U.S. patent application number 11/735376 was filed with the patent office on 2008-01-31 for anti-cd70 antibody-drug conjugates and their use for the treatment of cancer and immune disorders.
This patent application is currently assigned to Seattle Genetics, Inc.. Invention is credited to Jonathan G. Drachman, Che-Leung Law, Julie McEarchern.
Application Number | 20080025989 11/735376 |
Document ID | / |
Family ID | 46328663 |
Filed Date | 2008-01-31 |
United States Patent
Application |
20080025989 |
Kind Code |
A1 |
Law; Che-Leung ; et
al. |
January 31, 2008 |
ANTI-CD70 ANTIBODY-DRUG CONJUGATES AND THEIR USE FOR THE TREATMENT
OF CANCER AND IMMUNE DISORDERS
Abstract
Disclosed are anti-CD70 antibodies and derivatives thereof
conjugated to cytotoxic, immunosuppressive, or other therapeutic
agents, as well as pharmaceutical compositions and kits comprising
the antibody- and antibody derivative-drug conjugates. Also
disclosed are methods, for the treatment of CD70-expressing cancers
and immunological disorders, comprising administering to a subject
the disclosed pharmaceutical compositions.
Inventors: |
Law; Che-Leung; (Shoreline,
WA) ; McEarchern; Julie; (Mill Creek, WA) ;
Drachman; Jonathan G.; (Seattle, WA) |
Correspondence
Address: |
SEATTLE GENETICS, INC.
21823 30TH DRIVE SE
BOTHELL
WA
98021
US
|
Assignee: |
Seattle Genetics, Inc.
Bothell
WA
98021
|
Family ID: |
46328663 |
Appl. No.: |
11/735376 |
Filed: |
April 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10546304 |
Aug 19, 2005 |
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PCT/US04/05247 |
Feb 20, 2004 |
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11735376 |
Apr 13, 2007 |
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60449055 |
Feb 20, 2003 |
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60792127 |
Apr 13, 2006 |
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Current U.S.
Class: |
424/155.1 ;
424/178.1 |
Current CPC
Class: |
A61K 49/0041 20130101;
C07K 2317/565 20130101; C07K 16/2875 20130101; A61K 2039/505
20130101; C07K 2317/56 20130101; C07K 2317/73 20130101; A61K
49/0008 20130101; A61K 47/6849 20170801; A61K 47/6803 20170801;
A61K 47/65 20170801; A61P 35/00 20180101; A61K 49/0058
20130101 |
Class at
Publication: |
424/155.1 ;
424/178.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A method for the treatment of a CD70-expressing cancer in a
subject, the method comprising: administering to the subject, in an
amount effective for the treatment, an antibody-drug conjugate
comprising an antibody that binds to the CD70 and wherein the drug
is a cytotoxic agent; wherein the cancer is multiple myeloma,
Hodgkin's disease, a diffuse large B-cell lymphoma, a follicular
lymphoma, a mantle cell lymphoma, an Epstein Barr Virus positive B
cell lymphomas, a glioblastoma, a neuroblastoma, an astrocytoma, a
meningioma or Waldenstrom macroglobulinemia; wherein the cytotoxic
or cytostatic agent is not a peptide toxin.
2. The method of claim 1, wherein the antibody competes for binding
to CD70 with monoclonal antibody 1F6 or 2F2.
3. The method of claim 2, wherein the antibody comprises at least
one polypeptide region selected from the group consisting of (a) an
H1 region having at least 80% sequence identity to the amino acid
sequence set forth in SEQ ID NO:6; (b) an H2 region having at least
80% sequence identity to the amino acid sequence set forth in SEQ
ID NO:8; (c) an H3 region having at least 80% sequence identity to
the amino acid sequence set forth in SEQ ID NO:10; (d) an L1 region
having at least 80% sequence identity to the amino acid sequence
set forth in SEQ ID NO:16; (e) an L2 region having at least 80%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:18; (f) an L3 region having at least 80% sequence identity to
the amino acid sequence set forth in SEQ ID NO:20; (g) an H1 region
having at least 80% sequence identity to the amino acid sequence
set forth in SEQ ID NO:26; (h) an H2 region having at least 80%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:28; (i) an H3 region having at least 80% sequence identity to
the amino acid sequence set forth in SEQ ID NO:30; (j) an L1 region
having at least 80% sequence identity to the amino acid sequence
set forth in SEQ ID NO:36; (k) an L2 region having at least 80%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:38; and (l) an L3 region having at least 80% sequence identity
to the amino acid sequence set forth in SEQ ID NO:40.
