U.S. patent application number 15/170428 was filed with the patent office on 2016-12-22 for cytotoxic benzodiazepine derivatives.
The applicant listed for this patent is ImmunoGen, Inc.. Invention is credited to Ravi V.J. Chari, Michael Louis Miller, Manami Shizuka.
Application Number | 20160367698 15/170428 |
Document ID | / |
Family ID | 54140692 |
Filed Date | 2016-12-22 |
United States Patent
Application |
20160367698 |
Kind Code |
A1 |
Chari; Ravi V.J. ; et
al. |
December 22, 2016 |
CYTOTOXIC BENZODIAZEPINE DERIVATIVES
Abstract
The invention relates to novel benzodiazepine derivatives with
antiproliferative activity and more specifically to novel
benzodiazepine compounds of formula (I)-(VII). The invention also
provides conjugates of the benzodiazepine compounds linked to a
cell-binding agent. The invention further provides compositions and
methods useful for inhibiting abnormal cell growth or treating a
proliferative disorder in a mammal using the compounds or
conjugates of the invention.
Inventors: |
Chari; Ravi V.J.; (Newton,
MA) ; Miller; Michael Louis; (Framingham, MA)
; Shizuka; Manami; (Belmont, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ImmunoGen, Inc. |
Waltham |
MA |
US |
|
|
Family ID: |
54140692 |
Appl. No.: |
15/170428 |
Filed: |
June 1, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14843604 |
Sep 2, 2015 |
9381256 |
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15170428 |
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62164352 |
May 20, 2015 |
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62149409 |
Apr 17, 2015 |
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62087065 |
Dec 3, 2014 |
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62045236 |
Sep 3, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/92 20130101;
A61K 2039/505 20130101; C07K 16/2866 20130101; C07D 519/00
20130101; A61K 47/6803 20170801; A61K 47/6849 20170801; A61P 35/02
20180101; C07K 2317/24 20130101; A61K 47/6857 20170801; A61P 19/08
20180101; A61P 1/18 20180101; A61P 31/00 20180101; A61P 15/00
20180101; A61P 11/00 20180101; A61K 47/6845 20170801; C07D 487/04
20130101; C07K 16/2863 20130101; A61K 47/6851 20170801; A61P 29/00
20180101; A61P 13/12 20180101; C07K 2317/21 20130101; A61P 21/00
20180101; A61P 13/08 20180101; A61P 17/00 20180101; C07K 2317/73
20130101; A61P 37/06 20180101; C07K 16/28 20130101; A61P 25/00
20180101; A61P 31/04 20180101; A61P 31/12 20180101; A61K 45/06
20130101; A61P 35/00 20180101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; C07D 519/00 20060101 C07D519/00; C07K 16/28 20060101
C07K016/28; A61K 45/06 20060101 A61K045/06 |
Claims
1. A compound represented by any one of the following formulas:
##STR00097## or a pharmaceutically acceptable salt thereof,
wherein: the double line between N and C represents a single bond
or a double bond, provided that when it is a double bond, X is
absent and Y is --H, and when it is a single bond, X is selected
from --H; Y is --H, --SO.sub.3M or --OH wherein M is --H or a
cation; X' is --H; Y' is --H; R.sup.x is
--(CH.sub.2).sub.p--(CR.sup.fR.sup.g)--, wherein R.sup.f and
R.sup.g are each independently selected from --H or a linear or
branched alkyl having 1 to 4 carbon atoms; and p is 0, 1, 2 or 3;
R.sup.e is --H or a linear or branched alkyl having 1 to 6 carbon
atoms; G is --CH--; Z.sup.s is --H, --SR.sup.d, or is selected from
any one of the following formulas: ##STR00098## ##STR00099##
wherein: q is an integer from 1 to 5; R.sup.d is a linear or
branched alkyl having 1 to 6 carbon atoms or is selected from
phenyl, nitrophenyl, dinitrophenyl, carboxynitrophenyl, pyridyl and
nitropyridyl; n' is an integer from 2 to 6; U is --H or
--SO.sub.3M; and M is --H or a cation.
2-3. (canceled)
4. The compound of claim 1, wherein Z.sup.s is --H or --SR.sup.d,
wherein R.sup.d is -Me or pyridyl.
5. (canceled)
6. The compound of claim 4, wherein Z.sup.s is --H.
7. The compound of claim 1, wherein R.sup.e is --H or -Me.
8. (canceled)
9. The compound of claim 1, wherein R.sup.f and R.sup.g are the
same or different, and are selected from --H and -Me.
10-16. (canceled)
17. The compound of claim 1, wherein M is --H, Na.sup.+ or
K.sup.+.
18-23. (canceled)
24. The compound of claim 1, wherein the compound is represented by
the following formula: ##STR00100## or a pharmaceutically
acceptable salt thereof, wherein M is --H, Na.sup.+ or K.sup.+.
25. A conjugate comprising a cytotoxic compound and a cell-binding
agent (CBA), wherein the cytotoxic compound is covalently linked to
the CBA, and wherein said cytotoxic compound is represented by any
one of the following formulas: ##STR00101## or a pharmaceutically
acceptable salt thereof, wherein: CBA is an antibody or an antibody
fragment that specifically binds to a target cell; the double line
between N and C represents a single bond or a double bond, provided
that when it is a double bond, X is absent and Y is --H, and when
it is a single bond, X is selected from --H; Y is --H, --SO.sub.3M
or --OH, wherein M is --H or a cation; X' is --H; Y' is --H;
R.sup.x is --(CH.sub.2).sub.p--(CR.sup.fR.sup.g)--, wherein R.sup.f
and R.sup.g are each independently selected from --H or a linear or
branched alkyl having 1 to 4 carbon atoms; and p is 0, 1, 2 or 3;
R.sup.e is --H or a linear or branched alkyl having 1 to 6 carbon
atoms; G is --CH--; Z.sup.s1 is selected from any one of the
following formulas: ##STR00102## ##STR00103## wherein: q is an
integer from 1 to 5; R.sup.d is a linear or branched alkyl having 1
to 6 carbon atoms or is selected from phenyl, nitrophenyl,
dinitrophenyl, carboxynitrophenyl, pyridyl and nitropyridyl; n is
an integer from 2 to 6; U is --H or --SO.sub.3M; and M is --H.sup.+
or a cation.
26. (canceled)
27. The conjugate of claim 25, wherein Z.sup.s1 is represented by
the following formula: ##STR00104##
28. The conjugate of claim 25, wherein R.sup.e is --H or -Me.
29. (canceled)
30. The conjugate of claim 25, wherein R.sup.f and R.sup.g are the
same or different, and are selected from --H and -Me.
31-37. (canceled)
38. The conjugate of claim 25, wherein M is --H, Na.sup.+ or
K.sup.+.
39-44. (canceled)
45. The conjugate of claim 25, wherein the conjugate is represented
by any one of the following formulas: ##STR00105## ##STR00106##
##STR00107## or a pharmaceutically acceptable salt thereof, wherein
M is --H, Na.sup.+ or K.sup.+.
46-47. (canceled)
48. The conjugate of claim 25, wherein the cell-binding agent is an
anti-folate receptor antibody or an antibody fragment thereof, an
anti-EGFR antibody or an antibody fragment thereof, an anti-CD33
antibody or an antibody fragment thereof, an anti-CD19 antibody or
an antibody fragment thereof, an anti-Muc1 antibody or an antibody
fragment thereof, or an anti-CD37 antibody or an antibody fragment
thereof.
49-56. (canceled)
57. The conjugate of claim 25, wherein the antibody is huMOV19,
huML66, huMy9-6, huB4, huDS6 or huCD37-3 antibody.
58-76. (canceled)
77. A pharmaceutical composition comprising the conjugate of claim
25 and a pharmaceutically acceptable carrier.
78. A method of treating a cancer in a mammal, comprising
administering to said mammal a therapeutically effective amount of
a conjugate of claim 25, and optionally, a chemotherapeutic
agent.
79-80. (canceled)
81. The method of claim 78, wherein the cancer is ovarian cancer,
pancreatic cancer, melanoma, lung cancer (e.g., non-small cell lung
cancer), cervical cancer, breast cancer, squamous cell carcinoma of
the head and neck, prostate cancer, endometrial cancer, lymphoma
(e.g., non-Hodgkin lymphoma), myelodysplastic syndrome (MDS),
peritoneal cancer, or leukemia (e.g., acute myeloid leukemia (AML),
acute monocytic leukemia, promyelocytic leukemia, eosinophilic
leukaemia, acute lymphoblastic leukemia (e.g., B-ALL), chronic
lymphocytic leukemia (CLL), and chronic myeloid leukemia
(CML)).
82. The method of claim 81, wherein the cancer is acute myeloid
leukemia (AML), non-small cell lung cancer or ovarian cancer.
83-84. (canceled)
85. The method of claim 78, wherein the conjugate is represented by
##STR00108## ##STR00109## ##STR00110## or a pharmaceutically
acceptable salt thereof, wherein M is --H, Na.sup.+ or K.sup.+.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 14/843,604, filed Sep. 2, 2015, which
claims the benefit of the filing date under 35 U.S.C. .sctn.119(e),
of U.S. Provisional Application No. 62/045,236, filed on Sep. 3,
2014, U.S. Provisional Application No. 62/087,065, filed on Dec. 3,
2014, U.S. Provisional Application No. 62/149,409, filed on Apr.
17, 2015, and U.S. Provisional Application No. 62/164,352, filed on
May 20, 2015, the entire contents of each of which, including all
drawings, formulae, specifications, and claims, are incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to novel cytotoxic compounds,
and cytotoxic conjugates comprising these cytotoxic compounds and
cell-binding agents. More specifically, this invention relates to
novel benzodiazepine compounds, derivatives thereof, intermediates
thereof, conjugates thereof, and pharmaceutically acceptable salts
thereof, which are useful as medicaments, in particular as
anti-proliferative agents.
BACKGROUND OF THE INVENTION
[0003] Benzodiazepine derivatives are useful compounds for treating
various disorders, and include medicaments such as, antiepileptics
(imidazo [2,1-b][1,3,5] benzothiadiazepines, U.S. Pat. No.
4,444,688; U.S. Pat. No. 4,062,852), antibacterials
(pyrimido[1,2-c][1,3,5]benzothiadiazepines, GB 1476684), diuretics
and hypotensives (pyrrolo(1,2-b)[1,2,5]benzothiadiazepine 5,5
dioxide, U.S. Pat. No. 3,506,646), hypolipidemics (WO 03091232),
anti-depressants (U.S. Pat. No. 3,453,266), osteoporosis (JP
2138272).
[0004] It has been shown in animal tumor models that benzodiazepine
derivatives, such as pyrrolobenzodiazepines (PBDs), act as
anti-tumor agents (N-2-imidazolyl alkyl substituted
1,2,5-benzothiadiazepine-1,1-dioxide, U.S. Pat. No. 6,156,746),
benzo-pyrido or dipyrido thiadiazepine (WO 2004/069843), pyrrolo
[1,2-b] [1,2,5] benzothiadiazepines and pyrrolo[1,2-b][1,2,5]
benzodiazepine derivatives (WO2007/015280), tomaymycin derivatives
(e.g., pyrrolo[1,4]benzodiazepines), such as those described in WO
00/12508, WO2005/085260, WO2007/085930, and EP 2019104.
Benzodiazepines are also known to affect cell growth and
differentiation (Kamal A., et al., Bioorg Med Chem. 2008 Aug. 15;
16(16):7804-10 (and references cited therein); Kumar R, Mini Rev
Med Chem. 2003 June; 3(4):323-39 (and references cited therein);
Bednarski J J, et al., 2004; Sutter A. P, et al., 2002; Blatt N B,
et al., 2002), Kamal A. et al., Current Med. Chem., 2002; 2;
215-254, Wang J-J., J. Med. Chem., 2206; 49:1442-1449, Alley M. C.
et al., Cancer Res. 2004; 64:6700-6706, Pepper C. J., Cancer Res
2004; 74:6750-6755, Thurston D. E. and Bose D. S., Chem Rev 1994;
94:433-465; and Tozuka, Z., et al., Journal of Antibiotics, (1983)
36; 1699-1708. General structure of PBDs is described in US
Publication Number 20070072846. The PBDs differ in the number, type
and position of substituents, in both their aromatic A rings and
pyrrolo C rings, and in the degree of saturation of the C ring.
Their ability to form an adduct in the minor groove and crosslink
DNA enables them to interfere with DNA processing, hence their
potential for use as antiproliferative agents.
[0005] The first pyrrolobenzodiazepine to enter the clinic, SJG-136
(NSC 694501) is a potent cytotoxic agent that causes DNA
inter-strand crosslinks (S. G Gregson et al., 2001, J. Med. Chem.,
44: 737-748; M. C. Alley et al., 2004, Cancer Res., 64: 6700-6706;
J. A. Hartley et al., 2004, Cancer Res., 64: 6693-6699; C. Martin
et al., 2005, Biochemistry., 44: 4135-4147; S. Arnould et al.,
2006, Mol. Cancer Ther., 5: 1602-1509). Results from a Phase I
clinical evaluation of SJG-136 revealed that this drug was toxic at
extremely low doses (maximum tolerated dose of 45 .mu.g/m.sup.2,
and several adverse effects were noted, including vascular leak
syndrome, peripheral edema, liver toxicity and fatigue. DNA damage
was noted at all doses in circulating lymphocytes (D. Hochhauser et
al., 2009, Clin. Cancer Res., 15: 2140-2147). Thus, there exists a
need for improved benzodiazepine derivatives that are less toxic
and still therapeutically active for treating a variety of
proliferative disease states, such as cancer.
SUMMARY OF THE INVENTION
[0006] The novel cytotoxic benzodiazepine dimer compounds described
herein and conjugates thereof have unexpectedly higher therapeutic
index (ratio of maximum tolerated dose to minimum effective dose)
in vivo compared to benzodiazepine derivatives and conjugates
thereof described in the art.
[0007] Thus, provided herein are novel cytotoxic benzodiazepine
dimer compounds represented by any one of the following
formulas:
##STR00001## ##STR00002##
or a pharmaceutically acceptable salt thereof, wherein:
[0008] the double line between N and C represents a single bond or
a double bond, provided that when it is a double bond, X is absent
and Y is --H, and when it is a single bond, X is selected from --H,
or an amine protecting group;
[0009] Y is selected from --H, --OR, --OCOR', --SR, --NR'R'',
--SO.sub.3M, --SO.sub.2M or --OSO.sub.3M, wherein M is --H or a
cation;
[0010] R is --H, an optionally substituted linear, branched or
cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms
or a PEG group --(CH.sub.2CH.sub.2O).sub.n--R.sup.c, wherein n is
an integer from 1 to 24, and R.sup.c is a linear or branched alkyl
having 1 to 4 carbon atoms;
[0011] R' and R'' are the same or different, and are selected from
--H, --OR, --NRR.sup.g', --COR, an optionally substituted linear,
branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10
carbon atoms, an optionally substituted aryl having from 6 to 18
carbon atoms, an optionally substituted 3- to 18-membered
heterocyclic ring having 1 to 6 heteroatoms selected from O, S, N
and P, a PEG group --(CH.sub.2CH.sub.2O).sub.n--R.sup.c, wherein n
is an integer from 1 to 24, preferably n is 2, 4 or 8; and R.sup.g'
is --H, an optionally substituted linear, branched or cyclic alkyl,
alkenyl or alkynyl having from 1 to 10 carbon atoms or a PEG group
--(CH.sub.2CH.sub.2O).sub.n--R.sup.c;
[0012] X' is selected from the group consisting of --H, --OH, an
optionally substituted linear, branched or cyclic alkyl, alkenyl or
alkynyl having from 1 to 10 carbon atoms, phenyl, and an
amine-protecting group;
[0013] Y' is selected from the group consisting of --H, an oxo
group, an optionally substituted linear, branched or cyclic alkyl,
alkenyl or alkynyl having from 1 to 10 carbon atoms;
[0014] R.sup.x is a linear or branched alkylene having 1 to 6
carbon atoms, optionally substituted with a charged substituent or
an ionizable group Q;
[0015] R.sup.e is --H or a linear or branched alkyl having 1 to 6
carbon atoms;
[0016] G is selected from --CH-- or --N--;
[0017] Z.sup.s is --H, --SR.sup.d, --COR.sup.d' or is selected from
any one of the following formulas:
##STR00003##
wherein:
[0018] q is an integer from 1 to 5;
[0019] R.sup.d is a linear or branched alkyl having 1 to 6 carbon
atoms or is selected from phenyl, nitrophenyl, dinitrophenyl,
carboxynitrophenyl, pyridyl and nitropyridyl;
[0020] R.sup.d' is a linear or branched alkyl having 1 to 6 carbon
atoms;
[0021] n' is an integer from 2 to 6;
[0022] --C(.dbd.O)OE represents a reactive ester group; and
[0023] M is H.sup.+ or a cation, provided that the compound is
not
##STR00004##
[0024] In one embodiment, the reactive ester group represented by
--C(.dbd.O)OE is selected from N-hydroxysuccinimide ester,
N-hydroxysulfosuccinimide ester, nitrophenyl (e.g., 2 or
4-nitrophenyl) ester, dinitrophenyl (e.g., 2,4-dinitrophenyl)
ester, sulfo-tetrafluorophenyl (e.g.,
4-sulfo-2,3,5,6-tetrafluorophenyl) ester, and pentafluorophenyl
ester.
[0025] In one embodiment, for compounds of structural formulas (I),
(II), (III), (IV), (V) and (VI), Z.sup.s is represented by the
following structural formulas:
##STR00005##
wherein U is --H or --SO.sub.3M; and the remaining variables are as
described above for (a1')-(a15').
[0026] In one embodiment, for compound of structural formula (I) or
a pharmaceutically acceptable salt thereof, Z.sup.s is selected
from formulas (a1')-(a8') and (a10')-(a15'); or is selected from
formulas (a1)-(a8) and (a10)-(a15).
[0027] A second object of the invention is to provide conjugates of
cell binding agents with the novel benzodiazepine compounds or
derivatives thereof of the present invention. These conjugates are
useful as therapeutic agents, which are delivered specifically to
target cells and are cytotoxic.
[0028] Specifically, a conjugate of the invention can comprise: a
cytotoxic compound and a cell binding agent (CBA), wherein the
cytotoxic compound is covalently linked to the CBA, and wherein the
cytotoxic compound is represented by any one of the following
formulas:
##STR00006## ##STR00007##
or a pharmaceutically acceptable salt thereof, wherein:
[0029] the double line between N and C represents a single bond or
a double bond, provided that when it is a double bond, X is absent
and Y is --H, and when it is a single bond, X is selected from --H,
or an amine protecting group;
[0030] Y is selected from --H, --OR, --OCOR', --SR, --NR'R'',
--SO.sub.3M, --SO.sub.2M or --OSO.sub.3M, wherein M is --H or a
cation;
[0031] R is --H, an optionally substituted linear, branched or
cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms
or a PEG group --(CH.sub.2CH.sub.2O).sub.n--R.sup.c, wherein n is
an integer from 1 to 24, and R.sup.c is a linear or branched alkyl
having 1 to 4 carbon atoms;
[0032] R' and R'' are the same or different, and are selected from
--H, --OR, --NRR.sup.g', --COR, an optionally substituted linear,
branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10
carbon atoms, an optionally substituted aryl having from 6 to 18
carbon atoms, an optionally substituted 3- to 18-membered
heterocyclic ring having 1 to 6 heteroatoms selected from O, S, N
and P, a PEG group --(CH.sub.2CH.sub.2O).sub.n--R.sup.c, wherein n
is an integer from 1 to 24, preferably n is 2, 4 or 8; and R.sup.g'
is --H, an optionally substituted linear, branched or cyclic alkyl,
alkenyl or alkynyl having from 1 to 10 carbon atoms or a PEG group
--(CH.sub.2CH.sub.2O).sub.n--R.sup.c;
[0033] X' is selected from the group consisting of --H, --OH, an
optionally substituted linear, branched or cyclic alkyl, alkenyl or
alkynyl having from 1 to 10 carbon atoms, phenyl, and an
amine-protecting group;
[0034] Y' is selected from the group consisting of --H, an oxo
group, an optionally substituted linear, branched or cyclic alkyl,
alkenyl or alkynyl having from 1 to 10 carbon atoms;
[0035] R.sup.x is a linear or branched alkylene having 1 to 6
carbon atoms, optionally substituted with a charged substituent or
an ionizable group Q;
[0036] R.sup.e is --H or a linear or branched alkyl having 1 to 6
carbon atoms;
[0037] G is selected from --CH-- or --N--;
[0038] Z.sup.s1 is selected from any one of the following
formulas:
##STR00008##
wherein:
[0039] q is an integer from 1 to 5;
[0040] R.sup.d is a linear or branched alkyl having 1 to 6 carbon
atoms or is selected from phenyl, nitrophenyl, dinitrophenyl,
carboxynitrophenyl, pyridyl and nitropyridyl;
[0041] n is an integer from 2 to 6; and
[0042] M is H.sup.+ or a cation.
[0043] In one embodiment, for conjugates of the present invention,
the cell-binding agent is an anti-folate receptor antibody or an
antibody fragment thereof. More specifically, the anti-folate
receptor antibody is huMOV19 antibody.
[0044] In yet another embodiment, for conjugates of the present
invention, the cell-binding agent is an anti-EGFR antibody or an
antibody fragment thereof. In one embodiment, the anti-EGFR
antibody is a non-antagonist antibody, including, for example, the
antibodies described in WO2012058592, herein incorporated by
reference. In another embodiment, the anti-EGFR antibody is a
non-functional antibody, for example, humanized ML66. More
specifically, the anti-EGFR antibody is huML66.
[0045] The present invention also includes a composition (e.g., a
pharmaceutical composition) comprising novel benzodiazepine
compounds, derivatives thereof, or conjugates thereof, (and/or
solvates, hydrates and/or salts thereof) and a carrier (a
pharmaceutically acceptable carrier). The present invention
additionally includes a composition (e.g., a pharmaceutical
composition) comprising novel benzodiazepine compounds, derivatives
thereof, or conjugates thereof (and/or solvates, hydrates and/or
salts thereof), and a carrier (a pharmaceutically acceptable
carrier), further comprising a second therapeutic agent. The
present compositions are useful for inhibiting abnormal cell growth
or treating a proliferative disorder in a mammal (e.g., human) The
present compositions are useful for treating conditions such as
cancer, rheumatoid arthritis, multiple sclerosis, graft versus host
disease (GVHD), transplant rejection, lupus, myositis, infection,
immune deficiency such as AIDS, and inflammatory diseases in a
mammal (e.g., human).
[0046] The present invention includes a method of inhibiting
abnormal cell growth or treating a proliferative disorder in a
mammal (e.g., human) comprising administering to said mammal a
therapeutically effective amount of novel benzodiazepine compounds,
derivatives thereof, or conjugates thereof, (and/or solvates and
salts thereof) or a composition thereof, alone or in combination
with a second therapeutic agent. The present invention includes a
method of synthesizing and using novel benzodiazepine compounds,
derivatives thereof, and conjugates thereof for in vitro, in situ,
and in vivo diagnosis or treatment of mammalian cells, organisms,
or associated pathological conditions.
[0047] The compounds of this invention, derivatives thereof, or
conjugates thereof, and compositions comprising them, are useful
for treating or lessening the severity of disorders, such as,
characterized by abnormal growth of cells (e.g., cancer). Other
applications for compounds and conjugates of this invention
include, but are not limited to, treating conditions such as
cancer, rheumatoid arthritis, multiple sclerosis, graft versus host
disease (GVHD), transplant rejection, lupus, myositis, infection,
immune deficiency such as AIDS and inflammatory diseases in a
mammal (e.g., human).
BRIEF DESCRIPTION OF THE FIGURES
[0048] FIG. 1 shows MS spectrometry data for huMov19-sulfo-SPDB-1d
conjugate.
[0049] FIG. 2 shows in vivo efficacy of huMov19-sulfo-SPDB-1d
conjugate in NCI-H2110 tumor bearing SCID mice.
[0050] FIG. 3 shows MS spectrometry data for huML66-sulfo-SPDB-1d
conjugate.
[0051] FIG. 4 shows in vitro cytotoxicity of huML66-sulfo-SPDB-1d
conjugate against various cancer cell lines.
[0052] FIGS. 5-7 show in vitro cytotoxicity of
huMOV19-sulfo-SPDB-1d conjugate against various cancer cell
lines.
[0053] FIG. 8 shows that huMOV19-sulfo-SPDB-1d conjugate exhibits
strong cytotoxic effect on the neighboring antigen-negative
cells.
[0054] FIGS. 9A and 9B show binding affinity of
huMOV19-sulfo-SPDB-1d conjugate as compared to unconjugated
antibody huMOV19 on T47D cells.
[0055] FIG. 10 shows in vivo efficacy of huMOV19-sulfo-SPDB-1d
conjugate in NCI-H2110 tumor bearing SCID mice at 1, 3 and 5
.mu.g/kg doses.
[0056] FIG. 11 shows in vivo efficacy of huML66-sulfo-SPDB-1d
conjugate in NCI-H1703 tumor bearing SCID mice at 5, 20 and 50
.mu.g/kg doses.
[0057] FIG. 12 shows pharmacokinetics of huMOV19-sulfo-SPDB-1d
conjugate in CD-1 Mice.
[0058] FIG. 13 shows the scheme for incubation, purification, and
isolation of catabolites from huMOV19-sulfo-SPDB-1d conjugate
formed in KB cervical cancer cells in vitro. The six catabolites
identified by LC-MS are shown along with the calculated mass.
[0059] FIGS. 14A, 14B, 14C and 14D shows in vitro cytotoxicity of
huMOV19-sulfo-SPDB-1d conjugate against various cancer cell
lines.
[0060] FIG. 15 shows in vivo efficacy of huMov19-sulfo-SPDB-1d in
SCID mice bearing NCI-H2110 NSCLC xenografts.
[0061] FIG. 16 shows in vivo efficacy of huMov19-sulfo-SPDB-1d in
SCID mice bearing Hec-1b endometrial xenografts.
[0062] FIG. 17 shows in vivo efficacy of huMov19-sulfo-SPDB-1d in
SCID mice bearing Ishikawa endometrial xenografts.
[0063] FIG. 18 shows binding affinity of huCD123-6Gv4.7S3-sSPDB-1d
conjugate as compared to the unconjugated antibody on HNT-34
cells.
DETAILED DESCRIPTION OF THE INVENTION
[0064] Reference will now be made in detail to certain embodiments
of the invention, examples of which are illustrated in the
accompanying structures and formulas. While the invention will be
described in conjunction with the enumerated embodiments, it will
be understood that they are not intended to limit the invention to
those embodiments. On the contrary, the invention is intended to
cover all alternatives, modifications, and equivalents that can be
included within the scope of the present invention as defined by
the claims. One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention.
