variable regions). In one embodiment,
the antibody is 227D3.
[0093] 227D3 is secreted by the hybridoma deposited at the CNCM on
Nov. 18, 2009, under number 1-4247.
TABLE-US-00003 CDR Antibody numbering Heavy chain Light chain SEQ
ID NO. 227D3 IMGT CDR-L1 159 CDR-L2 160 CDR-L3 161 CDR-H1 162
CDR-H2 163 CDR-H3 164 227D3 Kabat CDR-L1 165 CDR-L2 166 CDR-L3 161
CDR-H1 167 CDR-H2 168 CDR-H3 169
[0094] In other embodiments, the anti-cMet antibodies are
administered for treatment purposes, either as components of
antibody drug conjugates (ADCs), or before/after/concurrently with
administration of the ADCs.
[0095] 5.6.1 ABT-700 and Related Antibodies for Treatment
Purposes
[0096] For purposes of the antibodies of this section, the CDRs
have been identified according to the IMGT numbering system.
[0097] ABBV-399 is an ADC comprised of the cMet targeting antibody
ABT-700 (PR-1266688, h224G11) conjugated to the potent cytotoxin
MMAE through a valine citrulline (vc) linker. The ADC binds to cMet
on the surface of tumor cells, is internalized, and then releases
MMAE leading to the inhibition of microtubule function and the
disruption of critical cellular processes and death. ABBV-399 is
potently cytotoxic to cancer cells with overexpress cMet or
amplified MET and demonstrates antitumor activity in human tumor
xenografts. Activity of ABBV-399 against ABT-700-refractory tumors
has also been demonstrated (see e.g., Example 14).
ABT-700
[0098] ABT-700 is a humanized version of mouse monoclonal antibody
224G11, which was first disclosed and embodimented in U.S. Pat. No.
8,329,173. ABT-700 is a "humanized" recombinant IgG1.kappa.
(disclosed as 224G11 [TH7 Hz3] in U.S. Pat. No. 8,741,290) that
targets a unique epitope of cMet located within the
immunoglobulin-plexin-transcription factor homology (IPT) domain 1,
resulting in blockade of both HGF-dependent and HGF-independent
cMet signaling. ABT-700 competes for binding to cMet with
antibodies directed against SEMA blade 5 (and vice versa), but not
with antibodies directed against blades 1-3 or IPT 2-3. In
contrast, 5D5 (the bivalent progenitor of one armed onartuzumab,
discussed below) binds to blade 5 of the SEMA domain.
[0099] The cMet-ADCs of this disclosure encompass any antibody that
comprises a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3
comprising respectively the amino acid sequence SEQ ID Nos. 1, 2
and 3; and a light chain comprising CDR-L1, CDR-L2 and CDR-L3
comprising respectively the amino acid sequence SEQ ID Nos. 5, 6,
and 7, according to U.S. Pat. No. 8,741,290. These are the CDRs of
the original murine 224G11 antibody, as defined based on the IMGT
numbering system.
[0100] As defined under the IMGT nomenclature, the CDR sequences of
ABT-700 comprise the following sequences:
TABLE-US-00004 CDR-H1: (SEQ ID NO: 72) GYIFTAYT CDR-H2: (SEQ ID NO:
73) IKPNNGLA CDR-H3: (SEQ ID NO: 74) ARSEITTEFDY CDR-L1: (SEQ ID
NO: 75) ESVDSYANSF CDR-L2: (SEQ ID NO: 76) RAS CDR-L3: (SEQ ID NO:
77) QQSKEDPLT
[0101] In one embodiment, the heavy chain variable region of 224G11
[TH7 Hz3] comprises SEQ ID No. 4 of U.S. Pat. No. 8,741,290:
TABLE-US-00005 (SEQ ID NO: 78)
QVQLVQSGAEVKKPGASVKVSCKASGYIFTAYTMHWVRQAPGQGLEWMG
WIKPNNGLANYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR
SEITTEFDYWGQGTLVTVSS;
[0102] and the light chain variable region comprises SEQ ID No. 10
of U.S. Pat. No. 8,741,290:
DIVMTQSPDSLAVSLGERATINCKSSESVDSYANSFLHWYQQKPGQPPK
LLIYRASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKED PLTFGGGTKVEIKR
(SEQ ID NO: 79)
[0103] In another embodiment, the heavy chain variable region of
224G11 [TH7 Hz3] comprises:
TABLE-US-00006 (SEQ ID NO: 80)
QVQLVQSGAEVKKPGASVKVSCKASGYIFTAYTMHWVRQAPGQGLEWMG
WIKPNNGLANYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR
SEITTEFDYWGQGTLVTVSS;
[0104] and the light chain variable region comprises:
TABLE-US-00007 (SEQ ID NO: 81)
DIVMTQSPDSLAVSLGERATINCKSSESVDSYANSFLHWYQQKPGQPPK
LLIYRASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKED PLTFGGGTKVEIK
[0105] In another embodiment, the antibody [224G11] [TH7 Hz3]
comprises a complete heavy chain comprising the amino acid sequence
SEQ ID No. 37 of U.S. Pat. No. 8,741,290 and a complete light chain
comprising the amino acid sequence SEQ ID No. 40 of U.S. Pat. No.
8,741,290. The modified hinge region has the sequence of SEQ ID
NO:170.
[0106] In some embodiments, the anti-cMet antibody comprises a
heavy chain variable region comprising SEQ ID No. 4 of U.S. Pat.
No. 8,741,290:
[0107] QVQLVQSGAEVKKPGASVKVSCKASGYIFTAYTMHWVRQAPGQGLEWMG
WIKPNNGLANYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR
SEITTEFDYWGQGTLVTVSS (SEQ ID NO: 78) linked to any heavy chain
constant region;
[0108] and a light chain variable region comprising SEQ ID No. 10
of U.S. Pat. No. 8,741,290:
DIVMTQSPDSLAVSLGERATINCKSSESVDSYANSFLHWYQQKPGQPPK
LLIYRASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKED PLTFGGGTKVEIKR
(SEQ ID NO: 79) linked to any light chain constant region. Examples
of suitable heavy and light chain constant regions are provided
below.
[0109] In some embodiments, the anti-cMet antibody comprises a
heavy chain variable region comprising SEQ ID No. 4 of U.S. Pat.
No. 8,741,290:
[0110] QVQLVQSGAEVKKPGASVKVSCKASGYIFTAYTMHWVRQAPGQGLEWMG
WIKPNNGLANYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR
SEITTEFDYWGQGTLVTVSS (SEQ ID NO: 80) linked to any heavy chain
constant region;
[0111] and a light chain variable region comprising:
DIVMTQSPDSLAVSLGERATINCKSSESVDSYANSFLHWYQQKPGQPPK
LLIYRASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKED PLTFGGGTKVEIK
(SEQ ID NO: 81) linked to any light chain constant region. Examples
of suitable heavy and light chain constant regions are provided
below.
[0112] In some embodiments, an anti-cMet antibody and/or binding
fragment composing an anti-cMet ADC is an IgG.sub.1.
[0113] In some embodiments, an anti-cMet antibody composing an
anti-cMet ADC comprises a heavy chain having a constant region
comprising or consisting of:
TABLE-US-00008 (SEQ ID NO: 82)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDCHCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPG
[0114] In some embodiments, an anti-cMet antibody composing an
anti-cMet ADC comprises a light chain having a constant region
comprising or consisting of:
TABLE-US-00009 (SEQ ID NO: 83)
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC
[0115] In some embodiments, an anti-cMet antibody composing an
anti-cMet ADC comprises a heavy chain having a constant region
comprising or consisting of:
TABLE-US-00010 (SEQ ID NO: 84)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK
SCDCHCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK
[0116] and a light chain having a constant region comprising or
consisting of:
TABLE-US-00011 (SEQ ID NO: 85)
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKFIKVYACEVTHQGLSSPVTK SFNRGEC
[0117] In some embodiments, the heavy chain of an anti-cMet
antibody (ABT-700) composing an anti-cMet ADC comprises or consists
of (constant regions are bold; CDRs are underlined (Kabat-numbered
CDR sequences disclosed as SEQ ID NOS 112-114, respectively, in
order of appearance)):
TABLE-US-00012 QVQLVQSGAE VKKPGASVKV SCKASGYIFT AYTMHWVRQA
PGQGLEWMGW 050 IKPNNGLANY AQKFQGRVTM TRDTSISTAY MELSRLRSDD
TAVYYCARSE 100 ITTEFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT
AALGCLVKDY 150 FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP
SSSLGTQTYI 200 CNVNHKPSNT KVDKRVEPKS CDCHCPPCPA PELLGGPSVF
LFPPKPKDTL 250 MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVENAKTKP
REEQYNSTYR 300 VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG
QPREPQVYTL 350 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY
KTTPPVLDSD 400 GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG
445 (full-length sequence disclosed as SEQ ID NO: 86)
[0118] and the light chain comprises or consists of (CDR sequences
disclosed as SEQ ID NOS 115-117, respectively, in order of
appearance):
TABLE-US-00013 DIVMTQSPDS LAVSLGERAT INCKSSESVD SYANSFLHWY
QQKPGQPPKL 050 LIYRASTRES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY
YCQQSKEDPL 100 TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL
NNFYPREAKV 150 QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY
EKHKVYACEV 200 THQGLSSPVT KSFNRGEC 218 (full-length sequence
disclosed as SEQ ID NO: 87)
[0119] In some embodiments, the heavy chain of an anti-cMet
antibody composing an anti-cMet ADC comprises or consists of a
variable region (amino acids 1-118 of SEQ ID NO: 88), a constant
region (shown in bold) and CDRs (underlined; CDR sequences
disclosed as SEQ ID NOS 118-120, respectively, in order of
appearance):
TABLE-US-00014 QVQLVQSGAE VKKPGASVKV SCKASGYIFT AYTMHWVRQA
PGQGLEWMGW 050 IKPNNGLANY AQKFQGRVTM TRDTSISTAY MELSRLRSDD
TAVYYCARSE 100 ITTEFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT
AALGCLVKDY 150 FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP
SSSLGTQTYI 200 CNVNHKPSNT KVDKRVEPKS CDCHCPPCPA PELLGGPSVF
LFPPKPKDTL 250 MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVENAKTKP
REEQYNSTYR 300 VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG
QPREPQVYTL 350 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY
KTTPPVLDSD 400 GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK
446 (full-length heavy chain sequence disclosed as SEQ ID NO:
88)
[0120] and the light chain comprises or consists of a variable
region (amino acids 1-110 in SEQ ID NO: 89), a constant region
(shown in bold), and CDR sequences (underlined and disclosed as SEQ
ID NOS 121-123, respectively, in order of appearance):
TABLE-US-00015 DIVMTQSPDS LAVSLGERAT INCKSSESVD SYANSFLHWY
QQKPGQPPKL 050 LIYRASTRES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY
YCQQSKEDPL 100 TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL
NNFYPREAKV 150 QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY
EKHKVYACEV 200 THQGLSSPVT KSFNRGEC 218 (full-length light chain
sequence disclosed as SEQ ID NO: 89)
[0121] In one embodiment, the antibody is ABT-700 and the heavy
chain is encoded by the following nucleotide sequence (full-length
sequence disclosed as SEQ ID NO: 90):
TABLE-US-00016 ATGGGATGGTCTTGGATCTTTCTGCTGTTTCTGTCTGGTACTGCTGGTGT
GCTGAGCcaggtccagctggtgcaatccggcgcagaggtgaagaagccag
gcgcttccgtgaaggtgagctgtaaggcctctggctacatcttcacagca
tacaccatgcactgggtgaggcaagctcctgggcagggactggagtggat
gggatggattaaacccaacaatgggctggccaactacgcccagaaattcc
agggtagggtcactatgacaagggataccagcatcagcaccgcatatatg
gagctgagcaggctgaggtctgacgacactgctgtctattattgcgccag
gagcgaaattacaacagaattcgattactgggggcagggcaccctggtga
ccgtgtcctctgccagcaccaagggcccaagcgtgttccccctggccccc
agcagcaagagcaccagcggcggcacagccgccctgggctgcctggtgaa
ggactacttccccgagcccgtgaccgtgtcctggaacagcggagccctca
cttctggagttcataccttcccagcagtattgcagagcagtggcctgtat
tcactgtcttccgtcgtaacagttccatcctccagcctcgggacacagac
ttacatttgtaacgtgaatcacaagcctagcaacaccaaggtcgacaaga
gagttgaaccaaagagttgtgattgccactgtcctccctgcccagctcct
gagctgcttggcggtcccagtgtcttcttgtttccccctaaacccaaaga
caccctgatgatctcaaggactcccgaggtgacatgcgtggtggtggatg
tgtctcatgaggacccagaggtgaagttcaactggtacgtggacggcgtg
gaggtgcacaacgccaagaccaagcccagagaggagcagtacaacagcac
ctacagggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacg
gcaaggagtacaagtgtaaggtgtccaacaaggccctgccagccccaatc
gaaaagaccatcagcaaggccaagggccagccaagagagccccaggtgta
caccctgccacccagcagggaggagatgaccaagaaccaggtgtccctga
cctgtctggtgaagggcttctacccaagcgacatcgccgtggagtgggag
agcaacggccagcccgagaacaactacaagaccacccccccagtgctgga
cagcgacggcagcttcttcctgtacagcaagctgaccgtggacaagagca
gatggcagcagggcaacgtgttcagctgctccgtgatgcacgaggccctg
cacaaccactacacccagaagagcctgagcctgtccccaggctga
[0122] Secretion signal peptide in bold CAPITAL letters.
[0123] Includes final stop codon (TGA)
[0124] Constant region is bold
[0125] CDRs are underlined (CDR sequences disclosed as SEQ ID NOS
124-126, respectively, in order of appearance)
[0126] In one embodiment, the antibody is ABT-700 and the light
chain is encoded by the following nucleotide sequence (full-length
sequence disclosed as SEQ ID NO: 91):
TABLE-US-00017 ATGGAAACTGATACACTGCTGCTGTGGGTCCTGCTGCTGTGGGTCCCTG
GAAGCACAGGGgacattgtgatgacccagtctcccgatagcctggccgtg
tccctgggcgagagggctaccatcaactgtaaaagctccgaatctgtgga
ctcttacgcaaacagctttctgcactggtatcagcaaaagccaggccaac
ctccaaagctgctgatttacagggcttctaccagggagagcggcgtgccc
gataggttcagcggatctggcagcggcaccgactttacactgaccatctc
cagcctgcaggccgaagatgtggcagtctattactgccagcagtccaagg
aggaccccctgactttcgggggtggtactaaagtggagatcaagcgtacgg
tggccgctcccagcgtgttcatcttccccccaagcgacgagcagctgaag
agcggcaccgccagcgtggtgtgtctgctgaacaacttctaccccaggga
ggccaaggtgcagtggaaggtggacaacgccctgcagagcggcaacagcc
aggagagcgtcaccgagcaggacagcaaggactccacctacagcctgagc
agcaccctgaccctgagcaaggccgactacgagaagcacaaggtgtacgc
ctgtgaggtgacccaccagggcctgtccagccccgtgaccaagagcttca
acaggggcgagtgctga
[0127] Secretion signal peptide in bold CAPITAL letters.
[0128] Includes final stop codon (tga)
[0129] Constant region is bold
[0130] CDRs are underlined (CDR sequences disclosed as SEQ ID NOS
127-129, respectively, in order of appearance)
[0131] In one embodiment, herein referred to as ABBV399, the
antibody heavy chain sequence is represented by SEQ ID NO:88, the
light chain sequence is represented by SEQ ID NO:89 conjugated to
monomethyl auristatin E (MMAE) through a valine citrulline (vc)
linker.
[0132] The sequence of ABT-700 PBD, comprising the sequence of
ABT-700 carrying a S238C mutation (also referred to herein as
ABT-700 (S238C)-PBD) according to Kabat numbering, is as follows
(CDRs are underlined; the numbering system is Kabat; and the S238C
mutation is represented by C (bold, italics, and underlined):
[0133] Amino Acid Sequence (10 AA Per Group, 5 Groups Per Line)
Heavy Chain (SEQ ID NO: 171) (underlined CDR sequences disclosed as
SEQ ID NOS 173-175, respectively, in order of appearance):
TABLE-US-00018 QVQLVQSGAE VKKPGASVKV SCKASGYIFT AYTMHWVRQA
PGQGLEWMGW 50 IKPNNGLANY AQKFQGRVTM TRDTSISTAY MELSRLRSDD
TAVYYCARSE 100 ITTEFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT
AALGCLVKDY 150 FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP
SSSLGTQTYI 200 CNVNHKPSNT KVDKRVEPKS CDCHCPPCPA PELLGGP VF
LFPPKPKDTL 250 MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP
REEQYNSTYR 300 VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG
QPREPQVYTL 350 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY
KTTPPVLDSD 400 GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG
445
Light Chain (SEQ ID NO:172) (underlined CDR sequences disclosed as
SEQ ID NOS 176-178, respectively, in order of appearance):
TABLE-US-00019 DIVMTQSPDS LAVSLGERAT INCKSSESVD SYANSFLHWY
QQKPGQPPKL 50 LIYRASTRES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY
YCQQSKEDPL 100 TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL
NNFYPREAKV 150 QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY
EKHKVYACEV 200 THQGLSSPVT KSFNRGEC 218
[0134] Accordingly, the antibody ABT-700 PBD comprises two PBD
drug-linker molecules conjugated to a cys engineered mAb ABT-700
(S238C), and has a heavy chain of SEQ ID NO: 171 and a light chain
of SEQ ID NO:172.
[0135] In one embodiment, the C-terminal lysine amino acid on the
heavy chain of 224G11 [TH7 Hz3] was engineered out to eliminate
heterogeneity at the C-terminus due to incomplete cleavage of the
lysine. In ABT-700, the heavy chain is post-translationally
modified by addition of N-linked glycans to asparagine-296. The
major glycans are fucosylated biantennary oligosaccharides
containing zero, one, or two galactose residues. In addition, at
the N-terminus of the heavy chain is a glutamine residue, which can
undergo spontaneous cyclization to form a pyroglutamate
residue.
[0136] The original murine 224G11 antibody has been further
chimerized and humanized. The chimerization and humanization
processes are described in detail in U.S. Pat. No. 8,741,290 and
those process are incorporated herein by reference in their
entirety, as are the descriptions of the biological and structural
properties of all of the antibodies described therein. During the
humanization process of the murine 224G11 antibody, the chimeric
form of 224G11 Mab (224G11chim/IgG1), meaning variable domain
(VH+VL) from m224G11 combined with human constant domain IgG1/kappa
yielded strong (17% of maximal HGF effect) agonist activity
associated with a reduced antagonist efficacy (54% inhibition of
HGF maximal effect compared to the m224G11 that yields 75%
inhibition of HGF maximum effect). Three humanized forms of 224G11
Mab, [224G11]Hz1/IgG1, [224G11]Hz2/IgG1 and [224G11]Hz3/IgG1, also
constructed on a human IgG1/kappa backbone, yielded also decreased
antagonist efficacy and significant agonist activity (11 to 24% of
maximal HGF level) as compared to mouse 224G11.
[0137] The hinges of some of the humanized forms of the 224G11
antibody were modified, as described in detail in the U.S. Pat. No.
8,741,290, and incorporated herein by reference. The resulting
antibodies, whose ADCs are also within the scope of this
disclosure, included 224G11 [TH7Hz3].
[0138] The antibody h224G11/ABT-700 refers to the humanized form
224G11 [TH7 Hz3]. This antibody represents the ABT-700 antibody
that is part of the ABBV-399 of this disclosure. The biological
activities of the antibody ABT-700, or h224G11, were extensively
characterized in U.S. Pat. No. 8,741,290. Its biological
characterizations therein are incorporated herein by reference in
their entireties. The entire description of U.S. Pat. No. 8,741,290
is incorporated herein by reference.
[0139] Exemplary versions of other chimerized and humanized
versions of 224G11 antibody drug conjugates that fall within the
scope of this disclosure are those referred to in the U.S. Pat. No.
8,741,290 as the antibodies [224G11] [IgG2Hz1], [224G11] [IgG2Hz2];
[224G11] [IgG2Hz3]; [224G11] [TH7Hz1]; [224G11] [TH7z2]; [224G11]
[TH7Hz3]; [224G11] [IgG2chim]; [224G11] [TH7chim]; [224G11] [C1];
[224G11] [C2]; [224G11] [C3]; [224G11] [C5]; [224G11] [C6];
[224G11] [C7]; [224G11] [C8]; and [224G11] [C9].
[0140] Other examples include the antibodies [224G11] [.DELTA.1-3];
[224G11] [C7.DELTA.6]; [224G11] [C6.DELTA.9]; [224G11] [C245-7];
[224G11] [C5.DELTA.2-6]; [224G11] [C9.DELTA.2-7]; [224G11]
[.DELTA.5-6-7-8]; [224G11] [IgG1/IgG2]; [224G11] [IgG2Hz1];
[224G11] [IgG2Hz2]; [224G11] [IgG2Hz3]; [224G11] [TH7Hz1]; [224G11]
[TH7Hz2]; [224G11] [TH7Hz3]; [224G11] [TH7chim]; [224G11] [MHchim];
[224G11] [MUP9Hchim]; and [224G11] [MMCHchim].
[0141] In both of these series of antibodies, the first bracket
refers to the name of the antibody that is modified (i.e., 224G11)
and the second bracket identifies the specific modification of the
antibody, most of which correspond to changes to the hinge region,
according to the IMGT unique numbering for C-domains. The symbol 4
means deletion. The specific details of each modification can be
found in U.S. Pat. No. 8,741,290.
[0142] Accordingly, in some embodiments, an anti-cMet antibody
and/or binding fragment comprising an anti-cMet ADC is suitable for
administration to humans. In a specific embodiment, the anti-cMet
antibody is humanized.
[0143] In some embodiments, anti-cMet antibodies and/or binding
fragments comprising an anti-anti-cMet ADC compete for binding cMet
on cells expressing cMet, or the
immunoglobulin-plexin-transcription factor homology (IPT) of human
cMet, or to Met-Fc or engineered/recombinant cMet in solid phase,
in in vitro assays with a reference antibody. The reference
antibody may be any antibody that specifically binds the
immunoglobulin-plexin-transcription factor homology (IPT) of human
cMet. In one specific embodiment, the reference antibody is mouse
224G11. In another specific embodiment, the reference antibody is
ABT-700.
[0144] Assays for competition include, but are not limited to, a
radioactive material labeled immunoassay (RIA), an enzyme-linked
immunosorbent assay (ELISA), a sandwich ELISA, flow cytometry
assays and surface plasmon resonance assays. A preferred method is
that described in Basilico C, Hultberg A, Blanchetot C, de Jonge N,
Festjens E, Hanssens V, Osepa S I, De Boeck G, Mira A, Cazzanti M,
Morello V, Dreier T, Saunders M, de Haard H, Michieli P. Four
individually druggable MET hotspots mediate HGF-driven tumor
progression. J Clin Invest. 2014 July; 124(7):3172-86. doi:
10.1172/JCI72316. Epub 2014 May 27.
[0145] In one exemplary embodiment of conducting an antibody
competition assay between a reference antibody and a test antibody
(irrespective of species or isotype), one may first label the
reference with a detectable label, such as a fluorophore, biotin or
an enzymatic, or radioactive label to enable subsequent detection.
In this case, cells expressing cMet or the extracellular domain of
cMet (or a subpart thereof), are incubated with unlabeled test
antibody, labeled reference antibody is added, and the intensity of
the bound label is measured. If the test antibody competes with the
labeled reference antibody by binding to the same, proximal or
overlapping epitope, the intensity of the detection signal will be
decreased relative to a control reaction carried out without test
antibody.
[0146] In a specific embodiment of this assay, the concentration of
labeled reference antibody that yields 80% of maximal binding
("conc.sub.80%") under the assay conditions (e.g., a specified
density of cells or a specified concentration of cMet/cMet
extracellular domain or subpart thereof) is first determined, and a
competition assay is carried out with 10.times.
concentration.sub.80% of unlabeled test antibody and conc.sub.80%
of labeled reference antibody.
[0147] In another exemplary embodiment of conducting a flow
cytometry competition assay, cells expressing cMet are incubated
with a titration series of antibodies comprising increasing
concentrations of unlabeled test antibody versus fluorescently
labeled anti-cMet reference antibody. The labeled reference
anti-cMet antibody is used at a fixed concentration X (for example,
X=1 .mu.g/ml) and the unlabeled test antibody is used in a range of
concentrations (for example, from 10.sup.-4.times. to 100.times.).
Cells or cMet/cMet extracellular domain or subpart thereof is
incubated with both unlabeled test antibody and labeled reference
antibody concurrently. Flow cytometry data is normalized relative
to fluorescently labeled reference antibody alone, where the
fluorescence intensity of a sample carried out without unlabeled
test antibody is assigned 100% binding. If a test antibody competes
for binding cMet with the labeled reference antibody, an assay
carried out with equal concentration of each (for example, 1
.mu.g/mL of unlabeled test antibody and 1 .mu.g/mL of labeled
reference antibody) will yield an approximately 50% reduction in
fluorescence intensity as compared to the 100% control, indicating
approximately 50% binding. Use of a labeled reference antibody at a
concentration of X and unlabeled test antibody that competes for
binding cMet at a concentration of 10.times. would yield an
approximately 90% reduction in binding as compared to the 100%
control, indicating approximately 10% binding.
[0148] The inhibition can be expressed as an inhibition constant,
or which is calculated according to the following formula:
K=IC.sub.50/(1+[reference Ab concentration]/K.sub.d),
[0149] where IC.sub.50 is the concentration of test antibody that
yields a 50% reduction in binding of the reference antibody and
K.sub.d is the dissociation constant of the reference antibody, a
measure of its affinity for cMet. Antibodies that compete with
reference cMet antibodies can have a K.sub.i from 10 pM to 100 nM
under assay conditions described herein.
[0150] In various embodiments, a test antibody is considered to
compete with a reference antibody if it decreases binding of the
reference antibody to cells expressing cMet or cMet/cMet
extracellular domain or subpart thereof by at least about 20% or
more, for example, by at least about 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95% or even more, or by a percentage ranging between any
of the foregoing values, at a reference antibody concentration that
is 80% of maximal binding under the specific assay conditions used,
and a test antibody concentration that is 10-fold higher than the
reference antibody concentration.
[0151] In various embodiments of a flow cytometry competition
assay, a test antibody is considered to compete with a reference
antibody if it decreases binding of the reference antibody to cells
expressing cMet by at least about 20% or more, for example, by at
least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or even
more, or by a percentage ranging between any of the foregoing
values, at a concentration of test antibody that is 10.times.
greater than that of the reference antibody.
[0152] Detection of expression of cMet generally involves
contacting a biological sample (cells, tissue, or body fluid of an
individual) with one or more anti-cMet antibodies (optionally
conjugated to detectable moiety), and detecting whether or not the
sample is positive for cMet expression, or whether the sample has
altered (e.g., reduced or increased) expression as compared to a
control sample. Methods for doing so are well known to one of
ordinary skill in the art, including those described in the
Examples.
[0153] 5.6.2. Some Other Exemplary cMet Antibodies
[0154] Another anti-cMet antibody that can be used according to
this disclosure has been named 227H1, comprises a heavy chain
comprising CDR-H1, CDR-H2 and CDR-H3 comprising respectively the
amino acid sequence SEQ ID Nos. 4, 5 and 6; and a light chain
comprising CDR-L1, CDR-L2 and CDR-L3 comprising respectively the
amino acid sequence SEQ ID Nos. 13, 11 and 14 of U.S. Pat. No.
8,329,173 (SEQ ID NOS 4, 5, 6, 13, 11 and 14, respectively, of this
application). These antibodies have been described in detail in
U.S. Pat. No. 8,329,173, and their descriptions are incorporated
herein by reference in their entireties. The Sequence Listing
submitted concurrently with this application includes SEQ ID NOS
1-71 from U.S. Pat. No. 8,329,173 as SEQ ID NOS 1-71.
[0155] In one embodiment, the antibody 227H1 comprises a heavy
chain comprising the amino acid sequence SEQ ID No. 19 and a light
chain comprising the amino acid sequence SEQ ID No. 22 of U.S. Pat.
No. 8,329,173 (SEQ ID NOS 19 and 20, respectively, of this
application). These antibodies have been described in detail in
U.S. Pat. No. 8,329,173, and their descriptions are incorporated
herein by reference in their entireties.
[0156] Another anti-cMet antibody that can be used according to
this disclosure has been named 223C4, comprises a heavy chain
comprising CDR-H1, CDR-H2 and CDR-H3 comprising respectively the
amino acid sequence SEQ ID Nos. 7, 8 and 9; and a light chain
comprising CDR-L1, CDR-L2 and CDR-L3 comprising respectively the
amino acid sequence SEQ ID Nos. 15, 16 and 17 of U.S. Pat. No.
8,329,173 (SEQ ID NOS 7, 8, 9, 15, 16 and 17, respectively, of this
application). These antibodies have been described in detail in
U.S. Pat. No. 8,329,173, and their descriptions are incorporated
herein by reference in their entireties.
[0157] In one embodiment, the antibody 223C4 comprises a heavy
chain comprising the amino acid sequence SEQ ID No. 20 and a light
chain comprising the amino acid sequence SEQ ID No. 23 of U.S. Pat.
No. 8,329,173 (SEQ ID NOS 20 and 23, respectively). These
antibodies have been described in detail in U.S. Pat. No.
8,329,173, and their descriptions are incorporated herein by
reference in their entireties.
[0158] Another anti-cMet antibody that can be used according to
this disclosure has been named 11E1, comprises a heavy chain
comprising CDR-H1, CDR-H2 and CDR-H3 comprising respectively the
amino acid sequence SEQ ID Nos. 56, 57 and 58; and a light chain
comprising CDR-L1, CDR-L2 and CDR-L3 comprising respectively the
amino acid sequence SEQ ID Nos. 59, 60 and 61 of U.S. Pat. No.
8,329,173 (SEQ ID NOS 56, 57, 58, 59, 60 and 61, respectively).
These antibodies have been described in detail in U.S. Pat. No.
8,329,173, and their descriptions are incorporated herein by
reference in their entireties.
[0159] In one embodiment, the antibody 11E1 comprises a heavy chain
comprising the amino acid sequence SEQ ID No. 62 and a light chain
comprising the amino acid sequence SEQ ID No. 63 of U.S. Pat. No.
8,329,173 (SEQ ID NOS 62 and 63, respectively). These antibodies
have been described in detail in U.S. Pat. No. 8,329,173, and their
descriptions are incorporated herein by reference in their
entireties.
[0160] These first monoclonal antibodies disclosed above, or one of
their functional fragments or derivatives, are characterized in
that said antibodies are secreted by the hybridoma deposited at the
Collection Nationale de Cultures de Microorganismes (CNCM, National
Collection of Microorganism Cultures) (Institut Pasteur, Paris,
France) on Mar. 14, 2007 under the numbers CNCM 1-3724
(corresponding to 11E1), 1-3731 (corresponding to 224G11), 1-3732
(corresponding to 227H1) and on Jul. 6, 2007 under the number
1-3786 (corresponding to 223C4). These hybridomas consist of murine
hybridomas resulting in the cellular fusion of immunized mouse
splenocytes with a myeloma cell line (Sp20 Ag14).
[0161] These first antibodies, all of which were originally
disclosed in U.S. Pat. No. 8,329,173, and which are covered by
several patents, are thus summarized as follows (the SEQ ID NOs are
the same in the '173 patent and in this application):
TABLE-US-00020 224G11 227H1 223C4 11E1 I-3731 I-3732 I-3786 I-3724
Prot. Nucl. Prot; Nucl. Prot. Nucl. Prot. Nucl. SEQ ID SEQ ID SEQ
ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: NO:
NO: CDR-H1 1 24 4 27 7 30 56 64 CDR-H2 2 25 5 28 8 31 57 65 CDR-H3
3 26 6 29 9 32 58 66 H. chain 18 41 19 42 20 43 62 70 CDR-L1 10 33
13 36 15 38 59 67 CDR-L2 11 34 11 34 16 39 60 68 CDR-L3 12 35 14 37
17 40 61 69 L. chain 21 44 22 45 23 46 63 71
[0162] The antibodies 224G11, 227H1, and 223C4 do not bind the SEMA
domain of the cMet receptor. 11E1 is able to bind the SEMA
domain.
[0163] In one embodiment, the anti-cMet antibody comprises the CDRs
of the antibody STI-D0602 or STI-0602 (Sorrento Therapeutics). In
another embodiment, the anti-cMet antibody is STI-D0602 or
STI-0602, as described in Lingna Li, Cathrine Fells, Julia Guo, Pia
Muyot, Edwige Gros, Yanliang Zhang, Yingqing Sun, Hong, Zhang,
Yanwen Fu, Tong Zhu, Jian Cao, Gunnar Kaufmann, Gang Chen, Zhenwei
Miao, A novel cMet targeting antibody drug conjugate for NSCLC,
Abstract No. 3897, AACR Annual Meeting, April 16-20, New Orleans,
USA.
[0164] In one embodiment, the anti-cMet antibody comprises the CDRs
of the antibody 5D5 (Genentech) or the one-armed (monovalent)
derivative onartuzumab. In one embodiment, the anti-cMet antibody
is the antibody 5D5 (Genentech) or the one-armed (monovalent)
derivative onartuzumab (FIG. 1B). Additional information for
onartuzumab is as follows:
TABLE-US-00021 Heavy chain (SEQ ID NO: 92):
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGL
EWVGMIDPSNSDTRENPNEKDRETISADTSKNTAYLQMNSLRAED
TAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM
TKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FELVSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK Light chain (SEQ ID
NO: 93): DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQK
PGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFA
TYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGT
ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Hinge-CH2-CH3 (SEQ ID NO:
94): DKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
EEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK
[0165] In one embodiment, the anti-cMet antibody comprises the CDRs
of the antibody emibetuzumab/LY2875358. In one embodiment, the
anti-cMet antibody is emibetuzumab/LY2875358 (Eli Lilly and
Company, CAS Number 1365287-97-3) (FIG. 1A). Additional information
for emibetuzumab is as follows:
TABLE-US-00022 Heavy Chain (SEQ ID NO: 95):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGL
EWMGRVNPNRRGTTYNQKFEGRVTMTTDTSTSTAYMELRSLRSDD
TAVYYCARANWLDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSE
STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP
APEAAGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF
NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL
TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Light Chain (SEO ID NQ: 96)
DIQMTQSPSSLSASVGDRVTITCSVSSSVSSIYLHWYQQKPGKAP
KLLIYSTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ
VYSGYPLTEGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC
LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[0166] In one embodiment, the anti-cMet antibody comprises the CDRs
of the antibody AbF46 or SAIT301 (Samsung Electronics). In one
embodiment, the antibody is AbF46 (FIG. 1C). In another embodiment,
the anti-cMet antibody is SAIT301 (FIG. 1E).
[0167] In one embodiment, the anti-cMet antibody comprises the CDRs
of the antibody ARGX-111 (36C4) (arGEN-X BV). In another
embodiment, the anti-cMet antibody is ARGX-111 (FIG. 1D).
[0168] In one embodiment, the anti-cMet antibody comprises the CDRs
of one of the antibodies in Sym015 (Hu9006, Hu9338) (Symphogen
A/S). In another embodiment, the anti-cMet antibody is Hu9006. In
another embodiment, the anti-cMet antibody is Hu9338. The amino
acid sequences of these antibodies, including their CDRs, are
disclosed in WO2016042412.
[0169] 5.5. Expression Systems and Methods of Making the
Antibodies
[0170] Anti-cMet antibodies can be prepared by recombinant
expression of immunoglobulin light and heavy chain genes in a host
cell through methods well known to those of ordinary skill in the
art. To express an antibody recombinantly, a host cell is
transfected with one or more recombinant expression vectors
carrying DNA fragments encoding the immunoglobulin light and heavy
chains of the antibody such that the light and heavy chains are
expressed in the host cell and, optionally, secreted into the
medium in which the host cells are cultured, from which medium the
antibodies can be recovered. Standard recombinant DNA methodologies
are used to obtain antibody heavy and light chain genes,
incorporate these genes into recombinant expression vectors and
introduce the vectors into host cells, such as those described in
Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook,
Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989),
Current Protocols in Molecular Biology (Ausubel, F. M. et al.,
eds., Greene Publishing Associates, 1989) and in U.S. Pat. No.
4,816,397.
[0171] To generate nucleic acids encoding such anti-cMet
antibodies, DNA fragments encoding the light and heavy chain
variable regions are first obtained. These DNAs can be obtained by
amplification and modification of germline DNA or cDNA encoding
light and heavy chain variable sequences, for example using the
polymerase chain reaction (PCR). Germline DNA sequences for human
heavy and light chain variable region genes are known in the art
(See, e.g., the "VBASE" human germline sequence database; see also
Kabat et al., 1991, Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242; Tomlinson et al., 1992, J.
Mol. Biol. 22T:116-198; and Cox et al., 1994, Eur. J. Immunol.
24:827-836; the contents of each of which are incorporated herein
by reference). The nucleotides encoding the antibodies 224G11,
227H1, 223C4, and 11E11 have been described in detail in U.S. Pat.
No. 8,329,173, and their descriptions are incorporated herein by
reference in their entireties.
[0172] Once DNA fragments encoding anti-cMet antibody-related
V.sub.H and V.sub.L segments are obtained, these DNA fragments can
be further manipulated by standard recombinant DNA techniques, for
example to convert the variable region genes to full-length
antibody chain genes, to Fab fragment genes or to a scFv gene. In
these manipulations, a V.sub.L- or V.sub.H-encoding DNA fragment is
operatively linked to another DNA fragment encoding another
protein, such as an antibody constant region or a flexible linker.
The term "operatively linked," as used in this context, is intended
to mean that the two DNA fragments are joined such that the amino
acid sequences encoded by the two DNA fragments remain
in-frame.
[0173] The isolated DNA encoding the V.sub.H region can be
converted to a full-length heavy chain gene by operatively linking
the V.sub.H-encoding DNA to another DNA molecule encoding heavy
chain constant regions (CH.sub.1, CH.sub.2, CH.sub.3 and,
optionally, CH.sub.4). The sequences of human heavy chain constant
region genes are known in the art (See, e.g., Kabat et al., 1991,
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242) and DNA fragments encompassing these regions can be
obtained by standard PCR amplification. The heavy chain constant
region can be an IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA,
IgE, IgM or IgD constant region, but in certain embodiments is an
IgG.sub.1 or IgG.sub.4 constant region. For a Fab fragment heavy
chain gene, the V.sub.H-encoding DNA can be operatively linked to
another DNA molecule encoding only the heavy chain CH.sub.1
constant region.
[0174] The isolated DNA encoding the V.sub.L region can be
converted to a full-length light chain gene (as well as a Fab light
chain gene) by operatively linking the V.sub.L-encoding DNA to
another DNA molecule encoding the light chain constant region, CL.
The sequences of human light chain constant region genes are known
in the art (See, e.g., Kabat et al., 1991, Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The light chain constant region can be a kappa or
lambda constant region, but in certain embodiments is a kappa
constant region. To create a scFv gene, the V.sub.H- and
V.sub.L-encoding DNA fragments are operatively linked to another
fragment encoding a flexible linker, e.g., encoding the amino acid
sequence (Gly.sub.4.about.Ser).sub.3 (SEQ ID NO:97), such that the
V.sub.H and V.sub.L sequences can be expressed as a contiguous
single-chain protein, with the V.sub.L and V.sub.H regions joined
by the flexible linker (See, e.g., Bird et al., 1988, Science
242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).
[0175] To express the anti-cMet antibodies, DNAs encoding partial
or full-length light and heavy chains, obtained as described above,
are inserted into expression vectors such that the genes are
operatively linked to transcriptional and translational control
sequences. In this context, the term "operatively linked" is
intended to mean that an antibody gene is ligated into a vector
such that transcriptional and translational control sequences
within the vector serve their intended function of regulating the
transcription and translation of the antibody gene. The expression
vector and expression control sequences are chosen to be compatible
with the expression host cell used. The antibody light chain gene
and the antibody heavy chain gene can be inserted into separate
vectors or, more typically, both genes are inserted into the same
expression vector.
[0176] The antibody genes are inserted into the expression vector
by standard methods (e.g., ligation of complementary restriction
sites on the antibody gene fragment and vector, or blunt end
ligation if no restriction sites are present). Prior to insertion
of the anti-cMet antibody-related light or heavy chain sequences,
the expression vector can already carry antibody constant region
sequences. For example, one approach to converting the anti-cMet
monoclonal antibody-related V.sub.H and V.sub.L sequences to
full-length antibody genes is to insert them into expression
vectors already encoding heavy chain constant and light chain
constant regions, respectively, such that the V.sub.H segment is
operatively linked to the CH segment(s) within the vector and the
V.sub.L segment is operatively linked to the CL segment within the
vector. Additionally or alternatively, the recombinant expression
vector can encode a signal peptide that facilitates secretion of
the antibody chain from a host cell. The antibody chain gene can be
cloned into the vector such that the signal peptide is linked
in-frame to the amino terminus of the antibody chain gene. The
signal peptide can be an immunoglobulin signal peptide or a
heterologous signal peptide (i.e., a signal peptide from a
non-immunoglobulin protein).
[0177] In addition to the antibody chain genes, the recombinant
expression vectors carry regulatory sequences that control the
expression of the antibody chain genes in a host cell. The term
"regulatory sequence" is intended to include promoters, enhancers
and other expression control elements (e.g., polyadenylation
signals) that control the transcription or translation of the
antibody chain genes. Such regulatory sequences are described, for
example, in Goeddel, Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif., 1990. It will be
appreciated by those skilled in the art that the design of the
expression vector, including the selection of regulatory sequences
may depend on such factors as the choice of the host cell to be
transformed, the level of expression of protein desired, etc.
Suitable regulatory sequences for mammalian host cell expression
include viral elements that direct high levels of protein
expression in mammalian cells, such as promoters and/or enhancers
derived from cytomegalovirus (CMV) (such as the CMV
promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40
promoter/enhancer), adenovirus, (e.g., the adenovirus major late
promoter (AdMLP)) and polyoma. For further description of viral
regulatory elements, and sequences thereof, see, e.g., U.S. Pat.
No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al.,
and U.S. Pat. No. 4,968,615 by Schaffner et al.
[0178] Recombinant expression vectors of the disclosure can carry
sequences in addition to the antibody chain genes and regulatory
sequences, such as sequences that regulate replication of the
vector in host cells (e.g., origins of replication) and selectable
marker genes. Selectable marker genes facilitate selection of host
cells into which the vector has been introduced (See, e.g., U.S.
Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.).
For example, typically a selectable marker gene confers resistance
to drugs, such as G418, hygromycin or methotrexate, on a host cell
into which the vector has been introduced. Suitable selectable
marker genes include the dihydrofolate reductase (DHFR) gene (for
use in DHFR.sup.- host cells with methotrexate
selection/amplification) and the neo gene (for G418 selection). For
expression of the light and heavy chains, the expression vector(s)
encoding the heavy and light chains is transfected into a host cell
by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
lipofection, calcium-phosphate precipitation, DEAE--dextran
transfection and the like.
[0179] It is possible to express anti-cMet antibodies composing
anti-cMet ADCs in either prokaryotic or eukaryotic host cells. In
certain embodiments, expression of antibodies is performed in
eukaryotic cells, e.g., mammalian host cells, of optimal secretion
of a properly folded and immunologically active antibody. Exemplary
mammalian host cells for expressing the recombinant antibodies of
the disclosure include Chinese Hamster Ovary (CHO cells) (including
DHFR.sup.- CHO cells, described in Urlaub and Chasin, 1980, Proc.
Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable
marker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol.