4. The method of claim 3, wherein the antibody comprises the H1,
H2, and H3 regions of (a), (b), and (c); or the H1, H2, and H3
regions of (g), (h), and (i).
5. The method of claim 4, wherein the polypeptide regions of (a),
(b), (c), (d), (e), and (f) have, respectively, the amino acid
sequences set forth in SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:10; SEQ
ID NO:16, SEQ ID NO:18, and SEQ ID NO:20.
6. The method of claim 4, wherein the antibody comprising the H1,
H2, and H3 regions of (a), (b), and (c) further comprises the L1,
L2, and L3 regions of (d), (e), and (f); or the antibody comprising
the H1, H2, and H3 regions of (g), (h), and (i) further comprises
the L1, L2, and L3 regions of (j), (k), and (l).
7. The method of claim 4, wherein the antibody comprises a heavy
chain variable region having at least 80% sequence identity to the
amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO 22.
8. The method of claim 7, wherein the heavy chain variable region
has the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID
NO:22.
9. The method of claim 7, wherein the antibody, comprising the
heavy chain variable region having at least 80% sequence identity
to SEQ ID NO:2 or SEQ ID NO:22, further comprises, respectively, a
light chain variable region having at least 80% sequence identity
to the amino acid sequence set forth in SEQ ID NO:12 or SEQ ID
NO:32.
10. The method of claim 9, wherein the light chain variable region
has the amino acid sequence set forth in SEQ ID NO:12 or SEQ ID
NO:32.
11. The method of claim 10, wherein the antibody is monoclonal
antibody 1F6 or 2F2.
12. The method of claim 11, wherein the antibody-drug conjugate is
selected from the group consisting of humanized 1F6-val-cit-MMAE,
humanized 1F6-val-cit-MMAF, humanized 1F6-val-cit-AFP, humanized
2F2-val-cit-MMAE, humanized 2F2-val-cit-MMAF, and humanized
2F2-val-cit-AFP.
13. The method of claim 1, wherein the antibody is a chimeric,
humanized, or human antibody.
14. The method of claim 13, wherein the chimeric antibody comprises
a human constant region.
15. The method of claim 1, wherein the antibody is multivalent.
16. The method of claim 1, wherein the cytotoxic agent is selected
from the group consisting of an auristatin, a DNA minor groove
binding agent, a DNA minor groove alkylating agent, an enediyne, a
lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a
maytansinoid, and a vinca alkaloid.
17. The method of claim 1, wherein the cytotoxic agent is AFP,
MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel,
CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin,
cyanomorpholino-doxorubicin, dolastatin-10, echinomycin,
combretatstatin, chalicheamicin, maytansine, DM-1, or
netropsin.
18. The method of claim 1, wherein the cytotoxic agent is an
anti-tubulin agent.
19. The method of claim 18, wherein the anti-tubulin agent is an
auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a
baccatin derivative, a cryptophysin, a maytansinoid, a
combretastatin, or a dolastatin.
20. The method of claim 18, wherein the antitubulin agent is AFP,
MMAF, MMAE, AEB, AEVB, auristatin E, vincristine, vinblastine,
vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel,
epothilone A, epothilone B, nocodazole, colchicines, colcimid,
estramustine, cemadotin, discodermolide, maytansine, DM-1, or
eleutherobin.
21. The method of claim 20, wherein the cytotoxic agent is AFP,
MMAF, or MMAE.
22. The method of claim 1, wherein the antibody is conjugated to
the cytotoxic agent via a linker.
23. The method of claim 22, wherein the linker is cleavable under
intracellular conditions.
24. The method of claim 23, wherein the cleavable linker is a
peptide linker cleavable by an intracellular protease.
25. The method of claim 24, wherein the peptide linker is a
dipeptide linker.
26. The method of claim 25, wherein the dipeptide linker comprises
a val-cit or a phe-lys dipeptide.
27. The method of claim 23, wherein the cleavable linker is
hydrolyzable at a pH of less than 5.5.
28. The method of claim 27, wherein the hydrolyzable linker is a
hydrazone linker.
29. The method of claim 23, wherein the cleavable linker is a
disulfide linker.
30. The method of claim 1, wherein the subject is human.
31. A method for the treatment of a cancer in a subject, the method
comprising: administering to the subject in need thereof from about
one to about ten suboptimal dosages, over a period of about four to
about ten days, of an antibody-drug conjugate comprising an
antibody that binds CD70 expressed on the cancer and wherein the
antibody is conjugated to a cytotoxic agent or an immunosuppressive
agent. thymic carcinoma, a nasopharyngeal carcinoma, and a brain
tumor.