[0065] It should be understood that any of the embodiments
described herein, including those described under different aspects
of the invention (e.g., compounds, compound-linker molecules,
conjugates, compositions, methods of making and using) and
different parts of the specification (including embodiments
described only in the Examples) can be combined with one or more
other embodiments of the invention, unless explicitly disclaimed or
improper. Combination of embodiments are not limited to those
specific combinations claimed via the multiple dependent
claims.
DEFINITIONS
[0066] As used herein, the term "cell-binding agent" or "CBA"
refers to a compound that can bind a cell (e.g., on a cell-surface
ligand) or bind a ligand associated with or proximate to the cell,
preferably in a specific manner. In certain embodiments, binding to
the cell or a ligand on or near the cell is specific. The CBA may
include peptides and non-peptides.
[0067] "Linear or branched alkyl" as used herein refers to a
saturated linear or branched-chain monovalent hydrocarbon radical
of one to twenty carbon atoms. Examples of alkyl include, but are
not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl,
2-methyl-1-propyl, --CH.sub.2CH(CH.sub.3).sub.2), 2-butyl,
2-methyl-2-propyl, 1-pentyl, 2-pentyl 3-pentyl, 2-methyl-2-butyl,
3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl),
2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,
4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,
2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and
the like. Preferably, the alkyl has one to ten carbon atoms. More
preferably, the alkyl has one to four carbon atoms.
[0068] "Linear or branched alkenyl" refers to linear or
branched-chain monovalent hydrocarbon radical of two to twenty
carbon atoms with at least one site of unsaturation, i.e., a
carbon-carbon, double bond, wherein the alkenyl radical includes
radicals having "cis" and "trans" orientations, or alternatively,
"E" and "Z" orientations. Examples include, but are not limited to,
ethylenyl or vinyl (--CH.dbd.CH.sub.2), allyl
(--CH.sub.2CH.dbd.CH.sub.2), and the like. Preferably, the alkenyl
has two to ten carbon atoms. More preferably, the alkyl has two to
four carbon atoms.
[0069] "Linear or branched alkynyl" refers to a linear or branched
monovalent hydrocarbon radical of two to twenty carbon atoms with
at least one site of unsaturation, i.e., a carbon-carbon, triple
bond. Examples include, but are not limited to, ethynyl, propynyl,
1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, hexynyl,
and the like. Preferably, the alkynyl has two to ten carbon atoms.
More preferably, the alkynyl has two to four carbon atoms.
[0070] The term "carbocycle," "carbocyclyl" and "carbocyclic ring"
refer to a monovalent non-aromatic, saturated or partially
unsaturated ring having 3 to 12 carbon atoms as a monocyclic ring
or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycles
having 7 to 12 atoms can be arranged, for example, as a bicyclo
[4,5], [5,5], [5,6], or [6,6] system, and bicyclic carbocycles
having 9 or 10 ring atoms can be arranged as a bicyclo [5,6] or
[6,6] system, or as bridged systems such as bicyclo[2.2.1]heptane,
bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane. Examples of
monocyclic carbocycles include, but are not limited to,
cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl,
1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,
1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,
cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,
cycloundecyl, cyclododecyl, and the like.
[0071] The terms "cyclic alkyl" and "cycloalkyl" can be used
interchangeably. They refer to a monovalent saturated carbocyclic
ring radical. Preferably, the cyclic alkyl is 3 to 7 membered
monocyclic ring radical. More preferably, the cyclic alkyl is
cyclohexyl.
[0072] The term "cyclic alkenyl" refers to a carbocyclic ring
radical having at least one double bond in the ring structure.
[0073] The term "cyclic alkynyl" refers to a carbocyclic ring
radical having at least one triple bond in the ring structure.
[0074] "Aryl" means a monovalent aromatic hydrocarbon radical of
6-18 carbon atoms derived by the removal of one hydrogen atom from
a single carbon atom of a parent aromatic ring system. Some aryl
groups are represented in the exemplary structures as "Ar." Aryl
includes bicyclic radicals comprising an aromatic ring fused to a
saturated, partially unsaturated ring, or aromatic carbocyclic or
heterocyclic ring. Typical aryl groups include, but are not limited
to, radicals derived from benzene (phenyl), substituted benzenes,
naphthalene, anthracene, indenyl, indanyl, 1,2-dihydronapthalene,
1,2,3,4-tetrahydronapthyl, and the like. Preferably, aryl is phenyl
group.
[0075] The terms "heterocycle," "heterocyclyl," and "heterocyclic
ring" are used interchangeably herein and refer to a saturated or a
partially unsaturated (i.e., having one or more double and/or
triple bonds within the ring) carbocyclic radical of 3 to 18 ring
atoms in which at least one ring atom is a heteroatom selected from
nitrogen, oxygen, phosphorus, and sulfur, the remaining ring atoms
being C, where one or more ring atoms is optionally substituted
independently with one or more substituents described below. A
heterocycle can be a monocycle having 3 to 7 ring members (2 to 6
carbon atoms and 1 to 4 heteroatoms selected from N, O, P, and S)
or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1
to 6 heteroatoms selected from N, O, P, and S), for example: a
bicyclo [4,5], [5,5], [5,6], or [6,6] system. Heterocycles are
described in Paquette, Leo A.; "Principles of Modern Heterocyclic
Chemistry" (W. A. Benjamin, New York, 1968), particularly Chapters
1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A
series of Monographs" (John Wiley & Sons, New York, 1950 to
present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am.
Chem. Soc. (1960) 82:5566. "Heterocyclyl" also includes radicals
where heterocycle radicals are fused with a saturated, partially
unsaturated ring, or aromatic carbocyclic or heterocyclic ring.
Examples of heterocyclic rings include, but are not limited to,
pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl,
tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl,
piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl,
homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl,
oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl,
2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl,
dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl,
dihydropyranyl, dihydrothienyl, dihydrofuranyl,
pyrazolidinylimidazolinyl, imidazolidinyl,
3-azabicyco[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, and
azabicyclo[2.2.2]hexanyl. Spiro moieties are also included within
the scope of this definition. Examples of a heterocyclic group
wherein ring atoms are substituted with oxo (.dbd.O) moieties are
pyrimidinonyl and 1,1-dioxo-thiomorpholinyl.
[0076] The term "heteroaryl" refers to a monovalent aromatic
radical of 5- or 6-membered rings, and includes fused ring systems
(at least one of which is aromatic) of 5-18 atoms, containing one
or more heteroatoms independently selected from nitrogen, oxygen,
and sulfur. Examples of heteroaryl groups are pyridinyl (including,
for example, 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl,
pyrimidinyl (including, for example, 4-hydroxypyrimidinyl),
pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl,
isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl,
quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,
cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl,
triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl,
thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl,
benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl,
naphthyridinyl, and furopyridinyl.
[0077] The heterocycle or heteroaryl groups can be carbon
(carbon-linked) or nitrogen (nitrogen-linked) attached where such
is possible. By way of example and not limitation, carbon bonded
heterocycles or heteroaryls are bonded at position 2, 3, 4, 5, or 6
of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2,
4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine,
position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran,
thiophene, pyrrole or tetrahydropyrrole, position 2, 4, or 5 of an
oxazole, imidazole or thiazole, position 3, 4, or 5 of an
isoxazole, pyrazole, or isothiazole, position 2 or 3 of an
aziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4,
5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of
an isoquinoline.
[0078] By way of example and not limitation, nitrogen bonded
heterocycles or heteroaryls are bonded at position 1 of an
aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline,
3-pyrroline, imidazole, imidazolidine, 2-imidazoline,
3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,
piperidine, piperazine, indole, indoline, 1H-indazole, position 2
of a isoindole, or isoindoline, position 4 of a morpholine, and
position 9 of a carbazole, or O-carboline.
[0079] The heteroatoms present in heteroaryl or heterocyclyl
include the oxidized forms such as NO, SO, and SO.sub.2.
[0080] The term "halo" or "halogen" refers to F, Cl, Br or I.
[0081] The alkyl, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl,
cyclic alkynyl, carbocyclyl, aryl, heterocyclyl and heteroaryl
described above can be optionally substituted with one more (e.g.,
2, 3, 4, 5, 6 or more) substituents.
[0082] If a substituent is described as being "substituted," a
non-hydrogen substituent is in the place of a hydrogen substituent
on a carbon, oxygen, sulfur or nitrogen of the substituent. Thus,
for example, a substituted alkyl substituent is an alkyl
substituent wherein at least one non-hydrogen substituent is in the
place of a hydrogen substituent on the alkyl substituent. To
illustrate, monofluoroalkyl is alkyl substituted with a fluoro
substituent, and difluoroalkyl is alkyl substituted with two fluoro
substituents. It should be recognized that if there is more than
one substitution on a substituent, each non-hydrogen substituent
can be identical or different (unless otherwise stated).
[0083] If a substituent is described as being "optionally
substituted," the substituent can be either (1) not substituted, or
(2) substituted. If a carbon of a substituent is described as being
optionally substituted with one or more of a list of substituents,
one or more of the hydrogens on the carbon (to the extent there are
any) can separately and/or together be replaced with an
independently selected optional substituent. If a nitrogen of a
substituent is described as being optionally substituted with one
or more of a list of substituents, one or more of the hydrogens on
the nitrogen (to the extent there are any) can each be replaced
with an independently selected optional substituent. One exemplary
substituent can be depicted as --NR'R'', wherein R' and R''
together with the nitrogen atom to which they are attached, can
form a heterocyclic ring. The heterocyclic ring formed from R' and
R'' together with the nitrogen atom to which they are attached can
be partially or fully saturated. In one embodiment, the
heterocyclic ring consists of 3 to 7 atoms. In another embodiment,
the heterocyclic ring is selected from the group consisting of
pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl,
pyridyl and thiazolyl.
[0084] This specification uses the terms "substituent," "radical,"
and "group" interchangeably.
[0085] If a group of substituents are collectively described as
being optionally substituted by one or more of a list of
substituents, the group can include: (1) unsubstitutable
substituents, (2) substitutable substituents that are not
substituted by the optional substituents, and/or (3) substitutable
substituents that are substituted by one or more of the optional
substituents.
[0086] If a substituent is described as being optionally
substituted with up to a particular number of non-hydrogen
substituents, that substituent can be either (1) not substituted;
or (2) substituted by up to that particular number of non-hydrogen
substituents or by up to the maximum number of substitutable
positions on the substituent, whichever is less. Thus, for example,
if a substituent is described as a heteroaryl optionally
substituted with up to 3 non-hydrogen substituents, then any
heteroaryl with less than 3 substitutable positions would be
optionally substituted by up to only as many non-hydrogen
substituents as the heteroaryl has substitutable positions. Such
substituents, in non-limiting examples, can be selected from a
linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1
to 10 carbon atoms, aryl, heteroaryl, heterocycyclyl, halogen,
guanidinium [--NH(C.dbd.NH)NH.sub.2], --OR.sup.100,
NR.sup.101R.sup.102, --NO.sub.2, NR.sup.101COR.sup.102,
--SR.sup.100, a sulfoxide represented by --SOR.sup.101, a sulfone
represented by --SO.sub.2R.sup.101, a sulfonate --SO.sub.3M, a
sulfate --OSO.sub.3M, a sulfonamide represented by
--SO.sub.2NR.sup.101R.sup.102, cyano, an azido, --COR.sup.101,
--OCOR.sup.101, OCONR.sup.101R.sup.102 and a polyethylene glycol
unit (--OCH.sub.2CH.sub.2).sub.nR.sup.101 wherein M is H or a
cation (such as Na.sup.+ or K.sup.+); R.sup.101, R.sup.102 and
R.sup.103 are each independently selected from H, linear, branched
or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon
atoms, a polyethylene glycol unit
(--OCH.sub.2CH.sub.2).sub.n--R.sup.104, wherein n is an integer
from 1 to 24, an aryl having from 6 to 10 carbon atoms, a
heterocyclic ring having from 3 to 10 carbon atoms and a heteroaryl
having 5 to 10 carbon atoms; and R.sup.104 is H or a linear or
branched alkyl having 1 to 4 carbon atoms, wherein the alkyl,
alkenyl, alkynyl, aryl, heteroaryl and heterocycyl in the groups
represented by R.sup.100, R.sup.101, R.sup.102, R.sup.103 and
R.sup.104 are optionally substituted with one or more (e.g., 2, 3,
4, 5, 6 or more) substituents independently selected from halogen,
--OH, --CN, --NO.sub.2 and unsubstituted linear or branched alkyl
having 1 to 4 carbon atoms. Preferably, the substituents for the
optionally substituted alkyl, alkenyl, alkynyl, cyclic alkyl,
cyclic alkenyl, cyclic alkynyl, carbocyclyl, aryl, heterocyclyl and
heteroaryl described above include halogen, --CN,
--NR.sup.102R.sup.103, --CF.sub.3, --OR.sup.101, aryl, heteroaryl,
heterocycyl, --SR.sup.101, --SOR.sup.101, --SO.sub.2R.sup.101 and
--SO.sub.3M.
[0087] The term "compound" or "cytotoxic compound," "cytotoxic
dimer" and "cytotoxic dimer compound" are used interchangeably.
They are intended to include compounds for which a structure or
formula or any derivative thereof has been disclosed in the present
invention or a structure or formula or any derivative thereof that
has been incorporated by reference. The term also includes,
stereoisomers, geometric isomers, tautomers, solvates, metabolites,
salts (e.g., pharmaceutically acceptable salts) and prodrugs, and
prodrug salts of a compound of all the formulae disclosed in the
present invention. The term also includes any solvates, hydrates,
and polymorphs of any of the foregoing. The specific recitation of
"stereoisomers," "geometric isomers," "tautomers," "solvates,"
"metabolites," "salt" "prodrug," "prodrug salt," "conjugates,"
"conjugates salt," "solvate," "hydrate," or "polymorph" in certain
aspects of the invention described in this application shall not be
interpreted as an intended omission of these forms in other aspects
of the invention where the term "compound" is used without
recitation of these other forms.
[0088] The term "conjugate" as used herein refers to a compound
described herein or a derivative thereof that is linked to a cell
binding agent.
[0089] The term "linkable to a cell binding agent" as used herein
refers to the compounds described herein or derivates thereof
comprising at least one linking group or a precursor thereof
suitable to bond these compounds or derivatives thereof to a cell
binding agent.
[0090] The term "precursor" of a given group refers to any group
that can lead to that group by any deprotection, a chemical
modification, or a coupling reaction.
[0091] The term "linked to a cell binding agent" refers to a
conjugate molecule comprising at least one of the compounds
described herein (e.g., compounds of formula (I)-(IV) and
(VIII)-(XI) and drug-linker compounds describe herein), or
derivative thereof bound to a cell binding agent via a suitable
linking group or a precursor thereof.
[0092] The term "chiral" refers to molecules that have the property
of non-superimposability of the mirror image partner, while the
term "achiral" refers to molecules that are superimposable on their
mirror image partner.
[0093] The term "stereoisomer" refers to compounds that have
identical chemical constitution and connectivity, but different
orientations of their atoms in space that cannot be interconverted
by rotation about single bonds.
[0094] "Diastereomer" refers to a stereoisomer with two or more
centers of chirality and whose molecules are not mirror images of
one another. Diastereomers have different physical properties, e.g.
melting points, boiling points, spectral properties, and
reactivities. Mixtures of diastereomers can separate under high
resolution analytical procedures such as crystallization,
electrophoresis and chromatography.
[0095] "Enantiomers" refer to two stereoisomers of a compound that
are non-superimposable mirror images of one another.
[0096] Stereochemical definitions and conventions used herein
generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of
Chemical Terms (1984) McGraw-Hill Book Company, New York; and
Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds,"
John Wiley & Sons, Inc., New York, 1994. The compounds of the
invention can contain asymmetric or chiral centers, and therefore
exist in different stereoisomeric forms. It is intended that all
stereoisomeric forms of the compounds of the invention, including
but not limited to, diastereomers, enantiomers and atropisomers, as
well as mixtures thereof such as racemic mixtures, form part of the
present invention. Many organic compounds exist in optically active
forms, i.e., they have the ability to rotate the plane of
plane-polarized light. In describing an optically active compound,
the prefixes D and L, or R and S, are used to denote the absolute
configuration of the molecule about its chiral center(s). The
prefixes d and I or (+) and (-) are employed to designate the sign
of rotation of plane-polarized light by the compound, with (-) or 1
meaning that the compound is levorotatory. A compound prefixed with
(+) or d is dextrorotatory. For a given chemical structure, these
stereoisomers are identical except that they are mirror images of
one another. A specific stereoisomer can also be referred to as an
enantiomer, and a mixture of such isomers is often called an
enantiomeric mixture. A 50:50 mixture of enantiomers is referred to
as a racemic mixture or a racemate, which can occur where there has
been no stereoselection or stereospecificity in a chemical reaction
or process. The terms "racemic mixture" and "racemate" refer to an
equimolar mixture of two enantiomeric species, devoid of optical
activity.
[0097] The term "tautomer" or "tautomeric form" refers to
structural isomers of different energies that are interconvertible
via a low energy barrier. For example, proton tautomers (also known
as prototropic tautomers) include interconversions via migration of
a proton, such as keto-enol and imine-enamine isomerizations.
Valence tautomers include interconversions by reorganization of
some of the bonding electrons.
[0098] The term "prodrug" as used in this application refers to a
precursor or derivative form of a compound of the invention that is
capable of being enzymatically or hydrolytically activated or
converted into the more active parent form. See, e.g., Wilman,
"Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions,
14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al.,
"Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed
Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). The prodrugs of this invention include, but are not limited
to, ester-containing prodrugs, phosphate-containing prodrugs,
thiophosphate-containing prodrugs, sulfate-containing prodrugs,
peptide-containing prodrugs, D-amino acid-modified prodrugs,
glycosylated prodrugs, .beta.-lactam-containing prodrugs,
optionally substituted phenoxyacetamide-containing prodrugs,
optionally substituted phenylacetamide-containing prodrugs,
5-fluorocytosine and other 5-fluorouridine prodrugs that can be
converted into the more active cytotoxic free drug. Examples of
cytotoxic drugs that can be derivatized into a prodrug form for use
in this invention include, but are not limited to, compounds of the
invention and chemotherapeutic agents such as described above.
[0099] The term "prodrug" is also meant to include a derivative of
a compound that can hydrolyze, oxidize, or otherwise react under
biological conditions (in vitro or in vivo) to provide a compound
of this invention. Prodrugs can only become active upon such
reaction under biological conditions, or they can have activity in
their unreacted forms. Examples of prodrugs contemplated in this
invention include, but are not limited to, analogs or derivatives
of compounds of any one of the formulae disclosed herein that
comprise biohydrolyzable moieties such as biohydrolyzable amides,
biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable
carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate
analogues. Other examples of prodrugs include derivatives of
compounds of any one of the formulae disclosed herein that comprise
--NO, --NO.sub.2, --ONO, or --ONO.sub.2 moieties. Prodrugs can
typically be prepared using well-known methods, such as those
described by Burger's Medicinal Chemistry and Drug Discovery (1995)
172-178, 949-982 (Manfred E. Wolff ed., 5th ed); see also Goodman
and Gilman's, The Pharmacological basis of Therapeutics, 8th ed.,
McGraw-Hill, Int. Ed. 1992, "Biotransformation of Drugs."
[0100] One preferred form of prodrug of the invention includes
compounds (with or without any linker groups) and conjugates of the
invention comprising an adduct formed between an imine bond of the
compounds/conjugates and an imine reactive reagent. Another
preferred form of prodrug of the invention includes compounds such
as those of formula (I)-(IV), wherein when the double line between
N and C represents a single bond, X is H or an amine protecting
group, and the compound becomes a prodrug. A prodrug of the
invention can contain one or both forms of prodrugs described
herein (e.g., containing an adduct formed between an imine bond of
the compounds/conjugates and an imine reactive reagent, and/or
containing a Y leaving group when X is --H).
[0101] The term "imine reactive reagent" refers to a reagent that
is capable of reacting with an imine group. Examples of imine
reactive reagent includes, but is not limited to, sulfites
(H.sub.2SO.sub.3, H.sub.2SO.sub.2 or a salt of HSO.sub.3.sup.-,
SO.sub.3.sup.2- or HSO.sub.2.sup.- formed with a cation),
metabisulfite (H.sub.2S.sub.2O.sub.5 or a salt of
S.sub.2O.sub.5.sup.2- formed with a cation), mono, di, tri, and
tetra-thiophosphates (PO.sub.3SH.sub.3, PO.sub.2S.sub.2H.sub.3,
POS.sub.3H.sub.3, PS.sub.4H.sub.3 or a salt of PO.sub.3S.sup.3-,
PO.sub.2S.sub.2.sup.3-, POS.sub.3.sup.3- or PS.sub.4.sup.3- formed
with a cation), thio phosphate esters
((R.sup.iO).sub.2PS(OR.sup.i), P.sup.iSH, R.sup.iSOH,
R.sup.iSO.sub.2H, R.sup.iSO.sub.3H), various amines (hydroxyl amine
(e.g., NH.sub.2OH), hydrazine (e.g., NH.sub.2NH.sub.2),
NH.sub.2O--R.sup.i, R.sup.iNH--R.sup.i, NH.sub.2--R.sup.i),
NH.sub.2--CO--NH.sub.2, NH.sub.2--C(.dbd.S)--NH.sub.2.sup.-
thiosulfate (H.sub.2S.sub.2O.sub.3 or a salt of
S.sub.2O.sub.3.sup.2- formed with a cation), dithionite
(H.sub.2S.sub.2O.sub.4 or a salt of S.sub.2O.sub.4.sup.2- formed
with a cation), phosphorodithioate (P(.dbd.S)(OR.sup.k)(SH)(OH) or
a salt thereof formed with a cation), hydroxamic acid
(R.sup.kC(.dbd.O)NHOH or a salt formed with a cation), hydrazide
(R.sup.kCONHNH.sub.2), formaldehyde sulfoxylate
(HOCH.sub.2SO.sub.2H or a salt of HOCH.sub.2SO.sub.2.sup.- formed
with a cation, such as HOCH.sub.2SO.sub.2.sup.-Na.sup.+), glycated
nucleotide (such as GDP-mannose), fludarabine or a mixture thereof,
wherein R.sup.i and R.sup.i' are each independently a linear or
branched alkyl having 1 to 10 carbon atoms and are substituted with
at least one substituent selected from --N(R.sup.j).sub.2,
--CO.sub.2H, --SO.sub.3H, and --PO.sub.3H; R.sup.i and R.sup.i' can
be further optionally substituted with a substituent for an alkyl
described herein; R.sup.j is a linear or branched alkyl having 1 to
6 carbon atoms; and R.sup.k is a linear, branched or cyclic alkyl,
alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heterocyclyl
or heteroaryl (preferably, R.sup.k is a linear or branched alkyl
having 1 to 4 carbon atoms; more preferably, R.sup.k is methyl,
ethyl or propyl). Preferably, the cation is a monovalent cation,
such as Na.sup.+ or K.sup.+. Preferably, the imine reactive reagent
is selected from sulfites, hydroxyl amine, urea and hydrazine. More
preferably, the imine reactive reagent is NaHSO.sub.3 or
KHSO.sub.3.
[0102] As used herein and unless otherwise indicated, the terms
"biohydrolyzable amide," "biohydrolyzable ester," "biohydrolyzable
carbamate," "biohydrolyzable carbonate," "biohydrolyzable ureide"
and "biohydrolyzable phosphate analogue" mean an amide, ester,
carbamate, carbonate, ureide, or phosphate analogue, respectively,
that either: 1) does not destroy the biological activity of the
compound and confers upon that compound advantageous properties in
vivo, such as uptake, duration of action, or onset of action; or 2)
is itself biologically inactive but is converted in vivo to a
biologically active compound. Examples of biohydrolyzable amides
include, but are not limited to, lower alkyl amides, .alpha.-amino
acid amides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides.
Examples of biohydrolyzable esters include, but are not limited to,
lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl
esters, and choline esters. Examples of biohydrolyzable carbamates
include, but are not limited to, lower alkylamines, substituted
ethylenediamines, amino acids, hydroxyalkylamines, heterocyclic and
heteroaromatic amines, and polyether amines Particularly favored
prodrugs and prodrug salts are those that increase the
bioavailability of the compounds of this invention when such
compounds are administered to a mammal.
[0103] The phrase "pharmaceutically acceptable salt" as used
herein, refers to pharmaceutically acceptable organic or inorganic
salts of a compound of the invention. Exemplary salts include, but
are not limited, to sulfate, citrate, acetate, oxalate, chloride,
bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate,
isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate,
tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,
gentisinate, fumarate, gluconate, glucuronate, saccharate, formate,
benzoate, glutamate, methanesulfonate "mesylate," ethanesulfonate,
benzenesulfonate, p-toluenesulfonate, pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal
(e.g., sodium and potassium) salts, alkaline earth metal (e.g.,
magnesium) salts, and ammonium salts. A pharmaceutically acceptable
salt can involve the inclusion of another molecule such as an
acetate ion, a succinate ion or other counter ion. The counter ion
can be any organic or inorganic moiety that stabilizes the charge
on the parent compound. Furthermore, a pharmaceutically acceptable
salt can have more than one charged atom in its structure.
Instances where multiple charged atoms are part of the
pharmaceutically acceptable salt can have multiple counter ions.
Hence, a pharmaceutically acceptable salt can have one or more
charged atoms and/or one or more counter ion.
[0104] If the compound of the invention is a base, the desired
pharmaceutically acceptable salt can be prepared by any suitable
method available in the art, for example, treatment of the free
base with an inorganic acid, such as hydrochloric acid, hydrobromic
acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric
acid and the like, or with an organic acid, such as acetic acid,
maleic acid, succinic acid, mandelic acid, fumaric acid, malonic
acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a
pyranosidyl acid, such as glucuronic acid or galacturonic acid, an
alpha hydroxy acid, such as citric acid or tartaric acid, an amino
acid, such as aspartic acid or glutamic acid, an aromatic acid,
such as benzoic acid or cinnamic acid, a sulfonic acid, such as
p-toluenesulfonic acid or ethanesulfonic acid, or the like.
[0105] If the compound of the invention is an acid, the desired
pharmaceutically acceptable salt can be prepared by any suitable
method, for example, treatment of the free acid with an inorganic
or organic base, such as an amine (primary, secondary or tertiary),
an alkali metal hydroxide or alkaline earth metal hydroxide, or the
like. Illustrative examples of suitable salts include, but are not
limited to, organic salts derived from amino acids, such as glycine
and arginine, ammonia, primary, secondary, and tertiary amines, and
cyclic amines, such as piperidine, morpholine and piperazine, and
inorganic salts derived from sodium, calcium, potassium, magnesium,
manganese, iron, copper, zinc, aluminum and lithium.
[0106] As used herein, the term "solvate" means a compound that
further includes a stoichiometric or non-stoichiometric amount of
solvent such as water, isopropanol, acetone, ethanol, methanol,
DMSO, ethyl acetate, acetic acid, and ethanolamine dichloromethane,
2-propanol, or the like, bound by non-covalent intermolecular
forces. Solvates or hydrates of the compounds are readily prepared
by addition of at least one molar equivalent of a hydroxylic
solvent such as methanol, ethanol, 1-propanol, 2-propanol or water
to the compound to result in solvation or hydration of the imine
moiety.