159:601-621), NS0 myeloma cells, COS cells and SP2 cells. When
recombinant expression vectors encoding antibody genes are
introduced into mammalian host cells, the antibodies are produced
by culturing the host cells for a period of time sufficient to
allow for expression of the antibody in the host cells or secretion
of the antibody into the culture medium in which the host cells are
grown. Antibodies can be recovered from the culture medium using
standard protein purification methods. Host cells can also be used
to produce portions of intact antibodies, such as Fab fragments or
scFv molecules. It is understood that variations on the above
procedure are within the scope of the present disclosure. For
example, it can be desirable to transfect a host cell with DNA
encoding either the light chain or the heavy chain (but not both)
of an anti-cMet antibody.
[0180] Recombinant DNA technology can also be used to remove some
or all of the DNA encoding either or both of the light and heavy
chains that is not necessary for binding to cMet. The molecules
expressed from such truncated DNA molecules are also encompassed by
the antibodies of the disclosure.
[0181] For recombinant expression of an anti-cMet antibody, the
host cell can be co-transfected with two expression vectors, the
first vector encoding a heavy chain derived polypeptide and the
second vector encoding a light chain derived polypeptide. The two
vectors can contain identical selectable markers, or they can each
contain a separate selectable marker. Alternatively, a single
vector can be used which encodes both heavy and light chain
polypeptides.
[0182] Once a nucleic acid encoding one or more portions of an
anti-cMet antibody is obtained, further alterations or mutations
can be introduced into the coding sequence, for example to generate
nucleic acids encoding antibodies with different CDR sequences,
antibodies with reduced affinity to the Fc receptor, or antibodies
of different subclasses.
[0183] Antibodies and/or binding fragments composing anti-cMet ADCs
can also be produced by chemical synthesis (e.g., by the methods
described in Solid Phase Peptide Synthesis, 2.sup.nd ed., 1984 The
Pierce Chemical Co., Rockford, Ill.). Variant antibodies can also
be generated using a cell-free platform, See, e.g., Chu et al.,
Biochemia No. 2, 2001 (Roche Molecular Biologicals) and Murray et
al., 2013, Current Opinion in Chemical Biology, 17:420-426.
[0184] Once an anti-cMet antibody and/or binding fragment has been
produced by recombinant expression, it can be purified by any
method known in the art for purification of an immunoglobulin
molecule, for example, by chromatography (e.g., ion exchange,
affinity, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard technique for the
purification of proteins. Further, the anti-cMet antibodies and/or
binding fragments can be fused to heterologous polypeptide
sequences described herein or otherwise known in the art to
facilitate purification.
[0185] Once isolated, the anti-cMet antibody and/or binding
fragment can, if desired, be further purified, e.g., by column
chromatography. (see, e.g., Fisher, Laboratory Techniques In
Biochemistry And Molecular Biology, Work and Burdon, eds.,
Elsevier, 1980), or by gel filtration chromatography on a
Superdex.TM. 75 column (Pharmacia Biotech AB, Uppsala, Sweden).
[0186] 5.6. Specific Anti-cMet Antibody Drug Conjugates
[0187] As mentioned, anti-cMet ADCs generally comprise an anti-cMet
antigen binding moiety, such as an anti-cMet antibody and/or
binding fragment, having one or more cytotoxic and/or cytostatic
agents, which may be the same or different, linked thereto by way
of one or more linkers, which may also be the same or different.
Multiple different cytotoxic/cytostatic agents can be attached to
each Ab to make an ADC. These agents may target two or more
pathways to kill or arrest the growth of tumor cells, target
multiple nodes of the same pathway, or double up on same target
(i.e., inhibit growth and/or kill cells through two or more
different mechanisms).
[0188] In specific embodiments, the anti-cMet ADCs are compounds
according to structural formula (I):
[D-L-XY].sub.n-Ab (I)
[0189] or salts thereof, where each "D" represents, independently
of the others, a cytotoxic and/or cytostatic agent ("drug"); each
"L" represents, independently of the others, a linker; "Ab"
represents an anti-cMet antigen binding moiety, such as an
anti-cMet antibody or binding fragment; each "XY" represents a
linkage formed between a functional group R.sup.x on the linker and
a "complementary" functional group R.sup.y on the antigen binding
moiety; and n represents the number of drugs linked to Ab of the
ADC.
[0190] Specific embodiments of various antibodies or binding
fragments (Ab) that may compose ADCs according to structural
formula (I) include the various embodiments of anti-cMet antibodies
and/or binding fragments described above.
[0191] In some specific embodiments of the ADCs or salts of
structural formula (I), each D is the same and/or each L is the
same.
[0192] Specific embodiments of cytotoxic and/or cytostatic agents
(D) and linkers (L) that may compose the anti-cMet ADCs, as well as
the number of cytotoxic and/or cytostatic agents linked to the
anti-cMet ADCs, are described in more detail below.
[0193] In a specific exemplary embodiment, the anti-cMet ADCs are
compounds according to structural formula (I) in which each "D" is
the same and is either a cell-permeating auristatin (for example,
dolastatin-10 or MMAE) or a cell-permeating minor groove-binding
DNA cross-linking agent (for example, a PBD or a PBD dimer); each
"L" is the same and is a linker cleavable by a lysosomal enzyme;
each "XY" is a linkage formed between a maleimide and a sulfydryl
group; "Ab" is an antibody comprising six CDRs corresponding to the
six CDRs of antibody ABT-700 (224G11), or an antibody that competes
for binding cMet with such an antibody; and n is 2, 3 or 4. In a
specific embodiment of this exemplary embodiment or the anti-cMet
ADCs of structural formula (I), "Ab" is a humanized antibody, for
example, a humanized antibody comprising V.sub.H and V.sub.L chains
corresponding to the V.sub.H and V.sub.L chains of antibody 5D5. In
another specific embodiment of the anti-cMet ADCs of structural
formula (I), the Ab is the antibody STI-D0602 (Sorrento).
[0194] In a specific exemplary embodiment, the compound according
to structural formula (I) has the structure of formula (IIa):
##STR00001##
[0195] In one embodiment, the Ab in the compound of formula (IIa)
is ABT-700.
[0196] In a specific exemplary embodiment, the compound of
structural formula (I) has the following structure:
##STR00002##
[0197] or a pharmaceutically acceptable salt thereof, wherein n has
an average value ranging from 2-4, and the Ab is a full length
anti-cMet antibody.
[0198] In a specific embodiment, the Ab in the compound of this
particular formula is ABT-700.
[0199] In a specific embodiment, n has an average value ranging
from 2-4 and Ab is a full-length anti-cMet antibody.
[0200] 5.6.1. Cytotoxic and/or Cytostatic Agents
[0201] The cytotoxic and/or cytostatic agents may be any agents
known to inhibit the growth and/or replication of and/or kill
cells, and in particular cancer and/or tumor cells. Numerous agents
having cytotoxic and/or cytostatic properties are known in the
literature. Non-limiting examples of classes of cytotoxic and/or
cytostatic agents include, by way of example and not limitation,
radionuclides, alkylating agents, DNA cross-linking agents, DNA
intercalating agents (e.g., groove binding agents such as minor
groove binders), cell cycle modulators, apoptosis regulators,
kinase inhibitors, protein synthesis inhibitors, mitochondria
inhibitors, nuclear export inhibitors, topoisomerase I inhibitors,
topoisomerase II inhibitors, RNA/DNA antimetabolites and
antimitotic agents.
[0202] Specific non-limiting examples of agents within certain of
these various classes are provided below.
[0203] Alkylating Agents:
[0204] asaley (L-Leucine,
N--[N-acetyl-4-[bis-(2-chloroethy)amino]-DL-phenylalanyl]-,
ethylester); AZQ (1,4-cyclohexadiene-1,4-dicarbamic acid, 2,
5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ester); BCNU
(N,N'-Bis(2-chloroethyl)-N-nitrosourea); busulfan (1,4-butanediol
dimethanesulfonate); (carboxyphthalato)platinum; CBDCA
(cis-(1,1-cyclobutanedicarboxylato)diammineplatinum(II))); CCNU
(N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea); CHIP (iproplatin;
NSC 256927); chlorambucil; chlorozotocin (2-[[[(2-chloroethyl)
nitrosoamino]carbonyl]amino]-2-deoxy-D-glucopyranose); cis-platinum
(cisplatin); clomesone; cyanomorpholinodoxorubicin; cyclodisone;
dianhydrogalactitol (5,6-diepoxydulcitol); fluorodopan
((5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyl-uracil);
hepsulfam; hycanthone; indolinobenzodiazepine dimer DGN462;
melphalan; methyl CCNU
((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea);
mitomycin C; mitozolamide; nitrogen mustard ((bis(2-chloroethyl)
methylamine hydrochloride); PCNU
((1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea));
piperazine alkylator
((1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine
dihydrochloride)); piperazinedione; pipobroman
(N,N'-bis(3-bromopropionyl) piperazine); porfiromycin
(N-methylmitomycin C); spirohydantoin mustard; teroxirone
(triglycidylisocyanurate); tetraplatin; thio-tepa
(N,N',N''-tri-1,2-ethanediylthio phosphoramide);
triethylenemelamine; uracil nitrogen mustard (desmethyldopan);
Yoshi-864 ((bis(3-mesyloxy propyl)amine hydrochloride).
[0205] DNA Alkylating-Like Agents:
[0206] Cisplatin; Carboplatin; Nedaplatin; Oxaliplatin;
Satraplatin; Triplatin tetranitrate; Procarbazine; altretamine;
dacarbazine; mitozolomide; temozolomide.
[0207] Alkylating Antineoplastic Agents:
[0208] Carboquone; Carmustine; Chlornaphazine; Chlorozotocin;
Duocarmycin; Evofosfamide; Fotemustine; Glufosfamide; Lomustine;
Mannosulfan; Nimustine; Phenanthriplatin; Pipobroman; Ranimustine;
Semustine; Streptozotocin; ThioTEPA; Treosulfan; Triaziquone;
Triethylenemelamine; Triplatin tetranitrate.
[0209] DNA Replication and Repair Inhibitors:
[0210] Altretamine; Bleomycin; Dacarbazine; Dactinomycin;
Mitobronitol; Mitomycin; Pingyangmycin; Plicamycin; Procarbazine;
Temozolomide; ABT-888 (veliparib); olaparib; KU-59436; AZD-2281;
AG-014699; BSI-201; BGP-15; INO-1001; ONO-2231.
[0211] Cell Cycle Modulators:
[0212] Paclitaxel; Nab-Paclitaxel; Docetaxel; Vincristine;
Vinblastine; ABT-348; AZD-1152; MLN-8054; VX-680; Aurora A-specific
kinase inhibitors; Aurora B-specific kinase inhibitors and
pan-Aurora kinase inhibitors; AZD-5438; BMI-1040; BMS-032; BMS-387;
CVT-2584; flavopyridol; GPC-286199; MCS-5A; PD0332991; PHA-690509;
seliciclib (CYC-202, R-roscovitine); ZK-304709; AZ:D/4877,
ARRY-520; GSK923295A.
[0213] Apoptosis Regulators:
[0214] AT-101 ((-)gossypol); G3139 or oblimersen (Bcl-2-targeting
antisense oligonucleotide); IPI-194; IPI-565;
N-(4-(4-((4'-chloro(1,1'-biphenyl)-2-yl)methyl)piperazin-1-ylbenzoyl)-4-(-
((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitroben-
zene sulfonamide);
N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)pip-
erazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl-
)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide;
GX-070 (Obatoclax.RTM.; 1H-Indole,
2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-)-
); HGS1029; GDC-0145; GDC-0152; LCL-161; LBW-242; venetoclax;
agents that target TRAIL or death receptors (e.g., DR4 and DR5)
such as ETR2-ST01, GDC0145, HGS-1029, LBY-135, PRO-1762; drugs that
target caspases, caspase-regulators, BCL-2 family members, death
domain proteins, TNF family members, Toll family members, and/or
NF-kappa-B proteins.
[0215] Angiogenesis Inhibitors:
[0216] ABT-869; AEE-788; axitinib (AG-13736); AZD-2171; CP-547,632;
IM-862; pegaptamib; sorafenib; BAY43-9006; pazopanib (GW-786034);
vatalanib (PTK-787, ZK-222584); sunitinib; SU-11248; VEGF trap;
vandetanib; ABT-165; ZD-6474; DLL4 inhibitors.
[0217] Proteasome Inhibitors:
[0218] Bortezomib; Carfilzomib; Epoxomicin; Ixazomib;
Salinosporamide A.
[0219] Kinase Inhibitors:
[0220] Afatinib; Axitinib; Bosutinib; Crizotinib; Dasatinib;
Erlotinib; Fostamatinib; Gefitinib; Ibrutinib; Imatinib; Lapatinib;
Lenvatinib; Mubritinib; Nilotinib; Pazopanib; Pegaptanib;
Sorafenib; Sunitinib; SU6656; Vandetanib; Vemurafenib; CEP-701
(lesaurtinib); XL019; INCB018424 (ruxolitinib); ARRY-142886
(selemetinib); ARRY-438162 (binimetinib); PD-325901; PD-98059;
AP-23573; CCI-779; everolimus; RAD-001; rapamycin; temsirolimus;
ATP-competitive TORC1/TORC2 inhibitors including PI-103, PP242,
PP30, Torin 1; LY294002; XL-147; CAL-120; ONC-21; AEZS-127;
ETP-45658; PX-866; GDC-0941; BGT226; BEZ235; XL765.
[0221] Protein Synthesis Inhibitors:
[0222] Streptomycin; Dihydrostreptomycin; Neomycin; Framycetin;
Paromomycin; Ribostamycin; Kanamycin; Amikacin; Arbekacin;
Bekanamycin; Dibekacin; Tobramycin; Spectinomycin; Hygromycin B;
Paromomycin; Gentamicin; Netilmicin; Sisomicin; Isepamicin;
Verdamicin; Astromicin; Tetracycline; Doxycycline;
Chlortetracycline; Clomocycline; Demeclocycline; Lymecycline;
Meclocycline; Metacycline; Minocycline; Oxytetracycline;
Penimepicycline; Rolitetracycline; Tetracycline; Glycylcyclines;
Tigecycline; Oxazolidinone; Eperezolid; Linezolid; Posizolid;
Radezolid; Ranbezolid; Sutezolid; Tedizolid; Peptidyl transferase
inhibitors; Chloramphenicol; Azidamfenicol; Thiamphenicol;
Florfenicol; Pleuromutilins; Retapamulin; Tiamulin; Valnemulin;
Azithromycin; Clarithromycin; Dirithromycin; Erythromycin;
Flurithromycin; Josamycin; Midecamycin; Miocamycin; Oleandomycin;
Rokitamycin; Roxithromycin; Spiramycin; Troleandomycin; Tylosin;
Ketolides; Telithromycin; Cethromycin; Solithromycin; Clindamycin;
Lincomycin; Pirlimycin; Streptogramins; Pristinamycin;
Quinupristin/dalfopristin; Virginiamycin.
[0223] Histone Deacetylase Inhibitors:
[0224] Vorinostat; Romidepsin; Chidamide; Panobinostat; Valproic
acid; Belinostat; Mocetinostat; Abexinostat; Entinostat; SB939
(pracinostat); Resminostat; Givinostat; Quisinostat;
thioureidobutyronitrile (Kevetrin.TM.); CUDC-10; CHR-2845
(tefinostat); CHR-3996; 4SC-202; CG200745; ACY-1215 (rocilinostat);
ME-344; sulforaphane.
[0225] Topoisomerase I Inhibitors:
[0226] camptothecin; various camptothecin derivatives and analogs
(for example, NSC 100880, NSC 603071, NSC 107124, NSC 643833, NSC
629971, NSC 295500, NSC 249910, NSC 606985, NSC 74028, NSC 176323,
NSC 295501, NSC 606172, NSC 606173, NSC 610458, NSC 618939, NSC
610457, NSC 610459, NSC 606499, NSC 610456, NSC 364830, and NSC
606497); morpholinisoxorubicin; SN-38.
[0227] Topoisomerase II Inhibitors:
[0228] doxorubicin; amonafide (benzisoquinolinedione); m-AMSA
(4'(9-acridinylamino)-3'-methoxymethanesulfonanilide);
anthrapyrazole derivative ((NSC 355644); etoposide (VP-16);
pyrazoloacridine ((pyrazolo[3,4,5-k1]acridine-2(6H)-propanamine,
9-methoxy-N, N-dimethyl-5-nitro-, monomethanesulfonate); bisantrene
hydrochloride; daunorubicin; deoxydoxorubicin; mitoxantrone;
menogaril; N,N-dibenzyl daunomycin; oxanthrazole; rubidazone;
teniposide.
[0229] DNA Intercalating Agents:
[0230] anthramycin; chicamycin A; tomaymycin; DC-81; sibiromycin;
pyrrolobenzodiazepine derivative; SGD-1882
((S)-2-(4-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-
-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propo-
xy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one); SG2000
(SIG-136;
(11aS,11a'S)-8,8'-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-
-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one)).
[0231] RNA/DNA Antimetabolites:
[0232] L-alanosine; 5-azacytidine; 5-fluorouracil; acivicin;
aminopterin derivative N-[2-chloro-5-[[(2,
4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl] L-aspartic
acid (NSC 132483); aminopterin derivative N-[4-[[(2,
4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl] L-aspartic
acid; aminopterin derivative N-[2-chloro-4-[[(2,
4-diamino-6-pteridinyl)methyl]amino]benzoyl] L-aspartic acid
monohydrate; antifolate PT523
((N.sup..alpha.-(4-amino-4-deoxypteroyl)-N.sup..gamma.-hemiphthaloyl-L-or-
nithine)); Baker's soluble antifol (NSC 139105); dichlorallyl
lawsone ((2-(3, 3-dichloroallyl)-3-hydroxy-1,4-naphthoquinone);
brequinar; ftorafur ((pro-drug;
5-fluoro-1-(tetrahydro-2-furyl)-uracil); 5,6-dihydro-5-azacytidine;
methotrexate; methotrexate derivative (N-[[4-[[(2,
4-diamino-6-pteridinyl)methyl]methylamino]-1-naphthalenyl]carbonyl]
L-glutamic acid); PALA ((N-(phosphonoacetyl)-L-aspartate);
pyrazofurin; trimetrexate.
[0233] DNA Antimetabolites:
[0234] 3-HP; 2'-deoxy-5-fluorouridine; 5-HP; .alpha.-TGDR
(.alpha.-2'-deoxy-6-thioguanosine); aphidicolin glycinate; ara C
(cytosine arabinoside); 5-aza-2'-deoxycytidine; .beta.-TGDR
(.beta.-2'-deoxy-6-thioguanosine); cyclocytidine; guanazole;
hydroxyurea; inosine glycodialdehyde; macbecin II;
pyrazoloimidazole; thioguanine; thiopurine.
[0235] Mitochondria Inhibitors:
[0236] pancratistatin; phenpanstatin; rhodamine-123; edelfosine;
d-alpha-tocopherol succinate; compound 11.beta.; aspirin;
ellipticine; berberine; cerulenin; GX015-070 (Obatoclax.RTM.;
1H-Indole,
2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-)-
; celastrol (tripterine); metformin; Brilliant green; ME-344.
[0237] Antimitotic Agents:
[0238] allocolchicine; auristatins, such as MMAE (monomethyl
auristatin E) and MMAF (monomethyl auristatin F); halichondrin B;
cemadotin; colchicine; cholchicine derivative (N-benzoyl-deacetyl
benzamide); dolastatin-10; dolastatin-15; maytansine;
maytansinoids, such as DM1
(N.sub.2'-deacetyl-N.sub.2'-(3-mercapto-1-oxopropyl)-maytansine);
rhozoxin; paclitaxel; paclitaxel derivative
((2'-N-[3-(dimethylamino)propyl]glutaramate paclitaxel); docetaxel;
thiocolchicine; trityl cysteine; vinblastine sulfate; vincristine
sulfate.
[0239] Nuclear Export Inhibitors:
[0240] callystatin A; delactonmycin; KPT-185 (propan-2-yl
(Z)-3-[3-[3-methoxy-5-(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]prop-2--
enoate); kazusamycin A; leptolstatin; leptofuranin A; leptomycin B;
ratjadone; Verdinexor
((Z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-N'-pyridin-
-2-ylprop-2-enehydrazide).
[0241] Hormonal Therapies:
[0242] anastrozole; exemestane; arzoxifene; bicalutamide;
cetrorelix; degarelix; deslorelin; trilostane; dexamethasone;
flutamide; raloxifene; fadrozole; toremifene; fulvestrant;
letrozole; formestane; glucocorticoids; doxercalciferol; sevelamer
carbonate; lasofoxifene; leuprolide acetate; megesterol;
mifepristone; nilutamide; tamoxifen citrate; abarelix; prednisone;
finasteride; rilostane; buserelin; luteinizing hormone releasing
hormone (LHRH); Histrelin; trilostane or modrastane; fosrelin;
goserelin.
[0243] Any of these agents that include, or that may be modified to
include, a site of attachment to an antibody and/or binding
fragment may be included in an anti-cMet ADC.
[0244] Skilled artisans will also appreciate that the above
mechanisms of action are not mutually exclusive, and that in some
embodiments it may be desirable to utilize anti-cMet ADCs capable
of exerting antitumor activity against cMet-expressing (herein
referred to as cMet+ tumors) or cMet-overexpressing tumors via more
than one mechanism of action. As a specific example, such an
anti-cMet ADC may include a cell-permeating cytotoxic and/or
cytostatic agent that is cytotoxic and/or cytostatic to both
cMet+/overexpressing tumors and cMet-negative tumor cells linked to
an anti-cMet antibody by way of a cleavable linker.
[0245] Accordingly, in some embodiments, the cytotoxic and/or
cytostatic agents included in an anti-cMet ADC will, upon cleavage
of the ADC, be able to traverse cell membranes ("cell permeable
cytostatic and/or cytotoxic agents"). Specific cytotoxic and/or
cytostatic agents of interest, and/or cleavage products of ADCs
including such agents, may be tested for the ability to traverse
cell membranes using routine methods known to those of skill in the
art. Permeability (P) of molecules across a membrane can be
expressed as P=KD/.DELTA.x where K is the partition coefficient, D
is the diffusion coefficient, and .DELTA.x is the thickness of the
cell membrane. The diffusion coefficient (D) is a measure of the
rate of entry into the cytoplasm depending on the molecular weight
or size of a molecule. K is a measure of the solubility of the
substance in lipids. A low value of K describes a molecule like
water that is not soluble in lipid. Graphically, it is expected
that permeability (P) as a function of the partition coefficient
(K) will increase linearly when D and .DELTA.x are constants.
(Walter & Gutknecht, 1986, "Permeability of small
nonelectrolytes through lipid bilayer membranes," Journal of
Membrane Biology 90:207-217; Diamond & Katz, 1974,
"Interpretation of nonelectrolyte partition coefficients between
dimyristoyl lecithin and water," Journal of Membrane Biology
17:121-154).
[0246] In a specific embodiment, the cytotoxic and/or cytostatic
agent is a cell-permeable antimitotic agent.
[0247] In another specific embodiment, the cytotoxic and/or
cytostatic agent is a cell-permeable auristatin, such as, for
example, dolastatin-10 or MMAE.
[0248] In another specific embodiment, the cytotoxic and/or
cytostatic agent is a cell-permeable minor groove-binding DNA
cross-linking agent, such as, for example, a pyrrolobenzodiazepine
("PBD") dimer.
[0249] 5.6.2. Linkers
[0250] In the anti-cMet ADCs described herein, the cytotoxic and/or
cytostatic agents are linked to the antigen binding moiety by way
of linkers. The linkers may be short, long, hydrophobic,
hydrophilic, flexible or rigid, or may be composed of segments that
each independently have one or more of the above-mentioned
properties such that the linker may include segments having
different properties. The linkers may be polyvalent such that they
covalently link more than one agent to a single site on the
antibody, or monovalent such that covalently they link a single
agent to a single site on the antibody.
[0251] As will be appreciated by skilled artisans, the linkers link
the cytotoxic and/or cytostatic agents to the antigen binding
moiety by forming a covalent linkage to the cytotoxic and/or
cytostatic agent at one location and a covalent linkage to the
antigen binding moiety at another. The covalent linkages are formed
by reaction between functional groups on the linker and functional
groups on the agents and the antigen binding moiety. As used
herein, the expression "linker" is intended to include (i)
unconjugated forms of the linker that include a functional group
capable of covalently linking the linker to a cytotoxic and/or
cytostatic agent and a functional group capable of covalently
linking the linker to the antigen binding moiety such as an
antibody; (ii) partially conjugated forms of the linker that
includes a functional group capable of covalently linking the
linker to an antigen binding moiety such as an antibody and that is
covalently linked to a cytotoxic and/or cytostatic agent, or vice
versa; and (iii) fully conjugated forms of the linker that is
covalently linked to both a cytotoxic and/or cytostatic agent and
an antigen binding moiety such as an antibody. In some specific
embodiments of linkers and ADCs described herein, as well as
synthons used to conjugate linker-agents to antibodies, moieties
comprising the functional groups on the linker and covalent
linkages formed between the linker and antibody are specifically
illustrated as R.sup.x and XY, respectively.
[0252] The linkers linking the cytotoxic and/or cytostatic agents
to the antigen binding moiety of an anti-cMet ADC may be long,
short, flexible, rigid, hydrophilic or hydrophobic in nature, or
may comprise segments that have different characteristics, such as
segments of flexibility, segments of rigidity, etc. The linker may
be chemically stable to extracellular environments, for example,
chemically stable in the blood stream, or may include linkages that
are not stable and release the cytotoxic and/or cytostatic agents
in the extracellular milieu. In some embodiments, the linkers
include linkages that are designed to release the cytotoxic and/or
cytostatic agents upon internalization of the anti-cMet ADC within
the cell. In some specific embodiments, the linkers includes
linkages designed to cleave and/or immolate or otherwise breakdown
specifically or non-specifically inside cells. A wide variety of
linkers useful for linking drugs to antigen binding moieties such
as antibodies in the context of ADCs are known in the art. Any of
these linkers, as well as other linkers, may be used to link the
cytotoxic and/or cytostatic agents to the antigen binding moiety of
the anti-cMet ADCs described herein.
[0253] The number of cytotoxic and/or cytostatic agents linked to
the antigen binding moiety of an anti-cMet ADC can vary (called the
"drug-to-antibody ratio," or "DAR"), and will be limited only by
the number of available attachments sites on the antigen binding
moiety and the number of agents linked to a single linker.
Typically, a linker will link a single cytotoxic and/or cytostatic
agent to the antigen binding moiety of an anti-cMet ADC. In
embodiments of anti-cMet ADCs which include more than a single
cytotoxic and/or cytostatic agent, each agent may be the same or
different. As long as the anti-cMet ADC does not exhibit
unacceptable levels of aggregation under the conditions of use
and/or storage, anti-cMet ADCs with DARs of twenty, or even higher,
are contemplated. In some embodiments, the anti-cMet ADCs described
herein may have a DAR in the range of about 1-10, 1-8, 1-6, or 1-4.
In certain specific embodiments, the anti-cMet ADCs may have a DAR
of 2, 3 or 4. In certain embodiments, the anti-Cmet ADC has an
average DAR of 3.1.
[0254] The linkers are preferably, but need not be, chemically
stable to conditions outside the cell, and may be designed to
cleave, immolate and/or otherwise specifically degrade inside the
cell. Alternatively, linkers that are not designed to specifically
cleave or degrade inside the cell may be used. Choice of stable
versus unstable linker may depend upon the toxicity of the
cytotoxic and/or cytostatic agent. A wide variety of linkers useful
for linking drugs to antibodies in the context of ADCs are known in
the art. Any of these linkers, as well as other linkers, may be
used to link the cytotoxic and/or cytostatic agents to the antibody
of the ADCs described herein.
[0255] Exemplary polyvalent linkers that may be used to link many
cytotoxic and/or cytostatic agents to a single antibody molecule
are described, for example, in WO 2009/073445; WO 2010/068795; WO
2010/138719; WO 2011/120053; WO 2011/171020; WO 2013/096901; WO
2014/008375; WO 2014/093379; WO 2014/093394; WO 2014/093640, the
contents of which are incorporated herein by reference in their
entireties. For example, the Fleximer linker technology developed
by Mersana et al. has the potential to enable high-DAR ADCs with
good physicochemical properties. As shown below, the Mersana
technology is based on incorporating drug molecules into a
solubilizing poly-acetal backbone via a sequence of ester bonds.
The methodology renders highly-loaded ADCs (DAR up to 20) while
maintaining good physicochemical properties.
[0256] Additional examples of dendritic type linkers can be found
in US 2006/116422; US 2005/271615; de Groot et al (2003) Angew.
Chem. Int. Ed. 42:4490-4494; Amir et al (2003) Angew. Chem. Int.
Ed. 42:4494-4499; Shamis et al (2004) J. Am. Chem. Soc.
126:1726-1731; Sun et al (2002) Bioorganic & Medicinal
Chemistry Letters 12:2213-2215; Sun et al (2003) Bioorganic &
Medicinal Chemistry 11:1761-1768; King et al (2002) Tetrahedron
Letters 43:1987-1990, each of which is incorporated herein by
reference.
[0257] Exemplary monovalent linkers that may be used are described,
for example, in Nolting, 2013, Antibody-Drug Conjugates, Methods in
Molecular Biology 1045:71-100; Kitson et al., 2013,
CROs/CMOs--Chemica Oggi--Chemistry Today 31(4):30-38; Ducry et al.,
2010, Bioconjugate Chem. 21:5-13; Zhao et al., 2011, J. Med. Chem.
54:3606-3623; U.S. Pat. No. 7,223,837; U.S. Pat. No. 8,568,728;
U.S. Pat. No. 8,535,678; and WO2004010957, each of which is
incorporated herein by reference.
[0258] By way of example and not limitation, some cleavable and
noncleavable linkers that may be included in the anti-cMet ADCs
described herein are described below.
[0259] 5.6.2.1. Cleavable Linkers
[0260] In certain embodiments, the linker selected is cleavable in
vivo. Cleavable linkers may include chemically or enzymatically
unstable or degradable linkages. Cleavable linkers generally rely
on processes inside the cell to liberate the drug, such as
reduction in the cytoplasm, exposure to acidic conditions in the
lysosome, or cleavage by specific proteases or other enzymes within
the cell. Cleavable linkers generally incorporate one or more
chemical bonds that are either chemically or enzymatically
cleavable while the remainder of the linker is noncleavable. In
certain embodiments, a linker comprises a chemically labile group
such as hydrazone and/or disulfide groups. Linkers comprising
chemically labile groups exploit differential properties between
the plasma and some cytoplasmic compartments. The intracellular
conditions to facilitate drug release for hydrazone containing
linkers are the acidic environment of endosomes and lysosomes,
while the disulfide containing linkers are reduced in the cytosol,
which contains high thiol concentrations, e.g., glutathione. In
certain embodiments, the plasma stability of a linker comprising a
chemically labile group may be increased by introducing steric
hindrance using substituents near the chemically labile group.
[0261] Acid-labile groups, such as hydrazone, remain intact during
systemic circulation in the blood's neutral pH environment (pH
7.3-7.5) and undergo hydrolysis and release the drug once the ADC
is internalized into mildly acidic endosomal (pH 5.0-6.5) and
lysosomal (pH 4.5-5.0) compartments of the cell. This pH dependent
release mechanism has been associated with nonspecific release of
the drug. To increase the stability of the hydrazone group of the
linker, the linker may be varied by chemical modification, e.g.,
substitution, allowing tuning to achieve more efficient release in
the lysosome with a minimized loss in circulation.
[0262] Hydrazone-containing linkers may contain additional cleavage
sites, such as additional acid-labile cleavage sites and/or
enzymatically labile cleavage sites. ADCs including exemplary
hydrazone-containing linkers include the following structures:
##STR00003## [0263] wherein D and Ab represent the cytotoxic and/or
cytostatic agent (drug) and antibody, respectively, and n
represents the number of drug-linkers linked to the antibody. In
certain linkers such as linker (Ig), the linker comprises two
cleavable groups--a disulfide and a hydrazone moiety. For such
linkers, effective release of the unmodified free drug requires
acidic pH or disulfide reduction and acidic pH. Linkers such as
(Ih) and (Ii) have been shown to be effective with a single
hydrazone cleavage site.
[0264] Other acid-labile groups that may be included in linkers
include cis-aconityl-containing linkers. cis-Aconityl chemistry
uses a carboxylic acid juxtaposed to an amide bond to accelerate
amide hydrolysis under acidic conditions.
[0265] Cleavable linkers may also include a disulfide group.
Disulfides are thermodynamically stable at physiological pH and are
designed to release the drug upon internalization inside cells,
wherein the cytosol provides a significantly more reducing
environment compared to the extracellular environment. Scission of
disulfide bonds generally requires the presence of a cytoplasmic
thiol cofactor, such as (reduced) glutathione (GSH), such that
disulfide-containing linkers are reasonably stable in circulation,
selectively releasing the drug in the cytosol. The intracellular
enzyme protein disulfide isomerase, or similar enzymes capable of
cleaving disulfide bonds, may also contribute to the preferential
cleavage of disulfide bonds inside cells. GSH is reported to be
present in cells in the concentration range of 0.5-10 mM compared
with a significantly lower concentration of GSH or cysteine, the
most abundant low-molecular weight thiol, in circulation at
approximately 5 .mu.M. Tumor cells, where irregular blood flow
leads to a hypoxic state, result in enhanced activity of reductive
enzymes and therefore even higher glutathione concentrations. In
certain embodiments, the in vivo stability of a
disulfide-containing linker may be enhanced by chemical
modification of the linker, e.g., use of steric hinderance adjacent
to the disulfide bond.
[0266] ADCs including exemplary disulfide-containing linkers
include the following structures:
##STR00004## [0267] wherein D and Ab represent the drug and
antibody, respectively, n represents the number of drug-linkers
linked to the antibody, and R is independently selected at each
occurrence from hydrogen or alkyl, for example. In certain
embodiments, increasing steric hinderance adjacent to the disulfide
bond increases the stability of the linker. Structures such as (Ij)
and (Il) show increased in vivo stability when one or more R groups
is selected from a lower alkyl such as methyl.
[0268] Another type of cleavable linker that may be used is a
linker that is specifically cleaved by an enzyme. Such linkers are
typically peptide-based or include peptidic regions that act as
substrates for enzymes. Peptide based linkers tend to be more
stable in plasma and extracellular milieu than chemically labile
linkers. Peptide bonds generally have good serum stability, as
lysosomal proteolytic enzymes have very low activity in blood due
to endogenous inhibitors and the unfavorably high pH value of blood
compared to lysosomes. Release of a drug from an antibody occurs
specifically due to the action of lysosomal proteases, e.g.,
cathepsin and plasmin. These proteases may be present at elevated
levels in certain tumor cells.
[0269] In exemplary embodiments, the cleavable peptide is selected
from tetrapeptides such as Gly-Phe-Leu-Gly (SEQ ID NO:98),
Ala-Leu-Ala-Leu (SEQ ID NO:99) or dipeptides such as Val-Cit,
Val-Ala, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, Phe-Lys, Ile-Val,
Asp-Val, His-Val, NorVal-(D)Asp, Ala-(D)Asp, Met-Lys, Asn-Lys,
Ile-Pro, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys,
Pro-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Met-(D)Lys, Asn-(D)Lys. In
certain embodiments, dipeptides are preferred over longer
polypeptides due to hydrophobicity of the longer peptides.
[0270] A variety of dipeptide-based cleavable linkers useful for
linking drugs such as doxorubicin, mitomycin, campotothecin,
tallysomycin and auristatin/auristatin family members to antibodies
have been described (see, Dubowchik et al., 1998, 1 Org. Chem.
67:1866-1872; Dubowchik et al., 1998, Bioorg. Med. Chem. Lett.
8(21):3341-3346; Walker et al., 2002, Bioorg. Med. Chem. Lett.
12:217-219; Walker et al., 2004, Bioorg. Med. Chem. Lett.
14:4323-4327; and Francisco et al., 2003, Blood 102:1458-1465,
Dornina et al., 2008, Bioconjugate Chemistry 19:1960-1963, of each
of which is incorporated herein by reference). All of these
dipeptide linkers, or modified versions of these dipeptide linkers,
may be used in the ADCs described herein. Other dipeptide linkers
that may be used include those found in ADCs such as Seattle
Genetics' Brentuximab Vendotin SGN-35 (Adcetris.TM.), Seattle
Genetics SGN-75 (anti-CD-70, Val-Cit-MMAF), Celldex Therapeutics
glembatumumab (CDX-011) (anti-NMB, Val-Cit-MMAE), and Cytogen
PSMA-ADC (PSMA-ADC-1301) (anti-PSMA, Val-Cit-MMAE).
[0271] Enzymatically cleavable linkers may include a
self-immolative spacer to spatially separate the drug from the site
of enzymatic cleavage. The direct attachment of a drug to a peptide
linker can result in proteolytic release of an amino acid adduct of
the drug, thereby impairing its activity. The use of a
self-immolative spacer allows for the elimination of the fully
active, chemically unmodified drug upon amide bond hydrolysis.
[0272] One self-immolative spacer is the bifunctional
para-aminobenzyl alcohol group, which is linked to the peptide
through the amino group, forming an amide bond, while amine
containing drugs may be attached through carbamate functionalities
to the benzylic hydroxyl group of the linker (PABC). The resulting
prodrugs are activated upon protease-mediated cleavage, leading to
a 1,6-elimination reaction releasing the unmodified drug, carbon
dioxide, and remnants of the linker group. The following scheme
depicts the fragmentation of .beta.-amidobenzyl ether and release
of the drug:
##STR00005##
[0273] wherein X-D represents the unmodified drug.
[0274] Heterocyclic variants of this self-immolative group have
also been described. See for example, U.S. Pat. No. 7,989,434,
incorporated herein by reference.
[0275] In some embodiments, the enzymatically cleavable linker is a
.beta.-glucuronic acid-based linker. Facile release of the drug may
be realized through cleavage of the .beta.-glucuronide glycosidic
bond by the lysosomal enzyme .beta.-glucuronidase. This enzyme is
present abundantly within lysosomes and is overexpressed in some
tumor types, while the enzyme activity outside cells is low.
.beta.-Glucuronic acid-based linkers may be used to circumvent the
tendency of an ADC to undergo aggregation due to the hydrophilic
nature of .beta.-glucuronides. In some embodiments,
.beta.-glucuronic acid-based linkers are preferred as linkers for
ADCs linked to hydrophobic drugs. The following scheme depicts the
release of the drug from and ADC containing a .beta.-glucuronic
acid-based linker:
##STR00006##
[0276] A variety of cleavable .beta.-glucuronic acid-based linkers
useful for linking drugs such as auristatins, camptothecin and
doxorubicin analogues, CBI minor-groove binders, and psymberin to
antibodies have been described (see, see Nolting, Chapter 5 "Linker
Technology in Antibody-Drug Conjugates," In: Antibody-Drug
Conjugates: Methods in Molecular Biology, vol. 1045, pp. 71-100,
Laurent Ducry (Ed.), Springer Science & Business Medica, LLC,
2013; Jeffrey et al., 2006, Bioconjug. Chem. 17:831-840; Jeffrey et
al., 2007, Bioorg. Med. Chem. Lett. 17:2278-2280; and Jiang et al.,
2005, J. Am. Chem. Soc. 127:11254-11255, each of which is
incorporated herein by reference). All of these .beta.-glucuronic
acid-based linkers may be used in the anti-cMet ADCs described
herein.
[0277] Additionally, cytotoxic and/or cytostatic agents containing
a phenol group can be covalently bonded to a linker through the
phenolic oxygen. One such linker, described in WO 2007/089149,
relies on a methodogy in which a diamino-ethane "SpaceLink" is used
in conjunction with traditional "PABO"-based self-immolative groups
to deliver phenols. The cleavage of the linker is depicted
schematically below, where D represents a cytotoxic and/or
cytostatic agent having a phenolic hydroxyl group.
##STR00007##
[0278] Cleavable linkers may include noncleavable portions or
segments, and/or cleavable segments or portions may be included in
an otherwise non-cleavable linker to render it cleavable. By way of
example only, polyethylene glycol (PEG) and related polymers may
include cleavable groups in the polymer backbone. For example, a
polyethylene glycol or polymer linker may include one or more
cleavable groups such as a disulfide, a hydrazone or a
dipeptide.
[0279] Other degradable linkages that may be employed in linkers
include, but are not limited to, ester linkages formed by the
reaction of PEG carboxylic acids or activated PEG carboxylic acids
with alcohol groups on a biologically active agent, wherein such
ester groups generally hydrolyze under physiological conditions to
release the biologically active agent. Hydrolytically degradable
linkages include, but are not limited to, carbonate linkages; imine
linkages resulting from reaction of an amine and an aldehyde;
phosphate ester linkages formed by reacting an alcohol with a
phosphate group; acetal linkages that are the reaction product of
an aldehyde and an alcohol; orthoester linkages that are the
reaction product of a formate and an alcohol; and oligonucleotide
linkages formed by a phosphoramidite group, including but not
limited to, at the end of a polymer, and a 5'-hydroxyl group of an
oligonucleotide.
[0280] In certain embodiments, the linker comprises an
enzymatically cleavable peptide moiety, for example, a linker
comprising structural formula (IVa), (IVb), (IVc), or (IVd):
##STR00008##
[0281] or a salt thereof, wherein:
[0282] peptide represents a peptide (illustrated C.fwdarw.N and not
showing the carboxy and amino "termini") cleavable by a lysosomal
enzyme;
[0283] T represents a polymer comprising one or more ethylene
glycol units or an alkylene chain, or combinations thereof;
[0284] R.sup.a is selected from hydrogen, alkyl, sulfonate and
methyl sulfonate;
[0285] p is an integer ranging from 0 to 5;
[0286] q is 0 or 1;
[0287] x is 0 or 1;
[0288] y is 0 or 1;
[0289] represents the point of attachment of the linker to a
cytotoxic and/or cytostatic agent; and
[0290] * represents the point of attachment to the remainder of the
linker.
[0291] In certain embodiments, the lysosomal enzyme is selected
from Cathepsin B and .beta.-glucoronidase.
[0292] In certain embodiments, the peptide is selected from a
tripeptide or a dipeptide. In particular embodiments, the dipeptide
is selected from: Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala;
Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser;
Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Lys;
Val-Lys; Ala-Lys; Phe-Cit; Leu-Cit; Ile-Cit; Phe-Arg; and Trp-Cit.
In certain embodiments, the peptide is selected from: Val-Cit;
Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit;
Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit;
Cit-Asp; Ala-Val; and Val-Ala and salts thereof.
[0293] Specific exemplary embodiments of linkers according to
structural formula (IVa) that may be included in the ADCs described
herein include the linkers illustrated below (as illustrated, the
linkers include a group suitable for covalently linking the linker
to an antibody):
##STR00009## ##STR00010##
[0294] Specific exemplary embodiments of linkers according to
structural formula (IVb) that may be included in the ADCs described
herein include the linkers illustrated below (as illustrated, the
linkers include a group suitable for covalently linking the linker
to an antibody):
##STR00011## ##STR00012## ##STR00013## ##STR00014##
##STR00015##
[0295] Specific exemplary embodiments of linkers according to
structural formula (IVc) that may be included in the ADCs described
herein include the linkers illustrated below (as illustrated, the
linkers include a group suitable for covalently linking the linker
to an antibody):
##STR00016##
[0296] Specific exemplary embodiments of linkers according to
structural formula (IVd) that may be included in the ADCs described
herein include the linkers illustrated below (as illustrated, the
linkers include a group suitable for covalently linking the linker
to an antibody):
##STR00017## ##STR00018## ##STR00019## ##STR00020##
[0297] In certain embodiments, the linker comprising structural
formula (IVa), (IVb), (IVc), or (IVd) further comprises a carbonate
moiety cleavable by exposure to an acidic medium. In particular
embodiments, the linker is attached through an oxygen to a
cytotoxic and/or cytostatic agent.