32. The method of claim 30, wherein the kidney tumor is a renal
cell carcinoma.
33. The method of claim 35, wherein the suboptimal dosage is from
about 0.05 to 1 mg/kg.
34. The method of claim 1, wherein the antibody competes for
binding to CD70 with monoclonal antibody 1F6 or 2F2.
35. A method for the treatment of an immunological disorder in a
subject, the method comprising: administering to the subject, in an
amount effective for the treatment, an antibody-drug conjugate
comprising an antibody that binds to CD70 and wherein the antibody
is conjugated to a cytotoxic agent or an immunosuppressive
agent.
36. The method of claim 35, wherein the antibody competes for
binding to CD70 with monoclonal antibody 1F6 or 2F2.
37. The method of claim 36, wherein the antibody comprises at least
one polypeptide region selected from the group consisting of (a) an
H1 region having at least 80% sequence identity to the amino acid
sequence set forth in SEQ ID NO:6; (b) an H2 region having at least
80% sequence identity to the amino acid sequence set forth in SEQ
ID NO:8; (c) an H3 region having at least 80% sequence identity to
the amino acid sequence set forth in SEQ ID NO:10; (d) an L1 region
having at least 80% sequence identity to the amino acid sequence
set forth in SEQ ID NO:16; (e) an L2 region having at least 80%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:18; (f) an L3 region having at least 80% sequence identity to
the amino acid sequence set forth in SEQ ID NO:20; (g) an H1 region
having at least 80% sequence identity to the amino acid sequence
set forth in SEQ ID NO:26; (h) an H2 region having at least 80%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:28; (i) an H3 region having at least 80% sequence identity to
the amino acid sequence set forth in SEQ ID NO:30; (j) an L1 region
having at least 80% sequence identity to the amino acid sequence
set forth in SEQ ID NO:36; (k) an L2 region having at least 80%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:38; and (l) an L3 region having at least 80% sequence identity
to the amino acid sequence set forth in SEQ ID NO:40.
38. The method of claim 37, wherein the antibody comprises the H1,
H2, and H3 regions of (a), (b), and (c); or the H1, H2, and H3
regions of (g), (h), and (i).
39. The method of claim 38, wherein the polypeptide regions of (a),
(b), (c), (d), (e), and (f) have, respectively, the amino acid
sequences set forth in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10; SEQ
ID NO:16, SEQ ID NO:18, and SEQ ID NO:20.
40. The method of claim 38, wherein the antibody comprising the H1,
H2, and H3 regions of (a), (b), and (c) further comprises the L1,
L2, and L3 regions of (d), (e), and (f); or the antibody comprising
the H1, H2, and H3 regions of (g), (h), and (i) further comprises
the L1, L2, and L3 regions of (j), (k), and (l).
41. The method of claim 38, wherein the antibody comprises a heavy
chain variable region having at least 80% sequence identity to the
amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO 22.
42. The method of claim 41, wherein the heavy chain variable region
has the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID
NO:22.
43. The method of claim 41, wherein the antibody, comprising the
heavy chain variable region having at least 80% sequence identity
to SEQ ID NO:2 or SEQ ID NO:22, further comprises, respectively, a
light chain variable region having at least 80% sequence identity
to the amino acid sequence set forth in SEQ ID NO:12 or SEQ ID
NO:32.
44. The method of claim 43, wherein the light chain variable region
has the amino acid sequence set forth in SEQ ID NO:12 or SEQ ID
NO:32.
45. The method of claim 44, wherein the antibody is monoclonal
antibody 1F6 or 2F2.
46. The method of claim 45, wherein the antibody-drug conjugate is
selected from the group consisting of humanized 1F6-val-cit-AFP,
humanized 1F6-val-cit-MMAF, humanized 1F6-val-cit-MMAE, humanized
2F2-val-cit-AFP, humanized 2F2-val-cit-MMAF, and humanized
2F2-val-cit-MMAE.
47. The method of claim 35, wherein the antibody is a chimeric,
humanized, or human antibody.
48. The method of claim 47, wherein the chimeric antibody comprises
a human constant region.
49. The method of claim 35, wherein the antibody is
multivalent.
50. The method of claim 35, wherein the cytotoxic agent is selected
from the group consisting of an auristatin, a DNA minor groove
binding agent, a DNA minor groove alkylating agent, an enediyne, a
lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a
maytansinoid, and a vinca alkaloid.