[0107] The terms "abnormal cell growth" and "proliferative
disorder" are used interchangeably in this application. "Abnormal
cell growth," as used herein, unless otherwise indicated, refers to
cell growth that is independent of normal regulatory mechanisms
(e.g., loss of contact inhibition). This includes, for example, the
abnormal growth of: (1) tumor cells (tumors) that proliferate by
expressing a mutated tyrosine kinase or overexpression of a
receptor tyrosine kinase; (2) benign and malignant cells of other
proliferative diseases in which aberrant tyrosine kinase activation
occurs; (3) any tumors that proliferate by receptor tyrosine
kinases; (4) any tumors that proliferate by aberrant
serine/threonine kinase activation; and (5) benign and malignant
cells of other proliferative diseases in which aberrant
serine/threonine kinase activation occurs.
[0108] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. A "tumor" comprises one or more
cancerous cells, and/or benign or pre-cancerous cells.
[0109] A "therapeutic agent" encompasses both a biological agent
such as an antibody, a peptide, a protein, an enzyme or a
chemotherapeutic agent.
[0110] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer.
[0111] A "metabolite" is a product produced through metabolism in
the body of a specified compound, a derivative thereof, or a
conjugate thereof, or salt thereof. Metabolites of a compound, a
derivative thereof, or a conjugate thereof, can be identified using
routine techniques known in the art and their activities determined
using tests such as those described herein. Such products can
result for example from the oxidation, hydroxylation, reduction,
hydrolysis, amidation, deamidation, esterification,
deesterification, enzymatic cleavage, and the like, of the
administered compound. Accordingly, the invention includes
metabolites of compounds, a derivative thereof, or a conjugate
thereof, of the invention, including compounds, a derivative
thereof, or a conjugate thereof, produced by a process comprising
contacting a compound, a derivative thereof, or a conjugate
thereof, of this invention with a mammal for a period of time
sufficient to yield a metabolic product thereof.
[0112] The phrase "pharmaceutically acceptable" indicates that the
substance or composition must be compatible chemically and/or
toxicologically, with the other ingredients comprising a
formulation, and/or the mammal being treated therewith.
[0113] The term "protecting group" or "protecting moiety" refers to
a substituent that is commonly employed to block or protect a
particular functionality while reacting other functional groups on
the compound, a derivative thereof, or a conjugate thereof. For
example, an "amine-protecting group" or an "amino-protecting
moiety" is a substituent attached to an amino group that blocks or
protects the amino functionality in the compound. Such groups are
well known in the art (see for example P. Wuts and T. Greene, 2007,
Protective Groups in Organic Synthesis, Chapter 7, J. Wiley &
Sons, NJ) and exemplified by carbamates such as methyl and ethyl
carbamate, FMOC, substituted ethyl carbamates, carbamates cleaved
by 1,643-elimination (also termed "self immolative"), ureas,
amides, peptides, alkyl and aryl derivatives. Suitable
amino-protecting groups include acetyl, trifluoroacetyl,
t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and
9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description of
protecting groups and their use, see P. G. M. Wuts & T. W.
Greene, Protective Groups in Organic Synthesis, John Wiley &
Sons, New York, 2007.
[0114] The term "leaving group" refers to an group of charged or
uncharged moiety that departs during a substitution or
displacement. Such leaving groups are well known in the art and
include, but not limited to, halogens, esters, alkoxy, hydroxyl,
tosylates, triflates, mesylates, nitriles, azide, carbamate,
disulfides, thioesters, thioethers and diazonium compounds.
[0115] The term "bifunctional crosslinking agent," "bifunctional
linker" or "crosslinking agents" refers to modifying agents that
possess two reactive groups; one of which is capable of reacting
with a cell binding agent while the other one reacts with the
cytotoxic compound to link the two moieties together. Such
bifunctional crosslinkers are well known in the art (see, for
example, Isalm and Dent in Bioconjugation chapter 5, p 218-363,
Groves Dictionaries Inc. New York, 1999). For example, bifunctional
crosslinking agents that enable linkage via a thioether bond
include
N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate
(SMCC) to introduce maleimido groups, or with
N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB) to introduce
iodoacetyl groups. Other bifunctional crosslinking agents that
introduce maleimido groups or haloacetyl groups on to a cell
binding agent are well known in the art (see US Patent Applications
2008/0050310, 20050169933, available from Pierce Biotechnology Inc.
P.O. Box 117, Rockland, Ill. 61105, USA) and include, but not
limited to, bis-maleimidopolyethyleneglycol (BMPEO), BM(PEO).sub.2,
BM(PEO).sub.3, N-(.beta.-maleimidopropyloxy)succinimide ester
(BMPS), .gamma.-maleimidobutyric acid N-succinimidyl ester (GMBS),
.epsilon.-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS),
5-maleimidovaleric acid NHS, HBVS,
N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproa-
te), which is a "long chain" analog of SMCC (LC-SMCC),
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),
4-(4-N-maleimidophenyl)-butyric acid hydrazide or HCl salt (MPBH),
N-succinimidyl 3-(bromoacetamido)propionate (SBAP), N-succinimidyl
iodoacetate (SIA), .kappa.-maleimidoundecanoic acid N-succinimidyl
ester (KMUA), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB),
succinimidyl-6-(.beta.-maleimidopropionamido)hexanoate (SMPH),
succinimidyl-(4-vinylsulfonyl)benzoate (SVSB),
dithiobis-maleimidoethane (DTME), 1,4-bis-maleimidobutane (BMB),
1,4 bismaleimidyl-2,3-dihydroxybutane (BMDB), bis-maleimidohexane
(BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl
4-(N-maleimido-methyl)cyclohexane-1-carboxylate (sulfo-SMCC),
sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (sulfo-SIAB),
m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS),
N-(.gamma.-maleimidobutryloxy)sulfosuccinimide ester (sulfo-GMBS),
N-(.epsilon.-maleimidocaproyloxy)sulfosuccimido ester (sulfo-EMCS),
N-(.kappa.-maleimidoundecanoyloxy)sulfosuccinimide ester
(sulfo-KMUS), and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate
(sulfo-SMPB).
[0116] Heterobifunctional crosslinking agents are bifunctional
crosslinking agents having two different reactive groups.
Heterobifunctional crosslinking agents containing both an
amine-reactive N-hydroxysuccinimide group (NHS group) and a
carbonyl-reactive hydrazine group can also be used to link the
cytotoxic compounds described herein with a cell-binding agent
(e.g., antibody). Examples of such commercially available
heterobifunctional crosslinking agents include succinimidyl
6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl
4-hydrazidoterephthalate hydrochloride (SHTH) and succinimidyl
hydrazinium nicotinate hydrochloride (SHNH). Conjugates bearing an
acid-labile linkage can also be prepared using a hydrazine-bearing
benzodiazepine derivative of the present invention. Examples of
bifunctional crosslinking agents that can be used include
succinimidyl-p-formyl benzoate (SFB) and
succinimidyl-p-formylphenoxyacetate (SFPA).
[0117] Bifunctional crosslinking agents that enable the linkage of
cell binding agent with cytotoxic compounds via disulfide bonds are
known in the art and include
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP),
N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB),
N-succinimidyl-4-(2-pyridyldithio)2-sulfo butanoate (sulfo-SPDB) to
introduce dithiopyridyl groups. Other bifunctional crosslinking
agents that can be used to introduce disulfide groups are known in
the art and are disclosed in U.S. Pat. Nos. 6,913,748, 6,716,821
and US Patent Publications 20090274713 and 20100129314, all of
which are incorporated herein by reference. Alternatively,
crosslinking agents such as 2-iminothiolane, homocysteine
thiolactone or S-acetylsuccinic anhydride that introduce thiol
groups can also be used.
[0118] A "linker," "linker moiety," or "linking group" as defined
herein refers to a moiety that connects two groups, such as a cell
binding agent and a cytotoxic compound, together. Typically, the
linker is substantially inert under conditions for which the two
groups it is connecting are linked. A bifunctional crosslinking
agent can comprise two reactive groups, one at each ends of a
linker moiety, such that one reactive group can be first reacted
with the cytotoxic compound to provide a compound bearing the
linker moiety and a second reactive group, which can then react
with a cell binding agent. Alternatively, one end of the
bifunctional crosslinking agent can be first reacted with the cell
binding agent to provide a cell binding agent bearing a linker
moiety and a second reactive group, which can then react with a
cytotoxic compound. The linking moiety can contain a chemical bond
that allows for the release of the cytotoxic moiety at a particular
site. Suitable chemical bonds are well known in the art and include
disulfide bonds, thioether bonds, acid labile bonds, photolabile
bonds, peptidase labile bonds and esterase labile bonds (see for
example U.S. Pat. Nos. 5,208,020; 5,475,092; 6,441,163; 6,716,821;
6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026 and
7,414,073). Preferred are disulfide bonds, thioether and peptidase
labile bonds. Other linkers that can be used in the present
invention include non-cleavable linkers, such as those described in
are described in detail in U.S. publication number 20050169933, or
charged linkers or hydrophilic linkers and are described in US
2009/0274713, US 2010/01293140 and WO 2009/134976, each of which is
expressly incorporated herein by reference, each of which is
expressly incorporated herein by reference.
[0119] In one embodiment, the linking group with a reactive group
attached at one end, such as a reactive ester, is selected from the
following:
--O(CR.sub.20R.sub.21).sub.m(CR.sub.22R.sub.23).sub.n(OCH.sub.2CH.sub.2).-
sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).sub.q(CO).sub.tX'',
--O(CR.sub.20R.sub.21).sub.m(CR.sub.26.dbd.CR.sub.27).sub.m'(CR.sub.22R.s-
ub.23).sub.n(OCH.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub-
.24R.sub.25).sub.q(CO).sub.tX'',
--O(CR.sub.20R.sub.21).sub.m(alkynyl).sub.n'(CR.sub.22R.sub.23).sub.n(OCH-
.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).sub-
.q(CO).sub.tX'',
--O(CR.sub.20R.sub.21).sub.m(piperazino).sub.t'(CR.sub.22R.sub.23).sub.n(-
OCH.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).-
sub.q(CO).sub.tX'',
--O(CR.sub.20R.sub.21).sub.m(pyrrolo).sub.t'(CR.sub.22R.sub.23).sub.n(OCH-
.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).sub-
.q(CO).sub.tX'',
--O(CR.sub.20R.sub.21).sub.mA''.sub.m''(CR.sub.22R.sub.23).sub.n(OCH.sub.-
2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).sub.q(CO-
).sub.tX'',
--S(CR.sub.20R.sub.21).sub.m(CR.sub.22R.sub.23).sub.n(OCH.sub.2CH.sub.2).-
sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).sub.q(CO).sub.tX'',
--S(CR.sub.20R.sub.21).sub.m(CR.sub.26.dbd.CR.sub.27).sub.m'(CR.sub.22R.s-
ub.23).sub.n(OCH.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub-
.24R.sub.25).sub.q(CO).sub.tX'',
--S(CR.sub.20R.sub.21).sub.m(alkynyl).sub.n'(CR.sub.22R.sub.23).sub.n(OCH-
.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).sub-
.q(CO).sub.tX'',
--S(CR.sub.20R.sub.21).sub.m(piperazino).sub.t'(CR.sub.22R.sub.23).sub.n(-
OCH.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).-
sub.q(CO).sub.tX'',
--S(CR.sub.20R.sub.21).sub.m(pyrrolo).sub.t'(CR.sub.22R.sub.23).sub.n(OCH-
.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).sub-
.q(CO).sub.tX'',
--S(CR.sub.20R.sub.21).sub.mA''.sub.m''(CR.sub.22R.sub.23).sub.n(OCH.sub.-
2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).sub.q(CO-
).sub.tX'',
--NR.sub.33(C.dbd.O).sub.p''(CR.sub.20R.sub.21).sub.m(CR.sub.22R.sub.23).-
sub.n(OCH.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.su-
b.25).sub.q(CO).sub.tX'',
--NR.sub.33(C.dbd.O).sub.p''(CR.sub.20R.sub.21).sub.m(CR.sub.26.dbd.CR.su-
b.27).sub.m'(CR.sub.22R.sub.23).sub.n(OCH.sub.2CH.sub.2).sub.p(CR.sub.40R.-
sub.41).sub.p''Y''(CR.sub.24R.sub.25).sub.q(CO).sub.tX'',
--NR.sub.33(C.dbd.O).sub.p''(CR.sub.20R.sub.21).sub.m(alkynyl).sub.n'(CR.-
sub.22R.sub.23).sub.n(OCH.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y-
''(CR.sub.24R.sub.25).sub.q--(CO).sub.tX'',
--NR.sub.33(C.dbd.O).sub.p''(CR.sub.20R.sub.21).sub.m(piperazino).sub.t'(-
CR.sub.22R.sub.23).sub.n(OCH.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p-
''Y''(CR.sub.24R.sub.25).sub.q(CO).sub.tX'',
--NR.sub.33(C.dbd.O).sub.p''(CR.sub.20R.sub.21).sub.m(pyrrolo).sub.t'(CR.-
sub.22R.sub.23).sub.n(OCH.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y-
''(CR.sub.24R.sub.25).sub.q(CO).sub.tX'',
--NR.sub.33(C.dbd.O).sub.p''(CR.sub.20R.sub.21).sub.mA''.sub.m''(CR.sub.2-
2R.sub.23).sub.n(OCH.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR-
.sub.24R.sub.25).sub.q(CO).sub.tX'',
--(CR.sub.20R.sub.21).sub.m(CR.sub.22R.sub.23).sub.n(OCH.sub.2CH.sub.2).s-
ub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).sub.q(CO).sub.tX'',
--(CR.sub.20R.sub.21).sub.m(CR.sub.26CR.sub.27).sub.m'(CR.sub.22R.sub.23)-
.sub.n(OCH.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.s-
ub.25).sub.q(CO).sub.tX'',
--(CR.sub.20R.sub.21).sub.m(alkynyl).sub.n'(CR.sub.22R.sub.23).sub.n(OCH.-
sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).sub.-
q(CO).sub.tX'',
--(CR.sub.20R.sub.21).sub.m(piperazino).sub.t'(CR.sub.22R.sub.23).sub.n(O-
CH.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).s-
ub.q(CO).sub.tX'',
--(CR.sub.20R.sub.21).sub.mA''.sub.m''(CR.sub.22R.sub.23).sub.n(OCH.sub.2-
CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).sub.q(CO)-
.sub.tX'',
--(CR.sub.20R.sub.21).sub.m(CR.sub.29.dbd.N--NR.sub.30).sub.n''-
(CR.sub.22R.sub.23).sub.n(OCH.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub.-
p''Y''(CR.sub.24R.sub.25).sub.q(CO).sub.tX'',
--(CR.sub.20R.sub.21).sub.m(CR.sub.29.dbd.N--NR.sub.30).sub.n''(CR.sub.26-
.dbd.CR.sub.27).sub.m'(CR.sub.22R.sub.23).sub.n(OCH.sub.2CH.sub.2).sub.p(C-
R.sub.40R.sub.41).sub.p''Y''(CR.sub.24R.sub.25).sub.q(CO).sub.tX'',
--(CR.sub.20R.sub.21).sub.m(CR.sub.29.dbd.N--NR.sub.30).sub.n''(alkynyl).-
sub.n'(CR.sub.22R.sub.23).sub.n(OCH.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41-
).sub.p''Y''(CR.sub.24R.sub.25).sub.q (CO).sub.tX'',
--(CR.sub.20R.sub.21).sub.m(CR.sub.29.dbd.N--NR.sub.30).sub.n''A''.sub.m'-
'(CR.sub.22R.sub.23).sub.n(OCH.sub.2CH.sub.2).sub.p(CR.sub.40R.sub.41).sub-
.p''Y''(CR.sub.24R.sub.25).sub.q(CO).sub.tX'',
wherein:
[0120] m, n, p, q, m', n', t' are integer from 1 to 10, or are
optionally 0;
[0121] t, m'', n'', and p'' are 0 or 1;
[0122] X'' is selected from OR.sub.36, SR.sub.37,
NR.sub.38R.sub.39, wherein R.sub.36, R.sub.37, R.sub.38, R.sub.39
are H, or linear, branched or cyclic alkyl, alkenyl or alkynyl
having from 1 to 20 carbon atoms and, or, a polyethylene glycol
unit --(OCH.sub.2CH.sub.2).sub.n, R.sub.37, optionally, is a thiol
protecting group when t=1, COX'' forms a reactive ester selected
from N-hydroxysuccinimide esters, N-hydroxyphthalimide esters,
N-hydroxy sulfo-succinimide esters, para-nitrophenyl esters,
dinitrophenyl esters, pentafluorophenyl esters and their
derivatives, wherein said derivatives facilitate amide bond
formation;
[0123] Y'' is absent or is selected from O, S, S--S or NR.sub.32,
wherein R.sub.32 has the same definition as given above for R;
or
[0124] when Y'' is not S--S and t=0, X'' is selected from a
maleimido group, a haloacetyl group or SR.sub.37, wherein R.sub.37
has the same definition as above;
[0125] A'' is a residue of an amino acid or a polypeptide
containing between 2 to 20 amino acid units;
[0126] R.sub.20, R.sub.21, R.sub.22, R.sub.23, R.sub.24, R.sub.25,
R.sub.26, and R.sub.27 are the same or different, and are --H or a
linear or branched alkyl having from 1 to 5 carbon atoms;
[0127] R.sub.29 and R.sub.30 are the same or different, and are --H
or alkyl from 1 to 5 carbon atoms;
[0128] R.sub.33 is --H or linear, branched or cyclic alkyl, alkenyl
or alkynyl having from 1 to 12 carbon atoms, a polyethylene glycol
unit R--(OCH.sub.2CH.sub.2).sub.n--, or R.sub.33 is --COR.sub.34,
--CSR.sub.34, --SOR.sub.34, or --SO.sub.2R.sub.34, wherein R.sub.34
is H or linear, branched or cyclic alkyl, alkenyl or alkynyl having
from 1 to 20 carbon atoms or, a polyethylene glycol unit
--(OCH.sub.2CH.sub.2).sub.n; and
[0129] one of R.sub.40 and R.sub.41 is optionally a negatively or
positively charged functional group and the other is H or alkyl,
alkenyl, alkynyl having 1 to 4 carbon atoms. Any of the above
linking groups can be present in any of the compounds, drug-linker
compounds, or conjugates of the invention, including replacing the
linking groups of any of the formulas described herein.
[0130] The term "amino acid" refers to naturally occurring amino
acids or non-naturally occurring amino acid. In one embodiment, the
amino acid is represented by
NH.sub.2--C(R.sup.aa'R.sup.aa)--C(.dbd.O)OH, wherein R.sup.aa and
R.sup.aa' are each independently H, an optionally substituted
linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10
carbon atoms, aryl, heteroaryl or heterocyclyl or R.sup.aa and the
N-terminal nitrogen atom can together form a heterocyclic ring
(e.g., as in proline). The term "amino acid residue" refers to the
corresponding residue when one hydrogen atom is removed from the
amine and/or carboxy end of the amino acid, such as
--NH--C(R.sup.aa'R.sup.aa)--C(.dbd.O)O--.
[0131] The term "cation" refers to an ion with positive charge. The
cation can be monovalent (e.g., Na.sup.+, K.sup.+, etc.), bi-valent
(e.g., Ca.sup.2+, Mg.sup.2+, etc.) or multi-valent (e.g., Al.sup.3+
etc.). Preferably, the cation is monovalent.
[0132] The term "therapeutically effective amount" means that
amount of active compound or conjugate that elicits the desired
biological response in a subject. Such response includes
alleviation of the symptoms of the disease or disorder being
treated, prevention, inhibition or a delay in the recurrence of
symptom of the disease or of the disease itself, an increase in the
longevity of the subject compared with the absence of the
treatment, or prevention, inhibition or delay in the progression of
symptom of the disease or of the disease itself. Determination of
the effective amount is well within the capability of those skilled
in the art, especially in light of the detailed disclosure provided
herein. Toxicity and therapeutic efficacy of compound I can be
determined by standard pharmaceutical procedures in cell cultures
and in experimental animals. The effective amount of compound or
conjugate of the present invention or other therapeutic agent to be
administered to a subject will depend on the stage, category and
status of the multiple myeloma and characteristics of the subject,
such as general health, age, sex, body weight and drug tolerance.
The effective amount of compound or conjugate of the present
invention or other therapeutic agent to be administered will also
depend on administration route and dosage form. Dosage amount and
interval can be adjusted individually to provide plasma levels of
the active compound that are sufficient to maintain desired
therapeutic effects.
Cytotoxic Compounds
[0133] In a first embodiment, the present invention is directed to
cytotoxic compounds described herein (e.g., compounds of formulas
(I), (II), (III), (IV), (V), and (VI) describe above or a
pharmaceutically acceptable salt thereof).
[0134] In one embodiment, the cytotoxic dimer is a compound of
formula (I):
##STR00009##
[0135] or a pharmaceutically acceptable salt thereof.
[0136] In a 1.sup.st specific embodiment, Z.sup.s is represented by
either one of the following formulas:
##STR00010##
and the remaining variables are as described above in the first
embodiment.
[0137] In a 2.sup.nd specific embodiment, Z.sup.s is --H or
--SR.sup.d; and the remaining variables are as described above the
first embodiment.
[0138] In one embodiment, Z.sup.s is --H; and the remaining
variables are as described above in the 2.sup.nd specific
embodiment.
[0139] In another embodiments, Z.sup.s is --SR.sup.d; R.sup.d is
-Me or pyridyl; and the remaining variables are as described above
in the 2.sup.nd specific embodiment.
[0140] In a 3.sup.rd specific embodiment, R.sup.e is H or Me; and
the remaining variables are as described above in the first
embodiment or the 1.sup.st or 2.sup.nd specific embodiment.
[0141] In a 4.sup.th specific embodiment, R.sup.x is
--(CH.sub.2).sub.p--(CR.sup.fR.sup.g)--, wherein R.sup.f and
R.sup.g are each independently selected from H or a linear or
branched alkyl having 1 to 4 carbon atoms; p is 0, 1, 2 or 3; and
the remaining variables are as described above in the first
embodiment or the 1.sup.st, 2.sup.nd or 3.sup.rd embodiment.
[0142] In one embodiment, R.sup.f and R.sup.g are the same or
different, and are selected from --H and -Me; and the remaining
variables are as described above in the 4.sup.th specific
embodiment. More specifically, R.sup.f and R.sup.g are both -Me;
and p is 2.
[0143] In a 5.sup.th specific embodiment, R.sup.x is a linear or
branched alkylene having 1 to 4 carbon atoms substituted with a
charged substituent or an ionizable group Q; and the remaining
variables are as described above in the first embodiment or the
1.sup.st, 2.sup.nd or 3.sup.rd embodiment.
[0144] In one embodiment, Q is i) --SO.sub.3H, --Z'--SO.sub.3H,
--OPO.sub.3H.sub.2, --Z'--OPO.sub.3H.sub.2, --PO.sub.3H.sub.2,
--Z'--PO.sub.3H.sub.2, --CO.sub.2H, --Z'--CO.sub.2H,
--NR.sub.11R.sub.12, or --Z'--NR.sub.11R.sub.12, or a
pharmaceutically acceptable salt thereof; or, ii)
--N.sup.+R.sub.14R.sub.15R.sub.16X.sup.- or
--Z'--N.sup.+R.sub.14R.sub.15R.sub.16X.sup.-; Z' is an optionally
substituted alkylene, an optionally substituted cycloalkylene or an
optionally substituted phenylene; R.sub.14 to R.sub.16 are each
independently an optionally substituted alkyl; X.sup.- is a
pharmaceutically acceptable anion; and the remaining variables are
as described above as in the 5.sup.th specific embodiment. More
specifically, Q is SO.sub.3H or a pharmaceutically acceptable salt
thereof.
[0145] In a 6.sup.th specific embodiment, the double line between N
and C represents a double bond; and the remaining variables are as
described above in the first embodiment, or the 1.sup.st, 2.sup.nd,
3.sup.rd, 4.sup.th or 5.sup.th specific embodiment.
[0146] In a 7.sup.th embodiment, the double line between N and C
represents a single bond; X is --H or an amine protecting group; Y
is selected from --H, --SO.sub.3M, --OH, --OMe, --OEt or --NHOHY is
selected from --H, --SO.sub.3M, --OH, --OMe, --OEt or --NHOH; and
the remaining variables are as described above in the first
embodiment, or the 1.sup.st, 2.sup.nd, 3.sup.rd, 4.sup.th or
5.sup.th specific embodiment.
[0147] In one embodiment, Y is --H, --SO.sub.3M or --OH; and the
remaining variables are as described above in the 7.sup.th specific
embodiment. More specifically, M is H.sup.+, Na.sup.+ or
K.sup.+.
[0148] In a 8.sup.th specific embodiment, X' is --H, --OH or -Me;
and the remaining variables are as described above in the first
embodiment, or the 1.sup.st, 2.sup.nd, 3.sup.rd, 4.sup.th,
5.sup.th, 6.sup.th or 7.sup.th specific embodiment. More
specifically, X' is --H.
[0149] In a 9.sup.th specific embodiment, Y' is --H or oxo; and the
remaining variables are as described above in the first embodiment,
or the 1.sup.st, 2.sup.nd, 3.sup.rd, 4.sup.th, 5.sup.th, 6.sup.th,
7.sup.th or 8.sup.th specific embodiment. More specifically, Y' is
--H.
[0150] In a 10.sup.th specific embodiment, for the compounds of
formula (I), (II), (III), (IV), (V), and (VI),
[0151] the double line between N and C represents a single bond or
a double bond, provided that when it is a double bond X is absent
and Y is --H, and when it is a single bond, X is --H; Y is --OH or
--SO.sub.3M;
[0152] M is --H or a pharmaceutically acceptable cation;
[0153] X' and Y' are both --H; and
[0154] G is C; the remaining variables are as described above in
the first embodiment, or the 1.sup.st, 2.sup.nd, 3.sup.rd,
4.sup.th, or 5.sup.th specific embodiment.
[0155] In one embodiment, Y is --SO.sub.3M and M is H.sup.+,
Na.sup.+ or K.sup.+; and the remaining variables are as described
above in the 10.sup.th specific embodiment.
[0156] In a 11.sup.th specific embodiment, the compound is any one
of the following:
##STR00011## ##STR00012## ##STR00013## ##STR00014## ##STR00015##
##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020##
##STR00021## ##STR00022## ##STR00023## ##STR00024## ##STR00025##
##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030##
##STR00031## ##STR00032## ##STR00033## ##STR00034##
or a pharmaceutically acceptable salt thereof, wherein R.sup.d1 is
Me or Py; and M is a pharmaceutically acceptable cation. In one
embodiment, M is H.sup.+, Na.sup.+ or K.sup.+.