[0298] 5.6.2.2. Non-Cleavable Linkers
[0299] Although cleavable linkers may provide certain advantages,
the linkers composing the ADC described herein need not be
cleavable. For non-cleavable linkers, the release of drug does not
depend on the differential properties between the plasma and some
cytoplasmic compartments. The release of the drug is postulated to
occur after internalization of the ADC via antigen-mediated
endocytosis and delivery to lysosomal compartment, where the
antibody is degraded to the level of amino acids through
intracellular proteolytic degradation. This process releases a drug
derivative, which is formed by the drug, the linker, and the amino
acid residue to which the linker was covalently attached. The amino
acid drug metabolites from conjugates with non-cleavable linkers
are more hydrophilic and generally less membrane permeable, which
leads to less bystander effects and less nonspecific toxicities
compared to conjugates with a cleavable linker. In general, ADCs
with noncleavable linkers have greater stability in circulation
than ADCs with cleavable linkers. Non-cleavable linkers may be
alkylene chains, or may be polymeric in nature, such as, for
example, those based upon polyalkylene glycol polymers, amide
polymers, or may include segments of alkylene chains, polyalkylene
glycols and/or amide polymers.
[0300] A variety of non-cleavable linkers used to link drugs to
antibodies have been described. See, Jeffrey et al., 2006,
Bioconjug. Chem. 17; 831-840; Jeffrey et al., 2007, Bioorg. Med.
Chem. Lett. 17:2278-2280; and Jiang et al., 2005, J. Am. Chem. Soc.
127:11254-11255, each of which is incorporated herein by reference.
All of these linkers may be included in the ADCs described
herein.
[0301] In certain embodiments, the linker is non-cleavable in vivo,
for example a linker according to structural formula (VIa), (VIb),
(VIc) or (VId) (as illustrated, the linkers include a group
suitable for covalently linking the linker to an antibody:
##STR00021##
[0302] or salts thereof, wherein:
[0303] R.sup.a is selected from hydrogen, alkyl, sulfonate and
methyl sulfonate;
[0304] R.sup.x is a moiety including a functional group capable of
covalently linking the linker to an antibody; and
[0305] represents the point of attachment of the linker to a
cytotoxic and/or cytostatic agent.
[0306] Specific exemplary embodiments of linkers according to
structural formula (VIa)-(VId) that may be included in the ADCs
described herein include the linkers illustrated below (as
illustrated, the linkers include a group suitable for covalently
linking the linker to an antibody, and "" represents the point of
attachment to a cytotoxic and/or cytostatic agent):
##STR00022##
[0307] 5.6.2.3. Groups Used to Attach Linkers to Antibodies
[0308] A variety of groups may be used to attach linker-drug
synthons to antibodies to yield ADCs. Attachment groups can be
electrophilic in nature and include: maleimide groups, activated
disulfides, active esters such as NHS esters and HOBt esters,
haloformates, acid halides, alkyl and benzyl halides such as
haloacetamides. As discussed below, there are also emerging
technologies related to "self-stabilizing" maleimides and "bridging
disulfides" that can be used in accordance with the disclosure. The
specific group used will depend, in part, on the site of attachment
to the antibody.
[0309] One example of a "self-stabilizing" maleimide group that
hydrolyzes spontaneously under antibody conjugation conditions to
give an ADC species with improved stability is depicted in the
schematic below. See US20130309256 A1; also Lyon et al., Nature
Biotech published online, doi:10.1038/nbt.2968).
[0310] Normal System:
##STR00023##
[0311] SGN MalDPR (Maleimido Dipropylamino) System:
##STR00024##
[0312] Polytherics has disclosed a method for bridging a pair of
sulfhydryl groups derived from reduction of a native hinge
disulfide bond. See, Badescu et al., 2014, Bioconjugate Chem.
25:1124-1136. The reaction is depicted in the schematic below. An
advantage of this methodology is the ability to synthesize
homogeneous DAR4 ADCs by full reduction of IgGs (to give 4 pairs of
sulfhydryls) followed by reaction with 4 equivalents of the
alkylating agent. ADCs containing "bridged disulfides" are also
embodimented to have increased stability.
##STR00025##
[0313] Similarly, as depicted below, a maleimide derivative (1,
below) that is capable of bridging a pair of sulfhydryl groups has
been developed. See WO2013/085925.
##STR00026##
[0314] 5.6.2.4. Linker Selection Considerations
[0315] As is known by skilled artisans, the linker selected for a
particular ADC may be influenced by a variety of factors, including
but not limited to, the site of attachment to the antibody (e.g.,
Lys, Cys or other amino acid residues), structural constraints of
the drug pharmacophore and the lipophilicity of the drug. The
specific linker selected for an ADC should seek to balance these
different factors for the specific antibody/drug combination. For a
review of the factors that are influenced by choice of linkers in
ADCs, see Nolting, Chapter 5 "Linker Technology in Antibody-Drug
Conjugates," In: Antibody-Drug Conjugates: Methods in Molecular
Biology, vol. 1045, pp. 71-100, Laurent Ducry (Ed.), Springer
Science & Business Medica, LLC, 2013.
[0316] For example, anti-cMet ADCs can effect killing of bystander
cMet-negative tumor cells present in the vicinity of
cMet-expressing cancer cells. The mechanism of bystander cell
killing by ADCs has indicated that metabolic products formed during
intracellular processing of the ADCs may play a role.
Cell-permeable cytotoxic and/or cytostatic metabolites generated by
metabolism of the ADCs in cMet-expressing cells appear to play a
role in bystander cell killing, while non-cell-permeable
metabolites, which are incapable of traversing the cell membrane
and diffusing into the medium cannot effect bystander killing. In
certain embodiments, the linker is selected to effect, enhance or
increase the bystander killing effect of the anti-cMet ADCs.
[0317] The properties of the linker may also impact aggregation of
the ADC under conditions of use and/or storage. Typically, ADCs
reported in the literature contain no more than 3-4 drug molecules
per antigen-binding moiety, for example, per antibody molecule
(see, e.g., Chari, 2008, Acc Chem Res 41:98-107). Attempts to
obtain higher drug-to-antibody ratios ("DAR") often failed,
particularly if both the drug and the linker were hydrophobic, due
to aggregation of the ADC (King et al., 2002, J Med Chem
45:4336-4343; Hollander et al., 2008, Bioconjugate Chem 19:358-361;
Burke et al., 2009 Bioconjugate Chem 20:1242-1250). In many
instances, DARs higher than 3-4 could be beneficial as a means of
increasing potency. In instances where the cytotoxic and/or
cytostatic agent is hydrophobic in nature, it may be desirable to
select linkers that are relatively hydrophilic as a means of
reducing ADC aggregation, especially in instances where DARS
greater than 3-4 are desired. Thus, in certain embodiments, the
linker incorporates chemical moieties that reduce aggregation of
the ADCs during storage and/or use. A linker may incorporate polar
or hydrophilic groups such as charged groups or groups that become
charged under physiological pH to reduce the aggregation of the
ADCs. For example, a linker may incorporate charged groups such as
salts or groups that deprotonate, e.g., carboxylates, or protonate,
e.g., amines, at physiological pH.
[0318] Exemplary polyvalent linkers that have been reported to
yield DARs as high as 20 that may be used to link numerous
cytotoxic and/or cytostatic agents to an antibody are described in
WO 2009/073445; WO 2010/068795; WO 2010/138719; WO 2011/120053; WO
2011/171020; WO 2013/096901; WO 2014/008375; WO 2014/093379; WO
2014/093394; WO 2014/093640, the contents of which are incorporated
herein by reference in their entireties.
[0319] In particular embodiments, the aggregation of the ADCs
during storage or use is less than about 10% as determined by
size-exclusion chromatography (SEC). In particular embodiments, the
aggregation of the ADCs during storage or use is less than 10%,
such as less than about 5%, less than about 4%, less than about 3%,
less than about 2%, less than about 1%, less than about 0.5%, less
than about 0.1%, or even lower, as determined by size-exclusion
chromatography (SEC).
[0320] 5.6.3. ABBV-399
[0321] As described throughout the specification, ABBV-399 is an
ADC comprised of the cMet targeting antibody ABT-700 (PR-1266688,
h224G11) conjugated to the potent cytotoxin monomethyl auristatin E
(MMAE) through a valine citrulline (vc) linker. ABBV-399 has been
used in a Phase 1 clinical trial (see Example 16) with a DAR of
3.1.
[0322] In alternative embodiments, ABBV-399 can be used at a 1:1
E2/E4 ratio, which corresponds to an average DAR of 3.0. In other
words, the ABBV-399 is used as a composition comprising a 1:1 ratio
of the E2 and E4 purified fractions of antibody-drug conjugate.
[0323] 5.6.4. ABT-700 PBD
[0324] ABT-700 (S238C)-PBD (Kabat numbering) is the same as ABT-700
(S239C)-PBD (Eu numbering) and is comprised of two PBD drug-linker
molecules conjugated to a cys engineered mAb ABT-700. The
conjugation process consists of a quantitative reduction of the
engineered and interchain disulfides. The reduction mixture is then
purified to remove the excess reagent and its byproducts, followed
by quantitative oxidation of the interchain disulfides and then
conjugation with excess PBD drug-linker. After quenching, the
reaction mixture is purified and buffer-exchanged to yield ABT-700
(S238C)-PBD. Reaction parameters have been identified to provide a
conjugate with >80% DAR2 drug loading.
[0325] The sequence of ABT-700 PBD, which carries a S238C mutation
(Kabat numbering) (equivalent to S239C mutation in Eu numbering),
is as follows (CDRs are underlined; the numbering system is Kabat;
and the S238C mutation is represented by C (bold, underlined, and
italics):
[0326] Amino Acid Sequence (10 AA Per Group, 5 Groups Per Line)
[0327] Heavy Chain (SEQ ID NO: 171) (underlined CDR sequences
disclosed as SEQ ID NOS 173-175, respectively, in order of
appearance):
TABLE-US-00023 [0327] QVQLVQSGAE VKKPGASVKV SCKASGYIFT AYTMHWVRQA
PGQGLEWMGW 50 IKPNNGLANY AQKFQGRVTM TRDTSISTAY MELSRLRSDD
TAVYYCARSE 100 ITTEFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT
AALGCLVKDY 150 FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP
SSSLGTQTYI 200 CNVNHKPSNT KVDKRVEPKS CDCHCPPCPA PELLGGP VF
LFPPKPKDTL 250 MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP
REEQYNSTYR 300 VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG
QPREPQVYTL 350 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY
KTTPPVLDSD 400 GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG
445
[0328] Light Chain (SEQ ID NO: 172) (underlined CDR sequences
disclosed as SEQ ID NOS 176-178, respectively, in order of
appearance):
TABLE-US-00024 [0328] DIVMTQSPDS LAVSLGERAT INCKSSESVD SYANSFLHWY
QQKPGQPPKL 50 LIYRASTRES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY
YCQQSKEDPL 100 TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL
NNFYPREAKV 150 QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY
EKHKVYACEV 200 THQGLSSPVT KSFNRGEC 218
[0329] 5.7. Methods of Making Anti-cMet Antibody Drug
Conjugates
[0330] The ADCs described herein may be synthesized using
chemistries that are well-known. The chemistries selected will
depend upon, among other things, the identity of the cytotoxic
and/or cytostatic agent(s), the linker and the groups used to
attach linker to the antibody. Generally, ADCs according to formula
(I) may be prepared according to the following scheme:
D-L-R.sup.x+Ab-R.sup.y.fwdarw.(I)[D-L-XY].sub.n-Ab
[0331] where D, L, Ab, XY and n are as previously defined, and
R.sup.x and R.sup.y represent complementary groups capable of
forming covalent linkages with one another, as discussed above.
[0332] The identities of groups R.sup.x and R.sup.y will depend
upon the chemistry used to link synthon D-L-R.sup.x to the
antibody. Generally, the chemistry used should not alter the
integrity of the antibody, for example its ability to bind its
target. Preferably, the binding properties of the conjugated
antibody will closely resemble those of the unconjugated antibody.
A variety of chemistries and techniques for conjugating molecules
to biological molecules such as antibodies are known in the art and
in particular to antibodies, are well-known. See, e.g., Amon et
al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer
Therapy," in: Monoclonal Antibodies And Cancer Therapy, Reisfeld et
al. Eds., Alan R. Liss, Inc., 1985; Hellstrom et al., "Antibodies
For Drug Delivery," in: Controlled Drug Delivery, Robinson et al.
Eds., Marcel Dekker, Inc., 2nd Ed. 1987; Thorpe, "Antibody Carriers
Of Cytotoxic Agents In Cancer Therapy: A Review," in: Monoclonal
Antibodies '84: Biological And Clinical Applications, Pinchera et
al., Eds., 1985; "Analysis, Results, and Future Prospective of the
Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," in:
Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et
al., Eds., Academic Press, 1985; Thorpe et al., 1982, Immunol. Rev.
62:119-58; PCT publication WO 89/12624. Any of these chemistries
may be used to link the synthons to an antibody.
[0333] A number of functional groups R.sup.x and chemistries useful
for linking synthons to accessible lysine residues are known, and
include by way of example and not limitation NHS-esters and
isothiocyanates.
[0334] A number of functional groups R.sup.x and chemistries useful
for linking synthons to accessible free sulfhydryl groups of
cysteine residues are known, and include by way of example and not
limitation haloacetyls and maleimides.
[0335] However, conjugation chemistries are not limited to
available side chain groups. Side chains such as amines may be
converted to other useful groups, such as hydroxyls, by linking an
appropriate small molecule to the amine. This strategy can be used
to increase the number of available linking sites on the antibody
by conjugating multifunctional small molecules to side chains of
accessible amino acid residues of the antibody. Functional groups
R.sup.x suitable for covalently linking the synthons to these
"converted" functional groups are then included in the
synthons.
[0336] An antibody may also be engineered to include amino acid
residues for conjugation. An approach for engineering antibodies to
include non-genetically encoded amino acid residues useful for
conjugating drugs in the context of ADCs is described by Axup et
al., 2012, Proc Natl Acad Sci USA. 109(40):16101-16106, as are
chemistries and functional groups useful for linking synthons to
the non-encoded amino acids.
[0337] Typically, the synthons are linked to the side chains of
amino acid residues of the antibody, including, for example, the
primary amino group of accessible lysine residues or the sulfhydryl
group of accessible cysteine residues. Free sulfhydryl groups may
be obtained by reducing interchain disulfide bonds.
[0338] For linkages where R.sup.y is a sulfhydryl group (for
example, when R.sup.x is a maleimide), the antibody is generally
first fully or partially reduced to disrupt interchain disulfide
bridges between cysteine residues. Specific cysteine residues and
interchain disulfide bridges that may be reduced for attachment of
drug-linker synthons including a group suitable for conjugation to
a sulfhydryl group for exemplary antibody ABT-700, include by way
of example and not limitation, residues C221, C223, C225, and C228
on the human IgG.sub.1 heavy chain, and residue C218 on the human
Ig kappa light chain of the ABT-700 disclosed herein.
[0339] Cysteine residues for synthon attachment that do not
participate in disulfide bridges may be engineered into an antibody
by mutation of one or more codons. These unpaired cysteines provide
a sulfhydryl group suitable for conjugation. Preferred positions
for incorporating engineered cysteines include, by way of example
and not limitation, positions S112C, S113C, A114C, S115C, A176C,
5180C, S252C, V286C, V292C, S357C, A359C, S398C, S428C (Kabat
numbering) on the human IgG.sub.1 heavy chain and positions V110C,
S114C, S121C, S127C, S168C, V205C (Kabat numbering) on the human Ig
kappa light chain (see, e.g., U.S. Pat. No. 7,521,541, U.S. Pat.
No. 7,855,275 and U.S. Pat. No. 8,455,622).
[0340] As will be appreciated by skilled artisans, the number of
cytotoxic and/or cytostatic agents linked to an antibody molecule
may vary, such that an ADC preparation may be heterogeneous in
nature, where some antibodies in the preparation contain one linked
agent, some two, some three, etc. (and some none). The degree of
heterogeneity will depend upon, among other things, the chemistries
used for linking the cytotoxic and/or cytostatic agents. For
example, where the antibodies are reduced to yield sulfhydryl
groups for attachment, heterogenous mixtures of antibodies having
zero, 2, 4, 6 or 8 linked agents per molecule are often produced.
Furthermore, by limiting the molar ratio of attachment compound,
antibodies having zero, 1, 2, 3, 4, 5, 6, 7 or 8 linked agents per
molecule are often produced. Thus, it will be understood that
depending upon context, stated drug antibody ratios (DARs) may be
averages for a collection of antibodies. For example, "DAR4" refers
to an ADC preparation that has not been subjected to purification
to isolate specific DAR peaks and comprises a heterogeneous mixture
of ADC molecules having different numbers of cytostatic and/or
cytotoxic agents attached per antibody (e.g., 0, 2, 4, 6, 8 agents
per antibody), but has an average drug-to-antibody ratio of 4.
Similarly, "DARE" refers to a heterogeneous ADC preparation in
which the average drug-to-antibody ratio is 8.
[0341] Heterogeneous ADC preparations may be processed, for
example, by hydrophobic interaction chromatography ("HIC") to yield
preparations enriched in an ADC having a specified DAR of interest
(or a mixture of two or more specified DARS). Such enriched
preparations are designed herein as "EX," where "E" indicates the
ADC preparation has been processed and is enriched in an ADC having
a specific DAR and "X" represents the number of cytostatic and/or
cytotoxic agents linked per ADC molecule. Preparations enriched in
a mixture of ADCs having two specific DARs are designated "EX/EY,"
three specific DARs "EX/EY/EZ" etc., where "E" indicates the ADC
preparation has been processed to enrich the specified DARs and
"X," "Y" and "Z" represent the DARs enriched. As specific examples,
"E2" refers to an ADC preparation that has been enriched to contain
primarily ADCs having two cytostatic and/or cytotoxic agents linked
per ADC molecule. "E4" refers to an ADC preparation that has been
enriched to contain primarily ADCs having four cytostatic and/or
cytotoxic agents linked per ADC molecule. "E2/E4" refers to an ADC
preparation that has been enriched to contain primarily two ADC
populations, one having two cytostatic and/or cytotoxic agents
linked per ADC molecule and another having four cytostatic and/or
cytotoxic agents linked per ADC molecule.
[0342] As used herein, enriched "E" preparations will generally be
at least about 80% pure in the stated DAR ADCs, although higher
levels of purity, such as purities of at least about 85%, 90%, 95%,
98%, or even higher, may be obtainable and desirable. For example,
an "EX" preparation will generally be at least about 80% pure in
ADCs having X cytostatic and/or cytotoxic agents linked per ADC
molecule. For "higher order" enriched preparations, such as, for
example, "EX/EY" preparations, the sum total of ADCs having X and Y
cytostatic and/or cytotoxic agents linked per ADC molecule will
generally comprise at least about 80% of the total ADCs in the
preparation. Similarly, in an enriched "EX/EY/EZ" preparation, the
sum total of ADCs having X, Y and Z cytostatic and/or cytotoxic
agents linked per ADC molecule will comprise at least about 80% of
the total ADCs in the preparation.
[0343] Purity may be assessed by a variety of methods, as is known
in the art. As a specific example, an ADC preparation may be
analyzed via HPLC or other chromatography and the purity assessed
by analyzing areas under the curves of the resultant peaks.
Specific chromatography methods that may be employed to assess
purity of ADC preparations are provided in Example 6.
[0344] FIGS. 2A-2B illustrate Process I, which is used to obtain a
DAR of 3.1. FIGS. 3A-3B illustrate Process II, which was used to
obtain a 1:1 E2/E4 ratio.
[0345] Specific methods for obtaining heterogenous mixtures of ADCs
comprising humanized antibody huM25 having an average DAR of 4, as
well as highly purified preparations containing 2 and 4 linked
agents are provided in the Examples section. These specific methods
may be modified using routine skill to obtain heterogeous and/or
homogeneous ADCs comprising other anti-cMet antibodies, linkers
and/or cytotoxic and/or cytostatic agents.
[0346] After conjugation of vcMMAE to ABT-700, an additional
process step is used to reduce the average drug-to-antibody ratio
(DAR) from approximately 5 to approximately 3, which results in a
more homogeneous drug product with fewer MMAE molecules conjugated
to the antibody. This strategy was implemented to reduce the number
of drug molecules attached to ABBV-399, which may improve its
tolerability, since high order drug molecules may contribute
disproportionally to toxicity.
[0347] 5.8. Compositions
[0348] The ADCs described herein may be in the form of compositions
comprising the ADC and one or more carriers, excipients and/or
diluents. The compositions may be formulated for specific uses,
such as for veterinary uses or pharmaceutical uses in humans. The
form of the composition (e.g., dry powder, liquid formulation,
etc.) and the excipients, diluents and/or carriers used will depend
upon the intended uses of the antibody and/or ADC and, for
therapeutic uses, the mode of administration.
[0349] For therapeutic uses, the compositions may be supplied as
part of a sterile, pharmaceutical composition that includes a
pharmaceutically acceptable carrier. This composition can be in any
suitable form (depending upon the desired method of administering
it to a patient). The pharmaceutical composition can be
administered to a patient by a variety of routes such as orally,
transdermally, subcutaneously, intranasally, intravenously,
intramuscularly, intratumorally, intrathecally, topically or
locally. The most suitable route for administration in any given
case will depend on the particular antibody and/or ADC, the
subject, and the nature and severity of the disease and the
physical condition of the subject. Typically, the pharmaceutical
composition will be administered intravenously or
subcutaneously.
[0350] Pharmaceutical compositions can be conveniently presented in
unit dosage forms containing a predetermined amount of an antibody
and/or ADC described herein per dose. The quantity of antibody
and/or ADC included in a unit dose will depend on the disease being
treated, as well as other factors as are well known in the art.
Such unit dosages may be in the form of a lyophilized dry powder
containing an amount of antibody and/or ADC suitable for a single
administration, or in the form of a liquid. Dry powder unit dosage
forms may be packaged in a kit with a syringe, a suitable quantity
of diluent and/or other components useful for administration. Unit
dosages in liquid form may be conveniently supplied in the form of
a syringe pre-filled with a quantity of antibody and/or ADC
suitable for a single administration.
[0351] The pharmaceutical compositions may also be supplied in bulk
form containing quantities of ADC suitable for multiple
administrations.
[0352] Pharmaceutical compositions may be prepared for storage as
lyophilized formulations or aqueous solutions by mixing an antibody
and/or ADC having the desired degree of purity with optional
pharmaceutically-acceptable carriers, excipients or stabilizers
typically employed in the art (all of which are referred to herein
as "carriers"), i.e., buffering agents, stabilizing agents,
preservatives, isotonifiers, non-ionic detergents, antioxidants,
and other miscellaneous additives. See, Remington's Pharmaceutical
Sciences, 16th edition (Osol, ed. 1980) and Remington: The Science
and Practice of Pharmacy, 22.sup.nd Edition (Edited by Allen, Loyd
V. Jr., 2012). Such additives should be nontoxic to the recipients
at the dosages and concentrations employed.
[0353] Buffering agents help to maintain the pH in the range which
stabilizes the protein. They may be present at a wide variety of
concentrations, but will typically be present in concentrations
ranging from about 2 mM to about 50 mM. Suitable buffering agents
for use with the present disclosure include both organic and
inorganic acids and salts thereof such as citrate buffers (e.g.,
monosodium citrate-disodium citrate mixture, citric acid-trisodium
citrate mixture, citric acid-monosodium citrate mixture, etc.),
succinate buffers (e.g., succinic acid-monosodium succinate
mixture, succinic acid-sodium hydroxide mixture, succinic
acid-disodium succinate mixture, etc.), tartrate buffers (e.g.,
tartaric acid-sodium tartrate mixture, tartaric acid-potassium
tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.),
fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,
fumaric acid-disodium fumarate mixture, monosodium
fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g.,
gluconic acid-sodium gluconate mixture, gluconic acid-sodium
hydroxide mixture, gluconic acid-potassium gluconate mixture,
etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture,
oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate
mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate
mixture, lactic acid-sodium hydroxide mixture, lactic
acid-potassium lactate mixture, etc.) and acetate buffers (e.g.,
acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide
mixture, etc.). Additionally, phosphate buffers, histidine buffers
and trimethylamine salts such as Tris can be used.
[0354] Preservatives may be added to retard microbial growth, and
can be added in amounts ranging from about 0.2%-1% (w/v). Suitable
preservatives for use with the present disclosure include phenol,
benzyl alcohol, meta-cresol, methyl paraben, propyl paraben,
octadecyldimethylbenzyl ammonium chloride, benzalconium halides
(e.g., chloride, bromide, and iodide), hexamethonium chloride, and
alkyl parabens such as methyl or propyl paraben, catechol,
resorcinol, cyclohexanol, and 3-pentanol. Isotonicifiers sometimes
known as "stabilizers" can be added to ensure isotonicity of liquid
compositions of the present disclosure and include polyhydric sugar
alcohols, for example trihydric or higher sugar alcohols, such as
glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
Stabilizers refer to a broad category of excipients which can range
in function from a bulking agent to an additive which solubilizes
the therapeutic agent or helps to prevent denaturation or adherence
to the container wall. Typical stabilizers can be polyhydric sugar
alcohols (enumerated above); amino acids such as arginine, lysine,
glycine, glutamine, asparagine, histidine, alanine, ornithine,
L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic
sugars or sugar alcohols, such as lactose, trehalose, stachyose,
mannitol, sorbitol, xylitol, ribitol, myoinositol, galactitol,
glycerol and the like, including cyclitols such as inositol;
polyethylene glycol; amino acid polymers; sulfur containing
reducing agents, such as urea, glutathione, thioctic acid, sodium
thioglycolate, thioglycerol, .alpha.-monothioglycerol and sodium
thiosulfate; low molecular weight polypeptides (e.g., peptides of
10 residues or fewer); proteins such as human serum albumin, bovine
serum albumin, gelatin or immunoglobulins; hydrophilic polymers,
such as polyvinylpyrrolidone monosaccharides, such as xylose,
mannose, fructose, glucose; disaccharides such as lactose, maltose,
sucrose and trehalose; and trisaccacharides such as raffinose; and
polysaccharides such as dextran. Stabilizers may be present in
amounts ranging from 0.5 to 10 weight % per weight of ADC.
[0355] Non-ionic surfactants or detergents (also known as "wetting
agents") may be added to reduce adsorption to surfaces and to help
solubilize the glycoprotein as well as to protect the glycoprotein
against agitation-induced aggregation, which also permits the
formulation to be exposed to shear surface stress without causing
denaturation of the protein. Suitable non-ionic surfactants include
polysorbates (20, 80, etc.), poloxamers (184, 188 etc.), and
pluronic polyols. Non-ionic surfactants may be present in a range
of about 0.05 mg/mL to about 1.0 mg/mL, for example about 0.07
mg/mL to about 0.2 mg/mL.
[0356] Additional miscellaneous excipients include bulking agents
(e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g.,
ascorbic acid, methionine, vitamin E), and cosolvents.
[0357] A specific exemplary embodiment of an aqueous composition
suitable for administration via intravenous infusion comprises 20
mg/mL anti-cMet ADC, 10 mM histidine buffer, pH 6.0, 7% (w/v)
sucrose, 0.03% (w/v) polysorbate 80. The composition may be in the
form of a lyophilized powder that, upon reconstitution with 5.2 mL
sterile water or other solution suitable for injection or infusion
(for example, 0.9% saline, Ringer's solution, lactated Ringer's
solution, etc.) provides the above aqueous composition. It, or
other embodiments of compositions, may also be in the form of a
syringe or other device suitable for injection and/or infusion
pre-filled with a quantity of composition suitable for a single
administration of anti-cMet ADC.
[0358] In one embodiment, the composition comprises ABBV-399 in a
1:1 ratio of purified E1 and E4 fractions. Such fractions can be
obtained by any method known in the art for purifying ADCs,
including the method of Examples 2 and 3. In one embodiment, the
composition comprises ABBV-399 with a DAR in the range of 0-10. In
another embodiment, the composition comprises ABBV-399 with a DAR
in the range of 1-4. In another embodiment, the composition
comprises ABBV-399 with a DAR in the range of 2-4. In another
embodiment, the composition comprises ABBV-399 with a DAR of about
3.1. In another embodiment, the composition comprises ABBV-399 with
a DAR of about 3.0.
[0359] 5.9. Methods of Use
[0360] As discussed previously, for a variety of solid tumors, cMet
is expressed/overexpressed. Data provided herein demonstrate that
anti-cMet ADCs exert potent anti-tumor activity against these
cMet-expressing/overexpressing tumors in vivo. Accordingly, the
ADCs and/or pharmaceutical compositions comprising the ADCs may be
used therapeutically to treat cMet-expressing (i.e., cMet+ tumors)
and cMet-overexpressing tumors (i.e., cMet+/overexpressing
tumors).
[0361] Generally, the methods involve administering to a human
patient having a cMet-expressing or cMet-overexpressing tumor an
amount of an anti-cMet ADC effective to provide therapeutic
benefit. Any method known to one of ordinary skill in the art for
assessing the presence and/or expression level of the cMet receptor
protein in a cell can be used. In one embodiment, the cMet levels
are membranous. In another embodiment, the cMet levels are
cytoplasmic. In another embodiment, the overall cMet expression
level is measured. A preferred method for determining cMet
expression levels is described in detail in Example 17 and is
referred to herein as the "cMet ABBV-ADC staining protocol." The
H-scores (0-300) and the IHC score (0, 1+, 2+, and 3+) are assessed
based on methods known to a pathologist of ordinary skill in the
art. In one embodiment, patients with H-scores<150 and/or IHC
scores 0 and 1+ are selected for treatment. In one embodiment,
patients with H-scores.gtoreq.150 and/or IHC scores 2+ and 3+ are
selected for treatment.
[0362] Patients selected for the ADC treatments of this disclosure
include those with cMet-expressing and those with
cMet-overexpressing tumors, which include, but are not limited to,
any solid tumor (including also those that overexpress HGF and/or
have abnormal activation of HGF/cMet signaling or expression). More
specific examples include: lung cancers; breast cancers (e.g.,
invasive ductal carcinoma); head and neck cancers; pancreatic
cancers; gastric carcinomas; colorectal cancers (including
colorectal cancer lung metastases); ovarian cancers (e.g., serous
adenocarcinoma); stomach cancers; kidney cancers (e.g., renal cell
cancer such as papillary renal cell carcinoma, clear cell cancers,
hereditary papillary renal cell carcinomas); adrenal cancers;
gastro/oesophageal cancers; medulloblastomas; gliomas; liver
cancers (e.g., hepatocellular carcinomas (including advanced,
unresectable HCC)); prostate cancer (metastatic or nonmetastatic);
melanomas; salivary gland tumors; sarcomas; cervical cancers;
myxoid liposarcomas; adenocarcinomas of the paratyroid gland;
endometrial cancers; epithelioid mesotheliomas; appendix
carcinomas; goblet cell carcinomas; metastatic diffuse type gastric
adenocarcinoma with signet ring features; anaplastic large cell
lymphoma (ALCL); any advanced malignancy including, but not limited
to, advanced, relapsed, refractory subtypes of the cancers listed
herein.
[0363] Lung cancer can be classified using different systems. In
one system, lung cancer includes adenocarcinoma (mixed, acinar,
papillary, solid, micropapillary, lepidic nonmucinous and lepidic
mucinous), squamous cell carcinoma, large cell carcinoma (e.g,
non-small cell lung cancers or NSCLC (e.g., advanced or
non-advanced, LCNEC, LCNEM, NSCLC-not otherwise specified
(NOS)/adenosquamous carcinoma, sarcomatoid carcinoma, adenosquamous
carcinoma, and large-cell neuroendocrine carcinoma); and small cell
lung cancer/carcinoma or SCLC)).
[0364] Alternatively, in a different system, lung cancer can be
classified into preinvasive lesions, minimally invasive
adenocarcinoma, and invasive adenocarcinoma (invasive mucinous
adenocarcinoma, mucinous BAC, colloid, fetal (low and high grade),
and enteric).
[0365] More frequently, lung cancer may be categorized as either
small cell lung cancer ("SCLC") or non-small cell lung cancer
("NSCLC"). NSCLCs may be further categorized as squamous or
non-squamous. An example of a non-squamous NSCLC is
adenocarcinoma.
[0366] The cancer may be newly diagnosed and naive to treatment, or
may be relapsed, refractory, or relapsed and refractory, or a
metastasis or metastatic form of a cMet-expressing or of a
cMet-overexpressing tumors. As demonstrated in Example 14 of this
disclosure, cMet-overexpressing tumors that exhibit resistance to
other targeted or non-targeted chemotherapies, retain sensitivity
to ABBV-399.
[0367] Moreover, as shown in FIG. 12C, a cMet-overexpressing tumor
that eventually regrew following treatment with the anti-cMet
antibody ABT-700 remained sensitive to retreatment with the
anti-cMet ADC, ABBV-399. Accordingly, the anti-cMet ADCs described
herein provide significant benefits over current targeted and
non-targeted approaches toward the treatment of cMet-overexpressing
tumors.
[0368] Anti-cMet ADCs may be administered alone (monotherapy) or
adjunctive to, or with, other anti-cancer therapies and/or targeted
or non-targeted anti-cancer agents. When administered as anti-cMet
ADC monotherapy, one or more anti-cMet ADCs may be used. In certain
embodiments, an anti-cMet ADC is administered in conjunction with
an anti-cMet antibody that recognizes a different epitope on cMet
than that recognized by the ADC. This could be done, for example,
to stimulate internalization of the cMet receptor. Alternatively,
ABT-700 can be given prior to ABBV-399 (or another anti-cMet ADC)
in order to "block" endogenous cMet on normal tissues in an effort
to reduce possible toxicity associated with the activity of
ABBV-399 on normal tissues.
[0369] In another embodiment, the anti-cMet ADC recognizes two
different non-overlaping epitopes within cMet. Such ADCs, also
known as ADCs carrying a bivalent biparatopic antibody, can have
several advantages over monovalent antibodies. For example, they
can induce cMet receptor clustering, which in turn could promote
robust internalization, lysosomal trafficking, and degradation,
thereby improving the release of the drug portion of the ADC into
the cytoplasm as well as its availability for bystander effect.
[0370] Whether administered as monotherapy or adjunctive to, or
with, other therapies or agents, an amount of anti-cMet ADC is
administered such that the overall treatment regimen provides
therapeutic benefit. By therapeutic benefit is meant that the use
of anti-cMet ADCs to treat cancer in a patient results in any
demonstrated clinical benefit compared with no therapy (when
appropriate) or to a known standard of care. Clinical benefit can
be assessed by any method known to one of ordinary skill in the
art. In one embodiment, clinical benefit is assessed based on
objective response rate (ORR) (determined using RECIST version
1.1), duration of response (DOR), progression-free survival (PFS),
and/or overall survival (OS). In some embodiments, a complete
response indicates therapeutic benefit. In some embodiments, a
partial response indicates therapeutic benefit. In some
embodiments, stable disease indicates therapeutic benefit. In some
embodiments, an increase in overall survival indicates therapeutic
benefit. In some embodiments, therapeutic benefit may constitute an
improvement in time to disease progression and/or an improvement in
symptoms or quality of life. In other embodiments, therapeutic
benefit may not translate to an increased period of disease
control, but rather a markedly reduced symptom burden resulting in
improved quality of life. As will be apparent to those of skill in
the art, a therapeutic benefit may be observed using the anti-cMet
ADCs alone (monotherapy) or adjunctive to, or with, other
anti-cancer therapies and/or targeted or non-targeted anti-cancer
agents. Preferential methods for assessing therapeutic benefit are
described in detail in the Examples, as used in a Phase 1 clinical
trial with ABBV-399.
[0371] Typically, therapeutic benefit is assessed using standard
clinical tests designed to measure the response to a new treatment
for cancer. To assess the therapeutic benefits of the anti-cMet
ADCs described herein one or a combination of the following tests
can be used: (1) the Response Evaluation Criteria In Solid Tumors
(RECIST) version 1.1 (for details, see Example 16), (2) the Eastern
Cooperative Oncology Group (ECOG) Performance Status, (3)
immune-related response criteria (irRC), (4) disease evaluable by
assessment of tumor antigens, (5) validated patient reported
outcome scales, and/or (6) Kaplan-Meier estimates for overall
survival and progression free survival.
[0372] The ECOG Scale of Performance Status shown in TABLE 3 is
used to describe a patient's level of functioning in terms of their
ability to care for themselves, daily activity, and physical
ability. The scale was developed by the Eastern Cooperative
Oncology Group (ECOG), now part of the ECOG-ACRIN Cancer Research
Group, and published in 1982.
TABLE-US-00025 TABLE 3 Grade ECOG Performance Status 0 Fully
active, able to carry on all pre-disease performance without
restriction 1 Restricted in physically strenuous activity but
ambulatory and able to carry out work of a light or sedentary
nature, e.g., light house work, office work Ambulatory and capable
of all selfcare but unable to carry out any work activities; up and
about more than 50% of waking hours 3 Capable of only limited
selfcare; confined to bed or chair more than 50% of waking hours 4
Completely disabled; cannot carry on any selfcare; totally confined
to bed or chair 5 Dead
[0373] Assessment of the change in tumor burden is an important
feature of the clinical evaluation of cancer therapeutics. Both
tumor shrinkage (objective response) and time to the development of
disease progression are important endpoints in cancer clinical
trials. Standardized response criteria, known as RECIST (Response
Evaluation Criteria in Solid Tumors), were published in 2000. An
update (RECIST 1.1) was released in 2009. RECIST criteria are
typically used in clinical trials where objective response is the
primary study endpoint, as well as in trials where assessment of
stable disease, tumor progression or time to progression analyses
are undertaken because these outcome measures are based on an
assessment of anatomical tumor burden and its change over the
course of the trial. TABLE 4 provides the definitions of the
response criteria used to determine objective tumor response to a
study drug, such as the anti-cMet ADCs described herein.
TABLE-US-00026 TABLE 4 Response Criteria Complete Disappearance of
all target lesions. Any pathological Response lymph nodes (whether
target or non-target) must have (CR) reduction in short axis to
<10 mm. Partial At least a 30% decrease in the sum of diameters
of Response target lesions, taking as reference the baseline (PR)
sum diameters. Progressive At least a 20% increase in the sum of
diameters of Disease target lesions, taking as reference the
smallest (PD) sum on study (this includes the baseline sum if that
is the smallest on study). In addition to the relative increase of
20%, the sum must also demonstrate an absolute increase of at least
5 mm. (Note: the appearance of one or more new lesions is also
considered progression). Stable Neither sufficient shrinkage to
qualify for PR nor sufficient Disease increase to qualify for PD,
taking as reference the smallest (SD) sum diameters while on
study.
[0374] Secondary outcome measures that can be used to determine the
therapeutic benefit of the anti-cMet ADCs described herein include,
Objective Response Rate (ORR), Progression Free Survival (PFS),
Duration of Overall Response (DOR), and Depth of Response (DpR).
ORR is defined as the proportion of the participants who achieve a
complete response (CR) or partial response (PR). PFS is defined as
the time from the first dose date of an anti-cMet ADCs to either
disease progression or death, whichever occurs first. DOR is
defined as the time from the participant's initial CR or PR to the
time of disease progression. DpR is defined as the percentage of
tumor shrinkage observed at the maximal response point compared to
baseline tumor load. Clinical endpoints for both ORR and PFS can be
determined based on RECIST 1.1 criteria described above.
[0375] Another set of criteria that can be used to characterize
fully and to determine response to immunotherapeutic agents, such
as antibody-based cancer therapies, is the immune-related response
criteria (irRC), which was developed for measurement of solid
tumors in 2009, and updated in 2013 (Wolchok, et al. Clin. Cancer
Res. 2009; 15(23): 7412-7420 and Nishino, et al. Clin. Cancer Res.
2013; 19(14): 3936-3943, each of which is incorporated by reference
in its entirety). The updated irRC criteria are typically used to
assess the effect of an immunotherapeutic agent (e.g., an anti-PD1
antibody), and defines response according to TABLE 5.
TABLE-US-00027 TABLE 5 Response Criteria Complete Disappearance of
all target lesions in two consecutive Response observations not
less than 4 weeks apart (CR) Partial At least a 30% decrease in the
sum of the longest diameters Response of target lesions, taking as
reference the baseline sum (PR) diameters. Progressive At least a
20% increase in the sum of diameters of target Disease lesions,
taking as reference the smallest sum on study (PD) (this includes
the baseline sum if that is the smallest on study). (Note: the
appearance of one or more new lesions is not considered
progression. The measurement of new lesions is included in the sum
of the measurements). Stable Neither sufficient shrinkage to
qualify for PR nor sufficient Disease increase to qualify for PD,
taking as reference the smallest (SD) sum diameters while on
study.
[0376] Tumor antigens that can be used to evaluate the therapeutic
benefit of the anti-cMet ADCs described herein include ApoE, CD11c,
CD40, CD45 (PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86,
MS4A7, PIK3AP1, PIK3CD, CD74, CCL5, CCR5, CXCL10, IFNG, IL10RA1,
IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINE1,
STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN,
ANGPTL4, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2,
KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR1C2, CADM1,
CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNTSA, IGFBP3, MMP14,
CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CD11b, ADGRE1 (EMR1,
F4/80), CD86, CD68, MHC-Class II, CD3, HLA-DR, CD4, CD3, CD5, CD19,
CD7, CD8, CD16, TCR.alpha..beta., TCR.gamma..delta., PD-1, PDL-1,
CTLA-4, acid phosphatase, ACTH, alkaline phosphatase,
alpha-fetoprotein CA-125, CA15-3, CA19-9, CA-195, C-212, CA-549,
calcitonin, catecholamines, cathepsin-D, CEA, ERBB2 (HER2/neu),
chromagranin-A, c-Myc, EGFR, ERA (estrogen receptor assay),
ferritin, gastrin, 5-HIAA, hCG, alpha-HCG, beta-HCG, HVA, LDH1-5,
NSE (neuron specific enolase), pancreatic polypeptide, PLAP, PLP,
PRA (progesterone receptor A), proinsulin C-peptide, PSA, SMA, SCC,
thyroglobulin, TDT, TPA, and alpha-TSH. These antigens can be
assessed at the DNA, RNA or protein level using DNA sequencing
techniques, RNA sequencing techniques, gene chip microarray, PCR
based methods, flow cytometry or immunohistochemistry methods as
known to experts in the art.
[0377] One exemplary therapeutic benefit resulting from the use of
anti-cMet ADCs described herein to treat cMet-expressing and
cMet-overexpressing tumors, whether administered as monotherapy or
adjunctive to, or with, other therapies or agents, is a complete
response. Another exemplary therapeutic benefit resulting from the
use of anti-cMet ADCs described herein to cMet-overexpressing
tumors, whether administered as monotherapy or adjunctive to, or
with, other therapies or agents, is a partial response.
[0378] Validated patient reported outcome scales can also be used
to denote response provided by each patient through a specific
reporting system. Rather than being disease focused, such outcome
scales are concerned with retained function while managing a
chronic condition. One non-limiting example of a validated patient
reported outcome scale is PROMIS.RTM. (Patient Reported Outcomes
Measurement Information System) from the United States National
Institutes of Health. For example, PROMIS.RTM. Physical Function
Instrument for adult cancer patients can evaluate self-reported
capabilities for the functioning of upper extremities (e.g.,
dexterity), lower extremities (e.g., walking or mobility), and
central regions (e.g., neck, back mobility), and also includes
routine daily activities, such as running errands.
[0379] Kaplan-Meier curves (Kaplan and Meier, J. Am. Stat. Assoc.
1958; 53(282): 457-481) can also be used to estimate overall
survival and progression free survival for cancer patients
undergoing anti-cMet antibody or ADC therapy in comparison to
standard of care.