51. The method of claim 35, wherein the cytotoxic agent is AFP,
MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel,
CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin,
cyanomorpholino-doxorubicin, dolastatin-10, echinomycin,
combretatstatin, chalicheamicin, maytansine, DM-1, or
netropsin.
52. The method of claim 35, wherein the cytotoxic agent is an
anti-tubulin agent.
53. The method of claim 52, wherein the anti-tubulin agent is an
auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a
baccatin derivative, a cryptophysin, a maytansinoid, a
combretastatin, or a dolastatin.
54. The method of claim 52, wherein the antitubulin agent is AFP,
MMAF, MMAE, AEB, AEVB, auristatin E, vincristine, vinblastine,
vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel,
epothilone A, epothilone B, nocodazole, colchicines, colcimid,
estramustine, cemadotin, discodermolide, maytansine, DM-1, or
eleutherobin.
55. The method of claim 54, wherein the cytotoxic agent is AFP,
MMAF, or MMAE.
56. The method of claim 35, wherein the immunosuppressive agent is
gancyclovir, etanercept, cyclosporine, tacrolimus, rapamycin,
cyclophosphamide, azathioprine, mycophenolate mofetil,
methotrexate, cortisol, aldosterone, dexamethasone, a
cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, or a
leukotriene receptor antagonist.
57. The method of claim 35, wherein the antibody is conjugated to
the cytotoxic agent or the immunosuppressive agent via a
linker.
58. The method of claim 57, wherein the linker is cleavable under
intracellular conditions.
59. The method of claim 58, wherein the cleavable linker is a
peptide linker cleavable by an intracellular protease.
60. The method of claim 58, wherein the intracellular protease is a
lysosomal protease or an endosomal protease.
61. The method of claim 58, wherein the peptide linker is a
dipeptide linker.
62. The method of claim 61, wherein the dipeptide linker comprises
a val-cit linker or a phe-lys dipeptide.
63. The method of claim 57, wherein the cleavable linker is
hydrolyzable at a pH of less than 5.5.
64. The method of claim 63, wherein the hydrolyzable linker is a
hydrazone linker.
65. The method of claim 57, wherein the cleavable linker is a
disulfide linker.
66. The method of claim 35, wherein the immunological disorder is a
T cell-mediated immunological disorder.
67. The method of claim 66, wherein the T cell mediated
immunological disorder is a T-cell mediated and include activated T
cell expressing CD70.
68. The method of claim 67, wherein resting T cells are not
substantially depleted by administration of the antibody-drug
conjugate.
69. The method of claim 66, wherein the T cell-mediated
immunological disorder is rheumatoid arthritis, multiple sclerosis,
psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's
disease, primary biliary cirrhosis, Wegener's granulomatosis,
tuberculosis, or acute graft versus host disease.
70. The method of claim 35, wherein the immunological disorder is
an activated B-lymphocyte disorder.
71. The method of claim 35, wherein the subject is human.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 10/546,304, filed Aug. 19, 2005, which is a
national stage application of PCT Application No. PCT/US04/005247,
filed Feb. 20, 2004, which claims the benefit of U.S. Provisional
Patent Application No. 60/449,055, filed Feb. 20, 2003, all of
which are incorporated by reference herein in their entirety. This
application also claims the benefit of U.S. Provisional Patent
Application No. 60/792,127, filed Apr. 13, 2006, which is
incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0002] CD70 is a member of the tumor necrosis factor (TNF) family
of cell membrane-bound and secreted molecules that are expressed by
a variety of normal and malignant cell types. The primary amino
acid (AA) sequence of CD70 predicts a transmembrane type II protein
with its carboxyl terminus exposed to the outside of cells and its
amino terminus found in the cytosolic side of the plasma membrane
(Bowman et al., 1994, J Immunol 152:1756-61; Goodwin et al., 1993,
Cell 73:447-56). Human CD70 is composed of a 20 AA cytoplasmic
domain, an 18 AA transmembrane domain, and a 155 AA
extracytoplasmic domain with two potential N-linked glycosylation
sites (Bowman et al., supra; Goodwin et al., supra). Specific
immunoprecipitation of radioisotope-labeled CD70-expressing cells
by anti-CD70 antibodies yields polypeptides of 29 and 50 kDa
(Goodwin et al., supra; Hintzen et al., 1994, J Immunol
152:1762-73). Based on its homology to TNF-alpha and TNF-beta,
especially in structural strands C, D, H and I, a trimeric
structure is predicted for CD70 (Petsch et al., 1995, Mol Immunol
32:761-72).