Drug Compounds & Drug-Linker Compounds
[0157] Certain cytotoxic compounds described above (e.g., compounds
of formulas (I), (II), (III), (IV), (V) and (VI) or a
pharmaceutically acceptable salt thereof, wherein Z.sup.s is --H,
SSR.sup.d, --SC(O)R.sup.d1 or compounds described above having a
free thiol --SH group) can further react with a bifunctional
crosslinking reagent to form a drug-linker compound a reactive
group bonded thereto, wherein the reactive group can form a
covalent bond with a CBA.
[0158] The bifunctional crosslinking agents can be any bifunctional
linker known in the art. For example, the bifunctional linkers can
be used for making the drug-linker compounds are those that form
disulfide bonds, thioether bonds, acid labile bonds, photolabile
bonds, peptidase labile bonds and esterase labile bonds with the
cytotoxic compounds (see for example, U.S. Pat. Nos. 5,208,020;
5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497; 7,276,499;
7,368,565; 7,388,026 and 7,414,073, all of which are incorporated
herein by reference). Preferably, the bifunctional crosslinking
agents are those that form disulfide bonds, thioether and peptidase
labile bonds with the cytotoxic compounds. Other bifunctional
crosslinking agents that can be used in the present invention
include non-cleavable linkers, such as those described in U.S.
publication number US 2005/0169933, or charged linkers or
hydrophilic linkers and are described in US 2009/0274713, US
2010/01293140 and WO 2009/134976, each of which is expressly
incorporated herein by reference. The bifunctional crosslinking
agents that can be used for making the (drug-linker) compounds of
the present invention also include those described in Thermo
Scientific Pierce Crosslinking Technical Handbook, the entire
teaching of which is incorporated herein by reference.
[0159] In one embodiment, the bifunctional crosslinking agent is
N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP),
N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB),
N-succinimidyl-4-(2-pyridyldithio)2-sulfo butanoate
(sulfo-SPDB).
Synthesis of Cytotoxic Compounds
[0160] The cytotoxic compounds of the present invention can be
prepared according to methods described in U.S. Pat. No. 8,765,740
and U.S. Application Publication No. 2012/0238731.
[0161] Representative processes for preparing the cytotoxic dimer
compounds of the present invention are shown in Examples 1 and
2.
Cell-Binding Agents
[0162] The effectiveness of the conjugates of the invention as
therapeutic agents depends on the careful selection of an
appropriate cell-binding agent. Cell-binding agents can be of any
kind presently known, or that become known, including peptides and
non-peptides. Generally, these can be antibodies (such as
polyclonal antibodies and monoclonal antibodies, especially
monoclonal antibodies), lymphokines, hormones, growth factors,
vitamins (such as folate etc., which can bind to a cell surface
receptor thereof, e.g., a folate receptor), nutrient-transport
molecules (such as transferrin), or any other cell-binding molecule
or substance.
[0163] Selection of the appropriate cell-binding agent is a matter
of choice that partly depends upon the particular cell population
that is to be targeted, but in many (but not all) cases, human
monoclonal antibodies are a good choice if an appropriate one is
available. For example, the monoclonal antibody MY9 is a murine
IgG.sub.1 antibody that binds specifically to the CD33 Antigen (J.
D. Griffin et al., Leukemia Res., 8:521 (1984)), and can be used if
the target cells express CD33 as in the disease of acute
myelogenous leukemia (AML).
[0164] In certain embodiments, the cell-binding agent is not a
protein. For example, in certain embodiments, the cell binding
agent may be a vitamin that binds to a vitamin receptor, such as a
cell-surface receptor. In this regard, vitamin A binds to
retinol-binding protein (RBP) to form a complex, which complex in
turn binds the STRA6 receptor with high affinity and increases
vitamin A in-take. In another example, folic acid/folate/vitamin
B.sub.9 binds the cell-surface folate receptor (FR), for example,
FR.alpha., with high affinity. Folic acid or antibodies that bind
to FR.alpha. can be used to target the folate receptor expressed on
ovarian and other tumors. In addition, vitamin D and its analog
bind to vitamin D receptor.
[0165] In other embodiments, the cell-binding agent is a protein or
a polypeptide, or a compound comprising a protein or polypeptide,
including antibody, non-antibody protein, or polypeptide.
Preferably, the protein or polypeptides comprise one or more Lys
residues with side chain --NH.sub.2 group. The Lys side chain
--NH.sub.2 groups can be covalently linked to the bifunctional
crosslinkers, which in turn are linked to the dimer compounds of
the invention, thus conjugating the cell-binding agents to the
dimer compounds of the invention. Each protein-based cell-binding
agents can contain multiple Lys side chain --NH.sub.2 groups
available for linking the compounds of the invention through the
bifunctional crosslinkers.
[0166] In one embodiment, GM-CSF, a ligand/growth factor which
binds to myeloid cells can be used as a cell-binding agent to
diseased cells from acute myelogenous leukemia. IL-2 which binds to
activated T-cells can be used for prevention of transplant graft
rejection, for therapy and prevention of graft-versus-host disease,
and for treatment of acute T-cell leukemia. MSH, which binds to
melanocytes, can be used for the treatment of melanoma, as can
antibodies directed towards melanomas. Epidermal growth factor can
be used to target squamous cancers, such as lung and head and neck.
Somatostatin can be used to target neuroblastomas and other tumor
types. Estrogen (or estrogen analogues) can be used to target
breast cancer. Androgen (or androgen analogues) can be used to
target testes.
[0167] In certain embodiments, the cell-binding agent can be a
lymphokine, a hormone, a growth factor, a colony stimulating
factor, or a nutrient-transport molecule.
[0168] In certain embodiments, the cell-binding agent is an
antibody mimetic, such as an ankyrin repeat protein, a Centyrin, or
an adnectin/monobody.
[0169] In other embodiments, the cell-binding agent is an antibody,
a single chain antibody, an antibody fragment that specifically
binds to the target cell, a monoclonal antibody, a single chain
monoclonal antibody, a monoclonal antibody fragment (or
"antigen-binding portion") that specifically binds to a target
cell, a chimeric antibody, a chimeric antibody fragment (or
"antigen-binding portion") that specifically binds to the target
cell, a domain antibody (e.g., sdAb), or a domain antibody fragment
that specifically binds to the target cell.
[0170] In certain embodiments, the cell-binding agent is a
humanized antibody, a humanized single chain antibody, or a
humanized antibody fragment (or "antigen-binding portion"). In a
specific embodiment, the humanized antibody is huMy9-6 or another
related antibody, which is described in U.S. Pat. Nos. 7,342,110
and 7,557,189. In another specific embodiment, the humanized
antibody is an anti-folate receptor antibody described in U.S.
Provisional Application Nos. 61/307,797, 61/346,595, and 61/413,172
and U.S. application Ser. No. 13/033,723 (published as US
2012/0009181 A1). The teachings of all these applications are
incorporated herein by reference in its entirety.
[0171] In certain embodiments, the cell-binding agent is a
resurfaced antibody, a resurfaced single chain antibody, a
resurfaced antibody fragment (or "antigen-binding portion"), or a
bispecific antibody.
[0172] In certain embodiments, the cell-binding agent is a
minibody, an avibody, a diabody, a tribody, a tetrabody, a
nanobody, a probody, a domain antibody, or an unibody.
[0173] In other words, an exemplary cell binding agent may include
an antibody, a single chain antibody, an antibody fragment that
specifically binds to the target cell, a monoclonal antibody, a
single chain monoclonal antibody, a monoclonal antibody fragment
that specifically binds to a target cell, a chimeric antibody, a
chimeric antibody fragment that specifically binds to the target
cell, a bispecific antibody, a domain antibody, a domain antibody
fragment that specifically binds to the target cell, an interferon
(e.g., .alpha., .beta., .gamma.), a lymphokine (e.g., IL-2, IL-3,
IL-4, and IL-6), a hormone (e.g., insulin, thyrotropin releasing
hormone (TRH), melanocyte-stimulating hormone (MSH), and a steroid
hormone (e.g., androgen and estrogen)), a vitamin (e.g., folate), a
growth factor (e.g., EGF, TGF-alpha, FGF, VEGF), a colony
stimulating factor, a nutrient-transport molecule (e.g.,
transferrin; see O'Keefe et al. (1985) J. Biol. Chem. 260:932-937,
incorporated herein by reference), a Centyrin (a protein scaffold
based on a consensus sequence of fibronectin type III (FN3)
repeats; see U.S. Patent Publication Nos. 2010/0255056,
2010/0216708 and 2011/0274623 incorporated herein by reference), an
Ankyrin Repeat Protein (e.g., a designed ankyrin repeat protein,
known as DARPin; see U.S. Patent Publication Nos. 2004/0132028,
2009/0082274, 2011/0118146, and 2011/0224100, incorporated herein
by reference, and also see C. Zahnd et al., Cancer Res. (2010)
70:1595-1605; Zahnd et al., J. Biol. Chem. (2006)
281(46):35167-35175; and Binz, H. K., Amstutz, P. & Pluckthun,
A., Nature Biotechnology (2005) 23:1257-1268, incorporated herein
by reference), an ankyrin-like repeats protein or synthetic peptide
(see e.g., U.S. Patent Publication No. 2007/0238667; U.S. Pat. No.
7,101,675; WO 2007/147213; and WO 2007/062466, incorporated herein
by reference), an Adnectin (a fibronectin domain scaffold protein;
see US Patent Publication Nos. 2007/0082365; 2008/0139791,
incorporated herein by reference), Avibody (including diabodies,
triabodies, and tetrabodies; see U.S. Publication Nos. 2008/0152586
and 2012/0171115), dual receptor retargeting (DART) molecules (P.
A. Moore et al., Blood, 2011; 117(17):4542-4551; Veri M C, et al.,
Arthritis Rheum, 2010 Mar. 30; 62(7):1933-43; Johnson S, et al. J
Mol Biol, 2010 Apr. 9; 399(3):436-49), cell penetrating
supercharged proteins (Methods in Enzymol. 502, 293-319 (2012), and
other cell-binding molecules or substances.
[0174] In certain embodiments, the cell-binding agent may be a
ligand that binds to a moiety on the target cell, such as a
cell-surface receptor. For example, the ligand may be a growth
factor or a fragment thereof that binds to a growth factor
receptor; or may be a cytokine or a fragment thereof that binds to
a cytokine receptor. In certain embodiments, the growth factor
receptor or cytokine receptor is a cell-surface receptor.
[0175] In certain embodiments, wherein the cell-binding agent is an
antibody or an antigen-binding portion thereof (including antibody
derivatives), or certain antibody mimetics, the CBA may bind to a
ligand on the target cell, such as a cell-surface ligand, including
cell-surface receptors.
[0176] Specific exemplary antigens or ligands may include renin; a
growth hormone (e.g., human growth hormone and bovine growth
hormone); a growth hormone releasing factor; a parathyroid hormone;
a thyroid stimulating hormone; a lipoprotein; alpha-1-antitrypsin;
insulin A-chain; insulin B-chain; proinsulin; a follicle
stimulating hormone; calcitonin; a luteinizing hormone; glucagon; a
clotting factor (e.g., factor vmc, factor IX, tissue factor, and
von Willebrands factor); an anti-clotting factor (e.g., Protein C);
an atrial natriuretic factor; a lung surfactant; a plasminogen
activator (e.g., a urokinase, a human urine or tissue-type
plasminogen activator); bombesin; a thrombin; hemopoietic growth
factor; tumor necrosis factor-alpha and -beta; an enkephalinase;
RANTES (i.e., the regulated on activation normally T-cell expressed
and secreted); human macrophage inflammatory protein-1-alpha; a
serum albumin (human serum albumin); Muellerian-inhibiting
substance; relaxin A-chain; relaxin B-chain; prorelaxin; a mouse
gonadotropin-associated peptide; a microbial protein
(beta-lactamase); DNase; IgE; a cytotoxic T-lymphocyte associated
antigen (e.g., CTLA-4); inhibin; activin; a vascular endothelial
growth factor; a receptor for hormones or growth factors; protein A
or D; a rheumatoid factor; a neurotrophic factor (e.g.,
bone-derived neurotrophic factor, neurotrophin-3, -4, -5, or -6), a
nerve growth factor (e.g., NGF-.beta.); a platelet-derived growth
factor; a fibroblast growth factor (e.g., aFGF and bFGF);
fibroblast growth factor receptor 2; an epidermal growth factor; a
transforming growth factor (e.g., TGF-alpha, TGF-.beta.1,
TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, and TGF-.beta.5);
insulin-like growth factor-I and -II; des(1-3)-IGF-I (brain IGF-I);
an insulin-like growth factor binding protein; melanotransferrin;
EpCAM; GD3; FLT3; PSMA; PSCA; MUC1; MUC16; STEAP; CEA; TENB2; an
EphA receptor; an EphB receptor; a folate receptor; FOLR1;
mesothelin; cripto; an alpha.sub.vbeta.sub.6; integrins; VEGF;
VEGFR; EGFR; transferrin receptor; IRTA1; IRTA2; IRTA3; IRTA4;
IRTA5; CD proteins (e.g., CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14,
CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37,
CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80. CD81,
CD103, CD105, CD123, CD134, CD137, CD138, and CD152), one or more
tumor-associated antigens or cell-surface receptors (see US
Publication No. 20080171040 or US Publication No. 20080305044,
incorporated in their entirety by reference); erythropoietin; an
osteoinductive factor; an immunotoxin; a bone morphogenetic
protein; an interferon (e.g., interferon-alpha, -beta, and -gamma);
a colony stimulating factor (e.g., M-CSF, GM-CSF, and G-CSF);
interleukins (e.g., IL-1 to IL-10); a superoxide dismutase; a
T-cell receptor; a surface membrane protein; a decay accelerating
factor; a viral antigen s (e.g., a portion of the HIV envelope); a
transport protein, a homing receptor; an addressin; a regulatory
protein; an integrin (e.g., CD11a, CD11b, CD11c, CD18, an ICAM,
VLA-4, and VCAM;) a tumor associated antigen (e.g., HER2, HER3 and
HER4 receptor); endoglin; c-Met; c-kit; 1GF1R; PSGR; NGEP; PSMA;
PSCA; TMEFF2; LGRS; B7H4; and fragments of any of the above-listed
polypeptides.
[0177] As used herein, the term "antibody" includes immunoglobulin
(Ig) molecules. In certain embodiments, the antibody is a
full-length antibody that comprises four polypeptide chains, namely
two heavy chains (HC) and two light chains (LC) inter-connected by
disulfide bonds. Each heavy chain is comprised of a heavy chain
variable region (HCVR or VH) and a heavy chain constant region
(CH). The heavy chain constant region is comprised of three
domains, CH1, CH2, and CH3. Each light chain is comprised of a
light chain variable region (LCVR or VL) and a light chain constant
region, which is comprised of one domain, CL. The VH and VL regions
can be further subdivided into regions of hypervariability, termed
complementarity determining regions (CDRs). Interspersed with such
regions are the more conserved framework regions (FRs). Each VH and
VL is composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, and FR4.
[0178] In certain embodiments, the antibody is IgG, IgA, IgE, IgD,
or IgM. In certain embodiments, the antibody is IgG1, IgG2, IgG3,
or IgG4; or IgA1 or IgA2.
[0179] In certain embodiments, the cell-binding agent is an
"antigen-binding portion" of a monoclonal antibody, sharing
sequences critical for antigen-binding with an antibody (such as
huMy9-6 or its related antibodies described in U.S. Pat. Nos.
7,342,110 and 7,557,189, incorporated herein by reference).
[0180] As used herein, the term "antigen-binding portion" of an
antibody (or sometimes interchangeably referred to as "antibody
fragments"), include one or more fragments of an antibody that
retain the ability to specifically bind to an antigen. It has been
shown that the antigen-binding function of an antibody can be
performed by certain fragments of a full-length antibody. Examples
of binding fragments encompassed within the term "antigen-binding
portion" of an antibody include (without limitation): (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and
CH1 domains (e.g., an antibody digested by papain yields three
fragments: two antigen-binding Fab fragments, and one Fc fragment
that does not bind antigen); (ii) a F(ab').sub.2 fragment, a
bivalent fragment comprising two Fab fragments linked by a
disulfide bridge at the hinge region (e.g., an antibody digested by
pepsin yields two fragments: a bivalent antigen-binding
F(ab').sub.2 fragment, and a pFc' fragment that does not bind
antigen) and its related F(ab') monovalent unit; (iii) a Fd
fragment consisting of the VH and CH1 domains (i.e., that portion
of the heavy chain which is included in the Fab); (iv) a Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody, and the related disulfide linked Fv; (v) a dAb (domain
antibody) or sdAb (single domain antibody) fragment (Ward et al.,
Nature 341:544-546, 1989), which consists of a VH domain; and (vi)
an isolated complementarity determining region (CDR). In certain
embodiments, the antigen-binding portion is a sdAb (single domain
antibody).
[0181] In certain embodiments, antigen-binding portion also include
certain engineered or recombinant derivatives (or "derivative
antibodies") that also include one or more fragments of an antibody
that retain the ability to specifically bind to an antigen, in
addition to elements or sequences that may not be found in
naturally existing antibodies.
[0182] For example, although the two domains of the Fv fragment, VL
and VH, are coded for by separate genes, they can be joined, using
standard recombinant methods, by a synthetic linker that enables
them to be made as a single protein chain in which the VL and VH
regions pair to form monovalent molecules (known as single chain Fv
(scFv); see, e.g., Bird et al. Science 242:423-426, 1988: and
Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988).
[0183] In all embodiments described herein, the N-terminum of an
scFv may be a VH domain (i.e., N-VH-VL-C), or a VL domain (i.e.,
N-VL-VH-C).
[0184] Divalent (or bivalent) single-chain variable fragments
(di-scFvs, bi-scFvs) can be engineered by linking two scFvs. This
produces a single peptide chain with two VH and two VL regions,
yielding a tandem scFvs (tascFv). More tandem repeats, such as
tri-scFv, may be similarly produced by linking three or more scFv
in a head-to-tail fashion.
[0185] In certain embodiments, scFvs may be linked through linker
peptides that are too short (about five amino acids) for the two
variable regions to fold together, forcing scFvs to dimerize, and
form diabodies (see, e.g., Holliger et al., Proc. Natl. Acad. Sci.
USA 90:6444-6448, 1993; Poljak et al., Structure 2:1121-1123,
1994). Diabodies may be bi-specific or monospecific. Diabodies have
been shown to have dissociation constants up to 40-fold lower than
corresponding scFvs, i.e., having a much higher affinity to the
target.
[0186] Still shorter linkers (one or two amino acids) lead to the
formation of trimers, or so-called triabodies or tribodies.
Tetrabodies have also been produced similarly They exhibit an even
higher affinity to their targets than diabodies. Diabodies,
triabodies, and tetrabodies are sometimes collectively called
AVIBODY.TM. cell binding agents (or "AVIBODY" in short). That is,
AVIBODY having two, three, or four Target Binding Regions (TBRs)
are commonly known as Dia-, Tria- and Tetra-bodies. See, for
example, U.S. Publication Nos. 2008/0152586 and 2012/0171115 for
details, the entire teachings of which are incorporated herein by
reference.
[0187] All of these formats can be composed from variable fragments
with specificity for two or more different antigens, in which case
they are types of bi- or multi-specific antibodies. For example,
certain bispecific tandem di-scFvs, are known as bi-specific T-cell
engagers (BiTEs).
[0188] In certain embodiments, each scFv in the tandem scFv or
diabody/triabody/tetrabody may have the same or different binding
specificity, and each may independently have an N-terminal VH or
N-terminal VL.
[0189] Single chain Fv (scFv) can also be fused to an Fc moiety,
such as the human IgG Fc moiety to obtain IgG-like properties, but
nevertheless they are still encoded by a single gene. As transient
production of such scFv-Fc proteins in mammalians can easily
achieve milligram amounts, this derivative antibody format is
particularly suitable for many research applications.
[0190] Fcabs are antibody fragments engineered from the Fc constant
region of an antibody. Fcabs can be expressed as soluble proteins,
or they can be engineered back into a full-length antibody, such as
IgG, to create mAb2. A mAb2 is a full-length antibody with an Fcab
in place of the normal Fc region. With these additional binding
sites, mAb2 bispecific monoclonal antibodies can bind two different
targets at the same time.
[0191] In certain embodiments, the engineered antibody derivatives
have reduced size of the antigen-binding Ig-derived recombinant
proteins ("miniaturized" full-size mAbs), produced by removing
domains deemed non-essential for function. One of the best examples
is SMIPs.
[0192] A Small modular immunopharmaceutical, or SMIP, is an
artificial protein largely built from parts of antibodies
(immunoglobulins), and is intended for use as a pharmaceutical
drug. SMIPs have similar biological half-life as antibodies, but
are smaller than antibodies and hence may have better tissue
penetration properties. SMIPs are single-chain proteins that
comprise one binding region, one hinge region as a connector, and
one effector domain. The binding region comprises a modified
single-chain variable fragment (scFv), and the rest of the protein
can be constructed from the Fc (such as CH2, and CH3 as the
effector domain) and the hinge region of an antibody, such as IgG1.
Genetically modified cells produce SMIPs as antibody-like dimers
that are about 30% smaller than real antibodies.
[0193] Another example of such engineered miniaturized antibody is
"unibody," in which the hinge region has been removed from IgG4
molecules. IgG4 molecules are unstable and can exchange light-heavy
chain heterodimers with one another. Deletion of the hinge region
prevents heavy chain-heavy chain pairing entirely, leaving highly
specific monovalent light/heavy heterodimers, while retaining the
Fc region to ensure stability and half-life in vivo.
[0194] A single-domain antibody (sdAb, including but not limited to
those called nanobody by Ablynx) is an antibody fragment consisting
of a single monomeric variable antibody domain Like a whole
antibody, it is able to bind selectively to a specific antigen, but
is much smaller due to its molecular weight of only 12-15 kDa. In
certain embodiments, the single-domain antibody is engineered from
heavy-chain antibodies (hcIgG). The first such sdAb was engineered
based on an hcIgG found in camelids, called V.sub.HH fragments. In
certain embodiments, the single-domain antibody is engineered from
IgNAR ("immunoglobulin new antigen receptor," see below) using a
V.sub.NAR fragment. Cartilaginous fishes (such as shark) have such
heavy-chain IgNAR antibodies. In certain embodiments, the sdAb is
engineered by splitting the dimeric variable domains from common
immunoglobulin G (IgG), such as those from humans or mice, into
monomers. In certain embodiments, a nanobody is derived from a
heavy chain variable domain. In certain embodiments, a nanobody is
derived from light chain variable domain. In certain embodiments,
the sdAb is obtained by screening libraries of single domain heavy
chain sequences (e.g., human single domain HCs) for binders to a
target antigen.
[0195] The single variable new antigen receptor domain antibody
fragments (V.sub.NARS, or V.sub.NAR domains) are derived from
cartilaginous fish (e.g., shark) immunoglobulin new antigen
receptor antibodies (IgNARs). Being one of the smallest known
immunoglobulin-based protein scaffolds, such single domain proteins
demonstrate favorable size and cryptic epitope recognition
properties. Mature IgNAR antibodies consist of homodimers of one
variable new antigen receptor (V.sub.NAR) domain and five constant
new antigen receptor (C.sub.NAR) domains. This molecule is highly
stable, and possesses efficient binding characteristics. Its
inherent stability can likely be attributed to both (i) the
underlying Ig scaffold, which presents a considerable number of
charged and hydrophilic surface exposed residues compared to the
conventional antibody VH and VL domains found in murine antibodies;
and (ii) stabilizing structural features in the complementary
determining region (CDR) loops including inter-loop disulphide
bridges, and patterns of intra-loop hydrogen bonds.
[0196] A minibody is an engineered antibody fragment comprising an
scFv linked to a CH domain, such as the CH3.gamma.1 (CH3 domain of
IgG1) or CH4E (CH4 domain of IgE). For example, an scFv specific
for carcinoembryonic antigen (CEA) has been linked to the
CH3.gamma.1 to create a minibody, which has previously been
demonstrated to possess excellent tumor targeting coupled with
rapid clearance in vivo (Hu et al., Cancer Res. 56:3055-3061,
1996). The scFv may have a N-terminal VH or VL. The linkage may be
a short peptide (e.g., two amino acid linker, such as ValGlu) that
results in a non-covalent, hingeless minibody. Alternatively, the
linkage may be an IgG1 hinge and a GlySer linker peptide that
produces a covalent, hinge-minibody.
[0197] Natural antibodies are mono-specific, but bivalent, in that
they express two identical antigen-binding domains. In contrast, in
certain embodiments, certain engineered antibody derivatives are
bi- or multi-specific molecules possess two or more different
antigen-binding domains, each with different target specificity.
Bispecific antibodies can be generated by fusing two
antibody-producing cells, each with distinct specificity. These
"quadromas" produced multiple molecular species, as the two
distinct light chains and two distinct heavy chains were free to
recombine in the quadromas in multiple configurations. Since then,
bispecific Fabs, scFvs and full-size mAbs have been generated using
a variety of technologies (see above).
[0198] The dual variable domain immunoglobulin (DVD-Ig) protein is
a type of dual-specific IgG that simultaneously target two
antigens/epitopes (DiGiammarino et al., Methods Mol Biol.
899:145-56, 2012). The molecule contains an Fc region and constant
regions in a configuration similar to a conventional IgG. However,
the DVD-Ig protein is unique in that each arm of the molecule
contains two variable domains (VDs). The VDs within an arm are
linked in tandem and can possess different binding
specificities.
[0199] Trispecific antibody derivative molecules can also been
generated by, for example, expressing bispecific antibodies with
two distinct Fabs and an Fc. One example is a mouse IgG2a
anti-Ep-CAM, rat IgG2b anti-CD3 quadroma, called BiUII, which is
thought to permit the co-localization of tumor cells expressing
Ep-CAM, T-cells expressing CD3, and macrophages expressing
FC.gamma.RI, thus potentiating the costimulatory and anti-tumor
functions of the immune cells.
[0200] Probodies are fully recombinant, masked monoclonal
antibodies that remain inert in healthy tissue, but are activated
specifically in the disease microenvironment (e.g., through
protease cleavage by a protease enriched or specific in a disease
microenvironment). See Desnoyers et al., Sci Transl Med 5:207ra144,
2013. Similar masking techniques can be used for any of the
antibodies or antigen-binding portions thereof described
herein.
[0201] An intrabody is an antibody that has been modified for
intracellular localization, for working within the cell to bind to
an intracellular antigen. The intrabody may remain in the
cytoplasm, or may have a nuclear localization signal, or may have a
KDEL sequence for ER targeting. The intrabody may be a single-chain
antibody (scFv), modified immunoglobulin VL domains with
hyperstability, selected antibody resistant to the more reducing
intracellular environment, or expressed as a fusion protein with
maltose binding protein or other stable intracellular proteins.
Such optimizations have improved the stability and structure of
intrabodies, and may have general applicability to any of the
antibodies or antigen-binding portions thereof described
herein.