[0380] 5.9.1. Adjunctive Therapies
[0381] Anti-cMet ADCs may be used adjunctive to, or with, other
agents or treatments having anti-cancer properties. When used
adjunctively, the anti-cMet and other agent(s) may be formulated
together in a single pharmaceutical formulation, or may be
formulated and administered separately, either on a single
coordinated dosing regimen or on different dosing regimens. Agents
administered adjunctively with anti-cMet ADCs will typically have
complementary activities to the anti-cMet ADCs such that the ADCs
and other agents do not adversely affect each other.
[0382] Agents that may be used adjunctively with anti-cMet ADCs
include, but are not limited to, alkylating agents, angiogenesis
inhibitors, antibodies, antimetabolites, antimitotics,
antiproliferatives, antivirals, aurora kinase inhibitors, ALK
kinase inhibitors (for example, crizotinib (XALKORI.RTM.),
ceritinib (ZYKADIA.RTM.), and alectinib (ALECENSA.RTM.), apoptosis
promoters (for example, Bcl-2 family inhibitors), activators of
death receptor pathway, Bcr-Abl kinase inhibitors, BiTE
(Bi-Specific T cell Engager) antibodies, antibody drug conjugates,
biologic response modifiers, cyclin-dependent kinase inhibitors,
cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia
viral oncogene homolog (ErbB2) receptor inhibitors, growth factor
inhibitors, heat shock protein (HSP)-90 inhibitors, histone
deacetylase (HDAC) inhibitors, hormonal therapies, immunologicals,
inhibitors of inhibitors of apoptosis proteins (IAPs),
intercalating antibiotics, kinase inhibitors, kinesin inhibitors,
Jak2 inhibitors, mammalian target of rapamycin inhibitors,
microRNAs, mitogen-activated extracellular signal-regulated kinase
inhibitors, multivalent binding proteins, non-steroidal
anti-inflammatory drugs (NSAIDs), poly ADP (adenosine
diphosphate)-ribose polymerase (PARP) inhibitors, platinum
chemotherapeutics, polo-like kinase (Plk) inhibitors,
phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors,
purine analogs, pyrimidine analogs, receptor tyrosine kinase
inhibitors, retinoids/deltoids plant alkaloids, small inhibitory
ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin
ligase inhibitors, and the like, as well as combinations of one or
more of these agents.
[0383] BiTE antibodies are bispecific antibodies that direct
T-cells to attack cancer cells by simultaneously binding the two
cells. The T-cell then attacks the target cancer cell. Examples of
BiTE antibodies include adecatumumab (Micromet MT201), blinatumomab
(BLINCYTO.RTM., Amgen and Onyx Pharmaceuticals) and the like.
Without being limited by theory, one of the mechanisms by which
T-cells elicit apoptosis of the target cancer cell is by exocytosis
of cytolytic granule components, which include perforin and
granzyme B.
[0384] SiRNAs are molecules having endogenous RNA bases or
chemically modified nucleotides. The modifications do not abolish
cellular activity, but rather impart increased stability and/or
increased cellular potency. Examples of chemical modifications
include phosphorothioate groups, 2'-deoxynucleotide,
2'-OCH.sub.3-containing ribonucleotides, 2'-F-ribonucleotides,
2'-methoxyethyl ribonucleotides, combinations thereof and the like.
The siRNA can have varying lengths (e.g., 10-200 bps) and
structures (e.g., hairpins, single/double strands, bulges,
nicks/gaps, mismatches) and are processed in cells to provide
active gene silencing. A double-stranded siRNA (dsRNA) can have the
same number of nucleotides on each strand (blunt ends) or
asymmetric ends (overhangs). The overhang of 1-2 nucleotides can be
present on the sense and/or the antisense strand, as well as
present on the 5'- and/or the 3'-ends of a given strand.
[0385] Multivalent binding proteins are binding proteins comprising
two or more antigen binding sites. Multivalent binding proteins are
engineered to have the three or more antigen binding sites and are
generally not naturally occurring antibodies. The term
"multispecific binding protein" means a binding protein capable of
binding two or more related or unrelated targets. Dual variable
domain (DVD) binding proteins are tetravalent or multivalent
binding proteins binding proteins comprising two or more antigen
binding sites. Such DVDs may be monospecific (i.e., capable of
binding one antigen) or multispecific (i.e., capable of binding two
or more antigens). DVD binding proteins comprising two heavy chain
DVD polypeptides and two light chain DVD polypeptides are referred
to as DVD Ig's. Each half of a DVD Ig comprises a heavy chain DVD
polypeptide, a light chain DVD polypeptide, and two antigen binding
sites. Each binding site comprises a heavy chain variable domain
and a light chain variable domain with a total of 6 CDRs involved
in antigen binding per antigen binding site.
[0386] Alkylating agents include, but are not limited to,
altretamine, AMD-473, AP-5280, apaziquone, bendamustine,
brostallicin, busulfan, carboquone, carmustine (BCNU),
chlorambucil, CLORETAZINE.RTM. (laromustine, VNP 40101M),
cyclophosphamide, dacarbazine, estramustine, fotemustine,
glufosfamide, ifosfamide, KW-2170, lomustine (CCNU), mafosfamide,
melphalan, mitobronitol, mitolactol, nimustine, nitrogen mustard
N-oxide, ranimustine, temozolomide, thiotepa, TREANDA.RTM.
(bendamustine), treosulfan, and trofosfamide.
[0387] Angiogenesis inhibitors include, but are not limited to,
endothelial-specific receptor tyrosine kinase (Tie-2) inhibitors,
epidermal growth factor receptor (EGFR) inhibitors, insulin growth
factor-2 receptor (IGFR-2) inhibitors, matrix metalloproteinase-2
(MMP-2) inhibitors, matrix metalloproteinase-9 (MMP-9) inhibitors,
platelet-derived growth factor receptor (PDGFR) inhibitors,
thrombospondin analogs, and vascular endothelial growth factor
receptor tyrosine kinase (VEGFR) inhibitors.
[0388] Antimetabolites include, but are not limited to, ALIMTA.RTM.
(pemetrexed disodium, LY231514, MTA), 5-azacitidine, XELODA.RTM.
(capecitabine), carmofur, LEUSTAT.RTM. (cladribine), clofarabine,
cytarabine, cytarabine ocfosfate, cytosine arabinoside, decitabine,
deferoxamine, doxifluridine, eflornithine, EICAR
(5-ethynyl-1-.beta.-D-ribofuranosylimidazole-4-carboxamide),
enocitabine, ethnylcytidine, fludarabine, 5-fluorouracil alone or
in combination with leucovorin, GEMZAR.RTM. (gemcitabine),
hydroxyurea, ALKERAN.RTM. (melphalan), mercaptopurine,
6-mercaptopurine riboside, methotrexate, mycophenolic acid,
nelarabine, nolatrexed, ocfosfate, pelitrexol, pentostatin,
raltitrexed, Ribavirin, triapine, trimetrexate, S-1, tiazofurin,
tegafur, TS-1, vidarabine, and UFT.
[0389] Antivirals include, but are not limited to, ritonavir,
acyclovir, cidofovir, ganciclovir, foscarnet, zidovudine,
ribavirin, and hydroxychloroquine.
[0390] Aurora kinase inhibitors include, but are not limited to,
ABT-348, AZD-1152, MLN-8054, VX-680, Aurora A-specific kinase
inhibitors, Aurora B-specific kinase inhibitors and pan-Aurora
kinase inhibitors.
[0391] Bcl-2 protein inhibitors include, but are not limited to,
AT-101 ((-)gossypol), GENASENSE.RTM. (G3139 or oblimersen
(Bcl-2-targeting antisense oligonucleotide)), IPI-194, IPI-565,
N-(4-(4-((4'-chloro(1,1'-biphenyl)-2-yl)methyl)piperazin-1-yl)benzoyl)-4--
(((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitrobe-
nzenesulfonamide),
N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)pip-
erazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl-
)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide,
venetoclax and GX-070 (obatoclax).
[0392] Bcr-Abl kinase inhibitors include, but are not limited to,
DASATINIB.RTM. (BMS-354825) and GLEEVEC.RTM. (imatinib).
[0393] CDK inhibitors include, but are not limited to, AZD-5438,
BMI-1040, BMS-032, BMS-387, CVT-2584, flavopyridol, GPC-286199,
MCS-5A, PD0332991, PHA-690509, seliciclib (CYC-202, R-roscovitine),
and ZK-304709.
[0394] COX-2 inhibitors include, but are not limited to, ABT-963,
ARCOXIA.RTM. (etoricoxib), BEXTRA.RTM. (valdecoxib), BMS347070,
CELEBREX.RTM. (celecoxib), COX-189 (lumiracoxib), CT-3,
DERAMAXX.RTM. (deracoxib), JTE-522,
4-methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoylphenyl-1H-pyrrole),
MK-663 (etoricoxib), NS-398, parecoxib, RS-57067, SC-58125,
SD-8381, SVT-2016, S-2474, T-614, and VIOXX.RTM. (rofecoxib).
[0395] EGFR inhibitors include, but are not limited to, afatinib
(GILOTRIF.RTM.), ABX-EGF, anti-EGFR immunoliposomes, EGF-vaccine,
EMD-7200, ERBITUX.RTM. (cetuximab), HR3, IgA antibodies,
IRESSA.RTM. (gefitinib), TARCEVA.RTM. (erlotinib or OSI-774),
TP-38, EGFR fusion protein, PORTRAZZA.RTM. (necitumumab),
TAGRISSO.RTM. (osimertinib), TYKERB.RTM. (lapatinib), TARCEVA.RTM.
(erlotinib), and TAGRISSO.RTM. (osimertinib).
[0396] ErbB2 receptor inhibitors include, but are not limited to,
CP-724-714, CI-1033 (canertinib), HERCEPTIN.RTM. (trastuzumab),
TYKERB.RTM. (lapatinib), OMNITARG.RTM. (2C4, pertuzumab), TAK-165,
GW-572016 (ionafarnib), GW-282974, EKB-569, PI-166, dHER2 (HER2
vaccine), APC-8024 (HER-2 vaccine), anti-HER/2neu bispecific
antibody, B7.her2IgG3, AS HER2 trifunctional bispecific antibodies,
mAB AR-209, and mAB 2B-1.
[0397] Histone deacetylase inhibitors include, but are not limited
to, depsipeptide, LAQ-824, MS-275, trapoxin, suberoylanilide
hydroxamic acid (SAHA), TSA, and valproic acid.
[0398] HSP-90 inhibitors include, but are not limited to,
17-AAG-nab, 17-AAG, CNF-101, CNF-1010, CNF-2024, 17-DMAG,
geldanamycin, IPI-504, KOS-953, MYCOGRAB.RTM. (human recombinant
antibody to HSP-90), NCS-683664, PU24FC1, PU-3, radicicol,
SNX-2112, STA-9090, and VER49009.
[0399] Inhibitors of apoptosis proteins include, but are not
limited to, HGS1029, GDC-0145, GDC-0152, LCL-161, and LBW-242.
[0400] Activators of death receptor pathway include, but are not
limited to, TRAIL, antibodies or other agents that target TRAIL or
death receptors (e.g., DR4 and DR5) such as Apomab, conatumumab,
ETR2-ST01, GDC0145 (lexatumumab), HGS-1029, LBY-135, PRO-1762 and
trastuzumab.
[0401] Kinesin inhibitors include, but are not limited to, Eg5
inhibitors such as AZD4877, ARRY-520; and CENPE inhibitors such as
GSK923295A.
[0402] JAK-2 inhibitors include, but are not limited to, CEP-701
(lesaurtinib), XL019 and INCB018424.
[0403] MEK inhibitors include, but are not limited to, ARRY-142886,
ARRY-438162, PD-325901, PD-98059, and trametinib.
[0404] mTOR inhibitors include, but are not limited to, AP-23573,
CCI-779, everolimus, RAD-001, rapamycin, temsirolimus,
ATP-competitive TORC1/TORC2 inhibitors, including PI-103, PP242,
PP30, and Torin 1.
[0405] Non-steroidal anti-inflammatory drugs include, but are not
limited to, AMIGESIC.RTM. (salsalate), DOLOBID.RTM. (diflunisal),
MOTRIN.RTM. (ibuprofen), ORUDIS.RTM. (ketoprofen), RELAFEN.RTM.
(nabumetone), FELDENE.RTM. (piroxicam), ibuprofen cream, ALEVE.RTM.
(naproxen) and NAPROSYN.RTM. (naproxen), VOLTAREN.RTM.
(diclofenac), INDOCIN.RTM. (indomethacin), CLINORIL.RTM.
(sulindac), TOLECTIN.RTM. (tolmetin), LODINE.RTM. (etodolac),
TORADOL.RTM. (ketorolac), and DAYPRO.RTM. (oxaprozin).
[0406] PDGFR inhibitors include, but are not limited to, C-451,
CP-673 and CP-868596.
[0407] Platinum chemotherapeutics include, but are not limited to,
cisplatin, ELOXATIN.RTM. (oxaliplatin) eptaplatin, lobaplatin,
nedaplatin, PARAPLATIN.RTM. (carboplatin), satraplatin, and
picoplatin.
[0408] Polo-like kinase inhibitors include, but are not limited to,
BI-2536.
[0409] BRAF inhibitors vemurafenib, dabrafenib, cobimetinib.
[0410] Phosphoinositide-3 kinase (PI3K) inhibitors include, but are
not limited to, wortmannin, LY294002, XL-147, CAL-120, ONC-21,
AEZS-127, ETP-45658, PX-866, GDC-0941, BGT226, BEZ235, and
XL765.
[0411] Thrombospondin analogs include, but are not limited to,
ABT-510, ABT-567, ABT-898, and TSP-1.
[0412] VEGFR inhibitors include, but are not limited to,
AVASTIN.RTM. (bevacizumab), ABT-869, AEE-788, ANGIOZYME.TM. (a
ribozyme that inhibits angiogenesis (Ribozyme Pharmaceuticals
(Boulder, Colo.) and Chiron (Emeryville, Calif.)), axitinib
(AG-13736), AZD-2171, CP-547,632, IM-862, MACUGEN.RTM.
(pegaptamib), NEXAVAR.RTM. (sorafenib, BAY43-9006), pazopanib
(GW-786034), vatalanib (PTK-787, ZK-222584), SUTENT.RTM.
(sunitinib, SU-11248), VEGF trap, and ZACTIMA.TM. (vandetanib,
ZD-6474), cabozantanib (VEGFR2 and cMet inhibitor), ramucirumab
(anti-VEGFR2 inhibitory mAb).
[0413] Antibiotics include, but are not limited to, intercalating
antibiotics aclarubicin, actinomycin D, amrubicin, annamycin,
adriamycin, BLENOXANE.RTM. (bleomycin), daunorubicin, CAELYX.RTM.
or MYOCET.RTM. (liposomal doxorubicin), elsamitrucin, epirbucin,
glarbuicin, ZAVEDOS.RTM. (idarubicin), mitomycin C, nemorubicin,
neocarzinostatin, peplomycin, pirarubicin, rebeccamycin,
stimalamer, streptozocin, VALSTAR.RTM. (valrubicin), and
zinostatin.
[0414] Topoisomerase inhibitors include, but are not limited to,
aclarubicin, 9-aminocamptothecin, amonafide, amsacrine,
becatecarin, belotecan, BN-80915, CAMPTOSAR.RTM. (irinotecan
hydrochloride), camptothecin, CARDIOXANE.RTM. (dexrazoxine),
diflomotecan, edotecarin, ELLENCE.RTM. or PHARMORUBICIN.RTM.
(epirubicin), etoposide, exatecan, 10-hydroxycamptothecin,
gimatecan, lurtotecan, mitoxantrone, Onivyde.TM. (liposomal
irinotecan), orathecin, pirarbucin, pixantrone, rubitecan,
sobuzoxane, SN-38, tafluposide, and topotecan.
[0415] Antibodies include, but are not limited to, AVASTIN.RTM.
(bevacizumab), CD40-specific antibodies, chTNT-1/B, denosumab,
ERBITUX.RTM. (cetuximab), HUMAX-CD4.RTM. (zanolimumab),
IGF1R-specific antibodies, lintuzumab, PANOREX.RTM. (edrecolomab),
RENCAREX.RTM. (WX G250), RITUXAN.RTM. (rituximab), ticilimumab,
trastuzumab, pertuzumab, VECTIBIX.RTM. (panitumumab) and CD20
antibodies types I and II.
[0416] Hormonal therapies include, but are not limited to,
ARIMIDEX.RTM. (anastrozole), AROMASIN.RTM. (exemestane),
arzoxifene, CASODEX.RTM. (bicalutamide), CETROTIDE.RTM.
(cetrorelix), degarelix, deslorelin, DESOPAN.RTM. (trilostane),
dexamethasone, DROGENIL.RTM. (flutamide), EVISTA.RTM. (raloxifene),
AFEMA.TM. (fadrozole), FARESTON.RTM. (toremifene), FASLODEX.RTM.
(fulvestrant), FEMARA.RTM. (letrozole), formestane,
glucocorticoids, HECTOROL.RTM. (doxercalciferol), RENAGEL.RTM.
(sevelamer carbonate), lasofoxifene, leuprolide acetate,
MEGACE.RTM. (megesterol), MIFEPREX.RTM. (mifepristone),
NILANDRON.TM. (nilutamide), NOLVADEX.RTM. (tamoxifen citrate),
PLENAXIS.TM. (abarelix), prednisone, PROPECIA.RTM. (finasteride),
rilostane, SUPREFACT.RTM. (buserelin), TRELSTAR.RTM. (luteinizing
hormone releasing hormone (LHRH)), VANTAS.RTM. (Histrelin implant),
VETORYL.RTM. (trilostane or modrastane), XTANDI.RTM.
(enzalutamide), ZOLADEX.RTM. (fosrelin, goserelin), and ZYTIGA.RTM.
(abiratenone).
[0417] Deltoids and retinoids include, but are not limited to,
seocalcitol (EB1089, CB1093), lexacalcitrol (KH1060), fenretinide,
PANRETIN.RTM. (aliretinoin), ATRAGEN.RTM. (liposomal tretinoin),
TARGRETIN.RTM. (bexarotene), and LGD-1550.
[0418] PARP inhibitors include, but are not limited to, ABT-888
(veliparib), olaparib, KU-59436, AZD-2281, AG-014699, BSI-201,
BGP-15, INO-1001, and ONO-2231.
[0419] Plant alkaloids include, but are not limited to,
vincristine, vinblastine, vindesine, and vinorelbine.
[0420] Proteasome inhibitors include, but are not limited to,
VELCADE.RTM. (bortezomib), KYPROLIS.RTM. (carfilzomib), MG132,
NPI-0052, and PR-171.
[0421] Examples of immunologicals include, but are not limited to,
interferons, immune checkpoint inhibitors, co-stimulatory agents,
and other immune-enhancing agents. Interferons include interferon
alpha, interferon alpha-2a, interferon alpha-2b, interferon beta,
interferon gamma-1a, ACTIMMUNE.RTM. (interferon gamma-1b) or
interferon gamma-n1, combinations thereof and the like. Immune
check point inhibitors include antibodies that target PD-1 (e.g.,
pembrolizumab, nivolumab and pidilizumab), PD-L1 (e.g., durvalumab,
atezolizumab, avelumab, MEDI4736, MSB0010718C and MPDL3280A), and
CTLA4 (cytotoxic lymphocyte antigen 4; e.g., ipilimumab,
tremelimumab). Co-stimulatory agents include, but are not limited
to, antibodies against CD3, CD40, CD40L, CD27, CD28, CSF1R, CD137
(e.g., urelumab), B7H1, GITR, ICOS, CD80, CD86, OX40, OX40L, CD70,
HLA-DR, LIGHT, LIGHT-R, TIM3, A2AR, NKG2A, TIGIT (T cell
immunoreceptor with Ig and ITIM domains), VISTA (V-domain Ig
suppressor of T cell activation), B7-H3, B7-H4, CD47, CD73, CD39,
KIR (e.g., lirilumab), TGF-.beta. (e.g., fresolimumab) and
combinations thereof.
[0422] Other agents include, but are not limited to,
ALFAFERONE.RTM. (IFN-.alpha.), BAM-002 (oxidized glutathione),
BEROMUN.RTM. (tasonermin), BEXXAR.RTM. (tositumomab), CAMPATH.RTM.
(alemtuzumab), dacarbazine, denileukin, epratuzumab, GRANOCYTE.RTM.
(lenograstim), lentinan, leukocyte alpha interferon, imiquimod,
melanoma vaccine, mitumomab, molgramostim, MYLOTARG.TM. (gemtuzumab
ozogamicin), NEUPOGEN.RTM. (filgrastim), OncoVAC-CL, OVAREX.RTM.
(oregovomab), pemtumomab (Y-muHMFG1), PROVENGE.RTM. (sipuleucel-T),
sargaramostim, sizofilan, teceleukin, THERACYS.RTM. (Bacillus
Calmette-Guerin), ubenimex, VIRULIZIN.RTM. (immunotherapeutic,
Lorus Pharmaceuticals), Z-100 (Specific Substance of Maruyama
(SSM)), WF-10 (Tetrachlorodecaoxide (TCDO)), PROLEUKIN.RTM.
(aldesleukin), ZADAXIN.RTM. (thymalfasin), ZINBRYTA.RTM.
(daclizumab high-yield process), and ZEVALIN.RTM.
(.sup.90Y-Ibritumomab tiuxetan).
[0423] Biological response modifiers are agents that modify defense
mechanisms of living organisms or biological responses, such as
survival, growth or differentiation of tissue cells to direct them
to have anti-tumor activity and include, but are not limited to,
krestin, lentinan, sizofiran, picibanil PF-3512676 (CpG-8954), and
ubenimex.
[0424] Pyrimidine analogs include, but are not limited to,
cytarabine (ara C or Arabinoside C), cytosine arabinoside,
doxifluridine, FLUDARA.RTM. (fludarabine), 5-FU (5-fluorouracil),
floxuridine, GEMZAR.RTM. (gemcitabine), TOMUDEX.RTM. (ratitrexed),
and TROXATYL.TM. (triacetyluridine troxacitabine).
[0425] Purine analogs include, but are not limited to, LANVIS.RTM.
(thioguanine) and PURI-NETHOL.RTM. (mercaptopurine).
[0426] Antimitotic agents include, but are not limited to,
batabulin, epothilone D (KOS-862),
N-(2-((4-hydroxyphenyl)amino)pyridin-3-yl)-4-methoxybenzenesulfonamide,
ixabepilone (BMS 247550), TAXOL.RTM. (paclitaxel), TAXOTERE.RTM.
(docetaxel), PNU100940 (109881), patupilone, XRP-9881 (larotaxel),
vinflunine, and ZK-EPO (synthetic epothilone).
[0427] Ubiquitin ligase inhibitors include, but are not limited to,
MDM2 inhibitors, such as nutlins, and NEDD8 inhibitors such as
MLN4924.
[0428] Tyrosine Kinase inhibitors include imatinib (GLEEVEC.RTM.),
dasatinib (SPRYCE.RTM.), nilotinib (TASIGNA.RTM.), bosutinib
(BOSULIF.RTM.), ponatinib (ICLUSIG.RTM.), Afatinib (GIOTRIF.RTM.),
Axitinib (INLYTA.RTM.), Crizotinib (XALKORI.RTM.), Erlotinib
(TARCEVA.RTM.), Gefitinib (IRESSA.RTM.), Lapatinib (TYVERB.RTM.),
Nilotinib (TASIGNA.RTM.), Pazopanib (VOTRIENT.RTM.), Regorafenib
(STIVARGA.RTM.), Sorafenib (NEXAVAR.RTM.), Sunitinib (SUTENT.RTM.),
toceranib (PALLADIA.RTM.), vatalanib, and radotinib
(SUPECT.RTM.).
[0429] Anti-cMet ADCs may also be used to enhance the efficacy of
radiation therapy. Examples of radiation therapy include external
beam radiation therapy, internal radiation therapy (i.e.,
brachytherapy) and systemic radiation therapy.
[0430] Anti-cMet ADCs may be administered adjunctive to or with
other chemotherapeutic agents such as ABRAXANE.TM. (ABI-007),
ABT-100 (farnesyl transferase inhibitor), ADVEXIN.RTM. (Ad5CMV-p53
vaccine), ALTOCOR.RTM. or MEVACOR.RTM. (lovastatin), AMPLIGEN.RTM.
(poly I:poly C12U, a synthetic RNA), APTOSYN.RTM. (exisulind),
AREDIA.RTM. (pamidronic acid), arglabin, L-asparaginase, atamestane
(1-methyl-3,17-dione-androsta-1,4-diene), AVAGE.RTM. (tazarotene),
AVE-8062 (combreastatin derivative) BEC2 (mitumomab), cachectin or
cachexin (tumor necrosis factor), canvaxin (vaccine), CEAVAC.RTM.
(cancer vaccine), CELEUK.RTM. (celmoleukin), CEPLENE.RTM.
(histamine dihydrochloride), CERVARIX.RTM. (human papillomavirus
vaccine), CHOP.RTM. (C: CYTOXAN.RTM. (cyclophosphamide); H:
ADRIAMYCIN.RTM. (hydroxydoxorubicin); O: Vincristine
(ONCOVIN.RTM.); P: prednisone), CYPAT.TM. (cyproterone acetate),
combrestatin A4P, DAB(389)EGF (catalytic and translocation domains
of diphtheria toxin fused via a His-Ala linker to human epidermal
growth factor) or TransMID-107R.TM. (diphtheria toxins),
dacarbazine, dactinomycin, 5,6-dimethylxanthenone-4-acetic acid
(DMXAA), eniluracil, EVIZON.TM. (squalamine lactate),
DIMERICINE.RTM. (T4N5 liposome lotion), discodermolide, DX-8951f
(exatecan mesylate), enzastaurin, EP0906 (epithilone B),
GARDASIL.RTM. (quadrivalent human papillomavirus (Types 6, 11, 16,
18) recombinant vaccine), GASTRIMMUNE.RTM., GENASENSE.RTM., GMK
(ganglioside conjugate vaccine), GVAX.RTM. (prostate cancer
vaccine), halofuginone, histrelin, hydroxycarbamide, ibandronic
acid, IGN-101, IL-13-PE38, IL-13-PE38QQR (cintredekin besudotox),
IL-13-pseudomonas exotoxin, interferon-.alpha., interferon-.gamma.,
JUNOVAN.TM. or MEPACT.TM. (mifamurtide), lonafarnib,
5,10-methylenetetrahydrofolate, miltefosine
(hexadecylphosphocholine), NEOVASTAT.RTM. (AE-941), NEUTREXIN.RTM.
(trimetrexate glucuronate), NIPENT.RTM. (pentostatin),
ONCONASE.RTM. (a ribonuclease enzyme), ONCOPHAGE.RTM. (melanoma
vaccine treatment), ONCOVAX.RTM. (IL-2 Vaccine), ORATHECIN.TM.
(rubitecan), OSIDEM.RTM. (antibody-based cell drug), OVAREX.RTM.
MAb (murine monoclonal antibody), paclitaxel, PANDIMEX.TM.
(aglycone saponins from ginseng comprising 20(S)protopanaxadiol
(aPPD) and 20(S)protopanaxatriol (aPPT)), panitumumab,
PANVAC.RTM.-VF (investigational cancer vaccine), pegaspargase, PEG
Interferon A, phenoxodiol, procarbazine, rebimastat, REMOVAB.RTM.
(catumaxomab), REVLIMID.RTM. (lenalidomide), RSR13 (efaproxiral),
SOMATULINE.RTM. LA (lanreotide), SORIATANE.RTM. (acitretin),
staurosporine (Streptomyces staurospores), talabostat (PT100),
TARGRETIN.RTM. (bexarotene), TAXOPREXIN.RTM. (DHA-paclitaxel),
TELCYTA.RTM. (canfosfamide, TLK286), temilifene, TEMODAR.RTM.
(temozolomide), tesmilifene, thalidomide, THERATOPE.RTM. (STn-KLH),
thymitaq
(2-amino-3,4-dihydro-6-methyl-4-oxo-5-(4-pyridylthio)quinazoline
dihydrochloride), TNFERADE.TM. (adenovector: DNA carrier containing
the gene for tumor necrosis factor-.alpha.), TRACLEER.RTM. or
ZAVESCA.RTM. (bosentan), tretinoin (Retin-A), tetrandrine,
TRISENOX.RTM. (arsenic trioxide), VIRULIZIN.RTM., ukrain
(derivative of alkaloids from the greater celandine plant), vitaxin
(anti-alphavbeta3 antibody), XCYTRIN.RTM. (motexafin gadolinium),
XINLAY.TM. (atrasentan), XYOTAX.TM. (paclitaxel poliglumex),
YONDELIS.RTM. (trabectedin), ZD-6126, ZINECARD.RTM. (dexrazoxane),
ZOMETA.RTM. (zolendronic acid), and zorubicin, as well as
combinations of any of these agents.
[0431] Adjunctive therapies and/or therapeutic agents typically
will be used at their approved dose, route of administration, and
frequency of administration, but may be used at lower dosages
and/or less frequently. When administered as monotherapy, the
anti-cMet ADC will typically be administered on a schedule that
generates therapeutic benefit. It is contemplated that anti-cMet
ADCs administered once a week, once every two weeks, once every
three weeks, once every four weeks, once every five weeks, once
every six weeks, once every seven weeks or once every eight weeks
will provide therapeutic benefit, although more or less frequent
administration may be beneficial. When administered adjunctive to
or with another therapy and/or agent, the anti-cMet ADC may be
administered before treatment, after treatment or concurrently with
the treatment with the other therapy or agent.
[0432] 5.10. Dosages and Administration Regimens
[0433] The amount of anti-cMet ADC administered will depend upon a
variety of factors, including but not limited to, the particular
type of cMet+/overexpressing tumors treated, the stage of the
cMet+/overexpressing tumors being treated, the mode of
administration, the frequency of administration, the desired
therapeutic benefit, the drug component of the ADC (e.g., MMAE
versus PBD) and other parameters such as the age, weight and other
characteristics of the patient, etc. Determination of dosages
effective to provide therapeutic benefit for specific modes and
frequency of administration is within the capabilities of those
skilled in the art.
[0434] Dosages effective to provide therapeutic benefit may be
estimated initially from in vivo animal models or clinical.
Suitable animal models for a wide variety of diseases are known in
the art.
[0435] The anti-cMet ADCs may be administered by any route
appropriate to the condition to be treated. An anti-cMet ADC will
typically be administered parenterally, i.e., infusion,
subcutaneous, intramuscular, intravenous (IV), intradermal,
intrathecal, bolus, intratumor injection or epidural ((Shire et
al., 2004, J. Pharm. Sciences 93(6):1390-1402)). In one embodiment,
an anti-cMet ADC is provided as a lyophilized powder in a vial. The
vials may contain, for example, 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg,
100 mg, or 200 mg of anti-cMet ADC. In one embodiment, prior to
administration, the lyophilized powder is reconstituted with
sterile water for injection (SWFI) or other suitable medium to
provide a solution containing 20 mg/mL anti-cMet ADC. The resulting
reconstituted solution is further diluted with saline or other
suitable medium and administered via an IV infusion once every 7
days, once every 14 days, once every 21 days, or once every 28
days. In some embodiments, for the first cycle, the infusion occurs
over 180 minutes, subsequent infusions are over 90 minutes. In
other embodiments, the infusion occurs over 60 minutes. In some
embodiments, all infusions for every cycle occur over 30
minutes.
[0436] In one exemplary embodiment, an anti-cMet ADC is
administered once every 14 days at 0.15 mg/kg, 0.3 mg/kg, 0.6
mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.8 mg/kg, 1.9
mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3
mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1
mg/kg, 5.4 mg/kg, or 6.0 mg/kg of the subject's body weight. In one
embodiment, the anti-cMet ADC is administered once every 14 days at
1.6 mg/kg. In one embodiment, the anti-cMet ADC is administered
once every 14 days at 1.9 mg/kg. In one embodiment, the anti-cMet
ADC is administered once every 14 days at 2.2 mg/Kg. In one
embodiment, the anti-cMet ADC is administered once every 14 days at
2.5 mg/Kg. In one embodiment, administration proceeds until disease
progression or unacceptable toxicity.
[0437] In one embodiment, the cancer is a NSCLC adenocarcinoma, the
anti-cMet ADC is ABBV-399, administered at 1.6 or 1.9 mg/kg every
14 days, and the patient has an H-score of 225 and above or an IHC
score of 3+. In another embodiment, the cancer is a NSCLC squamous
cell carcinoma, the anti-cMet ADC is ABBV-399, administered at 1.6
or 1.9 mg/kg every 14 days, and the patient has an H-score between
150 to 224 or an IHCscore of 2+.
[0438] In another exemplary embodiment, an anti-cMet ADC is
administered once every 7 days at 0.15 mg/kg, 0.3 mg/kg, 0.45
mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1
mg/kg, 2.4 mg/kg, 2.7 mg/kg, or 3.0 mg/kg. In one embodiment,
administration proceeds until disease progression or unacceptable
toxicity.
[0439] In another exemplary embodiment, an anti-cMet ADC is
administered once every 28 days at 0.15 mg/kg, 0.3 mg/kg, 0.6
mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4
mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2
mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0
mg/kg. In one embodiment, administration proceeds until disease
progression or unacceptable toxicity.
[0440] In another exemplary embodiment, an anti-cMet ADC is
administered once every 28 days at 2.7 mg/kg. In one embodiment,
administration proceeds until disease progression or unacceptable
toxicity.
[0441] In another exemplary embodiment, an anti-cMet ADC is
administered once every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6
mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4
mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2
mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0
mg/kg. In one embodiment, administration proceeds until disease
progression or unacceptable toxicity.
[0442] In another exemplary embodiment, an anti-cMet ADC (e.g.,
ABBV-399) is administered once every 21 days at 2.7 mg/kg. In one
embodiment, administration proceeds until disease progression or
unacceptable toxicity. In one embodiment, the cancer is a NSCLC
adenocarcinoma, the anti-cMet ADC is ABBV-399, administered at 2.7
mg/kg every 21 days, and the patient has an H-score of 225 and
above or an IHC score of 3+. In another embodiment, the cancer is a
NSCLC squamous cell carcinoma, the anti-cMet ADC is ABBV-399,
administered at 2.7 mg/kg every 21 days, and the patient has an
H-score of at least 150 or greater and at least an IHCscore of
2+.
[0443] In another exemplary embodiment, an anti-cMet PBD ADC (e.g.,
ABT-700 PBD) is administered once every 14 days, once every 21
days, or once every 28 days, at a dose between 1.0 .mu.g/kg to 1.0
mg/kg, 1.0 .mu.g/kg to 500.0 .mu.g/kg, or 5.0 .mu.g/kg to 200.0
.mu.g/kg of the subject's body weight. As for any other ADC, the
dosage depends, for example, on the frequency of administration,
condition of the patient and response to prior treatment, if any.
The concentration of the ADC in a liquid formulation can be e.g.,
0.01-10 mg/ml, such as 1.0 mg/ml.
[0444] In one embodiment, an anti-cMet PBD ADC (e.g., ABT-700 PBD)
is administered once every 14 days, once every 21 days, or once
every 28 days at 10 .mu.g/kg, 50 .mu.g/kg, 75 .mu.g/kg, 100
.mu.g/kg, 110 .mu.g/kg, 120 .mu.g/kg, 130 .mu.g/kg, 140 .mu.g/kg,
150 .mu.g/kg, 160 .mu.g/kg, 170 .mu.g/kg, 180 .mu.g/kg, 190
.mu.g/kg, 200 .mu.g/kg, 250 .mu.g/kg, 300 .mu.g/kg, 350 .mu.g/kg,
400 .mu.g/kg, 450 .mu.g/kg, or 500 .mu.g/kg. In one embodiment, the
anti-cMet PBD ADC (e.g., ABT-700 PBD) is administered at 100
.mu.g/kg. In one embodiment, the anti-cMet PBD ADC (e.g., ABT-700
PBD) is administered at 200 .mu.g/kg. In one embodiment, the
anti-cMet PBD ADC (e.g., ABT-700 PBD) is administered at 300
.mu.g/kg. In one embodiment, the anti-cMet PBD ADC (e.g., ABT-700
PBD) is administered at 400 .mu.g/kg.
[0445] When administered adjunctive to, or with, other agents, such
as other chemotherapeutic agents, the ADCs may be administered on
the same schedule as the other agent(s), or on a different
schedule. When administered on the same schedule, the ADC may be
administered before, after, or concurrently with the other agent.
In some embodiments where an ADC is administered adjunctive to, or
with, standards of care, the ADC may be initiated prior to
commencement of the standard therapy, for example a day, several
days, a week, several weeks, a month, or even several months before
commencement of standard of care therapy.
[0446] In one set of exemplary embodiments, the additional
anti-cancer agent is selected from the group consisting of
cabazitaxel, colcemid, colchicine, cryptophycin, democolcine,
docetaxel, nocodazole, paclitaxel, taccalonolide, taxane and
vinblastine.
[0447] In one exemplary embodiment, an anti-cMet ADC is used
adjunctive to afatinib (GILOTRIF.RTM.) to treat NSCLC. The
anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion once
every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,
2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg,
4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg. GILOTRIF.RTM. is
administered at 40 mg orally once daily until disease progression
or no longer tolerated by the patient. In one embodiment, the
patients are selected for for the first-line treatment of
metastatic NSCLC with GILOTRIF.RTM. based on the presence of EGFR
exon 19 deletions or exon 21 (L858R) substitution mutations in
tumor specimens.
[0448] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to TARCEVA.RTM. (erlotinib) to treat non small cell
lung cancer (NSCLC). The anti-cMet ADC (e.g., ABBV-399) is
administered via IV infusion once every 14 days or every 21 days at
0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg,
1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg,
3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg,
5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, preferably once every 21 days at
2.7 mg/kg. The recommended dose and schedule for erlotinib is 150
mg orally, once daily. The adjunctive anti-cMet ADC/erlotinib
therapy is continued until disease progression or no longer
tolerated by the patient.
[0449] In one embodiment, the cancer is NSCLC, the anti-cMet ADC is
ABBV-399, administered at 2.7 mg/kg every 21 days, and the
erlotinib is administered at 150 mg orally, once daily. The
adjunctive anti-cMet ADC/erlotinib therapy is continued until
disease progression or no longer tolerated by the patient. In one
embodiment, the cancer is a NSCLC EGFR-mutated adenocarcinoma, the
anti-cMet ADC is ABBV-399, administered at 2.7 mg/kg every 21 days,
the erlotinib is administered at 150 mg orally, once daily, and the
patient has an H-score of 225 and above or an IHC score of 3+.
[0450] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to IRESSA.RTM. (gefitinib) to treat non small cell
lung cancer (NSCLC). The anti-cMet ADC (e.g., ABBV-399) is
administered via IV infusion once every 14 days or every 21 days at
0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg,
1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg,
3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg,
5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, preferably once every 21 days at
2.7 mg/kg. The recommended dose and schedule for gefitinib is 250
mg orally, once daily. The adjunctive anti-cMet ADC/gefitinib
therapy is continued until disease progression or no longer
tolerated by the patient.
[0451] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to afatinib to treat non small cell lung cancer
(NSCLC). The anti-cMet ADC (e.g., ABBV-399) is administered via IV
infusion once every 14 days or every 21 days at 0.15 mg/kg, 0.3
mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1
mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9
mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7
mg/kg or 6.0 mg/kg, preferably once every 21 days at 2.7 mg/kg. The
recommended dose and schedule for afatinib is 40 mg orally, once
daily. The adjunctive anti-cMet ADC/afatinib therapy is continued
until disease progression or no longer tolerated by the
patient.
[0452] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to OPDIVO.RTM. (nivolumab) to treat non small cell
lung cancer (NSCLC). The anti-cMet ADC (e.g., ABBV-399) is
administered via IV infusion once every 14 days or every 21 days at
0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg,
1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg,
3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg,
5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, preferably once every 21 days at
2.7 mg/kg. Nivolumab is administered an intravenous infusion at 3
mg/kg over 60 minutes every two weeks. The adjunctive anti-cMet
ADC/nivolumab treatment is continued until disease progression or
no longer tolerated by the patient.
[0453] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to OPDIVO.RTM. (nivolumab) and YERVOY.RTM.
(ipilimumab) to treat non small cell lung cancer (NSCLC). The
anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion once
every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,
2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg,
4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg, for four doses with
ipilimumab, then every 14 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg,
3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg,
4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, without
ipilimumab. Nivolumab is administered as an intravenous infusion at
3 mg/kg over 60 minutes every two weeks. Ipilimumab is administered
intravenously at 3 mg/kg over 90 minutes every three weeks in the
first four doses. The adjunctive anti-cMet ADC/nivolumab treatment
is continued until disease progression or no longer tolerated by
the patient.
[0454] In still another exemplary embodiment, an anti-cMet ADC can
be used adjunctive to pembrolizumab (KEYTRUDA.RTM.) to treat NSCLC.
The anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion
once every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6
mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4
mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2
mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0
mg/kg, preferably once every 21 days at 2.7 mg/kg. Pembrolizumab is
administered as an intravenous infusion at 2 mg/kg over 30 minutes
every 3 weeks. The adjunctive anti-cMet ADC and pembrolizumab
therapy is continued until disease progression or no longer
tolerated by the patient.
[0455] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to cisplatin to treat NSCLC. The anti-cMet ADC
(e.g., ABBV-399) is administered via IV infusion once every 14 days
or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg,
1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg,
3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg,
4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, preferably
once every 21 days at 2.7 mg/kg. Cisplatin is administered at 20
mg/m.sup.2 or more, once every 3 to 4 weeks. The adjunctive
anti-cMet ADC/cisplatin therapy is continued until disease
progression or no longer tolerated by the patient.
[0456] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to carboplatin to treat NSCLC. The anti-cMet ADC is
administered via IV infusion once every 14 days at 0.15 mg/kg, 0.3
mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1
mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9
mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7
mg/kg or 6.0 mg/kg. Carboplatin is administered at 300 mg/m.sup.2
or more, once every 4 weeks. The adjunctive anti-cMet
ADC/carboplatin therapy is continued until disease progression or
no longer tolerated by the patient.
[0457] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to veliparib to treat NSCLC. The anti-cMet ADC
(e.g., ABBV-399) is administered via IV infusion once every 14 days
or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg,
1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg,
3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg,
4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, preferably
once every 21 days at 2.7 mg/kg. Veliparib is administered orally,
twice a day. The adjunctive anti-cMet ADC/veliparib therapy is
continued until disease progression or no longer tolerated by the
patient.
[0458] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to veliparib and pemetrexed to treat NSCLC. The
anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion once
every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,
2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg,
4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg. Veliparib is
administered orally, twice a day. Pemetrexed is administered at 500
mg/m.sup.2 intravenously every 21 days. The adjunctive anti-cMet
ADC/veliparib/pemetrexed therapy is continued until disease
progression or no longer tolerated by the patient.
[0459] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to cetuximab to treat NSCLC. The anti-cMet ADC
(e.g., ABBV-399) is administered via IV infusion once every 14 days
or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg,
1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg,
3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg,
4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, preferably
once every 21 days at 2.7 mg/kg. Cetuximab is administered at an
initial dose of 400 mg/m.sup.2 over a 120-minute intravenous
infusion followed by 250 mg/m.sup.2 weekly infusion over 60
minutes. The adjunctive anti-cMet ADC/cetuximab therapy is
continued until disease progression or no longer tolerated by the
patient.
[0460] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to ipilimumab (YERVOY.RTM.) to treat NSCLC. The
anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion once
every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,
2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg,
4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg. Ipilimumab is
administered at 3 mg/kg intravenously over 90 minutes every 3 weeks
for 3 months. The anti-cMet ADC therapy is continued until disease
progression or no longer tolerated by the patient.