[0202] The antigen-binding portions or derivative antibodies of the
invention may have substantially the same or identical (1) light
chain and/or heavy chain CDR3 regions; (2) light chain and/or heavy
chain CDR1, CDR2, and CDR3 regions; or (3) light chain and/or heavy
chain regions, compared to an antibody from which they are
derived/engineered. Sequences within these regions may contain
conservative amino acid substitutions, including substitutions
within the CDR regions. In certain embodiments, there is no more
than 1, 2, 3, 4, or 5 conservative substitutions. In an
alternative, the antigen-binding portions or derivative antibodies
have a light chain region and/or a heavy chain region that is at
least about 90%, 95%, 99% or 100% identical to an antibody from
which they are derived/engineered. These antigen-binding portions
or derivative antibodies may have substantially the same binding
specificity and/or affinity to the target antigen compared to the
antibody. In certain embodiments, the K.sub.d and/or k.sub.off
values of the antigen-binding portions or derivative antibodies are
within 10-fold (either higher or lower), 5-fold (either higher or
lower), 3-fold (either higher or lower), or 2-fold (either higher
or lower) of an antibody described herein.
[0203] In certain embodiments, the antigen-binding portions or
derivative antibodies may be derived/engineered from fully human
antibodies, humanized antibodies, or chimeric antibodies, and may
be produced according to any art-recognized methods.
[0204] Monoclonal antibody techniques allow for the production of
extremely specific cell-binding agents in the form of specific
monoclonal antibodies. Particularly well known in the art are
techniques for creating monoclonal antibodies produced by
immunizing mice, rats, hamsters or any other mammal with the
antigen of interest such as the intact target cell, antigens
isolated from the target cell, whole virus, attenuated whole virus,
and viral proteins such as viral coat proteins. Sensitized human
cells can also be used. Another method of creating monoclonal
antibodies is the use of phage libraries of scFv (single chain
variable region), specifically human scFv (see e.g., Griffiths et
al., U.S. Pat. Nos. 5,885,793 and 5,969,108; McCafferty et al., WO
92/01047; Liming et al., WO 99/06587). In addition, resurfaced
antibodies disclosed in U.S. Pat. No. 5,639,641 may also be used,
as may chimeric antibodies and humanized antibodies.
[0205] Cell-binding agent can also be peptides derived from phage
display (see, for example, Wang et al., Proc. Natl. Acad. Sci. USA
(2011) 108(17), 6909-6914) or peptide library techniques (see, for
example, Dane et al., Mol. Cancer. Ther. (2009)
8(5):1312-1318).
[0206] In certain embodiments, the CBA of the invention also
includes an antibody mimetic, such as a DARPin, an affibody, an
affilin, an affitin, an anticalin, an avimer, a Fynomer, a Kunitz
domain peptide, a monobody, or a nanofitin.
[0207] As used herein, the terms "DARPin" and "(designed) ankyrin
repeat protein" are used interchangeably to refer to certain
genetically engineered antibody mimetic proteins typically
exhibiting preferential (sometimes specific) target binding. The
target may be protein, carbohydrate, or other chemical entities,
and the binding affinity can be quite high. The DARPins may be
derived from natural ankyrin repeat-containing proteins, and
preferably consist of at least three, usually four or five ankyrin
repeat motifs (typically about 33 residues in each ankyrin repeat
motif) of these proteins. In certain embodiments, a DARPin contains
about four- or five-repeats, and may have a molecular mass of about
14 or 18 kDa, respectively. Libraries of DARPins with randomized
potential target interaction residues with diversities of over
10.sup.12 variants can be generated at the DNA level, for use in
selecting DARPins that bind desired targets (e.g., acting as
receptor agonists or antagonists, inverse agonists, enzyme
inhibitors, or simple target protein binders) with picomolar
affinity and specificity, using a variety of technologies such as
ribosome display or signal recognition particle (SRP) phage
display. See, for example, U.S. Patent Publication Nos.
2004/0132028, 2009/0082274, 2011/0118146, and 2011/0224100, WO
02/20565 and WO 06/083275 for DARPin preparation (the entire
teachings of which are incorporated herein by reference), and also
see C. Zahnd et al. (2010) Cancer Res., 70:1595-1605; Zahnd et al.
(2006) J. Biol. Chem., 281(46):35167-35175; and Binz, H. K.,
Amstutz, P. & Pluckthun, A. (2005) Nature Biotechnology,
23:1257-1268 (all incorporated herein by reference). Also see U.S.
Patent Publication No. 2007/0238667; U.S. Pat. No. 7,101,675; WO
2007/147213; and WO 2007/062466 (the entire teachings of which are
incorporated herein by reference), for the related ankyrin-like
repeats protein or synthetic peptide.
[0208] Affibody molecules are small proteins engineered to bind to
a large number of target proteins or peptides with high affinity,
thus imitating monoclonal antibodies. An Affibody consists of three
alpha helices with 58 amino acids and has a molar mass of about 6
kDa. They have been shown to withstand high temperatures
(90.degree. C.) or acidic and alkaline conditions (pH 2.5 or pH
11), and binders with an affinity of down to sub-nanomolar range
have been obtained from naive library selections, and binders with
picomolar affinity have been obtained following affinity
maturation. In certain embodiments, affibodies are conjugated to
weak electrophiles for binding to targets covalently.
[0209] Monobodies (also known as Adnectins), are genetically
engineered antibody mimetic proteins capable of binding to
antigens. In certain embodiments, monobodies consist of 94 amino
acids and have a molecular mass of about 10 kDa. They are based on
the structure of human fibronectin, more specifically on its tenth
extracellular type III domain, which has a structure similar to
antibody variable domains, with seven beta sheets forming a barrel
and three exposed loops on each side corresponding to the three
complementarity determining regions. Monobodies with specificity
for different proteins can be tailored by modifying the loops BC
(between the second and third beta sheets) and FG (between the
sixth and seventh sheets).
[0210] A tribody is a self-assembly antibody mimetic designed based
on the C-terminal coiled-coil region of mouse and human cartilage
matrix protein (CMP), which self-assembles into a parallel trimeric
complex. It is a highly stable trimeric targeting ligand created by
fusing a specific target-binding moiety with the trimerization
domain derived from CMP. The resulting fusion proteins can
efficiently self-assemble into a well-defined parallel homotrimer
with high stability. Surface plasmon resonance (SPR) analysis of
the trimeric targeting ligands demonstrated significantly enhanced
target-binding strength compared with the corresponding monomers.
Cellular-binding studies confirmed that such tribodies have
superior binding strength toward their respective receptors.
[0211] A Centyrin is another antibody mimetic that can be obtained
using a library built upon the framework of a consensus FN3 domain
sequence (Diem et al., Protein Eng Des Sel., 2014). This library
employs diversified positions within the C-strand, CD-loop,
F-strand and FG-loop of the FN3 domain, and high-affinity Centyrin
variants can be selected against specific targets.
[0212] In one embodiment, the cell-binding agent is an anti-folate
receptor antibody. More specifically, the anti-folate receptor
antibody is a humanized antibody or antigen binding fragment
thereof that specifically binds a human folate receptor 1 (also
known as folate receptor alpha (FR-.alpha.)). The terms "human
folate receptor 1," "FOLR1," or "folate receptor alpha
(FR-.alpha.)", as used herein, refers to any native human FOLR1,
unless otherwise indicated. Thus, all of these terms can refer to
either a protein or nucleic acid sequence as indicated herein. The
term "FOLR1" encompasses "full-length," unprocessed FOLR1 as well
as any form of FOLR1 that results from processing within the cell.
The FOLR1 antibody comprises: (a) a heavy chain CDR1 comprising
GYFMN (SEQ ID NO: 1); a heavy chain CDR2 comprising
RIHPYDGDTFYNQXaa.sub.1FXaa.sub.2Xaa.sub.3 (SEQ ID NO: 2); and a
heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO: 3); and (b) a
light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO: 4); a light
chain CDR2 comprising RASNLEA (SEQ ID NO: 5); and a light chain
CDR3 comprising QQSREYPYT (SEQ ID NO: 6); wherein Xaa.sub.1 is
selected from K, Q, H, and R; Xaa.sub.2 is selected from Q, H, N,
and R; and Xaa.sub.3 is selected from G, E, T, S, A, and V.
Preferably, the heavy chain CDR2 sequence comprises
RIHPYDGDTFYNQKFQG (SEQ ID NO: 7).
[0213] In another embodiment, the anti-folate receptor antibody is
a humanized antibody or antigen binding fragment thereof that
specifically binds the human folate receptor 1 comprising the heavy
chain having the amino acid sequence of
TABLE-US-00001 (SEQ ID NO: 8)
QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGR
IHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYD
GSRAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
[0214] In another embodiment, the anti-folate receptor antibody is
a humanized antibody or antigen binding fragment thereof encoded by
the plasmid DNA deposited with the ATCC on Apr. 7, 2010 and having
ATCC deposit nos. PTA-10772 and PTA-10773 or PTA-10774.
[0215] In another embodiment, the anti-folate receptor antibody is
a humanized antibody or antigen binding fragment thereof that
specifically binds the human folate receptor 1 comprising the light
chain having the amino acid sequence of
TABLE-US-00002 (SEQ ID NO: 9)
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRL
LIYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPY
TFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
THQGLSSPVTKSFNRGEC; or (SEQ ID NO: 10)
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRL
LIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPY
TFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
THQGLSSPVTKSFNRGEC.
[0216] In another embodiment the anti-folate receptor antibody is a
humanized antibody or antigen binding fragment thereof that
specifically binds the human folate receptor 1 comprising the heavy
chain having the amino acid sequence of SEQ ID NO: 8, and the light
chain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO:
10. Preferably, the antibody comprises the heavy chain having the
amino acid sequence of SEQ ID NO: 8 and the light chain having the
amino acid sequence of SEQ ID NO: 10 (hu FOLR1).
[0217] In another embodiment, the anti-folate receptor antibody is
a humanized antibody or antigen binding fragment thereof encoded by
the plasmid DNA deposited with the ATCC on Apr. 7, 2010 and having
ATCC deposit nos. PTA-10772 and PTA-10773 or 10774.
[0218] In another embodiment, the anti-folate receptor antibody is
a humanized antibody or antigen binding fragment thereof that
specifically binds the human folate receptor 1, and comprising a
heavy chain variable domain at least about 90%, 95%, 99% or 100%
identical to QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHP
YDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAM DYWGQGTTVTVSS
(SEQ ID NO: 11), and a light chain variable domain at least about
90%, 95%, 99% or 100% identical to
TABLE-US-00003 (SEQ ID NO: 12)
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPG
QQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATY
YCQQSREYPYTFGGGTKLEIKR; or (SEQ ID NO: 13)
DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPG
QQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATY
YCQQSREYPYTFGGGTKLEIKR.
[0219] In another embodiment, the anti-folated receptor antibody is
huMov19 or M9346A (see, for example, U.S. Pat. No. 8,709,432, U.S.
Pat. No. 8,557,966, and WO2011106528, all incorporated herein by
reference).
[0220] In another embodiment, the cell-binding agent is an
anti-EGFR antibody or an antibody fragment thereof. In one
embodiment, the anti-EGFR antibody is a non-antagonist antibody,
including, for example, the antibodies described in WO2012058592,
herein incorporated by reference. In another embodiment, the
anti-EGFR antibody is a non-functional antibody, for example,
humanized ML66 or EGFR-8. More specifically, the anti-EGFR antibody
is huML66.
[0221] In yet another embodiment, the anti-EGFR antibody comprising
the heavy chain having the amino acid sequence of SEQ ID NO: 14,
and the light chain having the amino acid sequence of SEQ ID NO:
15. As used herein, double underlined sequences represent the
variable regions (i.e., heavy chain variable region or HCVR, and
light chain variable region or LCVR) of the heavy or light chain
sequences, while bold sequences represent the CDR regions (i.e.,
from N-terminal to C-terminal, CDR1, CDR2, and CDR3, respectively,
of the heavy chain or light chain sequences).
TABLE-US-00004 Full-Length Heavy/ Antibody Light Chain Amino Acid
Sequence huML66HC QVQLQESGPGLVKPSETLSLTCTVSGLSLAS
NSVSWIRQPPGKGLEWMGVIWNHGGTDYNPS IKSRLSISRDTSKSQVFLKMNSLTAADTAMY
FCVRKGGIYFDYWGQGVLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG (SEQ ID NO: 14)
huML66LC DTVLTQSPSLAVSPGERATISCRASESVSTL
MHWYQQKPGQQPKWYLASHRESGVPARFSGS GSGTDFTLTIDPMEAEDTATYYCQQSRNDPW
TFGQGTKLELKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID
NO: 15)
[0222] In yet another embodiment, the anti-EGFR antibody comprises
the heavy chain CDR1-CDR3 of SEQ ID NO: 14, and/or the light chain
CDR1-CDR3 of SEQ ID NO: 15, and preferably specifically binds
EGFR.
[0223] In yet another embodiment, the anti-EGFR antibody comprises
a heavy chain variable region (HCVR) sequence at least about 90%,
95%, 97%, 99%, or 100% identical to SEQ ID NO: 14, and/or a light
chain variable region (LCVR) sequence at least about 90%, 95%, 97%,
99%, or 100% identical to SEQ ID NO: 15, and preferably
specifically binds EGFR.
[0224] In another embodiment, the anti-EGFR antibody are antibodies
described in U.S. Pat. No. 8,790,649 and WO 2012/058588, herein
incorporated by reference. In one embodiment, the anti-EGFR
antibody is huEGFR-7R antibody.
[0225] In one embodiment, the anti-EGFR antibody comprises an
immunoglobulin heavy chain region having the amino acid sequence
of
TABLE-US-00005 (SEQ ID NO: 16)
QVQLVQSGAEVAKPGASVKLSCKASGYTFTSYWMQWVKQRPGQGL
ECIGTIYPGDGDTTYTQKFQGKATLTADKSSSTAYMQLSSLRSED
SAVYYCARYDAPGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
and an immunoglobulin light chain region having the amino acid
sequence of
TABLE-US-00006 (SEQ ID NO: 17)
DIQMTQSPSSLSASVGDRVTITCRASQDINNYLAWYQHKPGKGPK
LLIHYTSTLHPGIPSRFSGSGSGRDYSFSISSLEPEDIATYYCLQ
YDNLLYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC,
or an immunoglobulin light chain region having the amino acid
sequence of
TABLE-US-00007 (SEQ ID NO: 18)
DIQMTQSPSSLSASVGDRVTITCKASQDINNYLAWYQHKPGKGPK
LLIHYTSTLHPGIPSRFSGSGSGRDYSFSISSLEPEDIATYYCLQ
YDNLLYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
[0226] In another embodiment, the anti-EGFR antibody comprises an
immunoglobulin heavy chain region having the amino acid sequence
set forth in SEQ ID NO:16 and an immunoglobulin light chain region
having the amino acid sequence set forth in SEQ ID NO:17.
[0227] In another embodiment, the anti-EGFR antibody comprises an
immunoglobulin heavy chain region having the amino acid sequence
set forth in SEQ ID NO:16 and an immunoglobulin light chain region
having the amino acid sequence set forth in SEQ ID NO:18.
[0228] In yet another embodiment, the anti-EGFR antibody comprises
the heavy chain CDR1-CDR3 of SEQ ID NO: 16, and/or the light chain
CDR1-CDR3 of SEQ ID NO: 17 or 18, and preferably specifically binds
EGFR.
[0229] In yet another embodiment, the anti-EGFR antibody comprises
a heavy chain variable region (HCVR) sequence at least about 90%,
95%, 97%, 99%, or 100% identical to SEQ ID NO: 16, and/or a light
chain variable region (LCVR) sequence at least about 90%, 95%, 97%,
99%, or 100% identical to SEQ ID NO: 17 or 18, and preferably
specifically binds EGFR.
[0230] In another embodiment, the cell-binding agent is an
anti-CD19 antibody, such as those described in U.S. Pat. No.
8,435,528 and WO2004/103272, herein incorporated by reference. In
one embodiment, the anti-CD19 antibody comprises an immunoglobulin
heavy chain region having the amino acid sequence of
QVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWMHWVKQAPGQGLEWIGEID
PSDSYTNYNQNFQGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPYY
YAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:19) and an immunoglobulin light
chain region having the amino acid sequence of
TABLE-US-00008 (SEQ ID NO: 20)
EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGTSPRR
WIYDTSKLASGVPARFSGSGSGTDYSLTISSMEPEDAATYYCHQR
GSYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN
FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
[0231] In another embodiment, the anti-CD19 antibody is huB4
antibody.
[0232] In yet another embodiment, the anti-CD19 antibody comprises
the heavy chain CDR1-CDR3 of SEQ ID NO: 19, and/or the light chain
CDR1-CDR3 of SEQ ID NO: 20, and preferably specifically binds
CD19.
[0233] In yet another embodiment, the anti-CD19 antibody comprises
a heavy chain variable region (HCVR) sequence at least about 90%,
95%, 97%, 99%, or 100% identical to SEQ ID NO: 19, and/or a light
chain variable region (LCVR) sequence at least about 90%, 95%, 97%,
99%, or 100% identical to SEQ ID NO: 20, and preferably
specifically binds CD19.
[0234] In yet another embodiment, the cell-binding agent is an
anti-Muc1 antibody, such as those described in U.S. Pat. No.
7,834,155, WO 2005/009369 and WO 2007/024222, herein incorporated
by reference. In one embodiment, the anti-Muc1 antibody comprises
an immunoglobulin heavy chain region having the amino acid sequence
of
TABLE-US-00009 (SEQ ID NO: 21)
QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGL
EWIGYIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSED
SAVYFCARGDSVPFAYWGQGTLVTVSAASTKGPSVFPLAPSSKST
SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
and an immunoglobulin light chain region having the amino acid
sequence of
TABLE-US-00010 (SEQ ID NO: 22)
EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKL
WIYSTSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQR
SSFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLL
NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
[0235] In another embodiment, the anti-Muc1 antibody is huDS6
antibody.
[0236] In yet another embodiment, the anti-Muc1 antibody comprises
the heavy chain CDR1-CDR3 of SEQ ID NO: 21, and/or the light chain
CDR1-CDR3 of SEQ ID NO: 22, and preferably specifically binds
Muc1.
[0237] In yet another embodiment, the anti-Muc1 antibody comprises
a heavy chain variable region (HCVR) sequence at least about 90%,
95%, 97%, 99%, or 100% identical to SEQ ID NO: 21, and/or a light
chain variable region (LCVR) sequence at least about 90%, 95%, 97%,
99%, or 100% identical to SEQ ID NO: 22, and preferably
specifically binds Muc1.
[0238] In another embodiment, the cell-binding agent is an
anti-CD33 antibody or fragment thereof, such as the antibodies or
fragments thereof described in U.S. Pat. Nos. 7,557,189, 7,342,110,
8,119,787 and 8,337,855 and WO2004/043344, herein incorporated by
reference. In another embodiment, the anti-CD33 antibody is huMy9-6
antibody.
[0239] In one embodiment, the anti-CD33 antibody comprises an
immunoglobulin heavy chain region having the amino acid sequence
of
TABLE-US-00011 (SEQ ID NO: 23)
QVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGL
EWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSED
SAVYYCAREVRLRYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKS
TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH
TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,
and an immunoglobulin light chain region having the amino acid
sequence of
TABLE-US-00012 (SEQ ID NO: 24)
EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQI
PGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLA
IYYCHQYLSSRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
[0240] In yet another embodiment, the anti-CD33 antibody comprises
the heavy chain CDR1-CDR3 of SEQ ID NO: 23, and/or the light chain
CDR1-CDR3 of SEQ ID NO: 24, and preferably specifically binds
CD33.
[0241] In yet another embodiment, the anti-CD33 antibody comprises
a heavy chain variable region (HCVR) sequence at least about 90%,
95%, 97%, 99%, or 100% identical to SEQ ID NO: 23, and/or a light
chain variable region (LCVR) sequence at least about 90%, 95%, 97%,
99%, or 100% identical to SEQ ID NO: 24, and preferably
specifically binds CD33.
[0242] In another embodiment, the cell-binding agent is an
anti-CD37 antibody or an antibody fragment thereof, such as those
described in U.S. Pat. No. 8,765,917 and WO 2011/112978, herein
incorporated by reference. In one embodiment, the anti-CD37
antibody is huCD37-3 antibody.
[0243] In one embodiment, the anti-CD37 antibody comprises an
immunoglobulin light chain region having the amino acid sequence
of
TABLE-US-00013 (SEQ ID NO: 25)
DIQMTQSPSSLSVSVGERVTITCRASENIRSNLAWYQQKPGKSPK
LLVNVATNLADGVPSRFSGSGSGTDYSLKINSLQPEDFGTYYCQH
YWGTTWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
and an immunoglobulin heavy chain region having the amino acid
sequence of
TABLE-US-00014 (SEQ ID NO: 26)
QVQVQESGPGLVAPSQTLSITCTVSGFSLTTSGVSWVRQPPGKGL
EWLGVIWGDGSTNYHPSLKSRLSIKKDHSKSQVFLKLNSLTAADT
ATYYCAKGGYSLAHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,
or an immunoglobulin heavy chain region having the amino acid
sequence of
TABLE-US-00015 (SEQ ID NO: 27)
QVQVQESGPGLVAPSQTLSITCTVSGFSLTTSGVSWVRQPPGKGL
EWLGVIWGDGSTNYHSSLKSRLSIKKDHSKSQVFLKLNSLTAADT
ATYYCAKGGYSLAHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
[0244] In another embodiment, the anti-CD37 antibody comprises an
immunoglobulin light chain region having the amino acid sequence
set forth in SEQ ID NO:25 and an immunoglobulin heavy chain region
having the amino acid sequence set forth in SEQ ID NO:26.
[0245] In yet another embodiment, the anti-CD37 antibody comprises
an immunoglobulin light chain region having the amino acid sequence
set forth in SEQ ID NO:25 and an immunoglobulin heavy chain region
having the amino acid sequence set forth in SEQ ID NO:27.
[0246] In yet another embodiment, the anti-CD37 antibody comprises
the heavy chain CDR1-CDR3 of SEQ ID NO: 26 or 27, and/or the light
chain CDR1-CDR3 of SEQ ID NO: 25, and preferably specifically binds
CD37.
[0247] In yet another embodiment, the anti-CD37 antibody comprises
a heavy chain variable region (HCVR) sequence at least about 90%,
95%, 97%, 99%, or 100% identical to SEQ ID NO: 26 or 27, and/or a
light chain variable region (LCVR) sequence at least about 90%,
95%, 97%, 99%, or 100% identical to SEQ ID NO: 25, and preferably
specifically binds CD37.
[0248] In yet another embodiment, the anti-CD37 antibody comprises
an immunoglobulin light chain region having the amino acid sequence
of
TABLE-US-00016 (SEQ ID NO: 28)
EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPKR
WIYDTSNLPYGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQW
SDNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL
NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
and an immunoglobulin heavy chain region having the amino acid
sequence of
TABLE-US-00017 (SEQ ID NO: 29)
QVQLQESGPGLLKPSQSLSLTCTVSGYSITSGFAWHWIRQHPGNK
LEWMGYILYSGSTVYSPSLKSRISITRDTSKNHFFLQLNSVTAAD
TATYYCARGYYGYGAWFAYWGQGTLVTVSAASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.
[0249] In yet another embodiment, the anti-CD37 antibody comprises
the heavy chain CDR1-CDR3 of SEQ ID NO: 29, and/or the light chain
CDR1-CDR3 of SEQ ID NO: 28, and preferably specifically binds
CD37.
[0250] In yet another embodiment, the anti-CD37 antibody comprises
a heavy chain variable region (HCVR) sequence at least about 90%,
95%, 97%, 99%, or 100% identical to SEQ ID NO: 29, and/or a light
chain variable region (LCVR) sequence at least about 90%, 95%, 97%,
99%, or 100% identical to SEQ ID NO: 28, and preferrably
specifically binds CD37.
[0251] In yet another embodiment, the anti-CD37 antibody is
huCD37-50 antibody.
Cell-Binding Agent-Drug Conjugates
[0252] The present invention also provides cell-binding agent-drug
conjugates comprising a cell-binding agent linked to one or more
cytotoxic compounds of the present invention via a variety of
linkers, including, but not limited to, disulfide linkers,
thioether linkers, amide bonded linkers, peptidase-labile linkers,
acid-labile linkers, esterase-labile linkers.
[0253] Representative conjugates of the invention are
antibody/cytotoxic compound, antibody fragment/cytotoxic compound,
epidermal growth factor (EGF)/cytotoxic compound, melanocyte
stimulating hormone (MSH)/cytotoxic compound, thyroid stimulating
hormone (TSH)/cytotoxic compound, somatostatin/cytotoxic compound,
folate/cytotoxic compound, estrogen/cytotoxic compound, estrogen
analogue/cytotoxic compound, androgen/cytotoxic compound, and
androgen analogue/cytotoxic compound.
[0254] In a preferred embodiment, the present invention provides
conjugates comprising an indolinobenzodiazepine dimer compound
(e.g., compounds of formulas (I)-(VI) or pharmaceutically
acceptable salt thereof) and the cell-binding agent linked through
a covalent bond. The linker can be cleaved at the site of the
tumor/unwanted proliferating cells to deliver the cytotoxic agent
to its target in a number of ways. The linker can be cleaved, for
example, by low pH (hydrazone), reductive environment (disulfide),
proteolysis (amide/peptide link), or through an enzymatic reaction
(esterase/glycosidase).
[0255] Thus in a second embodiment, the invention provides a
conjugate comprising: a cytotoxic compound and a cell binding agent
(CBA), wherein the cytotoxic compound is covalently linked to the
CBA, and wherein the cytotoxic compound is represented by any one
of the following formulas (I'), (II'), (III'), (IV'), (V') or (VI')
or a pharmaceutically acceptable salt thereof described above.
[0256] In certain embodiments, the conjugate comprises a CBA and a
cytotoxic compound represented by the following formula:
##STR00035##
or a pharmaceutically acceptable salt thereof.
[0257] In a 1.sup.st specific embodiment, Z.sup.s1 is represented
by either one of the following formulas:
##STR00036##
and the remaining variables are as described above in the second
embodiment.
[0258] In a 2.sup.nd specific embodiment, R.sup.e is H or Me; the
remaining variables are as described above in the second embodiment
or the 1.sup.st specific embodiment.
[0259] In a 3.sup.rd specific embodiment, R.sup.x can be
--(CH.sub.2).sub.p--(CR.sup.fR.sup.g)--, wherein R.sup.f and
R.sup.g are each independently selected from H or a linear or
branched alkyl having 1 to 4 carbon atoms; p is 0, 1, 2 or 3; and
the remaining variables are as described above in the second
embodiment or the 1.sup.st or 2.sup.nd specific embodiment.
[0260] In one embodiment, R.sup.f and R.sup.g are the same or
different, and are selected from --H and -Me; and the remaining
variables are as described above in the 3.sup.rd specific
embodiment. More specifically, R.sup.f and R.sup.g are both -Me;
and p is 2.
[0261] In a 4.sup.th specific embodiment, R.sup.x is a linear or
branched alkylene having 1 to 4 carbon atoms substituted with a
charged substituent or an ionizable group Q; and the remaining
variables are as described above in the second embodiment or the
1.sup.st or 2.sup.nd specific embodiment.