[0461] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to radiation to treat NSCLC. The anti-cMet ADC
(e.g., ABBV-399) is administered via IV infusion once every 14 days
or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg,
1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg,
3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg,
4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, preferably
once every 21 days at 2.7 mg/kg. Typically, external beam radiation
therapy is applied for a few minutes up to 5 days a week for 5 to 7
weeks, but this will vary depending on the type of external beam
radiation therapy that is used. The adjunctive anti-cMet
ADC/radiation therapy is continued until disease progression or no
longer tolerated by the patient.
[0462] In yet another exemplary embodiment, an anti-cMet ADC is
used adjunctive to AVASTIN.RTM. (bevacizumab) to treat NSCLC. The
anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion once
every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,
2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg,
4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg. The recommended dose
and schedule for bevacizumab is 10 mg/kg every 14 days or 15 mg/kg
every 21 days. The adjunctive anti-cMet ADC/bevacizumab therapy is
continued until disease progression or no longer tolerated by the
patient.
[0463] In one exemplary embodiment, an anti-cMet ADC is used
adjunctive to gemcitabine (GEMZAR.RTM.) to NSCLC cancer. The
anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion once
every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,
2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg,
4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg. Gemcitabine is
administered by intravenous infusion at a dose of 1000 mg/m.sup.2
over 30 minutes on days 1, 8, and 15 over an every 4-week schedule.
Administer cisplatin intravenously at 100 mg/m.sup.2 on day 1 after
the infusion of gemcitabine. In another embodiment, gemcitabine is
administered by intravenous infusion at a dose of 1250 mg/m.sup.2
over 30 minutes on days 1 and 8 over an every 3-week schedule.
Administer cisplatin intravenously at 100 mg/m.sup.2 on day 1 after
the infusion of gemcitabine. If myelosuppression is observed, dose
modifications as provided in the prescribing information for
gemcitabine may be used. The adjunctive anti-cMet ADC/gemcitabine
therapy is continued until disease progression or no longer
tolerated by the patient.
[0464] In one exemplary embodiment, an anti-cMet ADC is used
adjunctive to gemcitabine (GEMZAR.RTM.) to treat pancreatic,
ovarian, breast, or NSCLC cancer. The anti-cMet ADC (e.g.,
ABBV-399) is administered via IV infusion once every 14 days or
every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2
mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0
mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8
mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, preferably
once every 21 days at 2.7 mg/kg. In treating pancreatic cancer,
gemcitabine is administered by intravenous infusion at a dose of
1000 mg/m.sup.2 over 30 minutes once weekly for up to 7 weeks,
followed by a week of rest from treatment. After week 8: weekly
dosing on days 1, 8, and 15 of 28-day cycles. In treating ovarian
cancer, gemcitabine is administered by intravenous infusion at a
dose of 1000 mg/m.sup.2 over 30 minutes on days 1 and 8 of each
21-day cycle, in combination with carboplatin AUC 4 intravenously
after Gemzar administration on day 1 of each 21-day cycle. Refer to
carboplatin prescribing information for additional information. In
treating breast cancer, gemcitabine is administered by intravenous
infusion at a dose of 1250 mg/m.sup.2 intravenously over 30 minutes
on days 1 and 8 of each 21-day cycle that includes paclitaxel.
Paclitaxel should be administered at 175 mg/m2 on day 1 as a 3 hour
intravenous infusion before Gemzar administration. If
myelosuppression is observed, dose modifications as provided in the
prescribing information for gemcitabine may be used. Subsequent
cycles should consist of infusions once weekly for 3 consecutive
weeks out of every 4 weeks. The adjunctive anti-cMet
ADC/gemcitabine therapy is continued until disease progression or
no longer tolerated by the patient.
[0465] In another exemplary embodiment, an anti-cMet ADC is used
adjunctive to paclitaxel albumin-stabilized nanoparticle
formulation (ABRAXANE.RTM.) to treat breast or lung cancer. The
anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion once
every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,
2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg,
4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg. The recommended dose
and schedule for paclitaxel albumin-stabilized nanoparticle
formulation is 125 mg/m.sup.2 administered as an intravenous
infusion over 30-40 minutes on days 1, 8, and 15 of each 28-day
cycle. The adjunctive anti-cMet ADC/ABRAXANE.RTM. therapy is
continued until disease progression or no longer tolerated by the
patient.
[0466] In another exemplary embodiment, an anti-cMet ADC is used
adjunctive to paclitaxel albumin-stabilized nanoparticle
formulation (ABRAXANE.RTM.) plus gemcitabine (GEMZAR.RTM.) to treat
pancreatic cancer. The anti-cMet ADC (e.g., ABBV-399) is
administered via IV infusion once every 14 days or every 21 days at
0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg,
1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg,
3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg,
5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, preferably once every 21 days at
2.7 mg/kg. The recommended dose and schedule for paclitaxel
albumin-stabilized nanoparticle formulation is 125 mg/m.sup.2
administered as an intravenous infusion over 30-40 minutes on days
1, 8, and 15 of each 28-day cycle. Gemcitabine is administered by
intravenous infusion at a dose of 1000 mg/m.sup.2 over 30 minutes
once weekly for up to 7 weeks (or until toxicity reducing or
holding a dose), followed by a week of rest from treatment.
Subsequent cycles should consist of infusions once weekly for 3
consecutive weeks out of every 4 weeks. The adjunctive anti-cMet
ADC/ABRAXANE.RTM./GEMZAR.RTM. therapy is continued until disease
progression or no longer tolerated by the patient.
[0467] In yet another exemplary embodiment, an anti-cMet ADC is
used adjunctive to AVASTIN.RTM. (bevacizumab) to treat colorectal
cancer or lung or ovarian. The anti-cMet ADC (e.g., ABBV-399) is
administered via IV infusion once every 14 days or every 21 days at
0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg,
1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg,
3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg,
5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, preferably once every 21 days at
2.7 mg/kg. The recommended dose and schedule for bevacizumab is 10
mg/kg every 14 days or 15 mg/kg every 21 days. The adjunctive
anti-cMet ADC/bevacizumab therapy is continued until disease
progression or no longer tolerated by the patient.
[0468] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to FOLFIRINOX (or FOLFIRI or FOLFOX or irinotecan
or 5-FU or capecitabine) to treat colorectal cancer. The anti-cMet
ADC (e.g., ABBV-399) is administered via IV infusion once every 14
days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 0.9
mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7
mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5
mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg. FOLFIRINOX is a
combination of four chemotherapy agents: fluorouracil [5-FU],
leucovorin, irinotecan and oxaliplatin. In some embodiments,
FOLFIRINOX is administered as follows: oxaliplatin, 85 mg/m.sup.2;
irinotecan, 180 mg/m.sup.2; leucovorin, 400 mg/m.sup.2; and
fluorouracil, 400 mg/m.sup.2 given as a bolus followed by 2400
mg/m.sup.2 given as a 46-hour continuous infusion, every 2 weeks.
The adjunctive anti-cMet ADC/FOLFIRINOX therapy is continued until
disease progression or no longer tolerated by the patient.
[0469] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to Onivyde.RTM. to treat pancreatic cancer. The
anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion once
every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,
2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg,
4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg. Onivyde.RTM. is a
liposomal irinotecan formulation. In some embodiments, Onivyde.RTM.
is administered at 70 mg/m.sup.2 by intravenous infusion over 90
minutes every 2 weeks. The adjunctive anti-cMet ADC/Onivyde.RTM.
therapy is continued until disease progression or no longer
tolerated by the patient.
[0470] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to Onivyde.RTM., fluorouracil, and leucovorin to
treat pancreatic. The anti-cMet ADC (e.g., ABBV-399) is
administered via IV infusion once every 14 days or every 21 days at
0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg,
1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg,
3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg,
5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, preferably once every 21 days at
2.7 mg/kg. Onivyde.RTM. is a liposomal irinotecan formulation. In
some embodiments, Onivyde.RTM. is administered at 70 mg/m.sup.2 by
intravenous infusion over 90 minutes every 2 weeks, with leucovorin
400 mg/m.sup.2 and fluorouracil 2400 mg/m.sup.2 over 46 hours every
2 weeks. The adjunctive anti-cMet
ADC/Onivyde.RTM./leucovorin/fluorouracil therapy is continued until
disease progression or no longer tolerated by the patient.
[0471] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to nivolumab (OPDIVO.RTM.) to treat lung cancer and
other cancers where nivolumab is utilized. The anti-cMet ADC (e.g.,
ABBV-399) is administered via IV infusion once every 14 days or
every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2
mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0
mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8
mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, preferably
once every 21 days at 2.7 mg/kg. Nivolumab is administered an
intravenous infusion at 3 mg/kg over 60 minutes every two weeks.
The adjunctive anti-cMet ADC/nivolumab therapy is continued until
disease progression or no longer tolerated by the patient.
[0472] In still another exemplary embodiment, an anti-cMet ADC can
be used adjunctive to pembrolizumab (KEYTRUDA.RTM.) to treat
colorectal cancer. The anti-cMet ADC (e.g., ABBV-399) is
administered via IV infusion once every 14 days or every 21 days at
0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg,
1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg,
3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg,
5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, preferably once every 21 days at
2.7 mg/kg. Pembrolizumab is administered as an intravenous infusion
at 2 mg/kg over 30 minutes every 3 weeks. The adjunctive anti-cMet
ADC/pembrolizumab therapy is continued until disease progression or
no longer tolerated by the patient.
[0473] In one embodiment, the cancer is pancreatic cancer, the
anti-cMet ADC is ABBV-399, administered at 2.7 mg/kg every 21 days,
and the erlotinib is administered at 150 mg orally, once daily. The
adjunctive anti-cMet ADC/erlotinib therapy is continued until
disease progression or no longer tolerated by the patient.
[0474] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to doxorubicin to treat breast cancer. The
anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion once
every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,
2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg,
4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg. When used adjunctively
with other drugs, the most commonly used dosage of doxorubicin is
40 to 60 mg/m.sup.2 given as a single intravenous injection every
21 to 28 days. The adjunctive anti-cMet ADC/doxorubicin therapy is
continued until disease progression or no longer tolerated by the
patient.
[0475] In yet another exemplary embodiment, an anti-cMet ADC is
used adjunctive to AVASTIN.RTM. (bevacizumab) to treat breast
cancer. The anti-cMet ADC (e.g., ABBV-399) is administered via IV
infusion once every 14 days or every 21 days at 0.15 mg/kg, 0.3
mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1
mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9
mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7
mg/kg or 6.0 mg/kg, preferably once every 21 days at 2.7 mg/kg. The
recommended dose and schedule for bevacizumab is 10 mg/kg every 14
days or 15 mg/kg every 21 days. The adjunctive anti-cMet
ADC/bevacizumab therapy is continued until disease progression or
no longer tolerated by the patient.
[0476] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to gemcitabine to treat breast cancer. The
anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion once
every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,
2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg,
4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg. Gemcitabine is
administered by intravenous infusion at a dose of 1000 mg/m.sup.2
over 30 minutes once weekly for up to 7 weeks (or until toxicity
reducing or holding a dose), followed by a week of rest from
treatment. Subsequent cycles should consist of infusions once
weekly for 3 consecutive weeks out of every 4 weeks. The adjunctive
anti-cMet ADC/gemcitabine therapy is continued until disease
progression or no longer tolerated by the patient.
[0477] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to trastuzumab (HERCEPTIN.RTM.) to treat breast
cancer. The anti-cMet ADC (e.g., ABBV-399) is administered via IV
infusion once every 14 days or every 21 days at 0.15 mg/kg, 0.3
mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1
mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9
mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7
mg/kg or 6.0 mg/kg, preferably once every 21 days at 2.7 mg/kg. The
recommended initial loading dose for trastuzumab is 4 mg/kg
administered as a 90-minute infusion. The recommended weekly
maintenance dose for trastuzumab is 2 mg/kg which can be
administered as a 30 minute infusion if the initial loading dose
was well tolerated. The adjunctive anti-cMet ADC/trastuzumab
therapy is continued until disease progression or no longer
tolerated by the patient.
[0478] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to capecitabine (XELODA.RTM.) to treat breast
cancer. The anti-cMet ADC (e.g., ABBV-399) is administered via IV
infusion once every 14 days or every 21 days at 0.15 mg/kg, 0.3
mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1
mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9
mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7
mg/kg or 6.0 mg/kg, preferably once every 21 days at 2.7 mg/kg.
Capecitabine can be administered at 1250 mg/m.sup.2 twice daily for
2 weeks followed by a one week rest period in 3 week cycles. The
adjunctive anti-cMet ADC/capecitabine therapy is continued until
disease progression or no longer tolerated by the patient.
[0479] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to nivolumab (OPDIVO.RTM.) to treat breast cancer.
The anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion
once every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6
mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4
mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2
mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0
mg/kg, preferably once every 21 days at 2.7 mg/kg. Nivolumab is
administered an intravenous infusion at 3 mg/kg over 60 minutes
every two weeks. The adjunctive anti-cMet ADC/nivolumab therapy is
continued until disease progression or no longer tolerated by the
patient.
[0480] In still another exemplary embodiment, an anti-cMet ADC can
be used adjunctive to pembrolizumab (KEYTRUDA.RTM.) to treat breast
cancer. The anti-cMet ADC (e.g., ABBV-399) is administered via IV
infusion once every 14 days or every 21 days at 0.15 mg/kg, 0.3
mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1
mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9
mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7
mg/kg or 6.0 mg/kg, preferably once every 21 days at 2.7 mg/kg.
Pembrolizumab is administered as an intravenous infusion at 2 mg/kg
over 30 minutes every 3 weeks. The adjunctive anti-cMet
ADC/pembrolizumab therapy is continued until disease progression or
no longer tolerated by the patient.
[0481] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to TARCEVA.RTM. (erlotinib) to treat Head and Neck
cancer. The anti-cMet ADC (e.g., ABBV-399) is administered via IV
infusion once every 14 days or every 21 days at 0.15 mg/kg, 0.3
mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1
mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9
mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7
mg/kg or 6.0 mg/kg, preferably once every 21 days at 2.7 mg/kg. The
recommended dose and schedule for erlotinib is 150 mg orally, once
daily. The adjunctive anti-cMet ADC/erlotinib therapy is continued
until disease progression or no longer tolerated by the
patient.
[0482] In one embodiment, the cancer is Head and Neck cancer, the
anti-cMet ADC is ABBV-399, administered at 2.7 mg/kg every 21 days,
and the erlotinib is administered at 150 mg orally, once daily. The
adjunctive anti-cMet ADC/erlotinib therapy is continued until
disease progression or no longer tolerated by the patient.
[0483] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctively to radiation to treat Head and Neck cancer. The
anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion once
every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,
2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg,
4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg. Typically, external
beam radiation therapy is applied for a few minutes up to 5 days a
week for 5 to 7 weeks, but this will vary depending on the type of
external beam radiation therapy that is used. The adjunctive
anti-cMet ADC/radiation therapy is continued until disease
progression or no longer tolerated by the patient.
[0484] In yet another exemplary embodiment, an anti-cMet ADC is
used adjunctive to AVASTIN.RTM. (bevacizumab) to treat Head and
Neck cancer. The anti-cMet ADC (e.g., ABBV-399) is administered via
IV infusion once every 14 days or every 21 days at 0.15 mg/kg, 0.3
mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1
mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9
mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7
mg/kg or 6.0 mg/kg, preferably once every 21 days at 2.7 mg/kg. The
recommended dose and schedule for bevacizumab is 10 mg/kg every 14
days or 15 mg/kg every 21 days. The adjunctive anti-cMet
ADC/bevacizumab therapy is continued until disease progression or
no longer tolerated by the patient.
[0485] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to cetuximab to treat Head and Neck cancer. The
anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion once
every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,
2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg,
4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg. Cetuximab is
administered at an initial dose of 400 mg/m.sup.2 over a 120-minute
intravenous infusion followed by 250 mg/m.sup.2 weekly infusion
over 60 minutes. The adjunctive anti-cMet ADC/cetuximab therapy is
continued until disease progression or no longer tolerated by the
patient.
[0486] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to carboplatin to treat Head and Neck cancer. The
anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion once
every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,
2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg,
4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg. Carboplatin is
administered at 300 mg/m.sup.2 or more, once every 4 weeks. The
adjunctive anti-cMet ADC/carboplatin therapy is continued until
disease progression or no longer tolerated by the patient.
[0487] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to nivolumab (OPDIVO.RTM.) to treat Head and Neck
cancer. The anti-cMet ADC (e.g., ABBV-399) is administered via IV
infusion once every 14 days or every 21 days at 0.15 mg/kg, 0.3
mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1
mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9
mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7
mg/kg or 6.0 mg/kg, preferably once every 21 days at 2.7 mg/kg.
Nivolumab is administered an intravenous infusion at 3 mg/kg over
60 minutes every two weeks. The adjunctive anti-cMet ADC/nivolumab
therapy is continued until disease progression or no longer
tolerated by the patient.
[0488] In still another exemplary embodiment, an anti-cMet ADC can
be used adjunctive to pembrolizumab (KEYTRUDA.RTM.) to treat Head
and Neck cancer. The anti-cMet ADC (e.g., ABBV-399) is administered
via IV infusion once every 14 days or every 21 days at 0.15 mg/kg,
0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg,
2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg,
3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg,
5.7 mg/kg or 6.0 mg/kg, preferably once every 21 days at 2.7 mg/kg.
Pembrolizumab is administered as an intravenous infusion at 2 mg/kg
over 30 minutes every 3 weeks. The adjunctive anti-cMet
ADC/pembrolizumab therapy is continued until disease progression or
no longer tolerated by the patient.
[0489] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to cisplatin to treat Head and Neck cancer. The
anti-cMet ADC (e.g., ABBV-399) is administered via IV infusion once
every 14 days or every 21 days at 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,
0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,
2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg,
4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg,
preferably once every 21 days at 2.7 mg/kg. The adjunctive
anti-LRRC15 ADC/cisplatin therapy is continued until disease
progression or no longer tolerated by the patient.
[0490] In still another exemplary embodiment, an anti-cMet ADC is
used adjunctive to TARCEVA.RTM. (erlotinib) to treat Head and Neck
cancer. The anti-cMet ADC (e.g., ABBV-399) is administered via IV
infusion once every 14 days or every 21 days at 0.15 mg/kg, 0.3
mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1
mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9
mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7
mg/kg or 6.0 mg/kg, preferably once every 21 days at 2.7 mg/kg. The
recommended dose and schedule for erlotinib is 150 mg orally, once
daily. The adjunctive anti-cMet ADC/erlotinib therapy is continued
until disease progression or no longer tolerated by the
patient.
[0491] In one embodiment, the cancer is Head and Neck cancer, the
anti-cMet ADC is ABBV-399, administered at 2.7 mg/kg every 21 days,
and the erlotinib is administered at 150 mg orally, once daily. The
adjunctive anti-cMet ADC/erlotinib therapy is continued until
disease progression or no longer tolerated by the patient.
[0492] As will be appreciated by those of skill in the art, the
recommended dosages for the various agents described above may need
to be adjusted to optimize patient response and maximize
therapeutic benefit.
[0493] In alternate embodiments, all numbers expressing quantities
of ingredients, % purity, and so forth, used in this disclosure,
are modified by the term "about."
[0494] 5.11. Patient Selection
[0495] Patients selected for the ADC treatments of this disclosure
include those with cMet-expressing tumors and those with
cMet-overexpressing tumors, which include, but are not limited to,
any solid tumor (including also those that overexpress HGF and/or
have abnormal activation of HGF/cMet signaling or expression).
Patients can be selected for treatment with the ADC treatments of
this disclosure on the basis of their level of cMet, which is
classified in terms of an immunohistochemistry (IHC) H-score.
Details on how to quantify and qualify the level of cMet
overexpression are presented in the Detailed Description (section
5.3.) and in Example 17. cMet overexpression can be defined by an
IHC H-score of greater than or equal to 150 when measured according
to the assay of Example 17 "cMet ABBV-ADC staining protocol."
Briefly, an IHC staining protocol for cMet overexpression has been
developed using the Ventana cMet CONFIRM (SP44) kit. Tissue samples
are stained with the Ventana antibody and then scored by
determining the percentages of target tissue cells staining at
various intensity levels of low to high. FIG. 20 depicts
representative H-scores using the assay described in Example 17.
Alternatively, cMet overexpressing tumor tissue using an IHC score
from 0 to 3+ is described in Example 21. FIG. 19 depicts
representative IHC scores using the assay described in Example
21.
[0496] For purposes of this disclosure, an H-score between 150 and
224 is equivalent to an IHC score of 2+ and an H-score of 225 and
above is equivalent to an IHC score of 3+. In one example, NSCLC
squamous cell carcinoma patients can be selected for treatment when
their cancer has an H-score of at least between 150 and 224, or an
IHC score of 2+. In another example, NSCLC adenocarcinoma patients
can be selected for treatment when their cancer has an H-score of
225 and above, or an IHC score of 3+.
[0497] The cancer may be newly diagnosed and naive to treatment, or
may be relapsed, refractory, or relapsed and refractory, or a
metastasis or metastatic form of a cMet-expressing (herein referred
to as cMet+ tumors) or cMet-overexpressing tumor, i.e.,
cMet+/overexpressing tumors. As demonstrated in the Examples of
this disclosure, cMet+/overexpressing tumors that exhibit
resistance to other targeted or non-targeted chemotherapies, retain
sensitivity to ABBV-399.
[0498] The anti-cMet ADCs have myriad uses, and in one embodiment
are useful therapeutically for the treatment of cMet overexpressing
tumors in humans, tumors where the MET gene has been amplified; and
tumors carrying mutations in or around Exon 14 of the MET gene,
among others. In another embodiment, the anti-cMet ADCs are useful
therapeutically for the treatment of cMet expressing tumors in
humans, where cMet is not overexpressed but still expressed.
[0499] Tumors carrying EGFR Exon 19 deletions or EGFR Exon 21
mutations (L858R) are also within the scope of this disclosure.
Amplification of the MET gene is considered one of the more common
causes of acquired resistance in EGFR-mutant NSCLC.
[0500] Response to ABBV-399 and other cMet-ADCs disclosed herein
can correlate with expression of cMet at both the protein and
genomic level (e.g., amplification, Exon 14 mutations).
Preferential methods for measuring both of these biomarkers are
described in detail in the Examples. However, one of ordinary skill
in the art would know how to use other methods to assess the same
and those methods are within the scope of this disclosure.
[0501] If different results are obtained with different methods,
then the results obtained with the methods described in the
Examples are those to be used in determining whether a particular
embodiment falls within the scope of the embodiments. For example,
for evaluating expression of the cMet protein one would use the
"cMet ABBV-ADC staining protocol." If the Ventana reagents used in
this protocol are no longer available, another FDA-approved
protocol for assessment of cMet expression levels by IHC can be
used. For evaluating MET gene copy number one would use the
"MET/CEP7 cMET amplification method."
[0502] MET is subject to alternative splicing. Multiple MET
transcripts of different size have been identified in human cell
lines and tissues. At least three 8-kb variants have been described
and presumed to be generated by alternative splicing. A cMet
isoform was described that lacks 18 amino acids in the
extracellular region (exon 10) and is the most abundant form in a
variety of tissues and cell lines. Alternative splicing of exon 14
generates another variant that has an in-frame deletion of 47 amino
acids in the juxtamembrane cytoplasmic domain of the receptor. A
possible mechanism of alternative splicing could be at the origin
of a 85 kDa, N-terminally truncated form of MET found in malignant
musculo-skeletal tumors, although this short form could also
originate from alternative transcription start or proteolitic
cleavage.
[0503] It has been demonstrated that MET mutants involving deletion
of exon 14 stabilize the cMet receptor, resulting in a gain of
function activity. MET Exon 14 contains the Cbl ubiquitin ligases
site on tyrosine residue 1003 (Y1003) where ubiquitin is otherwise
normally attached to the tyrosine residue and leads to the
lysosomal degradation of the cMet protein. Hence, missense mutation
of Y1003 residue or "skipping" of the protein region that is
encoded by MET Exon 14 results in a relative over-expression of MET
protein, enhanced cMet activation and subsequent oncogenesis.
Inhibition by MET Tyrosine Kinase Inhibitors (TKIs) can result in
clinical benefit in at least NSCLC patients harboring these MET
Exon 14 alterations. Patients carrying any of these mutations can
benefit from the treatments disclosed herein.
[0504] Accordingly, patients may also be selected for treatment if
they carry cells with a mutation in Exon 14 of the MET gene, the
result of which is an increased level of cMet protein in those
cancer cells. The Examples also provide various methods for
assessing this biomarker.
[0505] MET amplification is recognized as one of the potential
molecular mechanisms of acquired resistance in EGFR-mutated NSCLC
to EGFR-TKIs. The decision on whether or not to select a particular
patient for treatment with the ADCs disclosed herein may also
encompass determining whether the patient's cancer carries a
deletion in Exon 19 of the Epidermal Growth Factor Receptor (EGFR),
a substitution in Exon 21 (L858R), or both. Patients whose cancer
carries one or both of these genomic alterations in at least some
of its cells are preferentially selected for treatment with
ABBV-399 or any other ADC disclosed herein. Methods for assessing
these two biomarkers are provided in the Examples below.
6. EXAMPLES
[0506] The following Examples, which highlight certain features and
properties of exemplary embodiments of anti-cMet ADCs and methods
of using these ADS to treat patients are provided for purposes of
illustration, and not limitation.
Example 1. Preparation of ABT-700
[0507] ABBV-399 (ABT-700-vcMMAE) is an antibody drug conjugate
(ADC) comprised of the antibody ABT-700 conjugated to the cytotoxic
microtubule inhibitor monomethylauristatin E (MMAE) via a cleavable
valine-citrulline (vc) linker. ABT-700 is a "humanized" recombinant
immunoglobulin G kappa (IgG.sub.1.kappa.) that targets a unique
epitope of cMet resulting in blockade of both HGF-dependent and
HGF-independent cMet signaling.
[0508] ABT-700 is a humanized recombinant monoclonal antibody
directed against cMet. The antibody consists of 2 identical IgG1
heavy chains of 445 amino acids paired with 2 identical light
chains of 218 amino acids. The heavy chain was engineered to
introduce an extra cysteine at position 223 as well as deletion of
a lysine residue preceding Cys-223 and deletion of 2 threonine
residues flanking His-224. In addition, the C-terminal lysine amino
acid on the heavy chain was engineered to eliminate heterogeneity
at the C terminus due to incomplete cleavage of the lysine. The
antibody is glycosylated at asparagine 296 on each heavy chain.
[0509] The heavy chain contains 12 cysteine residues and the light
chain contains 5 cysteine residues. Each heavy chain contains 4
intra-chain disulfide bridges and each light chain contains 2
intra-chain disulfide bridges. In addition, the 2 heavy chains are
covalently linked by 3 inter-chain disulfide bridges. Each light
chain participates in 1 disulfide bond with a heavy chain.
[0510] ABT-700 for the in vitro studies described below was
prepared by routine techniques, essentially as described in U.S.
Pat. No. 8,741,290. Briefly, suspension-adapted HEK293 EBNA cells
(InVitrogen, US) were routinely grown in 250 ml flasks in 50 ml of
serum-free medium Excell 293 (SAFC Biosciences) supplemented with 6
mM glutamine on an orbital shaker (110 rpm rotation speed).
Transient transfection was performed with 2.times.10.sup.6 cells/ml
using linear 25 kDa polyethyleneimine (PEI) (Polysciences) prepared
in water at a final concentration of 1 mg/ml mixed and plasmid DNA
(final concentration of 1.25 .mu.g/ml for heavy to light chain
plasmid ratio of 1:1). At 4 hours post-transfection, the culture
was diluted with one volume of fresh culture medium to achieve a
final cell density of 10.sup.6 cells/ml. The cultivation process
was monitored on the basis of cell viability and Mab production.
Typically, cultures were maintained for 4 to 5 days. ABT-700 was
purified using a conventional chromatography approach on a Protein
A resin (GE Healthcare, US).
[0511] ABT-700 for the clinical studies described below was
prepared essentially as described next. First, the plasmid
pConPlus.gamma.1f.DELTA.K/.kappa.-hz224G11[TH7] was constructed for
high-level expression of ABT-700 monoclonal antibody in CHO cells
using the Glutamine Synthetase GS-CHO technology. The heavy and
light chain sequences were cloned into vectors
pConPlus.gamma.1f.DELTA.K-hz224G11/TH7VH0 and
pConPlus.kappa.2-hz224G11/VL4(4-39-84) respectively, creating
single-gene vectors (SGVs). The SGVs containing the heavy chain and
the light chain genes were then combined, together with the
Glutamine Synthetase (GS) selection gene, to generate the final
double-gene vector (DGV): pConPlus.gamma.1f.DELTA.K/.kappa.-
[0512] hz224G11[TH7]. The major components of
pConPlus.gamma.1f.DELTA.K/.kappa.-hz224G11[TH7] include the
following genes or regulatory elements in the following order:
hCMV-MIE promoter, 5' UTR with intron, ABT-700 light chain coding
sequence [224G11 (HzVL)], SV40 polyadenylation sequence, hCMV-MIE
promoter, 5' UTR with intron, ABT-700 heavy chain coding sequence
[224G11 (HzVH)], SV40 polyadenylation sequence, plasmid origin of
replication, beta-lactamase, and Glutamine Synthetase cDNA with its
regulatory sequences.
[0513] The expression system used for production of ABT-700 drug
substance was Lonza Biologics' proprietary Glutamine Synthetase
(GS) Gene Expression System in Chinese Hamster Ovary (CHO) cells.
The host cell line was derived from CHO-K1SV host working cell bank
designated 269-W3 (prepared from the host master cell bank
269-M).
[0514] The double-gene vector
pConPlus.gamma.1f.DELTA.K/.kappa.-hz224G11[TH7] was transfected
into CHO-K1SV cells by electroporation and then distributed into
96-well plates. Cells expressing GS, and hence those containing the
expression vector were selected by growth in protein-free and
glutamine-free medium. The plates were incubated until foci of
transfected cells began to appear. Only cell lines that came from
wells containing single colonies (as determined by visual
assessment) were progressed. Culture supernatants from wells
containing single colonies were screened for antibody production
using an ELISA for assembled antibody. Several clonal cell lines
were established and the one showing the most consistent
performance was selected for ABT-700 production. The cells were
tested to confirm the quality of the mRNA and the fidelity of the
coding transcripts.
[0515] A single frozen vial of cells is expanded by either shaker
culture or cell bags. A larger volume of culture medium is
inoculated with the expanded cultures and the cultures expanded
further in a bioreactor (comprising growth medium supplemented with
methionine sulphoximide) in a 5% CO.sub.2, 36.degree. C. incubator.
The cultures are harvested and filtered for the removal of cells
and debris. The ABT-700 is purified through a Protein A column,
followed by anion exchange membrane chromatography, cation exchange
column chromatography, viral filtration, ultrafiltration, and final
bulk filtration. All solutions are prepared according to cGMP.
Example 2. Preparation of Heterogeneous DAR ABT700-vcMMAE ADCs
[0516] ABBV-399 is an ADC comprised of ABT-700 (an anti-cMet IgG1
antibody) conjugated to MMAE via a vc linker.
[0517] ABBV399 is derived from the conjugation of vcMMAE to
inter-chain disulfide bonds in ABT-700 after mild reduction to the
sulfhydryl groups. After an additional process step to remove
higher order DAR species, the average DAR for ABBV-399 is
approximately 3.
[0518] Two different processes, Process I (FIG. 2A and FIG. 2B) and
Process II (FIG. 3A and FIG. 3B) were used to make ABBV-399
heterogeneous DAR compositions.
[0519] An ABBV399 composition heterogeneous in DAR was prepared by
a two-step chemical process: disulfide reduction of ABT-700
followed by alkylation (conjugation) with maleimidocaproyl
valine-citrulline ("val-cit") para-aminobenzyl alcohol ("PABA")
monomethyl auristatin E (referred to herein as "vcMMAE"),
illustrated below:
[0520] In the first step, a limited number of interchain disulfide
bonds of ABT700 are reduced with tris(2-carboxyethyl) phosphine
("TCEP") (.gtoreq.0.8 equiv). Partially-reduced ABT700 is then
conjugated to vcMMAE (.gtoreq.1.8 equiv) in DMSO. Residual
unreacted vcMMAE is quenched with N-acetyl-L-cysteine.
[0521] FIG. 2A and FIG. 3A show chromatographic resolutions of the
resultant crude ADC preparations obtained from Process I (FIG. 2A)
or Process II (FIG. 3A). As can be seen, the resultant ADC
preparation is a heterogeneous mixture containing antibodies having
zero MMAE molecules attached ("E0" peak), two MMAE molecules
attached ("E2" peak), four MMAE molecules attached ("E4" peak), six
MMAE molecules attached ("E6" peak), eight MMAE molecules attached
("E8" peak), and ten MMAE molecules attached ("E10" peak). For
process I, the average DAR of the crude product preparation is
approximately 4.3. For process II the average DAR of the crude
product preparation is approximately 3.2.
Example 3. Preparation of ABT700-vcMMAE ADCs Enriched in DAR3.1 and
ABBV-399 Enriched in a 1:1 E2/E4 Ratio
Preparation of ABBV-399 Enriched in DAR 3.1 Using Process I
[0522] To obtain an average DAR of 3.1, as depicted in FIG. 2B, a
batch chromatographic process was used. The ABBV-399 crude product
solution (FIG. 2A) is diluted with a potassium phosphate buffer and
treated with a HIC resin to reduce the DAR to approximately 3. The
HIC resin is removed by filtration, washed with a
phosphate-buffered saline solution and the wash is optionally
combined with the ABBV-399 DAR 3.1 product solution.
[0523] FIG. 2B shows an analytical HIC chromatogram of the final
product from process I after treatment with the HIC resin (As can
be seen, the resultant ADC preparation is a heterogeneous mixture
containing antibodies having zero MMAE molecules attached ("E0"
peak), two MMAE molecules attached ("E2" peak), four MMAE molecules
attached ("E4" peak), and six MMAE molecules attached ("E6" peak),
and has an average DAR of 3.1.
Preparation of ABBV-399 Enriched in a 1:1 E2/E4 Ratio
[0524] To obtain a 1:1 E2/E4 ratio, as depicted in FIG. 3B, a
column chromatographic process was used. The ABBV-399 crude product
solution (FIG. 3A) is diluted with an ammonium sulfate/sodium
phosphate solution to the target binding concentration. This
material is loaded on the column and binds to the HIC resin. A step
gradient elution using an ammonium sulfate/sodium phosphate buffer
is used to enrich the antibody drug conjugates and isolate the ADC
species with two or four vcMMAE molecules attached. These are
eluted off the column in one peak.
[0525] FIG. 3B shows an analytical HIC chromatogram of the final
product from Process II after enrichment using the HIC
chromatography column. As can be seen, the resultant ADC
preparation is a heterogeneous mixture containing antibodies having
zero MMAE molecules attached ("E0" peak), two MMAE molecules
attached ("E2" peak), and four MMAE molecules attached ("E4" peak),
and has an average DAR of 3.0.
[0526] As will be shown below in Example 16, ABBV-399 has shown
anti-cancer effects in a Phase I clinical trial at a dose of 2.7
mg/kg Q3W. A dose escalation to 3 mg/kg is also proposed herein to
identify the maximum tolerated dose for Phase II studies keeping in
mind that brentuximab vedotin and DCDT2980S (an MMAE ADC targeting
CD22) have been tolerated at 1.8 and 2.4 mg/kg but not 2.7 or 3.2
mg/kg, respectively. Based on considerations of drug antibody ratio
(DAR; MMAE loading per antibody molecule), ABBV-399 with a DAR of
3.1 may be potentially more tolerable than brentuximab vedotin
which has an approximate DAR of 4.
Example 4. Preparation of ABT700-PBD Antibody Drug Conjugate
[0527] ABT-700 (S238C)-PBD is comprised of two PBD drug-linker
molecules conjugated to cys engineered mAb ABT-700. The PBD synthon
vaPBD was conjugated to the ABT-700 (S238C if using Kabat, S239C if
using the EU numbering system) antibody. The conjugation process
consists of a quantitative reduction of the engineered and
interchain disulfides. This takes place through reduction of the
interchain disulfides, quantitative oxidation, and conjugation with
excess PBD drug linker. The reduction mixture is then purified to
remove the excess reagent and its byproducts, followed by
quantitative oxidation of the interchain disulfides and then
conjugation with excess PBD drug-linker. After quenching, the
reaction mixture is purified and buffer-exchanged to yield ABT-700
(S238C)-PBD. Reaction parameters have been identified to provide a
conjugate with >80% DAR2 drug loading.
##STR00027##
Example 5. ABBV-399 Binds to Recombinant and Cellular cMet In
Vitro
Binding ELISA, Cell Binding Assay and Fluorescence-Activated Cell
Sorting (FACS) Analysis
[0528] 96-well plates (Costar #3369) were coated with 100
.mu.L/well of mouse anti-His antibody (Invitrogen #37-2900) at 1
.mu.g/mL in PBS pH7.4 at 4.degree. C. overnight, and then blocked
using Superblock (Pierce, #37535) for one hour at room temperature.
Plates were washed 4 times with PBST and then incubated with 100
.mu.L of recombinant human cMet extracellular domain (rh-cMet
ECD-6His) ("6His" disclosed as SEQ ID NO:100) at 2 .mu.g/mL in 10%
Superblock in PBST for 1 h at room temperature. Plates were washed
4 times with PBST and then incubated with ABT-700 or control human
IgG in serial dilutions in 10% Superblock in triplicate wells at
room temperature for 1 h. Plates were washed 4 times with PBST and
then incubated with 100 .mu.L of 1:15,000 goat anti-human IgG-HRP
(Thermo-scientific Pierce, Cat#31412) at room temperature for 1 h.
Plates were washed 4 times in PBST and 100 .mu.L of TMB (Pierce,
#34028) was added to each well and incubated at room temperature
until color developed (approximately 10 min). Reactions were
stopped by addition of 2N sulfuric acid (Mallinckrodt chemicals,
Cat#H381-05) and optical density (OD) was read at 450 nm.
[0529] The binding of ABBV-399 to surface cMet on a panel of human
cancer cells was determined by fluorescence-assisted cell sorting
(FACS) analysis. For cellular cMet binding studies, cells were
harvested from flasks when approximately 80% confluent using Cell
Dissociation Buffer (Invitrogen #13151-014 or #13150-016). Cells
were washed once in PBS/1% FBS (FACS buffer), resuspended at
1.5-2.times.106 cells/mL in FACS buffer and transferred to a round
bottom 96-well plate (BD Falcon #3910) at 100 .mu.L/well. Ten .mu.L
of a 10.times. concentration of ABT-700, ABBV-399, or controls was
added and plates were incubated at 4.degree. C. for two hours.
Wells were washed twice with FACS buffer and resuspended in 50
.mu.L of 1:500 anti-human IgG Ab (AlexaFluor 488, Invitrogen
#11013) diluted in FACS buffer. Plates were incubated at 4.degree.
C. for one hour, washed twice with FACS buffer. Cells were
resuspended in 100 .mu.L of PBS/1% formaldehyde and analyzed on a
Becton Dickinson LSRII flow cytometer.
[0530] ABBV-399 is reactive with the recombinant form of the human
cMet extracellular domain (ECD, residues 25-932) as determined by
enzyme-linked immunosorbent assay (ELISA), using a routine method
for apparent affinity measurement, as known and available to one of
ordinary skill in the art. ABBV-399 binds the human cMet ECD with
an apparent affinity (EC.sub.50) of 0.30 nM (TABLE 6), similar to
ABT-700 (EC.sub.50 of 0.22 nM) (TABLE 6).
[0531] ABBV-399 displayed binding affinity of 0.2 to 1.5 nM (TABLE
6) to tumor cells including NCI-H441, NCI-H292, and NCI-H1650 lung
cancer cells and Hs746T, IM-95, and SNU-5 gastric cancer lines.
This assay was conducted by a routine for apparent affinity
measurement, as known and available to one of ordinary skill in the
art.
TABLE-US-00028 TABLE 6 Binding affinity of ABBV-399 to recombinant
and cellular cMet ABBV-399 ABT-700 (EC.sub.50 nmol/L) (EC.sub.50
nmol/L) cMet ECD.sup.a by ELISA.sup.b 0.30 0.22 Cellular cMet by
FACS.sup.c Hs746T 0.4 +/- 0.1 0.4 +/- 0.1 SNU-5 1.4 +/- 0.4 1.6 +/-
1.1 IM-95 1.5 +/- 0.9 1.8 +/- 0.4 NCI-H820 0.2 +/- 0.1 0.3 +/- 0.2
NCI-H441 1.0 +/- 0.6 1.1 +/- 1.1 NCI-H1573 0.6 +/- 0.1 0.4 +/- 0.1
NCI-H1650 0.3 +/- 0.2 0.4 +/- 0.2 .sup.aExtracellular domain
(residues 25-932 of cMet) .sup.bEC.sub.50 values derived from ELISA
in which cMet ECD was captured on the plate via a His tag. Values
are the average of six experiments. .sup.cEC.sub.50 values derived
from FACS analysis of ABBV-399 on cancer cell lines. Values are the
average of at least two experiments, +/- the standard
deviation.
Example 6. In Vitro Potency of ABBV-399 Against Tumor Cell
Lines
Cytotoxicity Assay
[0532] Tumor cells were plated at 2000-5000 cells/well in 180 .mu.L
growth medium containing 10% FBS in 96-well plates, and cultured at
37.degree. C. in a humidified incubator with 5% CO2. The following
day, titrations of antibodies or ADCs in 20 .mu.L were added and
cells were incubated for 6 days. Cell viability was determined
using a CellTiter-Glo Luminescent Cell Viability Assay (Promega)
according to the manufacturer's instructions. A non-binding,
irrelevant negative control ADC conjugated to MMAE was also
included in all assays to confirm that cell killing was antigen
dependent.
[0533] ABBV-399 inhibited proliferation of cancer cells that
over-express cMet, including the MET-amplified cell lines Hs746T
and SNU-5 gastric cancer cells (FIG. 4). As a comparison, ABT-700
inhibited proliferation of cells with MET amplification (FIG. 4A
and FIG. 4B) but not cell lines without MET amplification, i.e.,
the NCI-H820 and NCI-H441 (FIG. 4C and FIG. 4D).
Determination of Receptor Density
[0534] cMet cell surface density (antigen binding capacity per
cell) was determined by indirect immunofluorescence staining of
cell surface antigens on cultured cells using QIFIKIT (Dako).
Briefly, cells were harvested from a culture flask as described
above for FACS analysis, added to a round bottom 96-well plate at
100 .mu.L/well and incubated at 4.degree. C. with 3 .mu.g/mL cMet
antibody m224G11. Wells, treated with an irrelevant mouse
monocloncal antibody of the same isotype mIgG1 at 3 .mu.g/mL, were
included as controls. Following a one hour incubation with primary
antibody, cells were centrifuged for 3 minutes at 300.times.g,
washed twice with FACS buffer, and incubated for one hour at
4.degree. C. with 100 .mu.L of the QIFIT-provided FITC conjugated
antibody diluted 1:50 in FACS buffer. Cells were centrifuged for 3
minutes at 300.times.g, washed twice with FACS buffer, and fixed
with 100 .mu.L/well of 1% formaldehyde in PBS. For the indirect
immunofluorescence staining of the QIFIKIT beads, 100 .mu.L of
resuspended beads from Vial 1 and Vial 2 were added to separate
wells, centrifuged for 3 min at 300.times.g, washed once with FACS
buffer and fixed with 100 .mu.L/well of 1% formaldehyde in PBS.
Data was acquired on a Becton Dickinson LSRII flow cytometer and
Geomean values for the 5 bead populations were recorded and used to
calculate a standard curve based on the lot specific antibody
molecules per bead. The standard curve was used to assign ABC
(Antibody Binding Capacity or number of receptors) to stained cell
samples.