[0262] In one embodiment, Q is i) --SO.sub.3H, --Z'--SO.sub.3H,
--OPO.sub.3H.sub.2, --Z'--OPO.sub.3H.sub.2, --PO.sub.3H.sub.2,
--Z'--PO.sub.3H.sub.2, --CO.sub.2H, --Z'--CO.sub.2H,
--NR.sub.11R.sub.12, or --Z'--NR.sub.11R.sub.12, or a
pharmaceutically acceptable salt thereof; or, ii)
--N.sup.+R.sub.14R.sub.15R.sub.16X.sup.- or
--Z'--N.sup.+R.sub.14R.sub.15R.sub.16X.sup.-; Z' is an optionally
substituted alkylene, an optionally substituted cycloalkylene or an
optionally substituted phenylene; R.sub.14 to R.sub.16 are each
independently an optionally substituted alkyl; and X.sup.- is a
pharmaceutically acceptable anion; and the remaining variables are
as described above in the 4.sup.th specific embodiment. More
specifically, Q is SO.sub.3H or a pharmaceutically acceptable salt
thereof.
[0263] In a 5.sup.th specific embodiment, the double line between N
and C represents a double bond; and the remaining variables are as
described above in the second embodiment or the 1.sup.st, 2.sup.nd,
3.sup.rd or 4.sup.th specific embodiment.
[0264] In a 6.sup.th specific embodiment, the double line between N
and C represents a single bond; X is --H or an amine protecting
group; Y is selected from --H, --SO.sub.3M, --OH, --OMe, --OEt or
--NHOH; and the remaining variables are as described above in the
second embodiment or the 1.sup.st, 2.sup.nd, 3.sup.rd or 4.sup.th
specific embodiment.
[0265] In one embodiment, Y is --H, --SO.sub.3M or --OH; and the
remaining variables are as described in the 6.sup.th specific
embodiment. More specifically, M is H.sup.+, Na.sup.+ or
K.sup.+.
[0266] In a 7.sup.th embodiment, X' is --H, --OH or -Me; and the
remaining variables are as described above in the second embodiment
or the 1.sup.st, 2.sup.nd, 3.sup.rd, 4.sup.th, 5.sup.th or 6.sup.th
specific embodiment. More specifically, X' is --H.
[0267] In a 8.sup.th specific embodiment, Y' is --H or oxo; and the
remaining variables are as described above in the second embodiment
or the 1.sup.st, 2.sup.nd, 3.sup.rd, 4.sup.th, 5.sup.th, 6.sup.th
or 7.sup.th specific embodiment. More specifically, Y' is --H.
[0268] In a 9.sup.th specific embodiment, for formulas (I'), (II'),
(III'), (IV'), (V'), and (VI'),
[0269] the double line between N and C represents a single bond or
a double bond, provided that when it is a double bond X is absent
and Y is --H, and when it is a single bond, X is --H; Y is --OH or
--SO.sub.3M;
[0270] M is --H or a pharmaceutically acceptable cation;
[0271] X' and Y' are both --H;
[0272] G is C; and the remaining variables are as described above
in the second embodiment or the 1.sup.st, 2.sup.nd, 3.sup.rd or
4.sup.th specific embodiment.
[0273] In one embodiment, Y is --SO.sub.3M and M is H.sup.+,
Na.sup.+ or K.sup.+; and the remaining variables are as described
above the 9.sup.th specific embodiment.
[0274] In a 10.sup.th specific embodiment, the conjugates of the
invention include the following:
##STR00037## ##STR00038## ##STR00039## ##STR00040## ##STR00041##
##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046##
##STR00047## ##STR00048## ##STR00049## ##STR00050## ##STR00051##
##STR00052## ##STR00053## ##STR00054## ##STR00055## ##STR00056##
##STR00057## ##STR00058## ##STR00059## ##STR00060## ##STR00061##
##STR00062## ##STR00063## ##STR00064## ##STR00065## ##STR00066##
##STR00067## ##STR00068## ##STR00069## ##STR00070## ##STR00071##
##STR00072## ##STR00073## ##STR00074## ##STR00075## ##STR00076##
##STR00077## ##STR00078## ##STR00079## ##STR00080##
or a pharmaceutically acceptable salt thereof, wherein M is H.sup.+
or a pharmaceutically acceptable cation; and r is an integer from 1
to 10. More specifically, M is H.sup.+, Na.sup.+ or K.sup.+.
[0275] In a 11.sup.th specific embodiment, the conjugate is
represented by any one of the following formulas:
##STR00081## ##STR00082##
or a pharmaceutically acceptable salt thereof, wherein M is H.sup.+
or a pharmaceutically acceptable cation; and r is an integer from 1
to 10. More specifically, M is H.sup.+, Na.sup.+ or K.sup.+.
[0276] In a 11.sup.th specific embodiment, the conjugate is
represented by any one of the following formulas:
##STR00083## ##STR00084##
or a pharmaceutically acceptable salt thereof, wherein M is
H.sup.+, Na.sup.+ or K.sup.+; and r is an integer from 1 to 10.
[0277] In certain embodiments, the conjugate of any one of the
described embodiments, such as those described in the second
embodiment or the 1.sup.st to 11.sup.th specific embodiment,
comprises 1-10 cytotoxic compounds, 2-9 cytotoxic compounds, 3-8
cytotoxic compounds, 4-7 cytotoxic compounds, or 5-6 cytotoxic
compounds, each cytotoxic compound comprising the linking group
linking the cytotoxic compound to the CBA, and each cytotoxic
compound on the conjugate is the same.
[0278] In any of the above-described embodiments regarding
conjugates of the invention, such as those described in the second
embodiment or the 1.sup.st to 11.sup.th specific embodiment, the
cell-binding agent can bind to target cells selected from tumor
cells, virus infected cells, microorganism infected cells, parasite
infected cells, autoimmune cells, activated cells, myeloid cells,
activated T-cells, B cells, or melanocytes; cells expressing the
CD4, CD6, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD44,
CD56, EpCAM, CanAg, CALLA, or Her-2 antigens; Her-3 antigens; or
cells expressing insulin growth factor receptor, epidermal growth
factor receptor, and folate receptor.
[0279] In any of the conjugates embodiments, such as those
described in the second embodiment or the 1.sup.st to 11.sup.th
specific embodiment, the cell-binding agent can be an antibody, a
single chain antibody, an antibody fragment that specifically binds
to the target cell, a monoclonal antibody, a single chain
monoclonal antibody, or a monoclonal antibody fragment that
specifically binds to a target cell, a chimeric antibody, a
chimeric antibody fragment that specifically binds to the target
cell, a domain antibody, a domain antibody fragment that
specifically binds to the target cell, a lymphokine, a hormone, a
vitamin, a growth factor, a colony stimulating factor, or a
nutrient-transport molecule.
[0280] The antibody can be a resurfaced antibody, a resurfaced
single chain antibody, or a resurfaced antibody fragment.
[0281] The antibody can be a monoclonal antibody, a single chain
monoclonal antibody, or a monoclonal antibody fragment thereof.
[0282] The antibody can be a humanized antibody, a humanized single
chain antibody, or a humanized antibody fragment.
[0283] In any of the conjugates embodiments, such as those
described in the second embodiment or the 1.sup.st to 11.sup.th
specific embodiment, the cell-binding agent can be anti-folate
receptor antibody or an antibody fragment thereof. More
specifically, the anti-folate receptor antibody is huMOV19
antibody.
[0284] In any of the conjugates embodiments, such as those
described in the second embodiment or the 1.sup.st to 11.sup.th
specific embodiment, the cell-binding agent can be anti-EGFR
antibody or an antibody fragment thereof. In one embodiment, the
anti-EGFR antibody is a non-antagonist antibody, including, for
example, the antibodies described in WO2012058592, herein
incorporated by reference. In another embodiment, the anti-EGFR
antibody is a non-functional antibody, for example, humanized ML66.
More specifically, the anti-EGFR antibody is huML66.
[0285] The invention further provides a pharmaceutical composition
comprising any of the conjugates described herein, and a
pharmaceutically acceptable carrier.
[0286] The invention further provides a drug-linker compound
comprising any of the subject compound covalently linked to a
bifunctional linker.
[0287] The invention additional provides a conjugate comprising any
of the subject compounds, or the subject drug-linker compounds,
linked to a cell-binding agent.
[0288] The invention further provides a method of inhibiting
abnormal cell growth or treating a proliferative disorder, an
autoimmune disorder, destructive bone disorder, infectious disease,
viral disease, fibrotic disease, neurodegenerative disorder,
pancreatitis or kidney disease in a mammal comprising administering
to the mammal a therapeutically effective amount of any of the
compounds (with or without any linker group) or conjugates of the
invention, and, optionally, a second chemotherapeutic agent.
[0289] In certain embodiments, the second chemotherapeutic agent is
administered to the mammal sequentially or consecutively.
[0290] In certain embodiments, the method is for treating a
condition selected from cancer, rheumatoid arthritis, multiple
sclerosis, graft versus host disease (GVHD), transplant rejection,
lupus, myositis, infection, and immune deficiency.
[0291] In certain embodiments, the method or conjugate is for
treating a cancer.
[0292] In certain embodiments, the cancer is a hematological cancer
or a solid tumor. More specifically, the cancer is ovarian cancer,
pancreatic cancer, melanoma, lung cancer (e.g., non-small cell lung
cancer (NSCLC)), cervical cancer, breast cancer, squamous cell
carcinoma of the head and neck, prostate cancer, endometrial
cancer, lymphoma (e.g., non-Hodgkin lymphoma), myelodysplastic
syndrome (MDS), peritoneal cancer, or leukemia (e.g., acute myeloid
leukemia (AML), acute monocytic leukemia, promyelocytic leukemia,
eosinophilic leukaemia, acute lymphoblastic leukemia (e.g., B-ALL),
chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia
(CML)).
Production of Cell-Binding Agent-Drug Conjugates
[0293] In order to link the cytotoxic compounds or derivative
thereof of the present invention to the cell-binding agent, the
cytotoxic compound can comprise a linking moiety with a reactive
group bonded thereto. In one embodiment, a bifunctional
crosslinking reagent can be first reacted with the cytotoxic
compound to provide the compound bearing a linking moiety with one
reactive group bonded thereto (i.e., drug-linker compound), which
can then react with a cell binding agent. Alternatively, one end of
the bifunctional crosslinking reagent can first react with the cell
binding agent to provide the cell binding agent bearing a linking
moiety with one reactive group bonded thereto, which can then react
with a cytotoxic compound. The linking moiety can contain a
chemical bond that allows for the release of the cytotoxic moiety
at a particular site. Suitable chemical bonds are well known in the
art and include disulfide bonds, thioether bonds, acid labile
bonds, photolabile bonds, peptidase labile bonds and esterase
labile bonds (see for example U.S. Pat. Nos. 5,208,020; 5,475,092;
6,441,163; 6,716,821; 6,913,748; 7,276,497; 7,276,499; 7,368,565;
7,388,026 and 7,414,073). Preferred are disulfide bonds, thioether
and peptidase labile bonds. Other linkers that can be used in the
present invention include non-cleavable linkers, such as those
described in are described in detail in U.S. publication number
2005/0169933, or charged linkers or hydrophilic linkers and are
described in US 2009/0274713, US 2010/01293140 and WO 2009/134976,
each of which is expressly incorporated herein by reference, each
of which is expressly incorporated herein by reference.
[0294] In one embodiment, a solution of a cell-binding agent (e.g.,
an antibody) in aqueous buffer may be incubated with a molar excess
of a bifunctional crosslinking agent, such as
N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP),
N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB),
N-succinimidyl-4-(2-pyridyldithio)2-sulfo butanoate (sulfo-SPDB) to
introduce dithiopyridyl groups. The modified cell-binding agent
(e.g., modified antibody) is then reacted with the thiol-containing
cytotoxic compound described herein, such as compound 1d or 2k, to
produce a disulfide-linked cell-binding agent-cytotoxic agent
conjugate of the present invention.
[0295] In another embodiment, the thiol-containing cytotoxic
compound described herein, such as compound 1d or 2k can react with
a bifunctional crosslinking agent such as
N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP),
N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB),
N-succinimidyl-4-(2-pyridyldithio)2-sulfo butanoate (sulfo-SPDB) to
form a cytotoxic agent-linker compound, which can then react with a
cell-biding agent to produce a disulfide-linked cell-binding
agent-cytotoxic agent conjugate of the present invention. The
cytotoxic agent-linker compound can be prepared in situ without
purification before reacting with the cell-binding agent. A
representative process is described in Example 3. Alternatively,
the cytotoxic agent-linker compound can be purified prior to
reacting with the cell-binding agent.
[0296] The cell binding agent-cytotoxic agent conjugate may be
purified using any purification methods known in the art, such as
those described in U.S. Pat. No. 7,811,572 and US Publication No.
2006/0182750, both of which are incorporated herein by reference.
For example, the cell-binding agent-cytotoxic agent conjugate can
be purified using tangential flow filtration, adsorptive
chromatography, adsorptive filtration, selective precipitation,
non-absorptive filtration or combination thereof. Preferably,
tangential flow filtration (TFF, also known as cross flow
filtration, ultrafiltration and diafiltration) and/or adsorptive
chromatography resins are used for the purification of the
conjugates.
[0297] Alternatively, the cell-binding agent (e.g., an antibody)
may be incubated with a molar excess of an antibody modifying agent
such as 2-iminothiolane, L-homocysteine thiolactone (or
derivatives), or N-succinimidyl-S-acetylthioacetate (SATA) to
introduce sulfhydryl groups. The modified antibody is then reacted
with the appropriate disulfide-containing cytotoxic agent, to
produce a disulfide-linked antibody-cytotoxic agent conjugate. The
antibody-cytotoxic agent conjugate may then be purified by methods
described above. The cell binding agent may also be engineered to
introduce thiol moieties, such as cysteine-engineered antibodies
disclosed in U.S. Pat. Nos. 7,772,485 and 7,855,275.
[0298] In another embodiment, a solution of a cell-binding agent
(e.g., an antibody) in aqueous buffer may be incubated with a molar
excess of an antibody-modifying agent such as
N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate to
introduce maleimido groups, or with
N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB) to introduce
iodoacetyl groups. The modified cell-binding agent (e.g., modified
antibody) is then reacted with the thiol-containing cytotoxic agent
to produce a thioether-linked cell-binding agent-cytotoxic agent
conjugate. The conjugate may then be purified by methods described
above.
[0299] The number of cytotoxic molecules bound per antibody
molecule can be determined spectrophotometrically by measuring the
ratio of the absorbance at 280 nm and 330 nm .DELTA.n average of
1-10 cytotoxic compounds/antibody molecule(s) can be linked by the
methods described herein. The preferred average number of linked
cytotoxic compounds per antibody molecule is 2-5, and the most
preferred is 2.5-4.0.
[0300] Representative processes for preparing the cell-binding
agent-drug conjugates of the present invention are described in
U.S. Pat. No. 8,765,740 and U.S. Application Publication No.
2012/0238731. The entire teachings of these references are
incorporated herein by reference.
Cytotoxicity of Compounds and Conjugates
[0301] The cytotoxic compounds and cell-binding agent-drug
conjugates of the invention can be evaluated for their ability to
suppress proliferation of various cancer cell lines in vitro. Cells
to be evaluated can be exposed to the compounds or conjugates for
1-5 days and the surviving fractions of cells measured in direct
assays by known methods. IC.sub.50 values can then be calculated
from the results of the assays. Alternatively or in addition, an in
vitro cell line sensitivity screen, such as the one described by
the U.S. National Cancer Institute (see Voskoglou-Nomikos et al.,
2003, Clinical Cancer Res. 9: 42227-4239, incorporated herein by
reference) can be used as one of the guides to determine the types
of cancers that are sensitive to treatment with the compounds or
conjugates of the invention.
[0302] In one example, in vivo efficacy of a cell binding
agent/cytotoxic agent conjugate was measured. SCID mice bearing
NCI-H2110 tumor cells were treated with huMov19-sulfo-SPDB-1d
conjugate and significant tumor regression was observed at multiple
doses while untreated mice grew tumors rapidly (FIG. 2). Activity
was observed at doses as low as 5 .mu.g/kg.
Compositions and Methods of Use
[0303] The present invention includes a composition (e.g., a
pharmaceutical composition) comprising novel benzodiazepine
compounds described herein (e.g., indolinobenzodiazepine or
oxazolidinobenzodiazepine), derivatives thereof, or conjugates
thereof, (and/or solvates, hydrates and/or salts thereof) and a
carrier (a pharmaceutically acceptable carrier). The present
invention also includes a composition (e.g., a pharmaceutical
composition) comprising novel benzodiazepine compounds described
herein, derivatives thereof, or conjugates thereof, (and/or
solvates, hydrates and/or salts thereof) and a carrier (a
pharmaceutically acceptable carrier), further comprising a second
therapeutic agent. The present compositions are useful for
inhibiting abnormal cell growth or treating a proliferative
disorder in a mammal (e.g., human) The present compositions are
also useful for treating depression, anxiety, stress, phobias,
panic, dysphoria, psychiatric disorders, pain, and inflammatory
diseases in a mammal (e.g., human).
[0304] The present invention includes a method of inhibiting
abnormal cell growth or treating a proliferative disorder in a
mammal (e.g., human) comprising administering to said mammal a
therapeutically effective amount of novel benzodiazepine compounds
described herein (e.g., indolinobenzodiazepine or
oxazolidinobenzodiazepine), derivatives thereof, or conjugates
thereof, (and/or solvates and salts thereof) or a composition
thereof, alone or in combination with a second therapeutic
agent.
[0305] The present invention also provides methods of treatment
comprising administering to a subject in need of treatment an
effective amount of any of the conjugates described above.
[0306] Similarly, the present invention provides a method for
inducing cell death in selected cell populations comprising
contacting target cells or tissue containing target cells with an
effective amount of a cytotoxic agent comprising any of the
cytotoxic compound-cell-binding agents (e.g.,
indolinobenzodiazepine or oxazolidinobenzodiazepine dimer linked to
a cell binding agent) of the present invention, a salt or solvate
thereof. The target cells are cells to which the cell-binding agent
can bind.
[0307] If desired, other active agents, such as other anti-tumor
agents, can be administered along with the conjugate.
[0308] Suitable pharmaceutically acceptable carriers, diluents, and
excipients are well known and can be determined by those of
ordinary skill in the art as the clinical situation warrants.
[0309] Examples of suitable carriers, diluents and/or excipients
include: (1) Dulbecco's phosphate buffered saline, pH about 7.4,
containing or not containing about 1 mg/mL to 25 mg/mL human serum
albumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v)
dextrose; and can also contain an antioxidant such as tryptamine
and a stabilizing agent such as Tween 20.
[0310] The method for inducing cell death in selected cell
populations can be practiced in vitro, in vivo, or ex vivo.
[0311] Examples of in vitro uses include treatments of autologous
bone marrow prior to their transplant into the same patient in
order to kill diseased or malignant cells: treatments of bone
marrow prior to their transplantation in order to kill competent
T-cells and prevent graft-versus-host-disease (GVHD); treatments of
cell cultures in order to kill all cells except for desired
variants that do not express the target antigen; or to kill
variants that express undesired antigen.
[0312] The conditions of non-clinical in vitro use are readily
determined by one of ordinary skill in the art.
[0313] Examples of clinical ex vivo use are to remove tumor cells
or lymphoid cells from bone marrow prior to autologous
transplantation in cancer treatment or in treatment of autoimmune
disease, or to remove T cells and other lymphoid cells from
autologous or allogenic bone marrow or tissue prior to transplant
in order to prevent GVHD. Treatment can be carried out as follows.
Bone marrow is harvested from the patient or other individual and
then incubated in medium containing serum to which is added the
cytotoxic agent of the invention, concentrations range from about
10 .mu.M to 1 pM, for about 30 minutes to about 48 hours at about
37.degree. C. The exact conditions of concentration and time of
incubation, i.e., the dose, are readily determined by one of
ordinary skill in the art. After incubation the bone marrow cells
are washed with medium containing serum and returned to the patient
intravenously according to known methods. In circumstances where
the patient receives other treatment such as a course of ablative
chemotherapy or total-body irradiation between the time of harvest
of the marrow and reinfusion of the treated cells, the treated
marrow cells are stored frozen in liquid nitrogen using standard
medical equipment.
[0314] For clinical in vivo use, the cytotoxic agent of the
invention will be supplied as a solution or a lyophilized powder
that are tested for sterility and for endotoxin levels. Examples of
suitable protocols of conjugate administration are as follows.
Conjugates are given weekly for 4 weeks as an intravenous bolus
each week. Bolus doses are given in 50 to 1000 mL of normal saline
to which 5 to 10 mL of human serum albumin can be added. Dosages
will be 10 .mu.g to 2000 mg per administration, intravenously
(range of 100 ng to 20 mg/kg per day). After four weeks of
treatment, the patient can continue to receive treatment on a
weekly basis. Specific clinical protocols with regard to route of
administration, excipients, diluents, dosages, times, etc., can be
determined by one of ordinary skill in the art as the clinical
situation warrants.
[0315] Examples of medical conditions that can be treated according
to the in vivo or ex vivo methods of inducing cell death in
selected cell populations include malignancy of any type including,
for example, cancer; autoimmune diseases, such as systemic lupus,
rheumatoid arthritis, and multiple sclerosis; graft rejections,
such as renal transplant rejection, liver transplant rejection,
lung transplant rejection, cardiac transplant rejection, and bone
marrow transplant rejection; graft versus host disease; viral
infections, such as CMV infection, HIV infection, AIDS, etc.; and
parasite infections, such as giardiasis, amoebiasis,
schistosomiasis, and others as determined by one of ordinary skill
in the art.
[0316] Cancer therapies and their dosages, routes of administration
and recommended usage are known in the art and have been described
in such literature as the Physicians Desk Reference (PDR). The PDR
discloses dosages of the agents that have been used in treatment of
various cancers. The dosing regimen and dosages of these
aforementioned chemotherapeutic drugs that are therapeutically
effective will depend on the particular cancer being treated, the
extent of the disease and other factors familiar to the physician
of skill in the art and can be determined by the physician. The
contents of the PDR are expressly incorporated herein in its
entirety by reference. One of skill in the art can review the PDR,
using one or more of the following parameters, to determine dosing
regimen and dosages of the chemotherapeutic agents and conjugates
that can be used in accordance with the teachings of this
invention. These parameters include:
Comprehensive index
By Manufacturer
[0317] Products (by company's or trademarked drug name) Category
index Generic/chemical index (non-trademark common drug names)
Color images of medications Product information, consistent with
FDA labeling Chemical information
Function/action
Indications & Contraindications
[0318] Trial research, side effects, warnings
Analogues and Derivatives
[0319] One skilled in the art of cytotoxic agents will readily
understand that each of the cytotoxic agents described herein can
be modified in such a manner that the resulting compound still
retains the specificity and/or activity of the starting compound.
The skilled artisan will also understand that many of these
compounds can be used in place of the cytotoxic agents described
herein. Thus, the cytotoxic agents of the present invention include
analogues and derivatives of the compounds described herein.
[0320] All references cited herein and in the examples that follow
are expressly incorporated by reference in their entireties.
EXAMPLES
[0321] The invention will now be illustrated by reference to
non-limiting examples. Unless otherwise stated, all percents,
ratios, parts, etc. are by weight. All reagents were purchased from
the Aldrich Chemical Co., New Jersey, or other commercial sources.
Nuclear Magnetic Resonance (.sup.1H NMR) spectra were acquired on a
Bruker 400 MHz instrument. Mass spectra were acquired on a Bruker
Daltonics Esquire 3000 instrument and LCMS were acquired on an
Agilent 1260 Infinity LC with an Agilent 6120 single quadrupole MS
using electrospray ionization.
Example 1
##STR00085##
[0323] Compound 1a:
[0324] To a stirred solution of (5-amino-1,3-phenylene)dimethanol
(1.01 g, 6.59 mmol) in anhydrous dimethylformamide (16.48 mL) and
anhydrous tetrahydrofuran (16.48 ml) was added
4-methyl-4-(methyldisulfanyl)pentanoic acid (1.281 g, 6.59 mmol),
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (2.53
g, 13.19 mmol), and 4-dimethylaminopyridine (0.081 g, 0.659 mmol).
The resulting mixture was stirred for 18 hours at room temperature.
The reaction was quenched with saturated ammonium chloride solution
and extracted with ethyl acetate (3.times.50 mL). The organic
extracts were washed with water and brine, then dried over
anhydrous sodium sulfate. The solution was filtered and
concentrated in vacuo and the resulting residue was purified by
silica gel chromatography (Ethyl acetate/Hexanes) to obtain
compound 1a as a white solid (0.70 g, 32% yield). .sup.1H NMR (400
MHz, DMSO-d6: .delta. 9.90 (s, 1H), 7.43 (s, 2H), 6.93 (s, 1H),
5.16 (t, 2H, J=5.7 Hz), 4.44 (d, 4H, J=5.7 Hz), 2.43 (s, 3H),
2.41-2.38 (m, 2H), 1.92-1.88 (m, 2H), 1.29 (s, 6H). MS (m/z). found
330.0 (M+1).sup.+.
##STR00086##
[0325] Compound 1b:
[0326] To a cooled (-10.degree. C.) solution of compound 1a (219
mg, 0.665 mmol) in anhydrous dichloromethane (6.65 mL) was added
triethylamine (463 .mu.l, 3.32 mmol) followed by dropwise addition
of methanesulfonic anhydride (298 mg, 1.662 mmol). The mixture
stirred at -10.degree. C. for 2 hours, then the mixture was
quenched with ice water and extracted with cold ethyl acetate
(2.times.30 mL). The organic extracts were washed with ice water,
dried with anhydrous sodium sulfate, filtered and concentrated
under reduced pressure to obtain the crude dimesylate.
[0327] The crude dimesylate (227 mg, 0.467 mmol) and IGN monomer A
(303 mg, 1.028 mmol) were dissolved in anhydrous DMF (3.11 mL).
Potassium carbonate (161 mg, 1.169 mmol) was added and the mixture
stirred for 18 hours at room temperature. Deionized water was added
and the resulting precipitate was filtered and rinsed with water.
The solid was re-dissolved in dichloromethane and washed with
water. The organic layer was dried with anhydrous magnesium
sulfate, filtered, and concentrated. The crude residue was purified
by silica gel chromatography (Methanol/Dichloromethane) to give
compound 1b (227 mg, 36% yield). MS (m/z). found 882.5
(M+1).sup.+.
##STR00087##
[0328] Compound 1c:
[0329] To a suspension of compound 1b (227 mg, 0.167 mmol) in
anhydrous 1,2-dichloroethane (3.346 mL) was added sodium
triacetoxyborohydride (37.3 mg, 0.167 mmol). The mixture was
stirred at room temp for one hour upon which it was quenched with
saturated ammonium chloride solution. The mixture was extracted
with dichloromethane and washed with brine. The organic layer was
dried with anhydrous magnesium sulfate, filtered and concentrated.
The crude residue was purified by RP-HPLC (C18,
Water/Acetonitrile). Fractions containing desired product were
extracted with dichloromethane, dried with anhydrous magnesium
sulfate, filtered and concentrated to give compound 1c (35 mg, 19%
yield). MS (m/z). found 884.3 (M+1).sup.+.
##STR00088##
[0330] Compound 1d:
[0331] To a solution of compound 1c (18 mg, 0.017 mmol) in
acetonitrile (921 .mu.L) and methanol (658 .mu.L) was added
tris(2-carboxyethyl)phosphine hydrochloride (17.51 mg, 0.060 mmol)
(neutralized with saturated sodium bicarbonate solution (0.2 mL) in
sodium phosphate buffer (132 .mu.L, 0.75 M, pH 6.5). The mixture
was stirred at room temperature for 3.5 hours, then diluted with
dichloromethane and deionized water. The organic layer was
separated, washed with brine, dried with anhydrous sodium sulfate,
filtered and concentrated under reduced pressure to obtain the
crude thiol. MS (m/z). found 838.3 (M+1).sup.+.
[0332] The crude thiol from step 5 (15.5 mg, 0.018 mmol) was
dissolved in 2-propanol (1.23 mL). Deionized water (617 .mu.L) and
sodium bisulfite (5.77 mg, 0.055 mmol) were added and the mixture
stirred for five hours at room temperature. The reaction was frozen
in an acetone/dry ice bath, lyophilized, and purified by RP-HPLC
(C18, deionized water/acetonitrile). Fractions containing desired
product were frozen and lyophilized to give compound
(12S,12aS)-9-((3-(4-mercapto-4-methylpentanamido)-5-((((R)-8-met-
hoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-
-9-yl)oxy)methyl)benzyl)oxy)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-be-
nzo [5,6][1,4]diazepino[1,2-a]indole-12-sulfonic acid (compound 1d)
(6.6 mg, 39% yield). MS (m/z). found 918.2 (M-1).sup.-.
Example 2
##STR00089##
[0334] Compound 2b:
[0335] Cs.sub.2CO.sub.3 (8.54 g, 26.2 mmol) was added to a stirred
solution of aniline 1a (10.0 g, 26.2 mmol) in DMF (52.4 mL).
Methyliodide (1.47 mL, 23.58 mmol) was added and the reaction was
stirred at rt for 3 h. Water (10 mL) and EtOAc (30 mL) were added
to the reaction mixture. The layers were separated and was
extracted with EtOAc (2.times.). The organic layers were washed
with water (4.times.), dried over Na.sub.2SO.sub.4, filtered and
concentrated. The crude residue was purified by silica gel flash
chromatography (EtOAc/hexanes, gradient, 0% to 10%) to obtain
compound 2b (3.8 g, 37% yield). .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 6.629 (s, 1H), 6.515 (s, 2H), 4.673 (s, 4H), 2.838 (s, 3H),
0.942 (s, 18H), 0.102 (s, 12H).
##STR00090##
[0336] Compound 2d:
[0337] N-methyl aniline (compound 2b) (500 mg, 1.26 mmol) and
compound 2c (258 mg, 1.33 mmol) were dissolved in CH.sub.2Cl.sub.2
(6.32 ml). EDC (484 mg, 2.53 mmol) and DMAP (77.0 mg, 0.632 mmol)
were added and the reaction mixture was stirred overnight at room
temperature. The reaction was diluted with dichloromethane and was
washed with saturated NH.sub.4Cl and brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated. The crude residue was
purified by silica gel flash chromatography (EtOAc/hexanes,
gradient, 0% to 30% to 100%) to obtain compound 2d a colorless oil
(705 mg, 98% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
7.236 (s, 1H), 7.016 (s, 2H), 4.744 (s, 4H), 3.242 (s, 3H), 2.336
(s, 3H), 2.190-2.153 (m, 2H), 1.924-1.884 (m, 2H), 1.137 (s, 6H),
0.940 (s, 18H), 0.106 (s, 12H).
##STR00091##
[0338] Compound 2e:
[0339] Compound 2d (700 mg, 1.22 mmol) was dissolved in THF (6.12
mL). 5 M aqueous HCl (4.89 mL, 24.47 mmol) was added at rt and was
stirred for a total of 3.5 h. The reaction mixture was diluted with
EtOAc and washed with sat'd NaHCO.sub.3 and brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated. CH.sub.3CN (15 mL) was
added to the residue and was concentrated to dryness. This was
repeated 3.times. to obtain compound 2e as a colorless oil (450 mg,
100% yield). LCMS (8 min method)=0.757 min. Mass observed=344.25
(M+H). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.340 (s, 1H),
7.119 (s, 2H), 4.736 (s, 4H), 3.252 (s, 3H), 2.348 (s, 3H),
2.172-2.152 (m, 2H), 1.930-1.890 (m, 2H), 1.165 (s, 6H).
##STR00092##
[0340] Compound 2f:
[0341] Compound 2e (370 mg, 1.08 mmol) was dissolved in
dichloromethane (7.18 mL). The solution was cooled to -5.degree. C.
and triethylamine (0.375 mL, 2.69 mmol) was then added, followed by
slow dropwise addition of methanesulfonyl chloride (0.193 mL, 2.48
mmol) under an atmosphere of argon. The reaction mixture was
stirred at -5.degree. C. for 2.5 h. The reaction was quenched with
ice/water and was diluted with EtOAc (15 mL). The layers were
separated and the organic layer was washed with cold water
(2.times.), dried over Na.sub.2SO.sub.4, filtered and concentrated
to give the crude compound 2f as a colorless oil (530 mg, 98%
yield) and was taken onto the next step without purification. Mass
observed=522.68 (M+Na).
##STR00093##
[0342] Compound 2g:
[0343] Dimesylate 2f (530 mg, 1.06 mmol) and IGN monomer A (723 mg,
2.33 mmol) were dissolved in anhydrous dimethylformamide (10.61
mL). Potassium carbonate (586 mg, 4.24 mmol) was added and the
reaction was stirred overnight at room temperature. Water (20 mL)
was added to precipitate out the product. The slurry was stirred
for 5 min, filtered and dried under vacuum/N.sub.2 for 1 h. The
crude residue was purified by silica gel flash chromatography
(EtOAc/hexanes, 50% to 100%, then switched to 5%
MeOH/CH.sub.2Cl.sub.2) to obtain 2g as a brownish solid (715 mg,
56% yield, 75% purity). LCMS (8 min method)=5.891 min. Mass
observed=896.50 (M+H).
##STR00094##
[0344] Compound 2h:
[0345] Compound 2g (440 mg, 0.368 mmol) was dissolved in
1,2-dichloroethane (3.68 mL). Sodium triacetoxyborohydride (78 mg,
0.368 mmol) was added and the reaction was stirred at rt under an
atmosphere of argon for 1 h. The reaction mixture was diluted with
dichloromethane and was washed with sat'd NH.sub.4Cl, brine, dried
over Na.sub.2SO.sub.4, filtered and concentrated. The crude reside
was purified by RPHPLC (C18 column, CH.sub.3CN/H.sub.2O, gradient,
55% to 75%) to yield mono imine 2h as a white fluffy solid (125 mg,
34% yield). LCMS (15 min method)=8.847 min. Mass observed=898.6
(M+H).
##STR00095##
[0346] Compound 2j:
[0347] TCEP.HCl (108 mg, 0.376 mmol) was neutralized with water
(.about.100 .mu.L) and sat'd aq. NaHCO.sub.3 (.about.925 .mu.L).
0.1 M NaH.sub.2PO.sub.4 buffer pH=6.5 (193 .mu.L) was added to the
TCEP solution. In a separate flask, compound 2h (125 mg, 0.125
mmol) was dissolved in acetonitrile (1.35 mL) and tetrahydrofuran
(900 .mu.L). The TCEP/buffer mixture (pH=6.5-7) was added to the
solution of compound 2h in acetonitrile, followed by the addition
of methanol (964 .mu.L). An additional tetrahydrofuran (200 .mu.L)
was added to get a clear homogeneous solution. The reaction mixture
was stirred at rt for 3 h. The reaction was diluted with
dichloromethane and water. The layers were separated and the
organic layer was washed with brine, dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to give crude compound
2j, which was used in the next step without purification (118 mg,
100% yield). LCMS (8 min method)=5.880 min. Mass observed=852.30
(M+H).
##STR00096##
[0348] Compound 2k:
[0349] The crude compound 2j (118 mg, 0.125 mmol) was suspended in
2-propanol (5.54 mL) and water (2.77 mL) and was sonicated for a
few minutes. NaHSO.sub.3 (130 mg, 1.25 mmol) was added and the
reaction was stirred overnight at room temperature. The clear
solution was diluted with CH.sub.3CN/H.sub.2O (1:1, 15 mL) and was
frozen and lyophilized. The resulting fluffy white powder was
dissolved in CH.sub.3CN/H.sub.2O (1:1) and was purified by RPHPLC
(C18 column, CH.sub.3CN/H.sub.2O, gradient, 25% to 45%) to obtain
(12S,12aS)-9-((3-(4-mercapto-N,4-dimethylpentanamido)-5-((((S)-8-methoxy--
6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl-
)oxy)methyl)benzyl)oxy)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5-
,6][1,4]diazepino[1,2-a]indole-12-sulfonic acid (compound 2k) as a
white powder (65 mg, 56% yield, 98% purity). LCMS (15 min
method)=4.841 min. Mass observed=852.6 (ESI.sup.+, M-SO.sub.3H+H),
932.4 (ESP, M-H).
Example 3
Preparation of huMOV19-Sulfo-SPDB-1d
[0350] A reaction containing 2.0 mg/mL huMOV19 antibody and 6 molar
equivalents of sulfo-SPDB-1d in situ mixture by linker in 50 mM
HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH 8.5
buffer and 15% v/v DMA (N,N-Dimethylacetamide) cosolvent was
allowed to conjugate for 6 hours at 25.degree. C. The in situ
mixture was prepared by reacting 1.5 mM sulfo-SPDB linker with 1.95
mM of compound 1d in 100% DMA for 4 hours in the presence of 10 mM
N,N-Diisopropylethyl amine (DIPEA). Free thiol was then capped by
adding a 3-fold excess of maleimido-propionic acid.
[0351] Post-reaction, the conjugate was purified and buffer
exchanged into 100 mM Arginine, 20 mM Histidine, 2% sucrose, 0.01%
Tween-20, 50 .mu.M sodium bisulfite formulation buffer pH 6.1 using
NAP desalting columns (Illustra Sephadex G-25 DNA Grade, GE
Healthcare). Dialysis was performed in the same buffer for 20 hours
at 4.degree. C. utilizing Slide-a-Lyzer dialysis cassettes
(ThermoScientific 20,000 MWCO).
[0352] The purified conjugate was found to have an average of 2.5
molecules of compound 1d linked per antibody (by UV-Vis using molar
extinction coefficients .epsilon..sub.330 nm=15,280
cm.sup.-1M.sup.-1 and .epsilon..sub.280 nm.sup.=30, 115
cm.sup.-1M.sup.-1 for compound 1d, and .epsilon..sub.280
nm.sup.=201,400 cm.sup.-1M.sup.-1 for huMOV19 antibody), 95%
monomer (by size exclusion chromatography), <0.1% unconjugated
compound 1d (by acetone precipitation, reverse-phase HPLC analysis)
and a final protein concentration of 1.8 mg/ml. The conjugated
antibody was found to be >80% intact by gel chip analysis.
Example 4
Antitumor Activity of Single-Dose huMOV19-Sulfo-SPDB-1d Against
NCI-H2110 NSCLC Xenografts in Female SCID Mice
[0353] Female CB.17 SCID mice, 6 weeks old, were received from
Charles River Laboratories. Mice were inoculated with
1.times.10.sup.7 NCI-H2110 tumor cells suspended in 0.1 ml 50%
matrigel/serum free medium by subcutaneous injection in the right
flank. When tumor volumes reached approximately 100 mm.sup.3 (day 7
post inoculation), animals were randomized based on tumor volume
into 3 groups of 6 mice each. Mice received a single IV
administration of vehicle control (0.2 ml/mouse) or
huMOV19-sulfo-SPDB-1d at 5 and 25 .mu.g/kg based on concentration
of compound 1d on day 1 (day 8 post inoculation).
[0354] Tumor size was measured twice to three times weekly in three
dimensions using a caliper. The tumor volume was expressed in
mm.sup.3 using the formula
V=Length.times.Width.times.Height.times.1/2. A mouse was considered
to have a partial regression (PR) when tumor volume was reduced by
50% or greater, complete tumor regression (CR) when no palpable
tumor could be detected. Tumor volume was determined by StudyLog
software. Tumor growth inhibition (T/C Value) was determined using
the following formula:
T/C (%)=Median tumor volume of the treated/Median tumor volume of
the control.times.100.
[0355] Tumor volume was determined simultaneously for treated (T)
and the vehicle control (C) groups when tumor volume of the vehicle
control reached predetermined size of 1000 mm.sup.3. The daily
median tumor volume of each treated group was determined, including
tumor-free mice (0 mm.sup.3) According to NCI standards, a
T/C.ltoreq.42% is the minimum level of anti-tumor activity. A
T/C<10% is considered a high anti-tumor activity level.
[0356] As shown in FIG. 1, the conjugate is highly active at both 5
and 25 .mu.g/kg dose.
Example 5
Preparation of huML66-Sulfo-SPDB-1d Conjugate
[0357] Sulfo-SPDB-1d was formed in situ by incubating 3.0 mM
sulfo-SPDB, 3.9 mM compound 1d, and 20 mM DIPEA
(N,N-diisopropylethylamine) in DMA (N, N-dimethylacetamide) for 5
hours at 25.degree. C. A reaction containing 2.0 mg/mL huML66
antibody, an anti-EGFR antibody (see WO 2012/058592), and 5.8 molar
equivalents of sulfo-SPDB-1d in 15 mM HEPES
(4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH 8.5 buffer
and 15% v/v DMA cosolvent was incubated overnight at 25.degree.
C.
[0358] Post-reaction, the conjugate was purified into 10 mM
histidine, 250 mM glycine, 1% sucrose, 0.01% Tween-20, 50 .mu.M
sodium bisulfite pH 6.2 formulation buffer using NAP desalting
columns (Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis
was performed in the same buffer for 4 hours at room temperature
and then overnight at 4.degree. C. using Slide-a-Lyzer dialysis
cassettes (ThermoScientific 30,000 MWCO).
[0359] The purified conjugate was found to have a final protein
concentration of 2.9 mg/ml and an average of 3.0 molecules of
compound 1d linked per antibody (by UV-Vis using molar extinction
coefficients .epsilon..sub.330 nm=15,484 cm.sup.-1M.sup.-1 and
.epsilon..sub.280 nm=30, 115 cm.sup.-1M.sup.-1 for compound 1d, and
.epsilon..sub.280 nm=205,520 cm.sup.-1M.sup.-1 for huML66
antibody); 92.8% monomer (by size exclusion chromatography); and
0.8% unconjugated compound 1d (by acetone precipitation,
reverse-phase HPLC analysis). The MS spectrometry data is shown in
FIG. 3.
Example 6
In Vitro Cytotoxic Assays for Conjugates
[0360] The ability of huML66-sulfo-SPDB-1d conjugate to inhibit
cell growth was measured using in vitro cytotoxicity assays. Target
cells were plated at 1-2,000 cells per well in 100 .mu.L in
complete RPMI media (RPMI-1640, 10% fetal bovine serum, 2 mM
glutamine, 1% penicillin-streptomycin, all reagents from
Invitrogen). Antibodies were diluted into complete RPMI media using
3-fold dilution series and 100 .mu.L were added per well. The final
concentration typically ranged from 3.times.10.sup.-8M to
4.6.times.10.sup.-12M. Cells were incubated at 37.degree. C. in a
humidified 5% CO.sub.2 incubator for 5-6 days. Viability of
remaining cells was determined by colorimetric WST-8 assay (Dojindo
Molecular Technologies, Inc., Rockville, Md., US). WST-8 is reduced
by dehydrogenases in living cells to an orange formazan product
that is soluble in tissue culture medium. The amount of formazan
produced is directly proportional to the number of living cells.
WST-8 was added to 10% of the final volume and plates were
incubated at 37.degree. C. in a humidified 5% CO2 incubator for an
additional 2-4 hours. Plates were analyzed by measuring the
absorbance at 450 nm (A450) in a multiwell plate reader. Background
A450 absorbance of wells with media and WST-8 only was subtracted
from all values. The percent viability was calculated by dividing
each treated sample value by the average value of wells with
untreated cells. Percent viability=100*(A450 treated sample-A450
background)/(A450 untreated sample-A450 background). The percent
viability value was plotted against the antibody concentration in a
semi-log plot for each treatment. Dose-response curves were
generated by non-linear regression and the EC.sub.50 value of each
curve was calculated using GraphPad Prism (GraphPad software, San
Diego, Calif.). In vitro cytotoxic activity.
[0361] The in vitro cytotoxicity of huML66-sulfo-SPDB-1d conjugate
was evaluated in the presence and absence of excess unconjugated
antibody and compared to the activity of a non-specific
IgG-sulfo-SPDB-1d conjugate in EGFR-expressing cells and the
results from a typical cytotoxicity assay are shown in FIG. 4. The
huML66-sulfo-SPDB-1d conjugate resulted in specific cell killing of
Detroit-562 SCC-HN cells with an EC.sub.50 value of 110 pM. The
presence of excess unconjugated antibody significantly reduced
activity and resulting in an EC.sub.50 value of approximately 1
nM.
[0362] Likewise, the huML66-sulfo-SPDB-1d conjugate resulted in
specific cell killing of NCI-H292 NSCLC cells with an EC.sub.50
value of 20 pM. The presence of excess unconjugated antibody
significantly reduced activity and resulting in an EC.sub.50 value
of approximately 0.7 nM. Additionally, the huML66-sulfo-SPDB-1d
conjugate resulted in specific cell killing of NCI-H1703 NSCLC
cells with an EC.sub.50 value of 70 pM. The presence of excess
unconjugated antibody significantly reduced activity and resulting
in an EC50 value of approximately 1 nM.
TABLE-US-00018 TABLE 1 Detroit562 NCI-H292 NCI-H1703 Conjugate EC50
in pM EC50 in pM EC50 in pM huML66-sulfo-SPDB-1d 110 20 70
huML66-sulfo-SPDB-1d + 960 690 1,310 block
Example 7
Cytotoxicity Assay of huMOV19-Sulfo-SPDB-1d Conjugate
[0363] 100 .mu.l/well of huMOV19-sulfo-SPDB-1d conjugate was each
diluted in RPMI-1640 (Life Technologies) supplemented with
heat-inactivated 10% FBS (Life Technologies) and 0.1 mg/ml
gentamycin (Life Technologies) in a 96-well plate (Corning, flat
bottom) at starting concentrations of 3.5e-9 M and to 3.5 e-8 M in
triplicate and serially diluted 3-fold in media above at ambient
temperature. KB cells (buccal epithelial tumor), grown in EMEM
(ATCC) supplemented with heat-inactivated 10% FBS (Life
Technologies) and 0.1 mg/ml gentamycin (Life Technologies), were
washed once in PBS and removed with 0.05% trypsin-EDTA (Life
Technogies). Other cells tested were NCI-H2110 (NSCLC) and T47D
(breat epthelial) grown in RPMI-1640 (LifeTechnologies)
supplemented with heat-inactivated 10% FBS (Life Technologies) and
0.1 mg/ml gentamycin (Life Technologies). T47D media also was
supplemented with 0.2 IU/ml bovine insulin. All cells were
resuspended in growth media (see above) to neutralize trypsin and
counted using a hemacytometer. 100 .mu.l/ml of 1000 KB cells/well
or 2000 T47D and NCI-H2110 cells/well were added to wells
containing ADC or media only and incubated in a 37.degree. C.
incubator with 5% CO.sub.2 for 5 days with and without 1 .mu.M
blocking anti-FOLR1 antibody (M9346A). Total volume is 200
.mu.l/well. The starting concentration of each conjugate on KB
cells was 3.5e-9 M and for T47D and NCI-H2110 cells, the starting
concentration of each conjugate was 3.5e-8 M. After incubation,
cell viability was analyzed by addition of 20 .mu.l/well WST-8
(Dojindo) and allowed to develop for 2 hr. Absorbance was read on a
plate reader at 450 and 620 nm .DELTA.bsorbances at 620 nm were
subtracted from absorbances at 450 nm Background in wells
containing media only was further subtracted from corrected
absorbances and surviving fraction (SF) of untreated cells was
calculated in Excel. An XY graph of ADC concentration (M) vs. SF
was created using Graph Pad Prism.
[0364] As shown in FIGS. 5-7 and Table 2, the conjugate is highly
potent against KB cells, NCI-H2110 cells and T47D cells.
TABLE-US-00019 TABLE 2 huMOV19- sulfo- KB NCI-H2110 T47D SPDB-1d
-Block +Block -Block +Block -Block +Block IC.sub.50 8e-12M 6e-10M
3e-10M 2e-9M 1e-10M 9e-9M
[0365] In another experiment, the ability of the conjugate to
inhibit cell growth was measured using a WST-8-based in vitro
cytotoxicity assay. Cells in 96-well plates (typically,
1.times.10.sup.3 per well) were treated with the conjugate at
various concentrations in an appropriate cell culture medium with a
total volume of 0.2 ml. Control wells containing cells and the
medium but lacking test compounds, and wells containing medium
only, were included in each assay plate. The plates were incubated
for 4 to 6 days at 37.degree. C. in a humidified atmosphere
containing 6% CO.sub.2. WST-8 reagent (10%, volume/volume; Dojindo
Molecular Technologies) was then added to the wells, and the plates
were incubated at 37.degree. C. for 2 to 6 hours depending on a
cell line. Then, the absorbance was measured on a plate reader
spectrophotometer in the dual-wavelength mode 450 nm/620 nm, and
the absorbance at the 620 nm (nonspecific light scattering by
cells) was subtracted. The resulting OD.sub.450 values were
utilized to calculate apparent surviving fractions of cells using
GraphPad Prism v4 (GraphPad software, San Diego, Calif.). The
apparent surviving fraction of the cells in each well was
calculated by first correcting for the medium background absorbance
and then dividing each value by the average of the values in the
control wells (non-treated cells). Dose response curves were
generated by non-linear regression using a sigmoidal curve fit with
variable slope in Graph Pad Prism. IC.sub.50 (inhibitory
concentration 50%) was generated by the software.
[0366] The conjugate is active against the tested cell lines
Ishikawa (endometrial cancer), KB (cervical cancer) and NCI-H2110
(non-small cell lung carcinoma) and T47D (breast cancer) as shown
in FIG. 14. The cell-killing activity was FOLR1-dependent, since an
excess of unmodified huMOV19 antibody (1 .mu.M) markedly decreased
potency of the conjugate (from 10 to 100-fold), Table 3, FIG.
14.
TABLE-US-00020 TABLE 3 IC50, nM huMOV19-sulfo-SPDB-1d + Cell line
huMOV19-sulfo-SPDB-1d unmodified huMOV19 Ishikawa 0.04 0.4 KB 0.01
1.0 NCI-H2110 0.1 1.0 T47D 0.1 7.0
Example 8
Bystander Killing Activity
[0367] 100 .mu.l/well of huMOV19-sulfo-SPDB-1d conjugate were each
diluted in RPMI-1640 (Life Technologies) supplemented with
heat-inactivated 10% FBS (Life Technologies), 0.1 mg/ml gentamycin
(Life Technologies) and .beta.ME (Life Technologies) in a 96-well
plate (Falcon, round bottom) at concentrations of 1 e-10 M and 4
e-10 M in sextuplicate. Both 300.19 cells (mouse) expressing
recombinant FOLR1(FR1#14) or no expression vector (parental) were
counted on a hemacytometer. 50 .mu.l/ml of 1000 FR1#14 cells/well
were added to wells containing the conjugate or media only, 50
.mu.l/ml of 2000 parental cells/well were added to wells containing
the conjugate or media only and both FR1#14 and parental cells were
added together to wells containing ADC or media only. All plates
were incubated in a 37.degree. C. incubator with 5% CO.sub.2 for 4
days. Total volume was 150 .mu.l/well. After incubation, cell
viability was analyzed by addition of 75 .mu.l/well Cell Titer Glo
(Promega) and allowed to develop for 45 min Luminescence was read
on a luminometer and background in wells containing media only was
subtracted from all values. A bar graph of the average of each cell
treatment was graphed using Graph Pad Prism.
[0368] As shown in FIG. 8, huMov19-sulfo-SPDB-1d exhibits strong
bystander killing activity.
Example 9
Flow Cytometry Assay for Binding Affinity of huMOV19-sSPBD-1d
Conjugate
[0369] 100 .mu.l/well of the conjugate huMOV19-sulfo-SPDB-1d or the
antibody huMOV19 were diluted in FACS buffer (1% BSA, 1.times.PBS)
in a 96-well plate (Falcon, round bottom) at a starting
concentration of 3.times.10-8 M in duplicate and serially diluted
3-fold in FACS buffer at 4.degree. C. T47D cells (human breast
tumor) grown in RPMI-1640 (Life Technologies) supplemented with
heat-inactivated 10% FBS (Life Technologies), 0.1 mg/ml gentamycin
(Life Technologies) and 0.2 IU bovine insulin/ml (Sigma) were
washed once in PBS and removed with versene (Life Technogies). T47D
cells were resuspended in growth media (see above) to neutralize
versene and counted on a Coulter counter. Cells were then washed
twice in cold FACS buffer, centrifuging in between washes at 1200
rpm for 5 min. 100 .mu.l/ml of 2.times.10.sup.4 cells/well were
added to wells containing the conjugate, antibody or FACS buffer
only and incubated at 4.degree. C. for 2 hr. After incubation,
cells were centrifuged as before and washed once in 200 .mu.l/well
cold FACS buffer. Cells were then stained with 200 .mu.l/well
FITC-conjugated Goat Anti-Human-IgG-Fc.gamma. secondary antibody
(controls included were unstained cells and those stained with
secondary antibody only) for 40 min at 4.degree. C., centrifuged
and washed once in 200 .mu.l/well cold PBS. Cells were fixed in 200
.mu.l/well 1% formaldehyde/PBS and stored at 4.degree. C. After
storage, cellular surface staining of conjugate or antibody was
detected using flow cytometry on a FACS Calibur (BD Biosciences).
The geometric means were plotted against the log concentration of
the conjugate or antibody using GraphPad Prism and the EC.sub.50
was calculated via non-linear 4-parameter logistic regression
analysis.
[0370] As shown in FIG. 9A, the conjugate binds similarly to the
surface of T47D cells expressing the target antigen as the
unconjugated antibody in flow cytometry, thereby demonstrating that
binding is not affected by the conjugation process. The binding
assay was repeated and similar results are observed (see FIG.
9B).