[0535] ABBV-399 is cytotoxic to cancer cells that over-express
cMet. To determine the correlation of cMet expression level to
sensitivity to ABBV-399, the in vitro analysis was expanded to
include a panel of 16 cell lines. These included 6 NSCLC lines
(A549, NCI-H1573, NCI-H820, NCI-H441, and NCI-H1650, 4 gastro
esophageal cancer lines (Hs746T, SNU-5, SNU-620, and IM-95), 2 CRC
lines (SW-48 and HT-29), 2 breast cancer lines (MDA-MB-231 and
MCF-7), the KP4 pancreatic cancer line, and the U-87 MG
glioblastoma cancer line. Additional NSCLC cell lines (EBC-1,
NCI-H226, SW900, HCC15, SK-MES-1, and NCI-H1702) were also tested
and are shown in Table 7A.
TABLE-US-00029 TABLE 7A Cytotoxicity IC.sub.50 (nM) cMet ABBV- ABT
MMAE/ receptors/cell 399 700-PBD PBD NSCLC cell line H820 (Adeno)
320,000 0.1 0.02 5 H441 (Adeno) 197,000 0.06 0.003 20 H1573 (Adeno)
116,000 18.3 0.07 261 H1650 (Adeno) 55,000 47.9 0.4 120 A549
(Adeno) 43,000 1.6 0.1 16 EBC-1 (Squamous, amp) 233,000 0.06 0.095
0.6 H226 (Squamous) 114,000 same as 0.04 n/a control SW900
(Squamous) 63,000 7.5 0.02 375 HCC15 (Squamous) 59,000 same as
0.003 n/a control SK-MES-1 (Squamous) 39,000 same as 0.17 n/a
control H1703 (Squamous) 23,000 same as 0.7 n/a control Other cell
lines Hs746T (Ga, amp) 350,000 0.11 0.018 6.1 BT-20 (Br) 41,000
0.23 0.1 2.3 U87MG (GBM) 22,000 1.9 0.21 9 M059J (GBM) 87,000 3.6
0.03 120 U118MG (GBM) 12,500 0.54 0.2 2.7 KP4 (Pa) 15,000 2.9 0.02
145 SW48 (CRC) 26,000 same as 0.0029 >1000 control NHBE (normal
bronchial 40,000 none none n/a epithelial)
[0536] FACS analysis demonstrated that these cell lines possess a
range of cMet expression levels as quantified via cMet antibody
binding capacity representing the number of cell surface cMet
molecules (TABLE 7B). Sensitivity to ABBV-399 in the cell
proliferation assay was quantified as maximal killing and IC.sub.50
(TABLE 7B). These data suggest that there is a threshold level of
cMet expression required for significant killing by ABBV-399.
Exceptions to this were the cell lines known to have an autocrine
HGF loop, such as IM 95, KP4, and U-87 MG, in which lower cMet
expression levels were sufficient for ABBV-399 to exert significant
cytotoxicity.
TABLE-US-00030 TABLE 7B cMet Expression on Tumor Cells In Vitro and
Sensitivity to ABBV-399 cMet Maximal ABBV-399 Expression.sup.a
Killing.sup.b IC.sub.50.sup.c Lung Cancer A549 43,000 22% 1.6 +/-
1.1 NCI-H1573 115,667 18% 18 +/- 14 NCI-H820 320,000 87% 0.20 +/-
0.07 NCI-H441 197,000 56% 0.06 +/- 0.05 NCI-H1650 4,500 13% 47.9
+/- 8.5 EBC-1 233,231 96% 0.06 +/- 0.03 Gastric Cancer Hs746T
350,000 87% 0.11 +/- 0.06 SNU-620 230,000 80% 0.17 +/- 0.08 SNU-5
291,000 85% 0.28 +/- 0.07 IM-95 21,500 53% 1.7 +/- 0.9 Colorectal
Cancer SW48 25,500 0% NA HT-29 161,438 70% 9.0 +/- 1.4 Breast
Cancer MDA-MB-231 30,500 0% NA MCF-7 8,300 0% NA Pancreatic Cancer
KP4 15,300 53% 2.9 +/- 1.9 Glioblastoma U-87MG 22,000 30% 1.9 +/-
0.1 Non-tumor Cell Lines NHBE (bronchial 40,085 10% NA epithelial)
HUVEC (vascular 15,790 6% NA endothelial HMEC (mammary ND 0% NA
epithelial) PrEC (prostate 64,853 0% NA epithelial) NHDF (dermal
1,602 0% NA fibroblasts) .sup.aApproximate number of cMet molecules
on cell surface determined by FACS analysis as antibody binding
capacity for m224G11 (the murine parent of ABT-700) binding at 10
.mu.g/mL. .sup.bRelative to untreated control at .ltoreq.1 .mu.g/mL
in a six day proliferation.
Example 7. ABT700-PBD ADC Inhibits Tumor Cell Proliferation in a
Broad Panel of Cell Lines
[0537] Tumor cells were plated at 2000-5000 cells/well in 180 .mu.L
growth medium containing 10% FBS in 96-well plates, and cultured at
37.degree. C. in a humidified incubator with 5% CO2. The following
day, titrations of ADCs in 20 .mu.L were added and cells were
incubated for 6 days. Cell viability was determined using a
CellTiter-Glo Luminescent Cell Viability Assay (Promega) according
to the manufacturer's instructions. A non-binding, irrelevant
negative control ADC conjugated to MMAE was also included in all
assays to confirm that cell killing was antigen-dependent.
[0538] The results are shown in FIG. 5. Both cMet ADCs were active
against a diverse panel of tumor types, with varying levels of cMet
expression (high/low) and gene amplification (amp). The MMAE/PBD
column indicates how much more MMAE ADC is required to give the
same cytotoxic activity as that achieved with the PBD ADC. In most
cell lines, the PBD ADC is significantly more potent than the MMAE
conjugate.
Example 8. ABT700-PBD ADC is Active In Vitro Against Human
Colorectal Cancer Cell Lines
[0539] Tumor cells were plated at 2000-5000 cells/well in 180 .mu.L
growth medium containing 10% FBS in 96-well plates, and cultured at
37.degree. C. in a humidified incubator with 5% CO2. The following
day, titrations of ADCs and free drug (PBD and MMAE) in 20 .mu.L
were added and cells were incubated for 6 days. Cell viability was
determined using a CellTiter-Glo Luminescent Cell Viability Assay
(Promega) according to the manufacturer's instructions. A
non-binding, irrelevant negative control ADC conjugated to MMAF
(Ab095 MMAF) was also included in all assays to confirm that cell
killing was antigen-dependent. The cetuximab-MMAE ADC is a positive
control. Receptor density levels were calculated as described in
Example 6.
[0540] The results are show in FIG. 6A and FIG. 6B. ABT700-PBD is
active against a variety of colorectal cancer cell lines, including
those with low levels of cMet receptors on the cell surface (e.g.,
SW48, FIG. 6B). A cMet gene-amplified cell line on average has
200-300K receptors per cell. The activity of the ABBV-399 ADC is
also shown for comparative purposes. Where no results are entered,
no activity was observed. In general, ABT700-PBD is more active
than ABBV-300 in colorectal cancer cell lines.
Example 9. ABT700-PBD ADC is Active In Vitro Against Human Brain
Cancer Cell Lines
[0541] Tumor cells were plated at 2000-5000 cells/well in 180 .mu.L
growth medium containing 10% FBS in 96-well plates, and cultured at
37.degree. C. in a humidified incubator with 5% CO2. The following
day, titrations of ADCs and free drug (PBD and MMAE) in 20 .mu.L
were added and cells were incubated for 6 days. Cell viability was
determined using a CellTiter-Glo Luminescent Cell Viability Assay
(Promega) according to the manufacturer's instructions. A
non-binding, irrelevant negative control ADC conjugated to MMAF
(Ab095 MMAF) was also included in all assays to confirm that cell
killing was antigen-dependent. Receptor density levels were
calculated as described in Example 6. A cMet gene-amplified cell
line on average has 200-300K receptors per cell.
[0542] The results are show in FIG. 7. ABT700-PBD is active against
a variety of brain cancer cell lines, including those with low
levels of cMet receptors on the cell surface (e.g., SW48, FIG. 6B).
The activity of the ABBV-399 ADC is also shown for comparative
purposes. Where no results are entered, no activity was observed.
In general, ABT700-PBD is more active than ABBV-300 in brain cancer
cell lines.
Example 10. ABT700-PBD ADC is Active In Vivo Against Human
Colorectal Tumor Xenografts
[0543] The in vivo efficacy of ABT-700, ABBV-399 and ABT-700 PBD
were evaluated in mice transplanted with SW-48 colorectal cells
(cMet IHC 1+). The experiments were done essentially as described
in Example 13 below.
[0544] ADCs or antibodies were administered every seven days at the
doses shown (mg/kg). ABT-700 PBD is superior to ABBV-399 in low
cMet expressor SW-48 xenografts. See FIG. 8.
Example 11. ABBV-399 and ABT700-PBD ADCs are Active In Vivo Against
Human NSCLC Patient-Derived Xenografts
[0545] Efficacy of ABBV-399 ABT700-PBD ADCs was determined in
xenografts derived from non-small cell lung and colorectal cancer
patients. Tumor fragments of 3 to 5 mm.sup.3 at passage 3 (P3) were
implanted subcutaneously in the right rear flank of NSG mice (The
Jackson Laboratory) with a trochar. ABBV-399 and ABT-700 PBD were
administered every seven days for a total of six doses. Numbers in
parentheses represent dose administered in mg/kg. For all groups,
tumor volumes were plotted only for the duration that allowed the
full set of animal to remain on study. If animals had to be taken
off study, the remaining animals were monitored for tumor growth
until they reached defined end-points. Tumor growth delay (TGD)
results are shown in Table 8.
TABLE-US-00031 PDX TGD.sup.1 (%) TGD (%) # Indication Model
ABBV-399 ABT-700 PBD 1 Colorectal CTG-0440 12 0 2 Colorectal
CTG-0084 0 0 3 Colorectal CTG-0419 54 54 4 Colorectal CTG-0117 0 54
5 Colorectal CTG-0387 0 63 6 Colorectal CTG-0058 71 80 7 Colorectal
CTG-0115 114 114 8 Colorectal CTG-0796 78 122 9 Colorectal CTG-0382
126 126 10 Colorectal CTG-0062 0 186 11 Colorectal CTG-0358 94 250
12 Colorectal CTG-0406 0 271 13 Colorectal CTG-0652 350 350 14 NSCL
CTG-0176 71 79 15 NSCL CTG-0363 0 125 16 NSCL CTG-0164 25 136 17
NSCL CTG-0165 170 170 18 NSCL CTG-0178 0 200 19 NSCL CTG-0162 88
288 20 NSCL CTG-0159 288 288 21 NSCL CTG-0170 336 336 22 NSCL
CTG-0167 0 445 .sup.1Tumor growth delay (TGD), expressed as a
percentage, is the difference of the median time of the test
article treated group tumors to reach 1 cm.sup.3 as compared to the
control group.
[0546] Graphs are shown for three different human tumor xenografts
with relatively low (CTG-0363), intermediate (CTG-0159), and high
(CTG-0170) levels of expression of cMet mRNA, a surrogate for cMet
protein levels on the cell surface (FIG. 9A, FIG. 9B, and FIG. 9C,
respectively). The tumor response to each ADC is dependent on the
cMet levels. The ABT700-PBD ADC is more active than ABBV-399, at
about 1/10 of the dose.
Example 12. ABBV-399 are Active In Vivo Against Human NSCLC
Patient-Derived Xenografts
[0547] For the LG0703 and LG1049 patient-derived xenograft models
(The Jackson Laboratory, Sacramento, Calif.), efficacy of ABBV-399
was determined in xenografts derived from non-small cell lung
cancer patients. Tumor fragments of 3 to 5 mm.sup.3 at passage 3
(P3) were implanted subcutaneously in the right rear flank of NSG
mice (The Jackson Laboratory) with a trochar. For all groups, tumor
volumes were plotted only for the duration that allowed the full
set of animal to remain on study. If animals had to be taken off
study, the remaining animals were monitored for tumor growth until
they reached defined end-points. Efficacy is depicted on a
Kaplan-Meier plot for (A) LG0703 and (B) LG1049 models as fractions
reaching the indicated tumors volumes following therapy. In both
models, ABBV-399 and control agents were administered every four
days for a total of six doses. In the LG1049 model, ABT-700 was
administered every seven days for a total of six doses. Numbers in
parentheses represent dose administered in mg/kg.
[0548] The ABBV-399 ADC is more active than ABT-700 alone. See
FIGS. 10A-10B.
Example 13. ABBV399, Alone and in Combination, Inhibits Tumor
Growth in cMet-Overexpressing Tumors in Animal Models
[0549] ABBV-399 has shown robust and reproducible antitumor effects
in a variety of xenograft models including gastric cancer, NSCLC
and glioblastoma multiforme models. The activity in tumors is in
part based on delivery of the MMAE cytotoxic payload. In addition,
ABBV-399 may also have antitumor activity through inhibition of
both HGF-dependent and -independent cMet signaling and antibody
mediated effector function.
[0550] The in vivo efficacy of ABBV-399 was evaluated in mice
transplanted with (FIG. 11A) Hs746T gastric cancer, (FIG. 11B)
NCI-H441 lung cancer cells, and (FIG. 11C) SW-40 colorectal cancer
cells. Female SCID, SCID-Beige and nude mice were obtained from
Charles River (Wilmington, Mass.) and housed at ten mice per cage.
The body weight upon arrival was 20-22 g. Food and water were
available ad libitum. Mice were acclimated to the animal facilities
for a period of at least one week prior to commencement of
experiments. Animals were tested in the light phase of a 12-hr
light: 12-hr dark schedule (lights on at 06:00 hours). All
experiments were conducted in compliance with AbbVie's
Institutional Animal Care and Use Committee and the National
Institutes of Health Guide for Care and Use of Laboratory Animals
Guidelines in a facility accredited by the Association for the
Assessment and Accreditation of Laboratory Animal Care.
[0551] To generate xenografts, a suspension of viable tumors cells
mixed with an equal amount of Matrigel (BD Biosciences) was
injected subcutaneously into the flank of 6- to 8-week old mice.
The injection volume was 0.2 mL composed of a 1:1 mixture of S-MEM
and Matrigel (BD Biosciences). Tumors were size matched at
approximately 200-250 mm3 unless otherwise indicated. Therapy began
the day of or 24 h after size matching the tumors. Mice weighed
approximately 25 g at the onset of therapy. Each experimental group
included 8-10 animals. Tumors were measured two to three times
weekly. Measurements of the length (L) and width (W) of the tumor
were obtained via electronic calipers and the volume was calculated
according to the following equation: V=L.times.W2/2. Mice were
euthanized when tumor volume reached a maximum of 3,000 mm3 or upon
presentation of skin ulcerations or other morbidities, whichever
occurred first. For Hs746T, ABT-700 was administered every seven
days while the ABBV-399 was administered every four days. For
NCI-H441 xenografts, both ABT-700 and ABBV-399 were administered
every four days for a total of six doses. Numbers in parentheses
represent dose administered in mg/kg and arrows indicate days of
administration. In both cancer types, the ABBV-399 ADC is more
active than ABT-700 alone, and the effect is dose-dependent (FIG.
11A and FIG. 11B).
[0552] (FIG. 11C) Combination efficacy of ABBV-399 and FOLFIRI was
determined using SW-48 human colorectal cancer xenografts.
5-Fluorouracil (APP Pharmaceuticals, Schaumburg, Ill.), irinotecan
(Hospira, Lake Forest, Ill.) were obtained as solutions and diluted
with 0.9% Sodium Chloride for Injection (USP), and leucovorin
calcium (Fluka Chemical Corp., Milwaukee, Wis.) was obtained as a
salt and re-constituted with saline before dosing. Standard of care
agents 5-fluorouracil (50 mg/kg), and irinotecan (30 mg/kg) were
administered intravenously and leucovorin (25 mg/kg) was
administered orally on Q7Dx5 regimen (FOLFIRI). IgG control, Ig
MMAE and ABBV-399 were administered intraperitoneally every seven
days. Numbers in parentheses represent dose administered in mg/kg
and arrows indicate days of administration. The ABBV-399+ FOLFIRI
combination is effective in SW-48 colon cancer xenografts.
Example 14. ABBV-399 Efficacy Against Human Tumor Xenograft Models
Refractory to ABT-700
[0553] ABBV-399 efficacy was evaluated in mice xenotransplanted
with parental Hs746T alone (FIG. 12B) or following relapse upon
treatment with ABT-700 (FIG. 12A and FIG. 12B). FIG. 12C evaluates
ABBV-399 efficacy following relapse upon treatment with ABT-700 in
mice senografts transplated with EBC-1 xenograft tumors. Numbers in
parentheses represent dose administered in mg/kg and arrows
indicate days of administration. Tumor volumes are depicted as
mean.+-.S.E.M.
[0554] Efficacy of ABBV-399 was evaluated in a gastric carcinoma
model (Hs746T) and a lung squamous cell carcinoma model (EBC-1)
that were made refractory to ABT-700 by repeated exposure to the
antibody in vivo (Hs746T ABT-700R and EBC-1 ABT-700R). Initially,
treatment of xenografts derived from the parental Hs746T with
ABT-700 resulted in tumor stasis followed by relapse (FIG. 12A;
blue line). Treatment of these relapsed tumors (red line) with
ABBV-399 led to regression (FIG. 12A, red line). In contrast,
Hs746T ABT-700R xenografts were refractory to ABT-700 treatment
with quick tumor outgrowth on therapy (FIG. 12B; blue line). When
these refractory tumors reached a mean cohort size of approximately
1,000 mm.sup.3, treatment with ABBV-399 resulted in tumor
regression (FIG. 12B; red line) followed by eventual outgrowth.
Treatment of Hs746T ABT-700R of approximately 300 mm.sup.3 with
ABBV-399 resulted in complete tumor regression (FIG. 12B). Similar
results were observed subsequent to treatment of the
ABT-700-resistant cell line EBC-1 with ABT-700 followed by
ABBV-399. These results suggest that efficacy of ABBV-399 is
independent of response to ABT-700, at least for cell lines with
amplified cMet.
Example 15. Formulation of ABBV-399 for Clinical Use
[0555] ABBV-399 Drug Product is provided as a sterile lyophilized
powder for reconstitution. Each vial contains 100 mg of ABBV-399.
After reconstitution with 5.0 mL of sterile water for injection,
the final concentration of ABBV-399 is 20 mg/mL. In addition to
ABBV-399, the formulation contains sucrose, polysorbate 80, and is
in a histidine buffer. Prior to administration, ABBV-399 is further
diluted in normal saline to a concentration range between 1-10
mg/mL, depending on the weight of the subject.
Example 16. Phase I Open-Label, Dose-Escalation and Expansion Study
of ABBV-399, an Antibody Drug Conjugate (ADC) Targeting cMet, in
Patients (Pts) with Advanced Solid Tumors
16.1. Summary
[0556] An ongoing Phase 1/1b open-label study is evaluating the
safety, pharmacokinetics (PK), and preliminary efficacy of ABBV-399
in subjects with advanced solid tumors. The study consists of two
phases: (1) a Dose-Escalation/Expansion Phase (Monotherapy) and a
Combination Therapy Phase. Subjects with advanced solid tumors with
cMet overexpression, MET exon 14 mutation or MET amplification
possibly including, but not limited to NSCLC, esophageal/gastric,
CRC or head and neck cancer may be enrolled in the dose expansion
and combination therapy phases of the study.
[0557] The monotherapy phase of the study evaluated the safety and
pharmacokinetic profile of ABBV-399 when administered intravenously
in approximately 24 to 42 subjects following the dose-escalation
scheme depicted in FIG. 13. ABBV-399 was administered at escalating
dose levels starting from 0.15 mg/kg in 21-day dosing cycles. Based
on safety and PK data from dosing every 21 days, ABBV-399 will also
be administered every 14 days on a 28-day schedule. Three to 6
subjects will be enrolled in each cohort and dosed once every 21
(one dose per 21-day Cycle) or 14 (2 doses per 28-day Cycle) days
until disease progression or unacceptable toxicity to determine the
maximum tolerated dose (MTD) or maximally administered dose (MAD).
Dose limiting toxicity (DLT) definitions will be used to make
decisions regarding dose-escalation. Based on available safety, PK,
and pharmacodynamic (PDx) data, up to 40 subjects will be enrolled
in an expansion cohort that will further evaluate ABBV-399 at a
dose level which is at or below the MTD or MAD. On the
dose-expansion, subjects with advanced solid tumors with cMet
overexpression, MET exon 14 mutation or MET amplification will be
enrolled.
[0558] In the combination therapy phase, up to 18 subjects will be
enrolled into each of the combination therapy arms as described
below: [0559] Combination cohort A: Subjects eligible to receive
ABBV-399 plus erlotinib [0560] Combination cohort B: Subjects
eligible to receive ABBV-399 plus cetuximab [0561] Combination
cohort C: Subjects eligible to receive ABBV-399 plus bevacizumab
[0562] Combination cohort D: Subjects eligible to receive ABBV-399
plus nivolumab
[0563] All subjects will be evaluated for safety and tolerability
of the regimen, PK profile of ABBV-399 and preliminary evidence of
efficacy. On the combination therapy arms, subjects with advanced
solid tumors with cMet overexpression, MET exon 14 mutation or MET
amplification may be enrolled. Subjects on combination arms A, B or
C will be assigned to a 14-day or 21-day ABBV-399 dosing schedule
whereas subjects on arm D will receive ABBV-399 on a 14-day
schedule to coincide with nivolumab every 14-day dosing.
[0564] Archival tumor tissue is required for enrollment on this
study. Tumor tissue will be analyzed for cMet protein, MET copy
number and other biomarkers. Expression of cMet will be determined
by an immunohistochemistry assay; amplification of MET will be
determined by fluorescence in situ hybridization (FISH) or DNA
sequencing of tumor or circulating tumor DNA.
16.2. Patient Selection: Diagnosis and Main Criteria for
Inclusion/Exclusion
[0565] Some of the Criteria for Inclusion for ABBV-399 Monotherapy
Dose-Escalation/Expansion: [0566] Subject must be .gtoreq.18 years
of age [0567] Subject with advanced solid tumor including but not
limited to non-small cell lung cancer (NSCLC), colorectal, breast,
ovarian, esophageal/gastric and head and neck cancer. [0568]
Subject must have advanced solid tumor that is not amenable to
surgical resection or other approved therapeutic options that have
demonstrated clinical benefit. [0569] For dose-expansion: Subject
must have tumor with cMet overexpression, MET exon 14 mutation or
MET amplification. [0570] Subject has an Eastern Cooperative
Oncology Group (ECOG) Performance Status of 0 to 2. [0571] Subject
must have measurable disease per RECIST version 1.1
[0572] Additional Inclusion Criteria for Subjects Enrolled on the
Combination Therapy Phase [0573] Subjects in the combination
therapy arms must meet the above inclusion criteria and be eligible
to receive erlotinib, cetuximab, bevacizumab or nivolumab per most
current prescribing information, or at the discretion of the
Investigator.
[0574] Main Exclusion Criteria:
For All Cohorts:
[0575] Subject has received anticancer therapy including
chemotherapy, immunotherapy, radiation therapy, immunotherapy,
biologic, or any investigational therapy within a period of 21
days, or herbal therapy within 7 days prior to the first dose of
ABBV-399. [0576] Palliative radiation therapy for painful bone,
skin or subcutaneous metastases for 10 fractions or less is not
subject to a washout period. [0577] For approved targeted small
molecules, a washout period of 5 half-lives is adequate (no washout
period required for subjects currently on erlotinib). [0578]
Subject has known uncontrolled metastases to the central nervous
system (CNS). Subjects with brain metastases are eligible after
definitive therapy provided they are asymptomatic off steroids and
anticonvulsants for at least 2 weeks prior to first dose of
ABBV-399. [0579] Subject has unresolved clinically significant
adverse events .gtoreq.Grade 2 from prior anticancer therapy except
for alopecia or anemia. [0580] Subject has had major surgery within
21 days prior to the first dose of ABBV-399.
[0581] Additional Exclusion Criteria for Subjects Enrolled on the
Combination Therapy Phase [0582] Subjects enrolled on the
combination therapy phase must satisfy the above exclusion criteria
and also the following: [0583] Subjects may not receive ABBV-399 in
combination with erlotinib, cetuximab, bevacizumab or nivolumab if
they have any medical condition which in the opinion of the
Investigator places the subject at an unacceptably high risk for
toxicities from the combination. [0584] Subjects may not receive
cetuximab if they have K-ras mutation. [0585] Subjects may not
receive bevacizumab if they have squamous NSCLC.
[0586] It is also planned that, in certain studies and for future
clinical use of the anti-cMet ADCs disclosed herein, patients will
be selected on the basis of their cMet expression levels (gene
amplification, membrane cMet) and MET exon 14 mutation. Methods for
assessing each of these markers are provided below.
16.3. Dosing Regimen
Dose-Escalation/Expansion Phase:
[0587] ABBV-399 was administered as an intravenous infusion once
every 21 days until disease progression or intolerable toxicity.
Dosing began at 0.15 mg/kg and escalated to 0.3, 0.6, 1.2, 1.8,
2.4, 3.0 and 3.3 mg/kg in subsequent cohorts as tolerated.
Alternative doses (intermediate or higher) or dosing schedules may
be employed based on clinical safety and PK data. A dose of 2.7
mg/kg was also utilized based on the clinical safety and PK data.
Based on safety and PK data from dosing every 21 days, ABBV-399
will also be administered every 14-days on a 28-day schedule
(starting dose of 1.6 mg/kg). ABBV-399 has been given over 30.+-.10
minutes. It is not administered as an intravenous push or
bolus.
Combination Therapy Phase:
[0588] ABBV-399 will be combined with standard doses of erlotinib,
cetuximab, bevacizumab or nivolumab starting at an ABBV-399 dose
level below the MTD or MAD and then escalated no higher than MTD or
MAD determined in the monotherapy dose-escalation/expansion. Dose
limiting toxicity definitions will apply to the dose-escalation
portion of each Combination.
TABLE-US-00032 Investigational Product: ABBV-399 Dose: Current dose
2.7 mg/kg of ABBV- 399 for every 21-day dosing 1.6 mg/kg starting
dose of ABBV- 399 every 14-day dosing Dose for subjects with weight
> 100 kg should be calculated for 100 kg Mode of Administration:
IV infusion Frequency of Administration Every 21 days (21-day
Cycle) or Every 14 days (28-day Cycle) Reference Therapy: Erlotinib
Dose: 150 mg Mode of Administration: Oral Frequency of
Administration Every day Reference Therapy: Cetuximab Dose: 400
mg/m.sup.2 initial dose over 120 minutes; then 250 mg/m.sup.2 over
60 minutes Mode of Administration: IV infusion Frequency of
Administration Every 7 days Reference Therapy: Bevacizumab Dose:
10-15 mg/kg Mode of Administration: IV infusion Frequency of
Administration Every 21 days (15 mg/kg) or every 14 days (10 mg/kg)
Reference Therapy: Nivolumab Dose: 3 mg/kg Mode of Administration:
IV infusion Frequency of Administration Every 14 days Duration of
Treatment: Subjects with clinical benefit (CR, PR or SD) will be
allowed to continue study treatment with ABBV-399 until disease
progression, intolerable side effects or for up to 24 months.
Subjects with clinical benefit beyond 24 months and able to
tolerate the drug can continue treatment on an extension study.
16.4. Assessments
[0589] Study visits and evaluations will be performed at Screening,
and at least weekly during the first cycle and on Day 1 of each
subsequent cycle. Assessments will include limited physical
examination, hematology, and chemistry tests prior to all study
drug dosing and at Final Visit. ECGs will be collected at
Screening, Cycle 1 Day 1, Cycle 2 Day 1 and at the Final Visit.
Adverse events, laboratory data and vital signs will be assessed
throughout the study.
[0590] Baseline radiographic tumor assessments with CT (or MRI) of
the head, chest, abdomen, and pelvis will be obtained no more than
28 days prior to Cycle 1 Day 1. CT scan (or MRI) will then be
repeated approximately every 6 weeks after start of therapy to
evaluate the extent of tumor burden. Radiographic tumor assessments
will continue until disease progression documented by imaging,
start of a new anti cancer therapy, death or withdrawal of consent.
Response evaluation will be based on RECIST version 1.1. In
addition, the Investigator will evaluate the subject for evidence
of clinical disease progression at each visit.
16.4.1. Biomarker Assessments
[0591] Archival tumor tissue (most recent sample is preferred) is
required for enrollment on this study. If a subject has local or
central lab data showing cMet overexpression, MET exon 14 mutation
or MET amplification and no archival tumor tissue available, the
subject may be eligible after discussion with the Medical Monitor.
An optional pre- and on-treatment biopsy (any time after the start
of therapy) may be obtained from subjects who consent voluntarily
if it is safe to do so in the judgment of the Investigator.
Institutional procedures should be followed to fix and embed
freshly collected tissue in paraffin. Tumor tissue will be analyzed
for cMet protein, MET copy number and other biomarkers.
[0592] Expression of cMet will be determined by an
immunohistochemistry assay (see Example 17); amplification of MET
will be determined by fluorescence in situ hybridization (FISH) or
DNA sequencing of tumor or circulating tumor DNA (see Example 18).
Biospecimens will be collected at designated time points throughout
the study to conduct research with the intent of identifying
biomarkers associated with subject outcome or to better
characterize the disease.
16.4.2 Criteria for Evaluation
[0593] Efficacy: The efficacy endpoints include objective response
rate (ORR) (determined using RECIST version 1.1), progression-free
survival (PFS), and duration of overall response (DOR). Radiologic
assessments will consist of CT scans (or MRI in subjects who cannot
tolerate contrast) and be performed approximately every 6 weeks
after start of therapy to evaluate the extent of tumor burden.
Radiographic tumor assessments will continue until disease
progression documented by imaging, start of a new anti-cancer
therapy, death or withdrawal of consentResponse evaluations will be
based on Response Evaluation Criteria in Solid Tumors (RECIST) 1.1
Eisenhauer EA, Therasse P, Bogaerts B, et al. New response
evaluation criteria in solid tumors; Revised RECIST guideline
(version 1.1). Eur J Cancer. 2009;45:228-47. Pharmacokinetic: Blood
samples for assay of ABBV-399, Total ABT-700 and free MMAE drug
levels will be used to evaluate PK parameters. Blood samples for
antidrug antibody (ADA) and neutralizing ADA (nADA) will be
collected at designated time points throughout the study and
ADA/nADA will be correlated with PK and safety outcomes. Safety:
Adverse events, laboratory profiles, physical exams, and vital
signs will be assessed throughout the study. Adverse events will be
graded according the National Cancer Institute Common Terminology
Criteria for Adverse Events (NCI CTCAE). version 4.03.
Statistical Methods:
[0594] Efficacy: Analyses of ORR, PFS, and DOR will be performed
for all evaluable dosed subjects. Pharmacokinetic: Scrum
concentrations of ABBV-399 and PK parameter values will be
tabulated for each subject and each regimen, and summary statistics
will be computed for each sampling time and each parameter. Safety:
The safety of ABBV-399 will be assessed by evaluating the study
drug exposure, adverse events, serious adverse events, all deaths,
as well as changes in laboratory determinations and vital sign
parameters.
Efficacy
[0595] All efficacy analyses are exploratory in nature. The
exploratory efficacy endpoints include objective response rate
(ORR) (determined using RECIST version 1.1) progression-free
survival (PFS), and duration of response (DOR).
[0596] Objective Response Rate
[0597] Objective response rate (ORR) is defined as the proportion
of subjects with a confirmed partial or complete response to the
treatment. The ORR for each treatment cohort will be estimated with
all the sites pooled. The 2-sided 80% confidence intervals of ORR,
as well as of CR and PR rates, will be provided based on the
Clopper-Pearson (exact) Method.
[0598] Progression-Free Survival
[0599] For each subject, the PFS time is defined as the time from
the subject's first dose of ABBV-399 to either the subject's
disease progression or death, whichever occurs first. Under the
situation that neither event occurs, the PFS time will be censored
at the date of last disease assessment. All subjects will be
followed to disease progression or up to 24 months for those who
continue study drug.
[0600] The PFS time for the treatment cohorts will be summarized by
Kaplan-Meier estimates. The mean and median time with 2-sided 80%
confidence intervals will be calculated to describe the
time-to-event distributions.
[0601] Duration of Response
[0602] The duration of response (DOR) for a subject is defined as
the time from the subject's initial objective response to study
drug therapy to disease progression or death, whichever occurs
first. If the dates of disease progression or death are not
available, the DOR will be censored at the date of last tumor
assessment. The DOR will be analyzed in the same fashion as for
PFS.
[0603] Tumor Assessments
[0604] Baseline radiographic tumor assessment must be performed
within 28 days prior to Cycle 1 Day 1 and will consist of CT (or
MRI or non-contrast CT in subjects who cannot tolerate contrast) of
the head, chest, abdomen, and pelvis (and other tumor involved
regions as clinically indicated). In general, imaging while on
therapy with ABBV-399 will occur approximately every 6 weeks
(imaging may be obtained up to 7 days prior to the next dose of
drug). For the ABBV-399 combination with nivolumab, the first
planned on-therapy imaging will occur at approximately 9 weeks with
subsequent imaging approximately every 6 weeks. Imaging must be
done prior to administering the next scheduled dose of ABBV-399.
Subjects who discontinue study drug for any reason other than
progressive disease demonstrated by imaging will be followed until
they have progressive disease documented by imaging or start new
anti-cancer therapy, death or withdraw consent. Imaging will also
be performed at the Final Visit for subjects who have not had
documented radiographic progression by RECIST 1.1 criteria if
clinically warranted. Imaging may also be performed at other times
if the Investigator suspects tumor progression. Imaging of the
brain for metastatic disease will only be repeated if clinically
indicated. The same imaging technique should be used throughout the
study if possible. The tumor assessment performed at Screening will
serve as the baseline for clinical assessment. Changes in
measurable lesions over the course of therapy will be assessed
using RECIST version 1.1, as described below.
[0605] RECIST (Version 1.1) Criteria for Tumor Response
[0606] Response criteria will be assessed using RECIST (version
1.1). Changes in the measurable lesions over the course of therapy
must be evaluated using the criteria listed below.
[0607] a. Eligibility
[0608] Subjects with measurable disease at Baseline can have
objective tumor response evaluated by RECIST criteria. Measurable
disease is defined by the presence of at least one measurable
lesion. If the measurable disease is restricted to a solitary
lesion, its neoplastic nature should be confirmed by
cytology/histology if possible.
[0609] b. Measurability
TABLE-US-00033 Measurable Lesions accurately measured in at least
one Lesions dimension with a minimum size of: longest diameter
.gtoreq. 10 mm (CT scan slice thickness no greater than 5 mm) 10 mm
caliper measurement by clinical exam Non-Measurable All other
lesions, including small lesions (longest Lesions diameter < 10
mm) as well as truly non-measurable lesions. Lesions considered
truly non-measurable include: leptomeningeal disease, ascites,
pleural/pericardial effusion, inflammatory breast disease,
lymphangitic involvement of skin or lung and also abdominal masses
that are not confirmed and followed by imaging techniques.
Measurable To be considered pathologically enlarged and Malignant
measurable, a lymph node must be .gtoreq.15 mm in short Lymph Nodes
axis when assessed by CT scan (CT scan slice thickness recommended
to be no greater than 5 mm). At baseline and in follow-up, only the
short axis will be measured and followed. Non-Measurable
Pathological lymph nodes with .gtoreq.10 to <15 mm Malignant
short axis. Lymph Nodes
[0610] All measurements should be taken and recorded in metric
notation, using calipers if clinically assessed. All baseline
evaluations should be performed as closely as possible to the
beginning of treatment and not more than 4 weeks before the
beginning of the treatment.
[0611] The same method of assessment and the same technique should
be used to characterize each identified and reported lesion at
Baseline and during follow-up.
[0612] Clinical lesions will only be considered measurable when
they are superficial (e.g., skin nodules and palpable lymph nodes)
and .gtoreq.10 mm diameter as assessed using calipers. For the case
of skin lesions, documentation by color photography including a
ruler to estimate the size of the lesion is recommended.
[0613] c. Methods of Measurement
[0614] Conventional CT should be performed with cuts of 5 mm or
less in slice thickness contiguously. This applies to tumors of the
chest and abdomen. A scale should be incorporated into all
radiographic measurements.
[0615] Cytology and histology can be used to differentiate between
partial response (PR) and complete response (CR) in rare cases.
[0616] d. Baseline Documentation of "Target" and "Non-Target"
Lesions
[0617] All measurable lesions up to a maximum of 2 lesions per
organ and 5 lesions in total, representative of all involved organs
should be identified as target lesions and recorded and measured at
Baseline. Tumor lesions situated in a previously irradiated area,
or in an area subjected to other loco-regional therapy, are usually
not considered measurable unless there has been demonstrated
progression in the lesion.
[0618] Lymph nodes merit special mention since they are normal
anatomical structures which may be visible by imaging even if not
involved by tumor. Pathological nodes which are defined as
measurable and may be identified as target lesions must meet the
criterion of a short axis of .gtoreq.15 mm by CT scan. Only the
short axis of these nodes will contribute to the baseline sum. The
short axis of the node is the diameter normally used by
radiologists to judge if a node is involved by solid tumor. Nodal
size is normally reported as two dimensions in the plane in which
the image is obtained (for CT scan this is almost always the axial
plane). The smaller of these measures is the short axis. For
example, an abdominal node which is reported as being 20
mm.times.30 mm has a short axis of 20 mm and qualifies as a
malignant, measurable node. In this example, 20 mm should be
recorded as the node measurement. All other pathological nodes
(those with short axis .gtoreq.10 mm but <15 mm) should be
considered non-target lesions. Nodes that have a short axis <10
mm are considered non-pathological and should not be recorded or
followed.
[0619] A sum of diameters for all target lesions will be calculated
and reported as the baseline sum of diameters. If lymph nodes are
to be included in the sum, then as noted above, only the short axis
is added into the sum. The baseline sum diameters will be used as
reference by which to characterize the objective tumor
response.
[0620] All other lesions (or sites of disease) including
pathological lymph nodes should be identified as non-target lesions
and should also be recorded at Baseline. Measurements of these
lesions are not required, but the presence (stable, increasing or
decreasing) or absence of each should be noted throughout
follow-up.
[0621] e. Evaluation of Target Lesions
Complete Response (CR):
[0622] The disappearance of all target lesions. Any pathological
lymph nodes (whether target or non-target) must have reduction in
short axis to <10 mm.
Partial Response (PR):
[0623] At least a 30% decrease in the sum of diameters of target
lesions, taking as reference the baseline sum diameters.
Progressive Disease (PD):
[0624] At least a 20% increase in the sum of the diameters of
target lesions, taking as reference the smallest sum of diameters
recorded since the treatment started (baseline or after) or the
appearance of one or more new lesions. In addition to the relative
increase of 20%, the sum must also demonstrate an absolute increase
of at least 5 mm.
Stable Disease (SD):
[0625] Neither sufficient shrinkage to qualify for PR nor
sufficient increase to qualify for PD, taking as reference the
smallest sum of diameters since the treatment started (baseline or
after).
Assessment of Target Lesions:
[0626] Lymph nodes identified as target lesions should always have
the actual short axis measurement recorded (measured in the same
anatomical plane as the baseline examination), even if the nodes
regress to below 10 mm on study. This means that when lymph nodes
are included as target lesions, the `sum` of lesions may not be
zero even if complete response criteria are met, since a normal
lymph node is defined as having a short axis of <10 mm. For PR,
SD and PD, the actual short axis measurement of the nodes is to be
included in the sum of target lesions.
[0627] All lesions (nodal and non-nodal) recorded at Baseline
should have their actual measurements recorded at each subsequent
evaluation, even when very small (<5 mm). However, sometimes
target lesions or lymph nodes become too small to measure. If it is
in the opinion of the radiologist that the lesion has likely
disappeared, the measurement should be recorded as 0 mm. If the
lesion is believed to be present, but too small to measure, a
default value of 5 mm should be assigned (as derived from the 5 mm
CT slice thickness). The measurement of these lesions is
potentially non-reproducible; therefore providing this default
value will prevent false responses or progression based upon
measurement error.
[0628] f. Evaluation of Non-Target Lesions
Complete Response (CR):
[0629] The disappearance of all non-target lesions and
normalization of tumor marker level. All lymph nodes must be
non-pathological in size (<10 mm short axis).
Non-CR/Non-PD:
[0630] Persistence of one or more non-target lesion(s) and/or
maintenance of tumor marker level above the normal limits.
Progressive Disease (PD):
[0631] Unequivocal progression of existing non-target lesions.
[0632] In this setting, to achieve `unequivocal progression` on the
basis of non-target disease, there must be an overall level of
substantial worsening in non-target disease such that, even in the
presence of SD or PR in target disease, the overall tumor burden
has increased sufficiently to merit discontinuation of therapy. A
modest `increase` in the size of one or more non-target lesions is
usually not sufficient to qualify for unequivocal progression
status. The designation of overall progression solely on the basis
of change in non-target disease in the face of SD or PR of target
disease will therefore be extremely rare.
[0633] Note: If the subject discontinues treatment for symptomatic
deterioration, every effort should be made to document objective
progression even after discontinuation of treatment.
New Lesions
[0634] The appearance of new malignant lesions denotes disease
progression. While there are no specific criteria for the
identification of new radiographic lesions, the findings of a new
lesion should be unequivocal, i.e., not attributable to differences
in scanning technique, timing of scanning, phase of contrast
administration, change in imaging modality or finding thought to
represent something other than tumor (e.g., some `new` bone lesions
may be simply healing or flare of pre-existing lesions). A lesion
identified on a follow-up study in an anatomical location that was
not scanned at Baseline is considered a new lesion and will
indicate disease progression. An example of this is the subject who
has visceral disease at Baseline and while on study has a CT or MRI
brain ordered which reveals metastases. The subject's brain
metastases are considered evidence of progressive disease even if
he/she did not have brain imaging at Baseline.
[0635] If a new lesion is equivocal (i.e., too small to measure),
continued therapy and follow-up evaluation will clarify if it
represents truly new disease. If repeat scans confirm there is a
new lesion, then progression should be declared using the date of
the initial scan.
16.5. Results
16.5.1. ABBV-399 Monotherapy Dose-Escalation/Expansion Phase (Phase
1):
[0636] In the 3+3 dose escalation design, ABBV-399 was administered
at doses ranging from 0.15 to 3.3 mg/kg once every 21 days to pts
with metastatic solid tumors (NCT02099058). As depicted in FIG. 13,
ABBV-399 was administered as an intravenous infusion once every 21
days until disease progression or intolerable toxicity. Dosing
began at 0.15 mg/kg and escalated to 0.3, 0.6, 1.2, 1.8, 2.4, 3.0
and 3.3 mg/kg in subsequent cohorts as tolerated. A dose of 2.7
mg/kg of ABBV-399 given every 21 days was also evaluated and based
on safety and PK, was chosen as the dose for the expansion cohort.
Based on safety and PK data from dosing every 21 days, ABBV-399
will also be administered every 14-days on a 28-day schedule
(starting dose of 1.6 mg/kg to 2.5 mg/kg in 0.3 mg/kg incremental
increases, i.e., 1.6, 1.9, 2.2, and 2.5 mg/kg). For administration
at 14 or 21 days, ABBV-399 will be given over 30.+-.10 minutes. It
is not administered as an intravenous push or bolus.
[0637] As of Mar. 31, 2016, 48 pts received at least 1 dose of
ABBV-399. Dose-proportional increases of area under the curve for
ABBV-399 and total antibody were observed after single dose
administration. Half-lives for ABBV-399 and total antibody were
approximately 2-4 days. Dose-limiting toxicity of febrile
neutropenia occurred in 1 pt at 3 mg/kg and 1 pt (with septic
shock) at 3.3 mg/kg. The best percent change in target lesions in
patients with at least 1 post-baseline tumor assessment is shown in
FIG. 14. As shown in FIG. 14 (and from data not shown in the
figures), best responses to ABBV-399 monotherapy in all treated
patients were: 3/40 (7.5%) partial response, 20/40 (50%) patients
with stable disease, and 17/40 (42.5%) patients with progressive
disease. RECIST data was not available for eight patients due to
clinical progression (4), adverse events (2), withdrawal of consent
(1) and death due to pneumonia (1). The three patients with a
partial response had cMet overexpressing non-small cell lung cancer
(NSCLC).