Example 10
Antitumor Activity of Single-Dose huMOV19-Sulfo-SPDB-1d Against
NCI-H2110 NSCLC Xenografts in Female SCID Mice
[0371] Female CB.17 SCID mice, 6 weeks old, were received from
Charles River Laboratories. Mice were inoculated with 1.times.107
NCI-H2110 tumor cells suspended in 0.1 ml 50% matrigel/serum free
medium by subcutaneous injection in the right flank. When tumor
volumes reached approximately 100 mm3 (day 7 post inoculation),
animals were randomized based on tumor volume into 4 groups of 6
mice each. Mice received a single IV administration of vehicle
control (0.2 ml/mouse) or huMOV19-sulfo-SPDB-1d at 1, 3 or 5
.mu.g/kg based on concentration of compound 1d on day 1 (day 8 post
inoculation).
[0372] Tumor size was measured twice to three times weekly in three
dimensions using a caliper. The tumor volume was expressed in mm3
using the formula V=Length.times.Width.times.Height.times.1/2. A
mouse was considered to have a partial regression (PR) when tumor
volume was reduced by 50% or greater, complete tumor regression
(CR) when no palpable tumor could be detected. Tumor volume was
determined by StudyLog software.
[0373] Tumor growth inhibition (T/C Value) was determined using the
following formula:
T/C (%)=Median tumor volume of the treated/Median tumor volume of
the control.times.100.
[0374] Tumor volume was determined simultaneously for treated (T)
and the vehicle control (C) groups when tumor volume of the vehicle
control reached predetermined size of 1000 mm3 The daily median
tumor volume of each treated group was determined, including
tumor-free mice (0 mm3) According to NCI standards, a
T/C.ltoreq.42% is the minimum level of anti-tumor activity. A
T/C<10% is considered a high anti-tumor activity level.
[0375] As shown in FIG. 10, the conjugate is highly active at 5
.mu.g/kg dose and active at 3 .mu.g/kg dose.
Example 11
Antitumor Activity of Single-Dose huML66-Sulfo-SPDB-1d Against
NCI-H1703 NSCLC Xenografts in Female SCID Mice
[0376] Female CB.17 SCID mice, 6 weeks old, were received from
Charles River Laboratories. Mice were inoculated with
5.times.10.sup.6 NCI-H1703 tumor cells suspended in 0.2 ml 50%
matrigel/serum free medium by subcutaneous injection in the right
flank. When tumor volumes reached approximately 100 mm.sup.3 (day
16 post inoculation), animals were randomized based on tumor volume
into 4 groups of 6 mice each. Mice received a single IV
administration of vehicle control (0.1 ml/mouse) or
huML66-sulfo-SPDB-1d at 5, 20, or 50 .mu.g/kg based on compound 1d
concentration on day 1 (day 17 post inoculation).
[0377] Tumor size was measured twice to three times weekly in three
dimensions using a caliper. The tumor volume was expressed in
mm.sup.3 using the formula
V=Length.times.Width.times.Height.times.1/2. A mouse was considered
to have a partial regression (PR) when tumor volume was reduced by
50% or greater, complete tumor regression (CR) when no palpable
tumor could be detected. Tumor volume was determined by StudyLog
software. Tumor growth inhibition (T/C Value) was determined using
the following formula:
T/C (%)=Median tumor volume of the treated/Median tumor volume of
the control.times.100.
Tumor volume was determined simultaneously for treated (T) and the
vehicle control (C) groups when tumor volume of the vehicle control
reached predetermined size of 1000 mm.sup.3. The daily median tumor
volume of each treated group was determined, including tumor-free
mice (0 mm.sup.3) According to NCI standards, a T/C.ltoreq.42% is
the minimum level of anti-tumor activity. A T/C<10% is
considered a high anti-tumor activity level.
[0378] As shown in FIG. 11, the huML66-sulfo-SPDB-1d conjugate is
highly active at 20 .mu.g/kg and 50 .mu.g/kg doses, with 20
.mu.g/kg as the minimal effective dose (MED).
Example 12
Pharmacokinetics of Single-Dose huMov19-Sulfo-SPDB-1d in Female
CD-1 Mice
[0379] Female CD-1 mice, 7 weeks old, were received from Charles
River Laboratories. Mice received a single IV administration of
huMov19-sulfo-SPDB-1d conjugate as a single intravenous bolus
injection via a lateral tail vein. Each mouse received a dose of
2.5 mg/kg based on Ab. The dose and injected volume were
individualized on the basis of the body weight of each mouse.
Injections were carried out using a 1.0 mL syringe Fitted with a 27
gauge, 1/2 inch needle. At 2 and 30 min, and at 2, 4 and 8 hours,
and at 1, 2, 3, 5, 7, 10, 14, 21 and 28 days after administration
of huMov19-sulfo-SPDB-1d conjugate, mice were anesthetized by
isoflurane inhalation, and approximately 150 .mu.L of blood was
collected from mice via the right retro-orbital blood sinus into a
heparinized capillary tube. At each time point (from 0 to 21 days),
blood was collected from all three mice in one group. Groups were
bled in turn; so that the mice in the set were not bled more than
two times in a 24-hour period. At the final time point, 28 days
post-administration, all mice were included for sample collection.
Blood samples were centrifuged to separate the plasma. 30 .mu.l
plasma was transferred to individual labeled microcentrifuge tubes
for each sample and time point, and then stored frozen at
-80.degree. C. to allow subsequent analysis by ELISA to determine
concentrations of total Ab (both unconjugated Ab and intact ADC)
and intact conjugate.
[0380] As shown in FIG. 12, the huMov19-sulfo-SPDB-1d conjugate
shows slow time-dependent release of benzodiazepine compound.
Example 13
Catabolite Enrichment by Affinity Capture with Protein A Resin
[0381] KB cells expressing folate receptor a (FR.alpha.) were
cultured in 5.times.T150 tissue culture plates. Saturating amount
of FR.alpha.-targeting huMov19-sulfo-SPDB-1d conjugate was
incubated with KB cells for 24 hours at 37.degree. C. in a
humidified incubator buffered with 5% CO2. After 24 hours, the
media containing cell-effluxed catabolites were harvested and
pooled for the following assay.
[0382] Saturating amount of anti-indolinobenzodiazepine antibody
was bound to a slurry of protein A resins by overnight incubation
at 4.degree. C. 1 mL of pre-bound protein
A/anti-indolinobenzodiazepine antibody complex was incubated with
25 mL of media on an end-to-end rotator for several hours. The
resins were centrifuged gently at 1,000 rpm, and the supernatant
was decanted. The protein-A/anti-indolinobenzodiazepine antibody
resins bound to the catabolites were washed with PBS. The
catabolites were released into organic phase by acetone extraction.
The catabolites were vacuum-dried overnight until the organic
solution was completely evaporated. The catabolites were
reconstituted with 20% acetonitrile in water, and analyzed by
LC/MS.
MS Analysis
[0383] Cell catabolites were identified by UHPLC/MS/MS using
Q-Exactive high resolution mass spec (Thermo). Extracted
ion-chromatograms (XIC) were used to identify and characterize the
target cell catabolites. All catabolite species containing the
characteristic indolinobenzodiazepine (286 m/z) mass signatures
were identified (see FIG. 13).
Example 14
Antitumor Activity of Single-Dose huMov19-Sulfo-SPDB-1d Against
NCI-H2110 NSCLC Xenografts, Hec-1b Endometrial Xenografts and
Ishikawa Endometrial Xenografts in Female CB.17 SCID Mice
[0384] Female CB.17 SCID mice, 6 weeks old, were received from
Charles River Laboratories. One cohort of mice were inoculated with
1.times.10.sup.7 NCI-H2110 tumor cells suspended in 0.1 ml 50%
matrigel/serum free medium by subcutaneous injection in the right
flank. The second cohort of mice were inoculated with
1.times.10.sup.7 Hec-1b tumor cells suspended in 0.1 ml serum free
medium by subcutaneous injection in the right flank. The third
cohort of mice were inoculated with 1.times.10.sup.7 Ishikawa tumor
cells suspended in 0.1 ml 50% matrigel/serum free medium by
subcutaneous injection in the right flank. When tumor volumes
reached approximately 100 mm.sup.3 (NCI-H2110 on day 7, Hec-1b on
day 7, and Ishikawa on day 17 post inoculation), animals were
randomized based on tumor volume into groups of 6 mice each.
[0385] Mice in the NCI-H2110 xenograft experiment received a single
IV administration of vehicle control (0.2 ml/mouse) or
huMov19-sulfo-SPDB-1d at 1, 3, or 5 .mu.g/kg based on drug
concentration on day 1 (day 8 post inoculation). Mice in the Hec-1b
xenograft experiment received a single IV administration of vehicle
control (0.2 ml/mouse) or huMov19-sulfo-SPDB-1d at 10 or 30
.mu.g/kg or the non-targeting control conjugate chKTI-sulfo-SPDB-1d
at 30 .mu.g/kg based on drug concentration on day 1 (day 8 post
inoculation). Mice in the Ishikawa xenograft experiment received a
single IV administration of vehicle control (0.2 ml/mouse) or
huMov19-sulfo-SPDB-1d at 10 or 30 .mu.g/kg or the non-targeting
control conjugate chKTI-sulfo-SPDB-1d at 30 .mu.g/kg based on drug
concentration on day 1 (day 18 post inoculation).
[0386] For all experiments, tumor size was measured twice to three
times weekly in three dimensions using a caliper. The tumor volume
was expressed in mm.sup.3 using the formula
V=Length.times.Width.times.Height.times.1/2. A mouse was considered
to have a partial regression (PR) when tumor volume was reduced by
50% or greater, complete tumor regression (CR) when no palpable
tumor could be detected. Tumor volume was determined by StudyLog
software.
[0387] Tumor growth inhibition (T/C Value) was determined using the
following formula:
T/C (%)=Median tumor volume of the treated/Median tumor volume of
the control.times.100.
[0388] Tumor volume was determined simultaneously for treated (T)
and the vehicle control (C) groups when tumor volume of the vehicle
control reached predetermined size of 1000 mm.sup.3. The daily
median tumor volume of each treated group was determined, including
tumor-free mice (0 mm.sup.3) According to NCI standards, a
T/C.ltoreq.42% is the minimum level of anti-tumor activity. A
T/C<10% is considered a high anti-tumor activity level.
[0389] As shown in FIG. 15, the huMov19-sulfo-SPDB-1d conjugate was
inactive in the NCI-H2110 xenograft model at a dose of 1 .mu.g/kg,
active at a dose of 3 .mu.g/kg with a T/C of 12% and highly active
at a dose of 5 .mu.g/kg with a T/C of 4%, 6/6 PRs and 3/6 CRs.
[0390] As shown in FIG. 16, the huMov19-sulfo-SPDB-1d conjugate was
active in Hec-1b xenograft model at both 10 .mu.g/kg and 30
.mu.g/kg doses. As shown in FIG. 2, the huMov19-sulfo-SPDB-1d
conjugate was active in the Hec-1b xenograft model at a dose of 10
.mu.g/kg with a T/C of 22% and active at a dose of 30 .mu.g/kg with
a T/C of 13%, 1/6 PRs and 1/6 CRs. The non-targeting control
conjugate chKTI-sulfo-SPDB-1d was inactive at a dose of 30
.mu.g/kg.
[0391] As shown in FIG. 17, the huMov19-sulfo-SPDB-1d conjugate was
active in the Ishikawa xenograft model at a dose of 10 .mu.g/kg
with a T/C of 23%, 6/6 PRs and 6/6 CRs and active at a dose of 30
.mu.g/kg with a T/C of 11%, 6/6 PRs and 6/6 CRs. The non-targeting
control conjugate chKTI-sulfo-SPDB-1d was active at a dose of 30
.mu.g/kg with a T/C of 22% and 3/6 PRs.
Example 15
Binding Affinity of CD123-Sulfo-SPDB-1d Conjugate
[0392] Binding affinity of the ADC conjugate of an exemplary
humanized anti-CD123 antibody, huCD123-6Gv4.7S3 antibody, was
assayed and compared to the corresponding unconjugated antibody by
flow cytometry using HNT-34 cells. HNT-34 cells (5.times.10.sup.4
cells per sample) were incubated with varying concentrations of the
ADC and the unconjugated huCD123-6Gv4.7S3 antibody in 200 .mu.L
FACS buffer (DMEM medium supplemented with 2% normal goat serum).
The cells were then pelleted, washed twice, and incubated for 1 hr
with 100 .mu.L of phycoerythrin (PE)-conjugated goat anti-human
IgG-antibody (Jackson Laboratory). The cells were pelleted again,
washed with FACS buffer and resuspended in 200 .mu.L of PBS
containing 1% formaldehyde. Samples were acquired using a
FACSCalibur flow cytometer with the HTS multiwell sampler, or a
FACS array flow cytometer, and analyzed using CellQuest Pro (all
from BD Biosciences, San Diego, US). For each sample the geomean
fluorescence intensity for FL2 was calculated and plotted against
the antibody concentration in a semi-log plot. A dose-response
curve was generated by non-linear regression and the EC50 value of
each curve, which corresponds to the apparent dissociation constant
(Kd) of each antibody, was calculated using GraphPad Prism v4
(GraphPad software, San Diego, Calif.).
[0393] As shown in FIG. 18, conjugation only moderately affected
the binding affinity of the exemplary anti-CD123 antibody.
Example 16
In Vitro Cytotoxic Activity for huCD123-Sulfo-SPDB-1d Conjugate
[0394] The ability of antibody-drug conjugates (ADC) of huCD123-6,
an anti-CD123 antibody, to kill cells that express CD123 on their
cell surface was measured using in vitro cytotoxicity assays. The
cell lines were cultured in culture medium as recommended by the
cell supplier (ATCC or DSMZ). The cells, 2,000 to 10,000 in 100
.mu.L of the culture medium, were added to each well of flat bottom
96-well plates. To block Fc receptors on the cell surface, the
culture medium was supplemented with 100 nM chKTI antibody (an
antibody of the same isotype). Conjugates were diluted into the
culture medium using 3-fold dilution series and 100 .mu.L were
added per well. To determine the contribution of CD123-independent
cytotoxicity, CD123 block (e.g., 100 nM of chCD123-6 antibody) was
added to some wells prior to the conjugates. Control wells
containing cells and the medium but lacking the conjugates, as well
as wells contained medium only, were included in each assay plate.
Assays were performed in triplicate for each data point. The plates
were incubated at 37.degree. C. in a humidified 6% CO2 incubator
for 4 to 7 days. Then the relative number of viable cells in each
well was determined using the WST-8 based Cell Counting Kit-8
(Dojindo Molecular Technologies, Inc., Rockville, Md.). The
apparent surviving fraction of cells in each well was calculated by
first correcting for the medium background absorbance, and then
dividing each value by the average of the values in the control
wells (non-treated cells). The surviving fraction of cells was
plotted against conjugate concentration in semi-log plots.
[0395] Fifteen CD123-positive cell lines of different origin (AML,
B-ALL, CML and NHL) were used in the study (Table 4). The majority
of the cell lines were derived from patients carrying a malignancy
with at least one negative prognostic factor (e.g., overexpression
of P-glycoprotein, overexpression of EVIL p53 alterations, DNMT3A
mutation, FLT3 internal tandem duplication). The conjugates
demonstrated high potency on these cell lines with IC.sub.50 values
ranging from sub-pM to low nM (Table 4).
TABLE-US-00021 TABLE 4 In vitro cytotoxicity of huCD123-6-90
conjugate against CD123-positive cell lines of different origin
Cell Line Origin Negative Prognostic Factor IC.sub.50 (M) THP1 AML
p53 deletion 5.8E-11 SHI-1 AML p53 gene alterations 3.2E-11 KO52
AML p53 mutant, Pgp overexpression 4.1E-10 KASUMI-3 AML EVI1 and
Pgp overexpression 1.4E-10 KG-1 AML p53 mutant, Pgp overexpression
4.1E-09 OCI-AML2 AML DNMT3A mutation 2.1E-10 HNT-34 AML MECOM
(EVI1) overexpression 5.9E-12 MV4-11 AML FLT3 internal tadem
duplication 1.3E-12 MOLM-13 AML FLT3 internal tadem duplication
1.2E-12 EOL-1 AML 4.7E-12 MOLM-1 CML EVI1 and Pgp overexpression
2.1E-10 KOPN8 B-ALL 3.0E-11 JM-1 B-ALL 4.1E-10 KCL-22 CML
2.9E-10
[0396] All publications, patents, patent applications, internet
sites, and accession numbers/database sequences (including both
polynucleotide and polypeptide sequences) cited herein are hereby
incorporated by reference in their entirety for all purposes to the
same extent as if each individual publication, patent, patent
application, internet site, or accession number/database sequence
were specifically and individually indicated to be so incorporated
by reference.
Sequence CWU 1
1
2915PRTArtificialFOLR1 antibody heavy chain CDR1 1Gly Tyr Phe Met
Asn 1 5 217PRTArtificialFOLR1 antibody heavy chain CDR2 2Arg Ile
His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Xaa Phe Xaa 1 5 10 15
Xaa 39PRTArtificialFOLR1 antibody heavy chain CDR3 3Tyr Asp Gly Ser
Arg Ala Met Asp Tyr 1 5 415PRTArtificialFOLR1 antibody light chain
CDR1 4Lys Ala Ser Gln Ser Val Ser Phe Ala Gly Thr Ser Leu Met His 1
5 10 15 57PRTArtificialFOLR1 antibody light chain CDR2 5Arg Ala Ser
Asn Leu Glu Ala 1 5 69PRTArtificialFOLR1 antibody light chain CDR3
6Gln Gln Ser Arg Glu Tyr Pro Tyr Thr 1 5 717PRTArtificialFOLR1
antibody heavy chain CDR2 7Arg Ile His Pro Tyr Asp Gly Asp Thr Phe
Tyr Asn Gln Lys Phe Gln 1 5 10 15 Gly 8448PRTArtificialFOLR1
antibody heavy chain 8Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Phe Met Asn Trp Val Lys Gln
Ser Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Arg Ile His Pro
Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys
Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His 65 70 75 80 Met
Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys 85 90
95 Thr Arg Tyr Asp Gly Ser Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110 Thr 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 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215
220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 9
218PRTArtificialFOLR1 antibody light chain 9Asp Ile Val Leu Thr Gln
Ser Pro Leu Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Pro Ala Ile
Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala 20 25 30 Gly Thr
Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro 35 40 45
Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp 50
55 60 Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Asn Ile
Ser 65 70 75 80 Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
Gln Ser Arg 85 90 95 Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys
Leu 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
10218PRTArtificialFOLR1 antibody light chain 10Asp Ile Val Leu Thr
Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Pro Ala
Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala 20 25 30 Gly
Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro 35 40
45 Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp
50 55 60 Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Thr
Ile Ser 65 70 75 80 Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Ser Arg 85 90 95 Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr
Lys Leu 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
11118PRTArtificialFOLR1 antibody heavy chain variable domain 11Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
20 25 30 Phe Met Asn Trp Val Lys Gln Ser Pro Gly Gln Ser Leu Glu
Trp Ile 35 40 45 Gly Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr
Asn Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Val Asp Lys
Ser Ser Asn Thr Ala His 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser
Glu Asp Phe Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Asp Gly Ser
Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val
Ser Ser 115 12112PRTArtificialFOLR1 antibody light chain variable
domain 12Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Ala Val Ser
Leu Gly 1 5 10 15 Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser
Val Ser Phe Ala 20 25 30 Gly Thr Ser Leu Met His Trp Tyr His Gln
Lys Pro Gly Gln Gln Pro 35 40 45 Arg Leu Leu Ile Tyr Arg Ala Ser
Asn Leu Glu Ala Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly
Ser Lys Thr Asp Phe Thr Leu Asn Ile Ser 65 70 75 80 Pro Val Glu Ala
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg 85 90 95 Glu Tyr
Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110
13112PRTArtificialFOLR1 antibody light chain variable domain 13Asp
Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly 1 5 10
15 Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala
20 25 30 Gly Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln
Gln Pro 35 40 45 Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala
Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp
Phe Thr Leu Thr Ile Ser 65 70 75 80 Pro Val Glu Ala Glu Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln Ser Arg 85 90 95 Glu Tyr Pro Tyr Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110
14445PRTArtificialhuML66HC Full-Length Heavy Chain 14Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Ala Ser Asn 20 25
30 Ser Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met
35 40 45 Gly Val Ile Trp Asn His Gly Gly Thr Asp Tyr Asn Pro Ser
Ile Lys 50 55 60 Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Ser
Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Thr Ala Ala Asp Thr
Ala Met Tyr Phe Cys Val 85 90 95 Arg Lys Gly Gly Ile Tyr Phe Asp
Tyr Trp Gly Gln Gly Val Leu Val 100 105 110 Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155
160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr 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 Asp Glu Leu 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 15213PRTArtificialhuML66LC Full-Length Light Chain
15Asp Thr Val Leu Thr Gln Ser Pro Ser Leu Ala Val Ser Pro Gly Glu 1
5 10 15 Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Thr Leu
Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro Lys Leu
Leu Ile Tyr 35 40 45 Leu Ala Ser His Arg Glu Ser Gly Val Pro Ala
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Asp Pro Met Glu Ala Glu 65 70 75 80 Asp Thr Ala Thr Tyr Tyr Cys
Gln Gln Ser Arg Asn Asp Pro Trp Thr 85 90 95 Phe Gly Gln Gly Thr
Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135
140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210
16448PRTArtificialanti-EGFR antibody immunoglobulin heavy chain
16Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Glu Cys Ile 35 40 45 Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Thr
Tyr Thr Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Ala Asp
Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Arg
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Asp Ala
Pro Gly Tyr Ala Met 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 Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu 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 Tyr 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 17214PRTArtificialanti-EGFR antibody immunoglobulin
light chain 17Asp 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 Arg Ala Ser Gln
Asp Ile Asn Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln His Lys Pro Gly
Lys Gly Pro Lys Leu Leu Ile 35 40 45 His Tyr Thr Ser Thr Leu His
Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg
Asp Tyr Ser Phe Ser Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Ile
Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Tyr 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105
110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg
Gly Glu Cys 210 18214PRTArtificialanti-EGFR antibody immunoglobulin
light chain 18Asp 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 Ala Ser Gln
Asp Ile Asn Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln His Lys Pro Gly
Lys Gly Pro Lys Leu Leu Ile 35 40 45 His Tyr Thr Ser Thr Leu His
Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg
Asp Tyr Ser Phe Ser Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Ile
Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Tyr 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105
110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg
Gly Glu Cys 210 19450PRTArtificialanti-CD19 antibody heavy chain
19Gln Val Gln Leu Val Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser
Asn 20 25 30 Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn
Tyr Asn Gln Asn Phe 50 55 60 Gln Gly Lys Ala Lys Leu Thr Val Asp
Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Val Ser Ser Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Asn
Pro Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135
140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260
265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385
390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450
20211PRTArtificialanti-CD19 antibody light chain 20Glu Ile Val Leu
Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Arg
Val Thr Met Thr Cys Ser Ala Ser Ser Gly Val Asn Tyr Met 20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Arg Arg Trp Ile Tyr 35
40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Met
Glu Pro Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Gly
Ser Tyr Thr Phe Gly 85 90 95 Gly Gly Thr Lys Leu Glu Ile Lys Arg
Thr Val Ala Ala Pro Ser Val 100 105 110 Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly Thr Ala Ser 115 120 125 Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 130 135 140 Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 145 150 155 160
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 165
170 175 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys
Glu 180 185 190 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
Phe Asn Arg 195 200 205 Gly Glu Cys 210 21447PRTArtificialanti-Muc1
antibody heavy chain 21Gln Ala Gln Leu Val Gln Ser Gly Ala Glu Val
Val 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 Asn Met His Trp Val Lys Gln
Thr Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro
Gly Asn Gly Ala Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys
Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met
Gln Ile Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90
95 Ala Arg Gly Asp Ser Val Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215
220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340
345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445
22213PRTArtificialanti-Muc1 antibody light chain 22Glu Ile Val Leu
Thr Gln Ser Pro Ala Thr Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Arg
Val Thr Ile Thr Cys Ser Ala His Ser Ser Val Ser Phe Met 20 25 30
His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr 35
40 45 Ser Thr Ser Ser Leu Ala Ser Gly Val Pro Ala Arg Phe Gly Gly
Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met
Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser
Ser Phe Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu
Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165
170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210
23447PRTArtificialanti-CD33 antibody immunoglobulin heavy chain
23Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val 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 Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu
Glu Trp Val 35 40 45 Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser
Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Ala Asp
Lys Ser Ser Thr 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 Arg Glu Val Arg
Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr 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 Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala 275 280
285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405
410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly 435 440 445 24219PRTArtificialanti-CD33 antibody
immunoglobulin light chain 24Glu Ile Val Leu Thr Gln Ser Pro Gly
Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Met Ser Cys
Lys Ser Ser Gln Ser Val Phe Phe Ser 20 25 30 Ser Ser Gln Lys Asn
Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln 35 40 45 Ser Pro Arg
Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70
75 80 Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His
Gln 85 90 95 Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195
200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
25214PRTArtificialanti-CD37 antibody immunoglobulin light chain
25Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Val Gly 1
5 10 15 Glu Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Arg Ser
Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys
Leu Leu Val 35 40 45 Asn Val Ala Thr Asn Leu Ala Asp Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu
Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr
Cys Gln His Tyr Trp Gly Thr Thr Trp 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
26444PRTartificialanti-CD37 antibody immunoglobulin heavy chain
26Gln Val Gln Val Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1
5 10 15 Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr
Ser 20 25 30 Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Asp Gly Ser Thr Asn Tyr
His Pro Ser Leu Lys 50 55 60 Ser Arg Leu Ser Ile Lys Lys Asp His
Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Leu Asn Ser Leu Thr Ala
Ala Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Lys Gly Gly Tyr Ser
Leu Ala His Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser
Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260
265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385
390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly 435 440 27444PRTartificialanti-CD37 antibody immunoglobulin
heavy chain 27Gln Val Gln Val Gln Glu Ser Gly Pro Gly Leu Val Ala
Pro Ser Gln 1 5 10 15 Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe
Ser Leu Thr Thr Ser 20 25 30 Gly Val Ser Trp Val Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Asp Gly
Ser Thr Asn Tyr His Ser Ser Leu Lys 50 55 60 Ser Arg Leu Ser Ile
Lys Lys Asp His Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Leu Asn
Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Lys
Gly Gly Tyr Ser Leu Ala His Trp Gly Gln Gly Thr Leu Val Thr 100 105
110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230
235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355
360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 28213PRTartificialanti-CD37
antibody immunoglobulin light chain 28Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Met
Thr Cys Ser Ala Thr Ser Ser Val Thr Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp
Thr Ser Asn Leu Pro Tyr Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Asp Asn Pro
Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr
Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185
190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205 Asn Arg Gly Glu Cys 210 29449PRTartificialanti-CD37
antibody immunoglobulin heavy chain 29Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu Leu Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr
Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Phe Ala Trp
His Trp Ile Arg Gln His Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met
Gly Tyr Ile Leu Tyr Ser Gly Ser Thr Val Tyr Ser Pro Ser Leu 50 55
60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn His Phe Phe
65 70 75 80 Leu Gln Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Gly Tyr Tyr Gly Tyr Gly Ala Trp Phe Ala
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala Ala Ser
Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310
315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
440 445 Gly
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