[0638] A dose of 2.7 mg/kg was chosen for dose-expansion based
primarily on safety and tolerability. For enrollment in this phase
of the study, NSCLC subjects were screened for cMet overexpression
using an IHC assay utilizing the CONFIRM anti-total cMet (SP44)
Rabbit Monoclonal Primary Antibody kit purchased from Ventana (REF
#790-4430). Tissue samples were scored by determining the
percentages of target tissue cells staining at various intensity
levels of low to high, i.e. IHC score of 0, 1+, 2+ or 3+ or an
H-score of 0 to 149, 150-224, or 225-300. The scoring can be done
either manually or via the aid of a computer. Details of the IHC
assay and scoring are described in Example 17. The following table
shows the number of NSCLC patients prospectively screened and the
H-score used to assess cMet overexpression:
TABLE-US-00034 H-score H-score H-score Total cMet H- 0-149 150-224
225-300 score .gtoreq. 150 Screened N (%) N (%) N (%) N (%) 91 39
35 17 52 (43%) (38%) (19%) (57%)
[0639] There were no treatment-related deaths. Treatment-related
adverse events occurring in .gtoreq.10% of pts (including all dose
levels and all grades) were fatigue (22.9%), nausea (20.8%),
neuropathy (14.6%), decreased appetite (12.5%), vomiting (12.5%)
and hypoalbuminemia (10.4%). Among 16 patients with cMet+ NSCLC
treated with ABBV-399, the results from 11 are shown in FIG. 15.
FIG. 15 is a waterfall plot showing the best percent change in
target lesion in response to ABBV-399 monotherapy based on
radiographic data. As shown in FIG. 15 (and from data not shown in
the figure) 3/16 treated patients with a partial responses (19%),
6/16 treated patients with stable disease (37.5%), 2/16 treated
patients with radiographic progressive disease (12.5%), and 5
patients with no available imaging due to clinical progression (3),
withdrawal of consent (1) and death due to pneumonia (1).
[0640] FIG. 16 shows the number of weeks that the 16 patients were
on study before clinical progression.
16.5.2. Combination Therapy Phase (Phase 1b):
[0641] Results from a NSCLC combination therapy trial using
ABBV-399 at 2.7 mg/kg once every 21 days and erlotinib 150 mg
administered orally every day are shown in FIG. 17 and FIG. 18.
FIG. 17 is a waterfall plot showing the best percent change in
target lesions for 6 patients treated with ABBV-399 and erlotinib.
As shown in FIG. 17, 2/6 patients achieved a partial response, 1/6
with progressive disease as evidenced by new lesions. FIG. 18 shows
the number of weeks the 6 patients were on study before clinical
progression.
16.5.3. Pre-Treatment Selection for Patients Carrying cMet+ Tumors
with IHC2+/3+ Scores or H-Scores.gtoreq.150 May Significantly
Improve Treatment Outcome
[0642] The pre-clinical results with cell lines and xenograft
models suggest that those with an cMet IHC2+/IHC3+ score will be
more responsive than those with IHC 0/1+. The use of companion
diagnostics to aid in the pre-treatment selection of those patients
with cMet IHC2/3+ or H-score.gtoreq.150 cancers would significantly
improve overall treatment outcomes and spare patients from
treatment that is predicted to be ineffective. As used herein, the
term cMet+ encompasses all tumors that express cMet, regardless of
whether or not the cMet is overexpressed. In some cMet+
embodiments, the cMet is overexpressed. In some cMet+ embodiments,
the cMet is not overexpressed.
[0643] Similarly, the results of this ongoing Phase 1 clinical
trial suggest that H-scores of 150 and above, are linked to and can
be predictive of response to treatment with an anti-cMet ADC,
including ABBV-399.
[0644] Without being bound by any theory, preliminary results
suggest that tumor heterogeneity may be a limiting factor in the
efficacy of ABBV-399. Among those cMet+ tumors with IHC2+ and IHC3+
scores, there are cancer cells that show none to low cMet
expression. Of those, at least some of the cells that are not
killed by a "bystander effect" could repopulate the tumor and
impede tumor response. ABBV-399 could be combined with standard of
care treatments that inhibit or kill low cMet expressing tumor
cells, not limited to targeted agents like erlotinib and
immunotherapies like nivolumab but also standard of care
chemotherapy, preferably with non-overlapping toxicity.
[0645] Table 9 provides clinical results from the ongoing phase 1
trial correlating overall response with H score in NSCLC patients
treated with ABBV-399 as a monotherapy once every two (Q2W) or
three (Q3W) weeks or with ABBV-399 in combination with erlotinib.
The IHC score was obtained using the protocol described in Example
17.
TABLE-US-00035 TABLE 9 Dose (mg/kg) and Frequency of IHC Overall
Subject Administration Tumor Histology Score Response 1 2.7 Q3W
PLUS Adenocarcinoma 295 PR ERLOTINIB (150 mg QD) 2 2.7 Q3W PLUS
adenocarcinoma 250 PR ERLOTINIB (150 mg QD) 3 2.7 Q3W PLUS
adenocarcinoma 270 PR ERLOTINIB (150 mg QD) 4 1.6 Q2W
adenocarcinoma 280 PR 5 1.9 Q2W adenocarcinoma 250 CR 6 2.7 Q3W
PLUS adenocarcinoma 250 PR ERLOTINIB (150 mg QD) 7 2.7 MG/KG Q3W
squamous cell carcinoma 165 PR 8 2.7 MG/KG Q3W squamous cell
carcinoma 185 PR 9 2.7 MG/KG Q3W squamous cell carcinoma 170 PR
[0646] As shown in Table 9, four patients with NSCLC
adenocarcinomas treated with 2.7 mg/kg ABBV-399 once every 3 weeks
(Q3W) and erlotinib having IHC scores of 225 or greater achieved
partial responses (PR). Two patients with NSCLC adenocarcinomas
treated once every two weeks with ABBV-399 having IHC scores of 225
or greater achieved either a partial response or a complete
response. Three patients with NSCLC squamous cell carcinomas
treated with 2.7 mg/kg ABBV-399 once every 3 weeks having IHC
scores between 150 to 224 achieved partial responses.
Example 17. cMet Immunohistochemistry Assay and the H-Score: The
"cMet ABBV-ADC Staining Protocol"
[0647] There are various methods available in the art for
evaluating cMet protein expression levels by immunohistochemistry
(IHC). One of ordinary skill in the art would have routinely known
how to use them and adapt them to their particular study. Several
vendors provide cMet staining as a fee-for-service (see, e.g,
Flagship Biosciences L.L.C., ARUP Laboratories, PathGroup Inc.). In
this Phase I study, cMet expression levels were evaluated using the
SP44 anti-cMet mAb from Ventana Medical Systems, more specifically
Ventana's CONFIRM.RTM. anti-total cMet rabbit monoclonal antibody
(Ventana Medical Systems, Inc; cat no. 790-4430), in combination
with a Ventana.RTM. automated slide stainer (BenchMark ULTRA.RTM.)
and a Ventana ultraView.RTM. Universal DAB detection kit (cat. no.
760-500). The stainings and results were processed by Flagship
Biosciences L.L.C. in collaboration with ARUP Laboratories.
Positive control tissues include colon adenocarcinomas and lung
adenocarcinoma. Negative control tissues include Breast ER100
control, Breast ER13781 control, and Hodgkin's lymphoma CD15-5
control. For purposes of this application, including the claims,
the particular assay used in this Phase 1 study is herein referred
to as the "cMet ABBV-ADC staining protocol."
[0648] Patient tumor biopsies were fixed in formalin in PBS and
embedded in paraffin. Slides were cut at 4 microns, allowed to dry,
and then baked for 60 minutes at 60.degree. C. Slides were used
within 2 weeks of cutting. The slides were transferred to a
BenchMark ULTRA.RTM. instrument and the following parameters were
selected:
[0649] Procedure: ultraView.RTM. DAB
[0650] Name: cMet CONFIRM.RTM.
[0651] Paraffin [selected]
[0652] Deparaffinization [selected]
[0653] Cell Conditioning [selected]
[0654] Conditioner #1 [selected]
[0655] [short--8 min Conditioning]
[0656] Mild CC1 [selected]
[0657] [harsh--95 min Conditioning]
[0658] Ab Incubation Temperatures [selected]
[0659] 36.degree. C. Ab [selected
[0660] Antibody [selected]
[0661] PREP KIT # [4430] ** 0 H 16 min
[0662] Counterstain [selected]
[0663] HEMATOXILIN [2021] 4 minutes
[0664] Post Counterstain [selected]
[0665] BLUING REAGENT [2037] 4 minutes
[0666] When the staining was finished, the slides were removed from
the instrument and rinsed with tap water. The slides were
dehydrated as follows:
[0667] Immerse slides in 70% ethanol, 2 changes, 1-2 minutes
each.
[0668] Immerse slides in 95% ethanol, 1-2 minutes.
[0669] Immerse slides in 99% (or absolute) ethanol, 3-5
minutes.
[0670] Clear with xylene, 3 changes, 3-5 minutes each.
[0671] After dehydration, the slides were coversliped with
non-aqueous mounting medium using glass coverslips.
[0672] The following reagents were used in this automated
system:
[0673] Ventana.RTM. cMet CONFIRM.RTM. cat. no. 790-4430 (incubation
approximately 16 minutes at 36.degree. C.)
[0674] One 5 ml dispenser of CONFIRM.RTM. anti-Total cMet contains
approximately 48.75 .mu.g of the recombinant rabbit monoclonal
antibody SP44 (also available from other commercial vendors). The
antibody is diluted in 0.05 M Tris-HCl with 1% carrier protein and
0.10% ProClin 300.RTM. (preservative). Total protein concentration
of the reagent is approximately 10 mg/mL. Specific antibody
concentration is approximately 9.75 .mu.g/mL. There is no known non
specific antibody reactivity observed in this product.
[0675] Ventana Ultra CC1 buffer cat. no. 950-224
[0676] Cell conditioning in Ultra CC1 solution was done at
64.degree. C. for 95 minutes.
[0677] Ventana ultraView.RTM. Universtal Detection Kit cat. no.
760-500
[0678] Ventana Hematoxylin II cat. no. 760-2021
[0679] Ventana Bluing Reagent cat. no. 760-2037
[0680] H-Score and IHC Score Determinations
[0681] The processed slides were analysed by a board-certified MD
pathologist. A scoring guide was used, as provided by the
manufacturer (see, e.g., FIG. 19). 10-12 representative areas of
each slide were used to deduce the score. Upon evaluating the cMet
staining, it was determined that an H-score approach would be the
best approach for quantitating cMet expression. The H-score
approach provides optimal data resolution for determining variation
in intensity and tumor percentage of staining within and among
tumor types. It also provides a good tool for determining
thresholds for positive staining. In this method, the percentage of
cells (0-100) within a tumor with staining intensities ranging from
0-3+ are provided. This protocol results in staining of the cMet
protein both in the cytoplasm and in the cell surface/membrane. The
staining intensity for each cell in a fixed field of the processed
tumor biopsy is determined, and an individual value is attributed
to each cell as follows, depending on the cell surface/membrane
staining:
[0682] 0=no staining
[0683] 1+=weak staining
[0684] 2+=moderate staining
[0685] 3+=strong staining
[0686] To obtain an H-score, the percentage of tumor cells are
multiplied by each intensity and added together. The maximum
H-score is 300 if 100% of tumor cells label with 3+ intensity. The
H-score is calculated as follows:
H-score=[1.times.(% cells 1+)+2.times.(% cells 2+)+3.times.(% cells
3+)]
[0687] This protocol results both in cytoplasmic and membrane cMet
staining. For the H-score calculations referred to herein, membrane
staining was used. The final tumor H-score (0-300) score gives more
relative weight to higher-intensity membrane staining (3+
cell>2+ cell>1+ cell).
[0688] FIG. 20 shows exemplary staining results for various tumor
H-scores (15, 90, 180, and 290) obtained with the "cMet ABBV-ADC
staining protocol."
[0689] Each tumor can also be given an IHC score of IHC 0, IHC 1+,
IHC 2+, or IHC 3+. While both IHC scores involve 0, 1+, 2+, and 3+
values they are not to be confused. For the H-score, 0, 1+, 2+, and
3+ values refer to the intensity of staining of a particular
individual cell. For the IHC score, 0, 1+, 2+, and 3+ values refer
to the overall staining of a particular area of the tumor sample.
FIG. 21 shows exemplary staining results for various tumor
IHC0/1+/2+/3+ scores obtained with the "cMet ABBV-ADC staining
protocol."
[0690] For the purposes on this disclosure, and following the
protocol described herein, if none of the cells in a fixed field
are stained, the value attributed to the tumor is IHC 0. If the
overall level of staining in a fixed field is low, the value
attributed is IHC 1+. If most of the cells in a fixed field exhibit
moderate staining, the value attributed is IHC 2+. If most of the
cells in a fixed field exhibit strong staining, the value
attributed is IHC 3+.
[0691] In another embodiment, and for the purposes on this
disclosure, and following the protocol described herein, if none of
the cells in a fixed field are stained, the value attributed to the
tumor is IHC 0. If the overall level of staining in a fixed field
is low, the value attributed is IHC 1+. If at least 15% of the
cells in a fixed field exhibit moderate staining, the value
attributed is IHC 2+. If at least 15% of the cells in a fixed field
exhibit strong staining, the value attributed is IHC 3+.
Example 18. Measuring MET Gene Copy Number Amplification
[0692] Amplification of the MET gene can improve patient response
to cMet inhibitors, including the treatments disclosed herein. A
variety of methods for measuring MET Gene Amplification have been
described in the art. See, e.g., Cappuzzo F, Marchetti A, Skokan M,
Rossi E, Gajapathy S, Felicioni L, et al. Increased MET gene copy
number negatively affects survival of surgically resected
non-small-cell lung cancer patients. J Clin Oncol 2009; 27:1667-74;
Koeppen H, Yu W, Zha J, Pandita A, Penuel E, Rangell L, et al.
Biomarker analyses from a placebo-controlled phase II study
evaluating erlotinib {+/-} onartuzumab in advanced non-small-cell
lung cancer: MET expression levels are predictive of patient
benefit. Clin Cancer Res 2014; 20:4488-98.
[0693] The preferred method is described as follows and is referred
herein as the "MET/CEP7 cMET amplification method." Briefly,
formalin-fixed, paraffin-embedded tissue blocks, can be submitted
to dual-color FISH assays using a MET/CEP7 probe cocktail prepared
with a MET DNA (RP 11-95120 BAC clone) probe, or using a 319 kb
probe constructed from 3 bacterial artificial chromosome (BAC)
clones that spans the entire MET gene on 7q31.1, labeled with
SpectrumRed and the SpectrumGreen CEP7 (Abbott Molecular). The FISH
assays can be performed, for example, according a protocol
previously described (Cappuzzo F, Hirsch F R, Rossi E, et al.
(2005) Epidermal growth factor receptor gene and protein and
gefitinib sensitivity in non-small cell lung cancer. J Natl Cancer
Inst 97:643-655), including pretreatment with 2.times. sodium
chloride-sodium citrate buffer at 75.degree. C. and digestion with
proteinase K for 7 to 15 minutes each, codenaturation at 85.degree.
C. for 15 minutes, hybridization for approximately 36 hours, and
rapid posthybridization washes with 2.times. sodium chloride-sodium
citrate buffer/0.4 nonyl-phenoxyl-polyethoxylethanol. Signals are
enumerated in at least 50 tumor nuclei per core, using
epifluorescence microscope with single interference filters sets
for green (FITC), red (Texas red) and blue (DAPI) as well as dual
(red/green) and triple (blue, red, green) band pass filters. For
each core, the mean and standard deviation of copy number per cell
of each tested DNA sequence, the percentage of cells with
.ltoreq.2, 3, and .gtoreq.4 copies of the MET genes, and the ratio
of MET/CEP7 (a gene located near the centrosome of the same
chromosome). When heterogeneous results were detected among the
three tested cores, the core with the highest mean copy number was
used to represent the patient in the statistic analyses. For
documentation, images were captured using a CCD camera and merged
using dedicated software (CytoVision; Genetix USA, Boston, Mass.).
MET can be considered amplified when the MET: CEP7 signal ratio is
.gtoreq.2.0 or when this ratio is <2.0 but there are >20
copies of MET signals in more than 10% of the tumor nuclei counted,
according to the criteria established by MD Anderson Pathology
Department based on prior studies. Zeng, ZS, Weiser M R, Kuntz E,
Chen C T, Khan S A, Forslund A, et al. cMet gene amplification is
associated with advanced stage colorectal cancer and liver
metastases. Cancer Lett 2008; 265:258-69. In some studies, it has
been reported that the copy number of the MET gene in relation to
CEP7 ranged from 2.05 to 16.14 (median 3.48).
[0694] Another cMET amplication test is a blood-based test. This
can be done by any one of a variety of commercially available
reagents such as, for example, Biocept Liquid Biopsy MET
Amplication Test (Biocept), MET Detect-R.RTM. (Personal Genome
Diagnostics), and Guardant360.RTM. (Guardant Health.RTM.).
Example 19. Assessing the Presence of Exon 14 Mutation/Skipping of
the MET Gene
[0695] MET Exon 14 contains the Cbl ubiquitin ligases site on
tyrosine residue 1003 (Y1003) where ubiquitin is otherwise normally
attached to the tyrosine residue and leads to the lysosomal
degradation of the cMet protein. Hence, missense mutation of Y1003
residue or "skipping" of the protein region that is encoded by MET
Exon 14 results in a relative over-expression of MET protein,
enhanced cMet activation and subsequent oncogenesis. Inhibition by
MET Tyrosine Kinase Inhibitors (TKIs) can result in clinical
benefit in at least NSCLC patients harboring these MET Exon 14
alterations. Patients carrying any of these mutations can benefit
from the treatments disclosed herein.
[0696] Several methods are available to one or ordinary skill in
the art to detect mutations in the MET gene. Because a mutation is
either present or not (i.e., it is an absolute value and not a
matter of degree), its detection is not assay-dependent and any
method can be used to detect it in tumor samples. Multiple
mutations have been described in Exon 14 of the MET gene, many of
which have been summarized in Impaired cMet Receptor Degradation
Mediated by MET Exon 14 Mutations in Non-Small-Cell Lung Cancer,
Mark M. Awad J C O Mar. 10, 2016:879-881; published online on Jan.
19, 2016; 10.1200/JCO.2015.64.2777. This method and the methods
used in references cited therein for identifying additional
mutations in Exon 14 of cancer samples are incorporated herein by
reference in their entireties. These methods can be used for
identifying those particular mutations in cancer samples. Also,
this disclosure is directed to any known mutation in the Exon 14
gene and is not limited to those exemplified herein.
[0697] Several splice mutations of Exon 14 have been identified in
pulmonary adenocarcinoma.
[0698] For example:
[0699] MET amplification, protein expression, and mutations in
pulmonary adenocarcinoma. Park S, Koh J, Kim D W, Kim M, Keam B,
Kim T M, Jeon Y K, Chung D H, Heo D S. Lung Cancer. 2015 December;
90(3):381-7. doi: 10.1016/j.lungcan.2015.10.022. Epub 2015 Oct. 27.
PMID: 26791796. This method and the methods used in references
cited therein for identifying additional mutations in Exon 14 of
cancer samples are incorporated herein by reference in their
entireties. These methods can be used for identifying those
particular mutations in cancer samples.
[0700] Responses to the multitargeted MET/ALK/ROS1 inhibitor
crizotinib and co-occurring mutations in lung adenocarcinomas with
MET amplification or MET exon 14 skipping mutation. Jorge S E,
Schulman S, Freed J A, VanderLaan P A, Rangachari D, Kobayashi S S,
Huberman M S, Costa D B. Lung Cancer. 2015 December; 90(3):369-74.
doi: 10.1016/j.lungcan.2015.10.028. Epub 2015 Oct. 31. This method
and the methods used in references cited therein for identifying
additional mutations in Exon 14 of cancer samples are incorporated
herein by reference in their entireties. These methods can be used
for identifying those particular mutations in cancer samples.
[0701] Additional Exon 14 mutations in NSCLC can be detected by the
methods described in the following references:
[0702] Next-Generation Sequencing of Pulmonary Sarcomatoid
Carcinoma Reveals High Frequency of Actionable MET Gene Mutations
Exon 14 Xuewen Liu, Yuxia Jia, Mark B. Stoopler, Yufeng Shen,
Haiying Cheng, Jinli Chen, Mahesh Mansukhani, Sanjay Koul, Balazs
Halmos, and Alain C. Borczuk, J C O Mar. 10, 2016:794-802;
published online on Jul. 27, 2015. This method and the methods used
in references cited therein for identifying additional mutations in
Exon 14 of cancer samples are incorporated herein by reference in
their entireties. These methods can be used for identifying those
particular mutations in cancer samples.
[0703] MET Exon 14 Mutations in Non-Small-Cell Lung Cancer Are
Associated With Advanced Age and Stage-Dependent MET Genomic
Amplification and cMet Overexpression. Awad M M, Oxnard G R,
Jackman D M, Savukoski D O, Hall D, Shivdasani P, Heng J C,
Dahlberg S E, Janne P A, Verma S, Christensen J, Hammerman P S,
Sholl L M. J Clin Oncol. 2016 Mar. 1; 34(7):721-30. doi:
10.1200/JCO.2015.63.4600. Epub 2016 Jan. 4. This method and the
methods used in references cited therein for identifying additional
mutations in Exon 14 of cancer samples are incorporated herein by
reference in their entireties. These methods can be used for
identifying those particular mutations in cancer samples.
[0704] Another MET exon 14 deletion has been reported in
gastrointestinal malignancies. Oncotarget. 2015 Sep. 29;
6(29):28211-22. doi: 10.18632/oncotarget.4721. Gastrointestinal
malignancies harbor actionable MET exon 14 deletions. Lee J, Ou
SH3, Lee J M, Kim HC5, Hong M6, Kim SY1, Jang J1, Ahn S6, Kang SY6,
Lee S1, Kim ST1, Kim B4, Choi J4, Kim KA4, Lee J, Park C Park S H,
Park J O, Lim H Y, Kang W K, Park K, Park Y S, Kim K M. This method
and the methods used in references cited therein for identifying
additional mutations in Exon 14 of cancer samples are incorporated
herein by reference in their entireties. These methods can be used
for identifying those particular mutations in cancer samples.
Example 20. Assessing the Presence of Exon 19 Deletions and Exon 21
(L858R) Substitutions in the EGFR Gene of Cancer Patients
[0705] The two most common EGFR somatic mutations, exon 19
deletions and L858R missense mutations, have been associated with
in vitro and in vivo sensitivity to treatment with the EGFR
tyrosine kinase inhibitors (EGFR-TKI) gefitinib and erlotinib.
These two different types of mutations are responsible for
.about.85% of all EGFR somatic mutations identified in patients
with NSCLC. Benefits of the treatments disclosed herein can be
observed in patients with Exon 19 deletions and Exon 21 L858R
substitution.
[0706] Several methods have been described in the art for detection
Exon 19 deletions in cancer samples. Examples of such methods,
which are available to one of ordinary skill in the art are
provided below. Because a mutation is either present or not (i.e.,
it is an absolute value and not a matter of degree), its detection
is not assay-dependent and any method can be used to detect it in
tumor samples.
[0707] A recent review of the literature reporting on the effect of
these mutations in cancer patients is that in EGFR-TKIEGFR-tyrosine
kinase inhibitor treatment in a patient with advanced non-small
cell lung cancer and concurrent exon 19 and 21 EGFR mutations: A
case report and review of the literature. Yang Y, Zhang B, Li R,
Liu B, Wang L. Oncol Lett. 2016 May; 11(5):3546-3550. Epub 2016
Apr. 5. The method used in this report and the methods used in
references cited therein for identifying Exon 19 deletions and Exon
21 (L858R) substitutions in the EGFR gene in patients' cancer
samples are incorporated herein by reference in their
entireties.
[0708] It has been reported that patients with NSCLC and EGFR exon
19 deletions have a longer survival following treatment with
gefitinib or erlotinib compared with those with the L858R mutation.
Jackman D M, Yeap B Y, Sequist L V, et al. (2006) Exon 19 deletion
mutations of epidermal growth factor receptor are associated with
prolonged survival in non-small cell lung cancer patients treated
with gefitinib or erlotinib. Clin Cancer Res 12:3908-3914. This
reference provides two different methods for detecting EGFR Exon 19
deletions and L858R mutation. These methods and the methods used in
references cited therein for identifying Exon 19 deletions and Exon
21 (L858R) substitutions in the EGFR gene in patients' cancer
samples are incorporated herein by reference in their
entireties.
[0709] All publications, patents, patent applications and other
documents cited in this application are hereby incorporated by
reference in their entireties for all purposes to the same extent
as if each individual publication, patent, patent application or
other document were individually indicated to be incorporated by
reference for all purposes.
[0710] While various specific embodiments have been illustrated and
described, and some are represented below, it will be appreciated
that various changes can be made without departing from the spirit
and scope of the invention(s).
[0711] 1. A method of treating a solid tumor cancer that
overexpresses cMet, comprising administering to a human subject
having said cancer an anti-cMet antibody drug conjugate ("ADC") in
an amount and for a period of time sufficient to provide a
therapeutic benefit.
[0712] 2. The method of embodiment 1 in which the cMet
overexpressing cancer is of a cancer type in which cMet is
overexpressed in at least about 10% of a patient population having
the cancer type.
[0713] 3. The method of embodiment 1 in which a biopsy of the cMet
overexpressing tumor tissue from the subject has an IHC score of 2+
and/or an H-score from 150 to 224, when measured according to the
cMet ABBV-ADC staining protocol.
[0714] 4. The method of embodiment 1 in which a biopsy of the cMet
overexpressing tumor tissue from the subject has an IHC score of 3+
and/or an H-score greater than 225, when measured according to the
cMet ABBV-ADC staining protocol.
[0715] 5. The method according to any one of embodiments 1-4 in
which the cMet overexpressing cancer is non-small cell lung cancer
("NSCLC").
[0716] 6. The method of embodiment 5, in which the NSCLC is a
non-squamous NSCLC.
[0717] 7. The method of embodiment 5 in which the NSCLC is squamous
NSCLC.
[0718] 8. The method of embodiment 5 in which the histology of the
NSCLC is NSCLC-not otherwise specified (NSCLC-NOS).
[0719] 9. The method of embodiment 1 in which the cancer is
colorectal cancer ("CRC").
[0720] 10. The method of embodiment 9 in which the histology of the
CRC is not specified.
[0721] 11. The method of embodiment 10 in which the CRC is an
adenocarcinoma.
[0722] 12. The method of embodiment 1 in which the cancer is head
& neck ("H&N") cancer.
[0723] 13. The method of embodiment 12 in which the histology of
H&N cancer is not specified.
[0724] 14. The method of embodiment 1 in which the cancer is
pancreatic cancer.
[0725] 15. The method of embodiment 14 in which the pancreatic
cancer is an adenocarcinoma.
[0726] 16. The method of embodiment 5 in which the cMet
overexpressing cancer has epidermal growth factor receptor ("EGFR")
exon 19 deletions or exon 21 (L858R) substitutions as detected by
an FDA approved test.
[0727] 17. The method of embodiment 1 in which the cMet
overexpressing cancer is resistant to prior treatment with targeted
and/or non-targeted chemotherapy.
[0728] 18. The method of embodiment 1 in which the cMet
overexpressing cancer is resistant to prior treatment with an
anti-cMet antibody.
[0729] 19. The method of embodiment 1 in which the anti-cMet ADC is
administered as monotherapy.
[0730] 20. The method of embodiment 1 in which the anti-cMet ADC is
administered adjunctive to an additional anticancer agent, where
the additional agent is administered according to its FDA-approved
dosing regimen.
[0731] 21. The method of embodiment 20 in which the additional
anticancer agent is an inhibitor of epidermal growth factor
receptor ("EGFR").
[0732] 22. The method of embodiment 21 in which the additional
anticancer agent is erlotinib.
[0733] 23. The method of embodiment 20 in which the cMet
overexpressing cancer has EGFR exon 19 deletions or exon 21 (L858R)
substitutions as detected by an FDA-approved test and the
additional anticancer agent is an inhibitor of EGFRs having such
deletions or substitutions.
[0734] 24. The method of embodiment 23 in which the additional
anticancer agent is afatinib.
[0735] 25. The method of embodiment 20 in which the cancer is
NSCLC.
[0736] 26. The method of embodiment 25 in which the additional
anticancer agent is selected from imatinib (GLEEVEC.RTM.),
dasatinib (SPRYCE.RTM.), nilotinib (TASIGNA.RTM.), bosutinib
(BOSULIF.RTM.), ponatinib (ICLUSIG.RTM.), Afatinib (GIOTRIF.RTM.),
Axitinib (INLYTA.RTM.), Crizotinib (XALKORI.RTM.), Erlotinib
(TARCEVA.RTM.), Gefitinib (IRESSA.RTM.), Lapatinib (TYVERB.RTM.),
Nilotinib (TASIGNA.RTM.), Pazopanib (VOTRIENT.RTM.), Regorafenib
(STIVARGA.RTM.), Sorafenib (NEXAVAR.RTM.), Sunitinib (SUTENT.RTM.),
toceranib (PALLADIA.RTM.), vatalanib, and radotinib
(SUPECT.RTM.).
[0737] 27. The method of embodiment 26 in which the additional
anticancer agent is an inhibitor of PD1.
[0738] 28. The method of embodiment 27 in which the inhibitor of
PD1 is an anti-PD1 antibody.
[0739] 29. The method of embodiment 28 in which the anti-PD1
antibody is nivolumab.
[0740] 30. The method of any one of embodiments 1-29 in which the
anti-cMet ADC is administered in an amount ranging from about 0.15
mg/kg to about 3.3 mg/kg once every three weeks.
[0741] 31. The method of embodiment 30 in which the anti-cMet ADC
is administered in an amount of about 2.7 mg/kg.
[0742] 32. The method of any one of embodiments 1-29 in which the
anti-cMet ADC is administered in an amount ranging from about 0.15
mg/kg to about 3.3 mg/kg once every two weeks.
[0743] 33. The method of embodiment 32 in which the anti-cMet ADC
is administered in an amount of about 1.6 mg/kg once every two
weeks.
[0744] 34. The method of embodiment 32 in which the anti-cMet ADC
is administered in an amount of about 1.9 mg/kg once every two
weeks.
[0745] 35. The method of any one of embodiments 1-34 in which the
anti-cMet ADC comprises an anti-cMet antibody linked to a
cytostatic and/or cytotoxic agent by way of a linker.
[0746] 36. The method of embodiment 35 in which the anti-cMet
antibody is a full-length antibody.
[0747] 37. The method of embodiment 35 in which the anti-cMet
antibody is internalized and has an apparent affinity EC.sub.50
value lower than about 10 nanomol/L, preferably from about 1
picomol/L to 10 nanomol/L.
[0748] 38. The method of embodiment 35 in which the anti-cMet
antibody binds human cMet in vitro with an apparent affinity
EC.sub.50 value of about 0.3 nmol/L.
[0749] 39. The method of embodiment according to any one of
embodiments 35 through 38 in which the anti-cMet antibody comprises
a V.sub.H chain comprising three CDRs, namely V.sub.H CDR #1 (SEQ
ID NO:112), V.sub.H CDR #2 (SEQ ID NO:113) and V.sub.H CDR #3 (SEQ
ID NO: 114); a V.sub.L chain comprising three CDRs, namely V.sub.L
CDR #1 (SEQ ID NO: 115), V.sub.L CDR #2 (SEQ ID NO: 116) and
V.sub.L CDR #3 (SEQ ID NO: 117); and a modified hinge region of SEQ
ID NO: 170.
[0750] 40. The method of embodiment 39 in which the anti-cMet
antibody is an IgG1.
[0751] 41. The method of embodiment 38 in which the anti-cMet
antibody comprises a V.sub.H chain of SEQ ID NO: 78; a V.sub.L
chain of SEQ ID NO: 79; and a modified hinge region of SEQ ID NO:
170.
[0752] 42. The method of embodiment 41 in which the anti-cMet
antibody is an IgG1.
[0753] 43. The method of embodiment 39 in which the anti-cMet
antibody comprises a heavy chain of SEQ ID NO: 86 and a light chain
of SEQ ID NO: 87.
[0754] 44. The method of embodiment 39 in which the anti-cMet
antibody is ABBV399.
[0755] 45. The method of embodiment 39 in which the anti-cMet
antibody comprises a heavy chain of SEQ ID NO: 171 and a light
chain of SEQ ID NO: 172.
[0756] 46. The method of embodiment 39 in which the anti-cMet
antibody is ABT-700 (S238C)-PBD.
[0757] 47. The method of embodiment 38 in which the anti-cMet
antibody comprises the six CDRs of the antibody
STI-D0602/STI-0602.
[0758] 48. The method of embodiment 47 in which the anti-cMet
antibody is an IgG1.
[0759] 49. The method of embodiment 35 in which the anti-cMet
antibody comprises a V.sub.H chain of STI-D0602/STI-0602 and a
V.sub.L chain of STI-D0602/STI-0602.
[0760] 50. The method of embodiment 49 in which the anti-cMet
antibody is an IgG1.
[0761] 51. The method of embodiment 35 in which the linker is
cleavable by a lysosomal enzyme.
[0762] 52. The method of embodiment 51 in which the lysosomal
enzyme is Cathepsin B.
[0763] 53. The method of embodiment 52 in which the linker
comprises a segment according to one or more of structural formulae
(IVa), (IVb), (IVc) and (IVd):
##STR00028## [0764] or a salt thereof, in which: [0765] peptide
represents a peptide (illustrated C.fwdarw.N and not showing the
carboxy and amino "termini") cleavable by Cathepsin B; [0766] T
represents a polymer comprising one or more ethylene glycol units
or an alkylene chain, or combinations thereof; [0767] R.sup.a is
selected from hydrogen, alkyl, sulfonate and methyl sulfonate;
[0768] p is an integer ranging from 0 to 5; [0769] q is 0 or 1;
[0770] x is 0 or 1; [0771] y is 0 or 1; [0772] represents the point
of attachment of the linker to the cytotoxic and/or cytostatic
agent; and [0773] * represents the point of attachment to the
remainder of the linker.
[0774] 54. The method of embodiment 53 in which peptide is selected
from the group consisting of Val-Cit; Cit-Val; Ala-Ala; Ala-Cit;
Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit;
Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; and Val-Ala
and salts thereof.
[0775] 55. The method of embodiment 51 in which the lysosomal
enzyme is .beta.-glucuronidase.
[0776] 56. The method of embodiment 35 in which the anti-cMet ADC
has an average drug-to-antibody ratio ("DAR") in the range of
0-10.
[0777] 57. The method of embodiment 35 in which the anti-cMet ADC
has an average drug-to-antibody ratio ("DAR") in the range of
1-4.
[0778] 58. The method of embodiment 57 in which the anti-cMet ADC
has a DAR in the range of 2-4.
[0779] 59. The method of embodiment 57 in which the anti-cMet ADC
has a DAR of about 3.1.
[0780] 60. The method of embodiment 57 in which the anti-cMet ADC
has an about 1:1 ratio of E2 and E4 ADC.
[0781] 61. The method of embodiment 57 in which the anti-cMet ADC
has a DAR of 3.0.
[0782] 62. The method of embodiment 35 in which the cytostatic
and/or cytotoxic agent is a microtubule inhibitor.
[0783] 63. The method of embodiment 62 in which the microtubule
inhibitor is an auristatin.
[0784] 64. The method of embodiment 63 in which the auristatin is
MMAE or MMAF.
[0785] 65. The method of embodiment 63 in which the auristatin is
MMAE.
[0786] 66. The method of embodiment 35 in which the anti-cMet ADC
is a compound according to structural formula (I):
[D-L-XY-].sub.n-Ab (I) [0787] or a salt thereof, in which: [0788] D
is the cytotoxic and/or cytostatic agent; [0789] L is the linker;
[0790] Ab is the anti-cMet antibody; [0791] XY represents a
covalent linkage linking linker L to antibody Ab; and [0792] n has
a value ranging from 2 to 8.
[0793] 67. The method of embodiment 66 in which n has a value of 2,
3 or 4.
[0794] 68. The method of embodiment 66 in which XY is a linkage
formed with an amino group on anti-cMet antibody Ab.
[0795] 69. The method of embodiment 66 in which XY is an amide or a
thiourea.
[0796] 70. The method of embodiment 66 in which XY is a linkage
formed with a sulfhydryl group on anti-cMet antibody Ab.
[0797] 71. The method of embodiment 66 in which XY is a
thioether.
[0798] 72. The method of embodiment 66 in which the compound
according to structural formula (I) has the structure of formula
(IIa):
##STR00029##
[0799] 73. The method of embodiment 72 in which anti-cMet antibody
Ab is ABT-700.
[0800] 74. The method of embodiment 66 in which the compound of
structural formula (I) has the following structure:
##STR00030##
[0801] 75. The method of embodiment 74 in which anti-cMet antibody
Ab is ABT-700.
[0802] 76. The method of embodiment 66 in which the compound
according to structural formula (I) has the structure of formula
(IIb):
##STR00031##
[0803] 77. The method of embodiment 76 in which anti-cMet antibody
Ab is ABT-700.
[0804] 78. The method of embodiment 66 in which the compound
according to structural formula (I) has the following
structure:
##STR00032##
[0805] 79. The method of embodiment 78 in which anti-cMet antibody
Ab is ABT-700.
[0806] 80. A method of treating a human patient diagnosed with
non-small cell lung cancer ("NSCLC") comprising administering to
the patient an anti-cMet antibody drug conjugate ("ADC") in an
amount and for a period of time sufficient to provide therapeutic
benefit.
[0807] 81. The method of embodiment 80 in which the NSCLC tumor
tissue has an immunohistochemistry ("IHC") H-score of greater than
or equal to 150 when measured according to the cMet ABBV-ADC
staining protocol or an IHC score of 2+.
[0808] 82. The method of embodiment 80 in which the NSCLC tumor
tissue has an immunohistochemistry ("IHC") H-score of greater than
225 when measured according to the cMet ABBV-ADC staining protocol
or an IHC score of 3+.
[0809] 83. The method of embodiment 80 in which the NSCLC tumor
tissue has an IHC score of 2+ and/or an H-score from 150 to 224,
when measured according to the cMet ABBV-ADC staining protocol.
[0810] 84. The method of embodiment 80 in which the NSCLC tumor
tissue has an IHC score of 3+ and/or an H-score greater than 225,
when measured according to the cMet ABBV-ADC staining protocol.
[0811] 85. The method according to any one of embodiments 80, 81,
and 94 in which the NSCLC is a non-squamous cell carcinoma.
[0812] 86. The method according to any one of of embodiments 80,
81, and 83 in which the NSCLC is a squamous cell carcinoma.
[0813] 87. The method of embodiment 80 in which the histology of
the NSCLC is NSCLC-not otherwise specified (NSCLC-NOS).
[0814] 88. The method of embodiment 80 in which the NSCLC tumor has
epidermal growth factor receptor ("EGFR") exon 19 deletions or exon
21 (L858R) substitutions as detected by and FDA-approved test such
as Cobas.RTM. EGFR Mutation Test v2 or the Therascreen.RTM. EGFR
RGQ FCR Kit.
[0815] 89. The method according to any one of embodiments 80
through 88 in which the NSCLC tumor is resistant to prior treatment
with a microtubule inhibitor.
[0816] 90. The method according to any one of embodiments 80
through 89 in which the NSCLC tumor is resistant to prior treatment
with an anti-cMet antibody.
[0817] 91. The method according to any one of embodiments 80
through 90 in which the anti-cMet ADC is administered as
monotherapy.
[0818] 92. The method according to any one of embodiments 80
through 91 in which the anti-cMet ADC is administered adjunctive to
an additional anticancer agent, where the additional agent is
administered according to its FDA-approved dosing regimen.
[0819] 93. The method of embodiment 92 in which the additional
anticancer agent is an inhibitor of epidermal growth factor
receptor ("EGFR").
[0820] 94. The method of embodiment 93 in which the additional
anticancer agent is erlotinib, administered once daily.
[0821] 95. The method of embodiment 92 in which the NSCLC tumor has
EGFR exon 18 deletions or exon 21 (L858R) substitutions as detected
by an FDA-approved test and the additional anticancer agent is an
inhibitor of EGFRs having such deletions or substitutions.
[0822] 96. The method of embodiment 95 in which the additional
anticancer agent is afatinib.
[0823] 97. The method of embodiment 92 in which the additional
anticancer agent is a microtubule inhibitor.
[0824] 98. The method of embodiment 97 in which the additional
anticancer agent is selected from the group consisting of
cabazitaxel, colcemid, colchicine, cryptophycin, democolcine,
docetaxel, nocodazole, paclitaxel, taccalonolide, taxane and
vinblastine.
[0825] 99. The method of embodiment 92 in which the additional
anticancer agent is an inhibitor of PD1.
[0826] 100. The method of embodiment 99 in which the inhibitor of
PD1 is an anti-PD1 antibody.
[0827] 101. The method of embodiment 100 in which the anti-PD1
antibody is nivolumab.
[0828] 102. The method of any one of embodiments 80 through 101 in
which the anti-cMet ADC is administered in an amount ranging from
about 0.15 mg/kg to about 3.3 mg/kg, once every 3 weeks.
[0829] 103. The method of embodiment 102 in which the anti-cMet ADC
is administered in an amount of about 2.7 mg/kg once every 3
weeks.
[0830] 104. The method of any one of embodiments 80 through 101 in
which the anti-cMet ADC is administered in an amount ranging from
about 0.15 mg/kg to about 3.3 mg/kg, once every 2 weeks.
[0831] 105. The method of embodiment 104 in which the anti-cMet ADC
is administered in an amount of about 1.6 mg/kg, once every 2
weeks. Add dependent to 1.9
[0832] 106. The method of embodiment 104 in which the anti-cMet ADC
is administered in an amount of about 1.9 mg/kg, once every 2
weeks.
[0833] 107. The method of any one of embodiments 80 through 105 in
which the anti-cMet ADC comprises an anti-cMet antibody linked to a
cytostatic and/or cytotoxic agent by way of a linker.
[0834] 108. The method of embodiment 107 in which the anti-cMet
antibody is a full-length antibody.
[0835] 109. The method of embodiment 109 in which the anti-cMet
antibody is internalized and has an apparent affinity EC.sub.50
value lower than about 10 nanomol/L, preferably from about 1
picomol/L to 10 nanomol/L.
[0836] 110. The method of embodiment 109 in which the anti-cMet
antibody binds human cMet in vitro with an apparent affinity
EC.sub.50 value of about 0.3 nmol/L.
[0837] 111. The method of embodiment according to any one of
embodiments 107 through 110 in which the anti-cMet antibody
comprises a V.sub.H chain comprising three CDRs, namely V.sub.H CDR
#1 (SEQ ID NO:112), V.sub.H CDR #2 (SEQ ID NO:113) and V.sub.H CDR
#3 (SEQ ID NO: 114); a V.sub.L chain comprising three CDRs, namely
V.sub.L CDR #1 (SEQ ID NO: 115), V.sub.L CDR #2 (SEQ ID NO: 116)
and V.sub.L CDR #3 (SEQ ID NO: 117); and a modified hinge region of
SEQ ID NO: 170.
[0838] 112. The method of embodiment 111 in which the anti-cMet
antibody is an IgG1.
[0839] 113. The method of embodiment 111 in which the anti-cMet
antibody comprises a V.sub.H chain of SEQ ID NO: 78; a V.sub.L
chain of SEQ ID NO: 79; and a modified hinge region of SEQ ID NO:
170.
[0840] 114. The method of embodiment 113 in which the anti-cMet
antibody is an IgG1.
[0841] 115. The method of embodiment 111 in which the anti-cMet
antibody comprises a heavy chain of SEQ ID NO: 86 and a light chain
of SEQ ID NO: 87.
[0842] 116. The method of embodiment 111 in which the anti-cMet
antibody comprises a heavy chain of SEQ ID NO: 171 and a light
chain of SEQ ID NO: 172.
[0843] 117. The method of embodiment 110 in which the anti-cMet
antibody comprises comprises the six CDRs of the antibody
STI-D0602/STI-0602.
[0844] 118. The method of embodiment 117 in which the anti-cMet
antibody is an IgG1.
[0845] 119. The method of embodiment 104 in which the anti-cMet
antibody comprises a V.sub.H chain of STI-D0602/STI-0602 and a
V.sub.L chain of STI-D0602/STI-0602.
[0846] 120. The method of embodiment 119 in which the anti-cMet
antibody is an IgG1.
[0847] 121. The method of embodiment 107 in which the linker is
cleavable by a lysosomal enzyme.
[0848] 122. The method of embodiment 121 in which the lysosomal
enzyme is Cathepsin B.
[0849] 123. The method of embodiment 122 in which the linker
comprises a segment according to one or more of structural formulae
(IVa), (IVb), (IVc) and (IVd):
##STR00033## [0850] or a salt thereof, in which: [0851] peptide
represents a peptide (illustrated C.fwdarw.N and not showing the
carboxy and amino "termini") cleavable by Cathepsin B; [0852] T
represents a polymer comprising one or more ethylene glycol units
or an alkylene chain, or combinations thereof; [0853] R.sup.a is
selected from hydrogen, alkyl, sulfonate and methyl sulfonate;
[0854] p is an integer ranging from 0 to 5; [0855] q is 0 or 1;
[0856] x is 0 or 1; [0857] y is 0 or 1; [0858] represents the point
of attachment of the linker to the cytotoxic and/or cytostatic
agent; and [0859] * represents the point of attachment to the
remainder of the linker.
[0860] 124. The method of embodiment 123 in which peptide is
selected from the group consisting of Val-Cit; Cit-Val; Ala-Ala;
Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val;
Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; and
Val-Ala and salts thereof.
[0861] 125. The method of embodiment 121 in which the lysosomal
enzyme is .beta.-glucuronidase.
[0862] 126. The method of embodiment 107 in which the anti-cMet ADC
has an average drug-to-antibody ratio ("DAR") in the range of
0-10.
[0863] 127. The method of embodiment 107 in which the anti-cMet ADC
has an average drug-to-antibody ratio ("DAR") in the range of
1-4.
[0864] 128. The method of embodiment 127 in which the anti-cMet ADC
has a DAR in the range of 2-4.
[0865] 129. The method of embodiment 127 in which the anti-cMet ADC
has a DAR of about 3.1.
[0866] 130. The method of embodiment 127 in which the anti-cMet ADC
has an about 1:1 ratio of E2 and E4 ADC.
[0867] 131. The method of embodiment 127 in which the anti-cMet ADC
has a DAR of 3.0.
[0868] 132. The method according to any one of embodiments 107
through 131 in which the cytostatic and/or cytotoxic agent is a
microtubule inhibitor.
[0869] 133. The method of embodiment 132 in which the microtubule
inhibitor is an auristatin.
[0870] 134. The method of embodiment 133 in which the auristatin is
MMAE or MMAF.
[0871] 135. The method of embodiment 134 in which the auristatin is
MMAE.
[0872] 136. The method according to any one of embodiments 107
through 135 in which the anti-cMet ADC is a compound according to
structural formula (I):
[D-L-XY-].sub.n-Ab (I) [0873] or a salt thereof, in which: [0874] D
is the cytotoxic and/or cytostatic agent; [0875] L is the linker;
[0876] Ab is the anti-cMet antibody; [0877] XY represents a
covalent linkage linking linker L to antibody Ab; and [0878] n has
a value ranging from 2 to 8.
[0879] 137. The method of embodiment 136 in which n has a value of
2, 3 or 4.
[0880] 138. The method of embodiment 136 in which XY is a linkage
formed with an amino group on anti-cMet antibody Ab.
[0881] 139. The method of embodiment 136 in which XY is an amide or
a thiourea.
[0882] 140. The method of embodiment 136 in which XY is a linkage
formed with a sulfhydryl group on anti-cMet antibody Ab.
[0883] 141. The method of embodiment 136 in which XY is a
thioether.
[0884] 142. The method of embodiment 136 in which the compound
according to structural formula (I) has the structure of formula
(IIa):
##STR00034##
[0885] 143. The method of embodiment 142 in which anti-cMet
antibody Ab is ABT-700.
[0886] 144. The method of embodiment 136 in which the compound of
structural formula (I) has the following structure:
##STR00035##
[0887] 145. The method of embodiment 144 in which anti-cMet
antibody Ab is ABT-700.
[0888] 146. The method of embodiment 136 in which the compound
according to structural formula (I) has the structure of formula
(IIb):
##STR00036##
[0889] 147. The method of embodiment 146 in which anti-cMet
antibody Ab is ABT-700.
[0890] 148. The method of embodiment 136 in which the compound
according to structural formula (I) has the following
structure:
##STR00037##
[0891] 149. The method of embodiment 148 in which anti-cMet
antibody Ab is ABT-700.
[0892] 150. A method of treating a human subject having a NSCLC
tumor with an IHC score of at least 2+ in at least one tumor biopsy
from the subject, comprising administering to the subject an
anti-cMet ADC in an amount of about 2.7 mg/kg once every two weeks
or once every 3 weeks, in which the anti-cMet ADC is a compound
according to the following structure:
##STR00038##
[0893] or a pharmaceutically acceptable salt thereof, in which n
has a value ranging from 2-4 and Ab is a full-length anti-cMet
antibody.
[0894] 151. The method of embodiment 150 in which the anti-cMet
antibody is ABT-700.
[0895] 152. The method of embodiment 151 in which the anti-cMet ADC
is administered as monotherapy.
[0896] 153. The method of embodiment 150 in which the anti-cMetADC
is administered adjunctive to an additional anticancer agent.
[0897] 154. The method of embodiment 153 in which the additional
anticancer agent is erlotinib.
[0898] 155. The method of embodiment 153 in which the additional
anticancer agent is Nivolumab.
[0899] 156. The method of embodiment 153 in which the NSCLC tumor
has EGFR exon 19 deletions or exon 21 (L858R) substitutions as
detected by an FDA-approved test and the additional anticancer
agent is afatinib.
[0900] 157. The method of anyone of embodiments 1-34 in which the
drug is a pyrrolobenzodiazepine (PBD), preferably PBD
((S)-2-(4-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-
-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propo-
xy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one); SG2000
(SJG-136;
(11aS,11a'S)-8,8'-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-
-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one)) (or
SGD-1882).
[0901] 158. The method of anyone of embodiments 35 through 56 and
62 in which the drug is a pyrrolobenzodiazepine (PBD), preferably
SGD-1882.
[0902] 159. The method of anyone of embodiments 66 through 71 and
76 through 78 in which the drug is a pyrrolobenzodiazepine (PBD),
preferably SGD-1882.
[0903] 160. The method according to embodiment 66, in which the
compound of formula I has the following structure:
##STR00039##
[0904] in which Ab is the antibody and n is 2.
[0905] 161. The method according to embodiment 160, in which the
antibody is ABT-700 or ABT-700 (S238C).
[0906] 162. The method according to anyone of embodiments 80
through 131 in which the drug is a pyrrolobenzodiazepine (PBD),
preferably SGD-1882.
[0907] 163. The method according to embodiment 107 in which the
cytostatic and/or cytotoxic agent is a DNA minor grove binding
crosslinking agent.
[0908] 164. The method according to embodiment 163, in which the
DNA minor grove binding crosslinking agent is a
pyrrolobenzodiazepine (PBD), preferably SGD-1882.
[0909] 165. The method according to embodiment 107 in which the
cMet ADC is the compound of formula
##STR00040##
in which Ab is ABT-700 or ABT-700 (S238C) and n is 2.
[0910] 166. A method of treating a human subject having a NSCLC
tumor with an IHC score of at least 2+ in at least one tumor biopsy
from the subject, comprising administering to the subject an
anti-cMet ADC in an amount of about 2.7 mg/kg once every two weeks
or once every 3 weeks, in which the anti-cMet ADC is a compound
according to the following structure:
##STR00041##
[0911] or a pharmaceutically acceptable salt thereof, in which n is
2 and Ab is a full-length anti-cMet antibody.
[0912] 167. The method of embodiment 166 in which the anti-cMet
antibody is ABT-700 or ABT-700 (S238C).
[0913] 168. The method of embodiment 167 in which the anti-cMet ADC
is administered as monotherapy.
[0914] 169. The method of embodiment 166 in which the anti-cMetADC
is administered adjunctive to an additional anticancer agent.
[0915] 170. The method of embodiment 169 in which the additional
anticancer agent is erlotinib.
[0916] 171. The method of embodiment 169 in which the additional
anticancer agent is Nivolumab.
[0917] 172. The method of embodiment 169 in which the NSCLC tumor
has EGFR exon 19 deletions or exon 21 (L858R) substitutions as
detected by an FDA-approved test and the additional anticancer
agent is afatinib.
[0918] 173. A method of treating a human subject having a NSCLC
adenocarcinoma, comprising administering to the subject ABBV-399
once every 3 weeks in an amount of about 2.7 mg/kg, in which the
adenocarcinoma has an H-score of at least 225.
[0919] 174. A method of treating a human subject having a NSCLC
adenocarcinoma, comprising administering to the subject ABBV-399
once every 3 weeks in an amount of about 2.7 mg/kg, in which the
adenocarcinoma has an IHC score of 3+.
[0920] 175. The method according to any one of embodiments 173 and
174, in which ABBV-399 is administered adjunctive to erlotinib, in
which the the erlotinib is administered once daily at 150 mg.
[0921] 176. A method of treating a human subject having a NSCLC
squamous cell carcinoma, comprising administering to the subject
ABBV-399 once every 2 weeks in an amount of about 1.6 mg/kg, in
which the squamous cell carcinoma has an H-score of from 150 to
224.
[0922] 177. A method of treating a human subject having a NSCLC
adenocarcinoma, comprising administering to the subject ABBV-399
once every 2 weeks in an amount of about 1.6 mg/kg, in which the
adenocarcinoma has an IHC score of 2+.
[0923] 178. A method of treating a human subject having a NSCLC
squamous cell carcinoma, comprising administering to the subject
ABBV-399 once every 2 weeks in an amount of about 1.9 mg/kg, in
which the squamous cell carcinoma has an H-score of from 150 to
224.
[0924] 179. A method of treating a human subject having a NSCLC
squamous cell carcinoma, comprising administering to the subject
ABBV-399 once every 2 weeks in an amount of about 1.9 mg/kg, in
which the squamous cell carcinoma has an IHC score of 2+.
[0925] 180. A method of treating a human subject having a NSCLC
adenocarcinoma, comprising administering to the subject ABT-700
(S238C)-PBD once every 3 weeks in an amount of about 2.7 mg/kg, in
which the adenocarcinoma has an H-score of at least 225.
[0926] 181. A method of treating a human subject having a NSCLC
adenocarcinoma, comprising administering to the subject ABT-700
(S238C)-PBD once every 3 weeks in an amount of about 2.7 mg/kg, in
which the adenocarcinoma has an IHC score of 3+.
[0927] 182. The method according to any one of embodiments 180 and
181, in which ABT-700 (S238C)-PBD is administered adjunctive to
erlotinib, in which the the erlotinib is administered once daily at
150 mg.
[0928] 183. A method of treating a human subject having a NSCLC
squamous cell carcinoma, comprising administering to the subject
ABT-700 (S238C)-PBD once every 2 weeks in an amount of about 1.6
mg/kg, in which the squamous cell carcinoma has an H-score of from
150 to 224.
[0929] 184. A method of treating a human subject having a NSCLC
squamous cell carcinoma, comprising administering to the subject
ABT-700 (S238C)-PBD once every 2 weeks in an amount of about 1.6
mg/kg, in which the squamous cell carcinoma has an IHC score of
2+.
[0930] 185. A method of treating a human subject having a NSCLC
squamous cell carcinoma, comprising administering to the subject
ABT-700 (S238C)-PBD once every 2 weeks in an amount of about 1.6
mg/kg, in which the squamous cell carcinoma has an H-score of from
150 to 224.
[0931] 186. A method of treating a human subject having a NSCLC
squamous cell carcinoma, comprising administering to the subject
ABT-700 (S238C)-PBD once every 2 weeks in an amount of about 1.9
mg/kg, in which the squamous cell carcinoma has an IHC score of 2+.
Sequence CWU 1
1
17818PRTMus musculus 1Gly Tyr Ile Phe Thr Ala Tyr Thr 1 5 28PRTMus
musculus 2Ile Lys Pro Asn Asn Gly Leu Ala 1 5 311PRTMus musculus
3Ala Arg Ser Glu Ile Thr Thr Glu Phe Asp Tyr 1 5 10 48PRTMus
musculus 4Gly Tyr Ser Phe Thr Asp Tyr Thr 1 5 58PRTMus musculus
5Ile Asn Pro Tyr Asn Gly Gly Thr 1 5 611PRTMus musculus 6Ala Arg
Glu Glu Ile Thr Lys Asp Phe Asp Phe 1 5 10 78PRTMus musculus 7Gly
Tyr Thr Phe Thr Asp Tyr Asn 1 5 88PRTMus musculus 8Ile Asn Pro Asn
Asn Gly Gly Thr 1 5 914PRTMus musculus 9Ala Arg Gly Arg Tyr Val Gly
Tyr Tyr Tyr Ala Met Asp Tyr 1 5 10 1010PRTMus musculus 10Glu Ser
Val Asp Ser Tyr Ala Asn Ser Phe 1 5 10 113PRTMus musculus 11Arg Ala
Ser 1 129PRTMus musculus 12Gln Gln Ser Lys Glu Asp Pro Leu Thr 1 5
1310PRTMus musculus 13Glu Ser Ile Asp Thr Tyr Gly Asn Ser Phe 1 5
10 149PRTMus musculus 14Gln Gln Ser Asn Glu Asp Pro Phe Thr 1 5
156PRTMus musculus 15Glu Asn Ile Tyr Ser Asn 1 5 163PRTMus musculus
16Ala Ala Thr 1 179PRTMus musculus 17Gln His Phe Trp Gly Pro Pro
Tyr Thr 1 5 18118PRTMus musculus 18Glu Val Gln Leu Gln Gln Ser Gly
Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys
Lys Thr Ser Gly Tyr Ile Phe Thr Ala Tyr 20 25 30 Thr Met His Trp
Val Arg Gln Ser Leu Gly Glu Ser Leu Asp Trp Ile 35 40 45 Gly Gly
Ile Lys Pro Asn Asn Gly Leu Ala Asn Tyr Asn Gln Lys Phe 50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65
70 75 80 Met Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser Glu Ile Thr Thr Glu Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110 Ala Leu Thr Val Ser Ser 115 19118PRTMus
musculus 19Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro
Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser
Phe Thr Asp Tyr 20 25 30 Thr Leu Asn Trp Val Lys Gln Ser His Gly
Lys Thr Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly
Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Glu Ile Thr Lys Asp Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110
Thr Leu Thr Val Ser Ser 115 20121PRTMus musculus 20Glu Val Leu Leu
Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30
Asn Met Asp Trp Val Lys Gln Ser His Gly Met Ser Leu Glu Trp Ile 35
40 45 Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Ile Phe Asn Gln Lys
Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Tyr Val Gly Tyr Tyr
Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val
Ser Ser 115 120 21111PRTMus musculus 21Asp Ile Val Leu Thr Gln Ser
Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile
Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30 Ala Asn Ser
Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys
Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55
60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80 Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln
Ser Lys 85 90 95 Glu Asp Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu
Glu Met Lys 100 105 110 22111PRTMus musculus 22Gly Ile Val Leu Thr
Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala
Thr Ile Ser Cys Arg Val Ser Glu Ser Ile Asp Thr Tyr 20 25 30 Gly
Asn Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40
45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr
Ile Asn 65 70 75 80 Pro Val Glu Ala Asp Asp Ser Ala Thr Tyr Tyr Cys
Gln Gln Ser Asn 85 90 95 Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr
Lys Leu Glu Met Lys 100 105 110 23107PRTMus musculus 23Asp Ile Gln
Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Glu
Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45 Tyr Ala Ala Thr Asn Leu Val Asp Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn
Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His
Phe Trp Gly Pro Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105 2424DNAMus musculus 24ggatacatat tcactgcata
cacc 242524DNAMus musculus 25attaaaccaa acaatggtct tgct
242633DNAMus musculus 26gcaagatctg agattacgac ggaatttgac tac
332724DNAMus musculus 27ggttattcat tcactgacta cacc 242824DNAMus
musculus 28attaatcctt acaatggtgg tact 242933DNAMus musculus
29gcaagagagg aaattacgaa ggactttgat ttc 333024DNAMus musculus
30ggatacacat tcactgacta caac 243124DNAMus musculus 31attaatccta
acaatggtgg tact 243242DNAMus musculus 32gcaagaggga ggtatgttgg
ttactactat gctatggact ac 423330DNAMus musculus 33gaaagtgttg
atagttatgc caatagtttt 30349DNAMus musculus 34cgtgcatcc 93527DNAMus
musculus 35cagcaaagta aggaggatcc tctcacg 273630DNAMus musculus
36gaaagtattg atacttatgg caatagtttt 303727DNAMus musculus
37cagcaaagta atgaggatcc attcacg 273818DNAMus musculus 38gagaatattt
acagtaat 18399DNAMus musculus 39gctgcaaca 94027DNAMus musculus
40caacattttt ggggtcctcc gtacacg 2741354DNAMus musculus 41gaggtccagc
tgcaacagtc tggacctgag ctggtgaagc ctggggcttc agtgaagata 60tcctgcaaga
cttctggata catattcact gcatacacca tgcactgggt gaggcagagc
120cttggagaga gccttgactg gattggaggt attaaaccaa acaatggtct
tgctaactac 180aaccagaagt tcaagggcaa ggccacattg actgtagaca
agtcctccag cacagcctac 240atggacctcc gcagcctgac atctgaggat
tctgcagtct attactgtgc aagatctgag 300attacgacgg aatttgacta
ctggggccaa ggcaccgctc tcacagtctc ctca 35442354DNAMus musculus
42gaggtccagc tgcaacagtc tggacctgaa ctggtgaagc ctggagcttc aatgaagatt
60tcctgcaagg cttctggtta ttcattcact gactacaccc tgaactgggt gaagcagagc
120catggaaaga cccttgagtg gattggactt attaatcctt acaatggtgg
tactacctac 180aaccagaagt tcaagggcaa ggccacatta actgtagaca
agtcatccag cacagcctac 240atggagctcc tcagtctgac atctgaggac
tctgcagtct attactgtgc aagagaggaa 300attacgaagg actttgattt
ctggggccaa ggcaccactc tcacagtctc ctca 35443363DNAMus musculus
43gaggtcctgc tgcaacagtc tggacctgag ctggtgaagc ctggggcttc agtgaagata
60ccctgcaagg cttctggata cacattcact gactacaaca tggactgggt gaagcagagc
120catggaatga gccttgagtg gattggagat attaatccta acaatggtgg
tactatcttc 180aaccagaagt tcaagggcaa ggccacattg actgtagaca
agtcctccag cacagcctac 240atggagctcc gcagcctgac atctgaggac
actgcagtct attactgtgc aagagggagg 300tatgttggtt actactatgc
tatggactac tggggtcaag gaacctcagt caccgtctcc 360tca 36344333DNAMus
musculus 44gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca
gagggccacc 60atatcctgca gagccagtga aagtgttgat agttatgcca atagttttat
gcactggtac 120cagcagaaac caggacagcc acccaaactc ctcatctatc
gtgcatccaa cctagaatct 180gggatccctg ccaggttcag tggcagtggg
tctaggacag acttcaccct caccattaat 240cctgtggagg ctgatgatgt
tgcaacctat tactgtcagc aaagtaagga ggatcctctc 300acgttcggct
cggggacaaa attggaaatg aaa 33345333DNAMus musculus 45ggcattgtgt
tgacccaatc tccagcttct ttggctgtgt ctctaggaca gagggccacc 60atatcctgca
gagtcagtga aagtattgat acttatggca atagttttat acactggtac
120cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa
cctagaatct 180gggatccctg ccaggttcag tggcagtggg tctaggacag
acttcaccct caccattaat 240cctgtggagg ctgatgattc tgcaacctat
tactgtcagc aaagtaatga ggatccattc 300acgttcggct cggggacaaa
gttggaaatg aaa 33346321DNAMus musculus 46gacatccaga tgactcagtc
tccagcctcc ctatctgtat ctgtgggaga aactgtcacc 60atcacatgtc gagcaagtga
gaatatttac agtaatttag catggtatca gcagaaacag 120ggaaaatctc
ctcagctcct ggtctatgct gcaacaaact tagtagatgg tgtgccatca
180aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag
cctgcagtct 240gaagattttg ggagttatta ctgtcaacat ttttggggtc
ctccgtacac gttcggaggg 300gggaccaagc tggagataaa g
3214724DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for housekeeping gene Ribosomal protein, large, P0
(RPL0) 47gaaactctgc attctcgctt cctg 244822DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer for
housekeeping gene Ribosomal protein, large, P0 (RPL0) 48aggactcgtt
tgtacccgtt ga 224926DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer for housekeeping gene Ribosomal protein,
large, P0 (RPL0) 49tgcagattgg ctacccaact gttgca 265018DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer for HGF
50aacaatgcct ctggttcc 185120DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer for HGF 51cttgtagctg
cgtcctttac 205227DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe for HGF 52ccttcaatag catgtcaagt ggagtga
275328DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for c-Met 53cattaaagga gacctcacca tagctaat
285422DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer for c-Met 54cctgatcgag aaaccacaac ct
225525DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe for c-Met 55catgaagcga ccctctgatg tccca 25568PRTMus
musculus 56Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 578PRTMus musculus
57Ile Asn Pro Thr Thr Gly Ser Thr 1 5 5811PRTMus musculus 58Ala Ile
Gly Gly Tyr Gly Ser Trp Phe Ala Tyr 1 5 10 597PRTMus musculus 59Ser
Ser Val Ser Ser Thr Tyr 1 5 603PRTMus musculus 60Thr Thr Ser 1
619PRTMus musculus 61His Gln Trp Ser Ser Tyr Pro Phe Thr 1 5
62118PRTMus musculus 62Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro
Thr Thr Gly Ser Thr Asp Tyr Asn Gln Lys Leu 50 55 60 Lys Asp Lys
Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Ala Ile Gly Gly Tyr Gly Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ala 115 63108PRTMus musculus 63Gln
Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10
15 Glu Lys Val Thr Leu Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Thr
20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys
Leu Trp 35 40 45 Ile Tyr Thr Thr Ser Ile Leu Ala Ser Gly Val Pro
Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu
Thr Ile Ser Ser Met Glu 65 70 75 80 Thr Glu Asp Ala Ala Ser Tyr Phe
Cys His Gln Trp Ser Ser Tyr Pro 85 90 95 Phe Thr Phe Gly Ser Gly
Thr Lys Leu Asp Ile Lys 100 105 6424DNAMus musculus 64ggctacactt
ttacttccta ctgg 246524DNAMus musculus 65attaacccta ccactggttc tact
246633DNAMus musculus 66gcaataggag gatatgggtc ctggtttgct tac
336721DNAMus musculus 67tcaagtgtaa gttccaccta c 21689DNAMus
musculus 68accacatcc 96927DNAMus musculus 69catcagtgga gtagttaccc
attcacg 2770354DNAMus musculus 70caggtccagc ttcagcagtc tggggctgaa
ctggcaaaac ctggggcctc agtgaagatg 60tcctgcaagg cttctggcta cacttttact
tcctactgga tgaactgggt gaaacagagg 120cctggacagg gtctggaatg
gattggatac attaacccta ccactggttc tactgactac 180aatcagaagt
taaaggacaa ggccacattg actgcagaca aatcctccaa cacagcctac
240atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc
aataggagga 300tatgggtcct ggtttgctta ctggggccaa gggactctgg
tcactgtctc tgca 35471324DNAMus musculus 71caaattgttc tcacccagtc
tccagcaatc atgtctgcat ctcctgggga gaaggtcacc 60ttgacctgca gtgccagctc
aagtgtaagt tccacctact tgtactggta ccagcagaag 120ccaggatcct
cccccaaact ctggatttat accacatcca tcctggcttc tggagtccct
180gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag
cagcatggag 240actgaagatg ctgcctctta tttctgccat cagtggagta
gttacccatt cacgttcggc 300tcggggacaa agttggacat aaaa 324728PRTMus
musculus 72Gly Tyr Ile Phe Thr Ala Tyr Thr 1 5 738PRTMus musculus
73Ile Lys Pro Asn Asn Gly Leu Ala 1 5 7411PRTMus musculus 74Ala Arg
Ser Glu Ile Thr Thr Glu Phe Asp Tyr 1 5 10 7510PRTMus musculus
75Glu Ser Val Asp Ser Tyr Ala Asn Ser Phe 1 5 10 763PRTMus musculus
76Arg Ala Ser 1 779PRTMus musculus 77Gln Gln Ser Lys Glu Asp Pro
Leu Thr 1 5 78118PRTMus musculus 78Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Ile Phe Thr Ala Tyr 20 25 30 Thr Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp
Ile Lys Pro Asn Asn Gly Leu Ala Asn Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65
70
75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Ser Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly
Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 79112PRTMus
musculus 79Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser
Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Glu Ser
Val Asp Ser Tyr 20 25 30 Ala Asn Ser Phe Leu His Trp Tyr Gln Gln
Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser
Thr Arg Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala
Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Glu Asp
Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110
80118PRTMus musculus 80Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ile Phe Thr Ala Tyr 20 25 30 Thr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Lys Pro
Asn Asn Gly Leu Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ser 115 81111PRTMus musculus 81Asp
Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10
15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Glu Ser Val Asp Ser Tyr
20 25 30 Ala Asn Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Thr Arg Glu Ser
Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala
Val Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Glu Asp Pro Leu Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 82327PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
82Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1
5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys
Ser Cys Asp Cys His Cys Pro Pro Cys Pro Ala 100 105 110 Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 115 120 125 Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 130 135
140 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
145 150 155 160 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln 165 170 175 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln 180 185 190 Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala 195 200 205 Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro 210 215 220 Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 225 230 235 240 Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 245 250 255
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 260
265 270 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr 275 280 285 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe 290 295 300 Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys 305 310 315 320 Ser Leu Ser Leu Ser Pro Gly 325
83107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 83Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90
95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
84327PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 84Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Phe 20 25 30 Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly 35 40 45 Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 50 55 60 Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 65 70 75 80 Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 85 90
95 Val Glu Pro Lys Ser Cys Asp Cys His Cys Pro Pro Cys Pro Ala Pro
100 105 110 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val 130 135 140 Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165 170 175 Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 195 200 205 Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215
220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser
Leu Ser Pro Gly Lys 325 85106PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 85Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50
55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
105 86445PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 86Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ile Phe Thr Ala Tyr 20 25 30 Thr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Lys Pro
Asn Asn Gly Leu Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn
Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Cys His 210 215
220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340
345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 87218PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
87Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1
5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Glu Ser Val Asp Ser
Tyr 20 25 30 Ala Asn Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly
Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Thr Arg Glu
Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val
Ala Val Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Glu Asp Pro Leu Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135
140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys 210 215 88446PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 88Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ala Tyr 20 25 30 Thr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Trp Ile Lys Pro Asn Asn Gly Leu Ala Asn Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu Ile Thr Thr Glu Phe Asp Tyr
Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
Asp Cys His 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385
390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 435 440 445 89218PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 89Asp Ile Val Met Thr
Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala
Thr Ile Asn Cys Lys Ser Ser Glu Ser Val Asp Ser Tyr 20 25 30 Ala
Asn Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40
45 Lys Leu Leu Ile Tyr Arg Ala Ser Thr Arg Glu Ser Gly Val Pro Asp
50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys
Gln Gln Ser Lys 85 90 95 Glu Asp Pro Leu Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170
175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
901395DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 90atgggatggt cttggatctt tctgctgttt
ctgtctggta ctgctggtgt gctgagccag 60gtccagctgg tgcaatccgg cgcagaggtg
aagaagccag gcgcttccgt gaaggtgagc 120tgtaaggcct ctggctacat
cttcacagca tacaccatgc actgggtgag gcaagctcct 180gggcagggac
tggagtggat gggatggatt aaacccaaca atgggctggc caactacgcc
240cagaaattcc agggtagggt cactatgaca agggatacca gcatcagcac
cgcatatatg 300gagctgagca ggctgaggtc tgacgacact gctgtctatt
attgcgccag gagcgaaatt 360acaacagaat tcgattactg ggggcagggc
accctggtga ccgtgtcctc tgccagcacc 420aagggcccaa gcgtgttccc
cctggccccc agcagcaaga gcaccagcgg cggcacagcc 480gccctgggct
gcctggtgaa ggactacttc cccgagcccg tgaccgtgtc ctggaacagc
540ggagccctca cttctggagt tcataccttc ccagcagtat tgcagagcag
tggcctgtat 600tcactgtctt ccgtcgtaac agttccatcc tccagcctcg
ggacacagac ttacatttgt 660aacgtgaatc acaagcctag caacaccaag
gtcgacaaga gagttgaacc aaagagttgt 720gattgccact gtcctccctg
cccagctcct gagctgcttg gcggtcccag tgtcttcttg 780tttcccccta
aacccaaaga caccctgatg atctcaagga ctcccgaggt gacatgcgtg
840gtggtggatg tgtctcatga ggacccagag gtgaagttca actggtacgt
ggacggcgtg 900gaggtgcaca acgccaagac caagcccaga gaggagcagt
acaacagcac ctacagggtg 960gtgtccgtgc tgaccgtgct gcaccaggac
tggctgaacg gcaaggagta caagtgtaag 1020gtgtccaaca aggccctgcc
agccccaatc gaaaagacca tcagcaaggc caagggccag 1080ccaagagagc
cccaggtgta caccctgcca cccagcaggg aggagatgac caagaaccag
1140gtgtccctga cctgtctggt gaagggcttc tacccaagcg acatcgccgt
ggagtgggag 1200agcaacggcc agcccgagaa caactacaag accacccccc
cagtgctgga cagcgacggc 1260agcttcttcc tgtacagcaa gctgaccgtg
gacaagagca gatggcagca gggcaacgtg 1320ttcagctgct ccgtgatgca
cgaggccctg cacaaccact acacccagaa gagcctgagc 1380ctgtccccag gctga
139591717DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 91atggaaactg atacactgct gctgtgggtc
ctgctgctgt gggtccctgg aagcacaggg 60gacattgtga tgacccagtc tcccgatagc
ctggccgtgt ccctgggcga gagggctacc 120atcaactgta aaagctccga
atctgtggac tcttacgcaa acagctttct gcactggtat 180cagcaaaagc
caggccaacc tccaaagctg ctgatttaca gggcttctac cagggagagc
240ggcgtgcccg ataggttcag cggatctggc agcggcaccg actttacact
gaccatctcc 300agcctgcagg ccgaagatgt ggcagtctat tactgccagc
agtccaagga ggaccccctg 360actttcgggg gtggtactaa agtggagatc
aagcgtacgg tggccgctcc cagcgtgttc 420atcttccccc caagcgacga
gcagctgaag agcggcaccg ccagcgtggt gtgtctgctg 480aacaacttct
accccaggga ggccaaggtg cagtggaagg tggacaacgc cctgcagagc
540ggcaacagcc aggagagcgt caccgagcag gacagcaagg actccaccta
cagcctgagc 600agcaccctga ccctgagcaa ggccgactac gagaagcaca
aggtgtacgc ctgtgaggtg 660acccaccagg gcctgtccag ccccgtgacc
aagagcttca acaggggcga gtgctga 71792449PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
92Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30 Trp Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Gly Met Ile Asp Pro Ser Asn Ser Asp Thr Arg
Phe Asn Pro Asn Phe 50 55 60 Lys Asp Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Tyr Arg Ser
Tyr Val Thr Pro Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser 355 360 365 Cys Ala
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 445 Lys 93220PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
93Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr
Thr 20 25 30 Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ala Tyr Pro
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135
140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 215 220 94227PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
94Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1
5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135
140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
95441PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 95Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Val Asn Pro
Asn Arg Arg Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Glu Gly Arg
Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr
100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Lys Thr
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val
Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 210 215
220 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys 245 250 255 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu
Val Gln Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 340
345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Arg Leu Thr Val Asp
Lys Ser Arg Trp Gln Glu Gly Asn 405 410 415 Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu
Ser Leu Ser Leu Gly 435 440 96215PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 96Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Ser Val Ser Ser Ser Val Ser Ser Ile 20 25 30 Tyr
Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser
50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Val Tyr
Ser Gly Tyr Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165
170
175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215
9715PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 97Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 1 5 10 15 984PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 98Gly Phe Leu Gly 1
994PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 99Ala Leu Ala Leu 1 1006PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
100His His His His His His 1 5 101115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
101Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45 Gly Arg Val Asn Pro Asn Arg Arg Gly Thr
Thr Tyr Asn Gln Lys Phe 50 55 60 Glu Gly Arg Val Thr Met Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Asn
Trp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser
Ser 115 102109PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 102Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Ser Val Ser Ser Ser Val Ser Ser Ile 20 25 30 Tyr Leu His Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr
Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65
70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Val Tyr Ser Gly
Tyr Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg 100 105 103119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 103Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Leu His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Met
Ile Asp Pro Ser Asn Ser Asp Thr Arg Phe Asn Pro Asn Phe 50 55 60
Lys Asp Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Tyr Arg Ser Tyr Val Thr Pro Leu Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
104114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 104Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys
Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30 Ser Ser Gln Lys Asn Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu
Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85
90 95 Tyr Tyr Ala Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile 100 105 110 Lys Arg 105118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 105Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Tyr
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40
45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala
50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr
Val Val Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
106114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 106Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys
Ser Ser Gln Ser Leu Leu Ala Ser 20 25 30 Gly Asn Gln Asn Asn Tyr
Leu Ala Trp His Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Met
Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val 50 55 60 Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85
90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile 100 105 110 Lys Arg 107127PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 107Gln Val Gln Leu Val
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser
Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Thr Thr Asn 20 25 30 Tyr
Tyr Tyr Trp Ser Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu 35 40
45 Trp Met Gly Val Ile Ala Tyr Asp Gly Ser Thr Asp Tyr Ser Pro Ser
50 55 60 Leu Lys Ser Arg Thr Ser Ile Ser Arg Asp Thr Ser Lys Asn
Gln Phe 65 70 75 80 Ser Leu Gln Leu Ser Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Val Arg Val Ile Ala Thr
Gly Trp Ala Thr Ala Asn 100 105 110 Ala Leu Asp Ala Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 125 108111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
108Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Lys
1 5 10 15 Thr Val Thr Ile Ser Cys Ala Gly Thr Ser Ser Asp Val Gly
Tyr Gly 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr
Ala Pro Lys Leu 35 40 45 Leu Ile Phe Ala Val Ser Tyr Arg Ala Ser
Gly Ile Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr
Ala Phe Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ser Tyr Arg Ser Ser 85 90 95 Asn Asn Ala Ala
Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 110
109117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 109Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Phe Ile Arg
Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85
90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr
Leu 100 105 110 Val Thr Val Ser Ser 115 110115PRTMus musculus
110Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu
Ala Trp 20 25 30 Ser Asn Gln Asn Asn Tyr Leu Ala Trp Tyr Leu Gln
Lys Pro Gly Gln 35 40 45 Ser Pro Gln Met Leu Ile Ile Trp Ala Ile
Thr Arg Val Gly Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln 85 90 95 Ser Tyr Ser Arg
Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg
Thr 115 1119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 111Gln Gln Ser Tyr Ser Arg Pro Tyr Thr 1
5 11210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 112Gly Tyr Ile Phe Thr Ala Tyr Thr Met His 1 5 10
11317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 113Trp Ile Lys Pro Asn Asn Gly Leu Ala Asn Tyr
Ala Gln Lys Phe Gln 1 5 10 15 Gly 1149PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 114Ser
Glu Ile Thr Thr Glu Phe Asp Tyr 1 5 11515PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 115Lys
Ser Ser Glu Ser Val Asp Ser Tyr Ala Asn Ser Phe Leu His 1 5 10 15
1167PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 116Arg Ala Ser Thr Arg Glu Ser 1 5
1179PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 117Gln Gln Ser Lys Glu Asp Pro Leu Thr 1 5
11810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 118Gly Tyr Ile Phe Thr Ala Tyr Thr Met His 1 5 10
11917PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 119Trp Ile Lys Pro Asn Asn Gly Leu Ala Asn Tyr
Ala Gln Lys Phe Gln 1 5 10 15 Gly 1209PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 120Ser
Glu Ile Thr Thr Glu Phe Asp Tyr 1 5 12115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 121Lys
Ser Ser Glu Ser Val Asp Ser Tyr Ala Asn Ser Phe Leu His 1 5 10 15
1227PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 122Arg Ala Ser Thr Arg Glu Ser 1 5
1239PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 123Gln Gln Ser Lys Glu Asp Pro Leu Thr 1 5
12430DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 124ggctacatct tcacagcata caccatgcac
3012551DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 125tggattaaac ccaacaatgg gctggccaac
tacgcccaga aattccaggg t 5112627DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 126agcgaaatta
caacagaatt cgattac 2712745DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 127aaaagctccg
aatctgtgga ctcttacgca aacagctttc tgcac 4512821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 128agggcttcta ccagggagag c 2112927DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 129cagcagtcca aggaggaccc cctgact
2713010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 130Gly Tyr Thr Phe Thr Asp Tyr Tyr Met His 1 5 10
13117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 131Arg Val Asn Pro Asn Arg Arg Gly Thr Thr Tyr
Asn Gln Lys Phe Glu 1 5 10 15 Gly 1326PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 132Ala
Asn Trp Leu Asp Tyr 1 5 13312PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 133Ser Val Ser Ser Ser Val
Ser Ser Ile Tyr Leu His 1 5 10 1347PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 134Ser
Thr Ser Asn Leu Ala Ser 1 5 1359PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 135Gln Val Tyr Ser Gly Tyr
Pro Leu Thr 1 5 13610PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 136Gly Tyr Thr Phe Thr Ser
Tyr Trp Leu His 1 5 10 13717PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 137Met Ile Asp Pro Ser Asn
Ser Asp Thr Arg Phe Asn Pro Asn Phe Lys 1 5 10 15 Asp
13812PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 138Ala Thr Tyr Arg Ser Tyr Val Thr Pro Leu Asp
Tyr 1 5 10 13917PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 139Lys Ser Ser Gln Ser Leu Leu Tyr Thr
Ser Ser Gln Lys Asn Tyr Leu 1 5 10 15 Ala 1407PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 140Trp
Ala Ser Thr Arg Glu Ser 1 5 1419PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 141Gln Gln Tyr Tyr Ala Tyr
Pro Trp Thr 1 5 1425PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 142Asp Tyr Tyr Met Ser 1 5
14314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 143Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr
Glu Tyr Ser 1 5 10 1447PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 144Arg Asp Asn Trp Phe Ala
Tyr 1 5 14511PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 145Lys Ser Ser Gln Ser Leu Leu Ala Ser
Gly Asn 1 5 10 1467PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 146Trp Ala Ser Thr Arg Val Ser 1 5
1479PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 147Gln Gln Ser Tyr Ser Ala Pro Leu Thr 1 5
1487PRTHomo sapiens 148Thr Asn Tyr Tyr Tyr Trp Ser 1 5 14916PRTHomo
sapiens 149Val Ile Ala Tyr Asp Gly Ser Thr Asp Tyr Ser Pro Ser Leu
Lys Ser 1 5 10 15 15017PRTHomo sapiens 150Asp Val Arg Val Ile Ala
Thr Gly Trp Ala Thr Ala Asn Ala Leu Asp 1 5 10 15 Ala 15114PRTHomo
sapiens 151Ala Gly Thr Ser Ser Asp Val Gly Tyr Gly Asn Tyr Val Ser
1 5 10 1527PRTHomo sapiens 152Ala Val Ser Tyr Arg Ala Ser 1 5
15311PRTHomo sapiens 153Ala Ser Tyr Arg Ser Ser Asn Asn Ala Ala Val
1 5
10 1545PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 154Asp Tyr Tyr Ile Ser 1 5 15519PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 155Phe
Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala Ser 1 5 10
15 Val Lys Gly 1566PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 156Asp Asn Trp Phe Ala Tyr 1 5
15717PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 157Lys Ser Ser Gln Ser Leu Leu Ala Trp Ser Asn
Gln Asn Asn Tyr Leu 1 5 10 15 Ala 1587PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 158Trp
Ala Ile Thr Arg Val Gly 1 5 15911PRTMus musculus 159Lys Ser Leu Leu
His Ser Asn Gly Ile Thr Tyr 1 5 10 1603PRTMus musculus 160Gln Met
Ser 1 1619PRTMus musculus 161Ala Gln Asn Leu Glu Leu Pro Tyr Thr 1
5 1628PRTMus musculus 162Gly Phe Thr Phe Ser Ser Tyr Ala 1 5
1637PRTMus musculus 163Ile Met Gly Gly Gly Thr Thr 1 5 16415PRTMus
musculus 164Ala Arg Gly Arg Asp Tyr Gly Ile Arg Ser Tyr Ala Met Asp
Tyr 1 5 10 15 16515PRTMus musculus 165Ser Ser Lys Ser Leu Leu His
Ser Asn Gly Ile Thr Tyr Leu Tyr 1 5 10 15 1667PRTMus musculus
166Gln Met Ser Asn Leu Ala Ser 1 5 1674PRTMus musculus 167Ser Tyr
Ala Met 1 16816PRTMus musculus 168Ser Ile Met Gly Gly Gly Thr Thr
Tyr Tyr Pro Asp Ser Val Lys Gly 1 5 10 15 16913PRTMus musculus
169Gly Arg Asp Tyr Gly Ile Arg Ser Tyr Ala Met Asp Tyr 1 5 10
17012PRTArtificial SequenceDescription of Artificial Sequence
Synthetic hinge peptide derived from IgG1 170Pro Lys Ser Cys Asp
Cys His Cys Pro Pro Cys Pro 1 5 10 171445PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
171Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr
Ala Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45 Gly Trp Ile Lys Pro Asn Asn Gly Leu Ala
Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu
Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130
135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys
Arg Val Glu Pro Lys Ser Cys Asp Cys His 210 215 220 Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Cys Val Phe 225 230 235 240 Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250
255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375
380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440 445 172218PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 172Asp Ile Val Met Thr
Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala
Thr Ile Asn Cys Lys Ser Ser Glu Ser Val Asp Ser Tyr 20 25 30 Ala
Asn Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40
45 Lys Leu Leu Ile Tyr Arg Ala Ser Thr Arg Glu Ser Gly Val Pro Asp
50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys
Gln Gln Ser Lys 85 90 95 Glu Asp Pro Leu Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170
175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
17310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 173Gly Tyr Ile Phe Thr Ala Tyr Thr Met His 1 5 10
17417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 174Trp Ile Lys Pro Asn Asn Gly Leu Ala Asn Tyr
Ala Gln Lys Phe Gln 1 5 10 15 Gly 1759PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 175Ser
Glu Ile Thr Thr Glu Phe Asp Tyr 1 5 17615PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 176Lys
Ser Ser Glu Ser Val Asp Ser Tyr Ala Asn Ser Phe Leu His 1 5 10 15
1777PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 177Arg Ala Ser Thr Arg Glu Ser 1 5
1789PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 178Gln Gln Ser Lys Glu Asp Pro Leu Thr 1 5
* * * * *
References