[0003] Original immunohistological studies revealed that CD70 is
expressed on germinal center B cells and rare T cells in tonsils,
skin, and gut (Hintzen et al., 1994, Int Immunol 6:477-80).
Subsequently, CD70 was reported to be expressed on the cell surface
of recently antigen-activated T and B lymphocytes, and its
expression wanes after the removal of antigenic stimulation (Lens
et al., 1996, Eur J Immunol 26:2964-71; Lens et al., 1997,
Immunology 90:38-45). Within the lymphoid system, natural killer
cells (Orengo et al., 1997, Clin Exp Immunol 107:608-13) and mouse
mature peripheral dendritic cells (Akiba et al., 2000, J Exp Med
191:375-80) also express CD70. In non-lymphoid lineages, CD70 has
been detected on thymic medullar epithelial cells (Hintzen et al.,
1994, supra; Hishima et al., 2000, Am J Surg Pathol 24:742-46).
[0004] In addition to expression on normal cells, CD70 expression
has been reported in different types of cancers including
lymphomas, carcinomas, and tumors of neural origin. In malignant B
cells, 71% of diffuse large B-cell lymphomas, 33% of follicle
center lymphomas, 25% of mantle lymphomas, and 50% of B-CLL have
been reported to express CD70 (Lens et al., 1999, Br J Haematol
106:491-503). CD70 is frequently expressed together with other
lymphoid activation markers on the malignant Hodgkin and
Reed-Sternberg cells of Hodgkin's disease (Gruss and Kadin, 1996,
Bailieres Clin Haematol 9:417-46). One report demonstrates CD70
expression on 88% (7 of 8 cases) of thymic carcinomas and 20% (1 of
5 cases) of atypical thymomas (Hishima et al., 2000, supra). The
second type of carcinoma on which CD70 has been detected is
nasopharyngeal carcinoma. One study reports the presence of CD70 on
80% (16 of 20 cases) of snap-frozen tumor biopsies obtained from
undifferentiated nasopharyngeal carcinomas (Agathanggelou et al.,
1995, Am J Path 147:1152-60). CD70 has also been detected on brain
tumor cells, especially glioma cell lines, solid human gliomas, and
meningiomas (Held-Feindt and Mentlein, 2002, Int J Cancer
98:352-56; Wischlusen et al., 2002, Can Res 62:2592-99).
[0005] It has been proposed that transforming viruses including the
Epstein-Barr virus (EBV) and the human T leukemia virus-1 (HTLV-1)
can induce CD70 on cells such as epithelial cells that normally do
not express CD70 (Agathanggelou et al., supra; Stein et al., 1989,
Oxford University Press, p. 446). Therefore, expression of CD70 on
malignant B cells could be a reflection of oncogenic transformation
(Lens et al., 1999, supra). Also, since CD70 expression is induced
on B cells after antigen encounter (Maurer et al., 1990, Eur J
Immunol 20:2679-84; Lens et al., 1996, supra), stable expression of
CD70 might reflect prolonged antigenic stimulation. This has been
postulated to occur in follicular non-Hodgkin's lymphomas based on
ongoing somatic hypermutation (Bahler et al., 1992, Proc Natl Acad
Sci USA 89:6770-74; Bahler et al., 1992, Cancer Res 52:suppl.
5547S-51S).
[0006] The receptor for CD70 is CD27, a glycosylated type I
transmembrane protein of about 55 kDa (Goodwin et al., 1993, Cell
73:447-56; Hintzen et al., 1994, supra). CD70 is sometimes referred
to as CD27L. CD27, which exists as a homodimer on the cell surface
(Gravestein et al., 1993, Eur J Immunol 23:943-50), is a member of
the TNF receptor superfamily as defined by cysteine-rich repeats of
about 40 amino acids in the extracellular domain (Smith et al.,
1990, Science 248:1019-23; Locksley et al., 2001, Cell
104:487-501). CD27 is typically expressed by thymocytes, NK, T, and
B cells (Hintzen et al., 1994, Immunol Today 15:307-11; Lens et
al., 1998, Semin Immunol 10:491-99). On resting T cells, CD27 is
constitutively expressed, yet antigenic triggering further
upregulates CD27 expression (de Jong et al., 1991, J Immunol
146:2488-94; Hintzen et al., 1993, J Immunol. 151:2426-35).
Further, triggering of T cells via their T cell antigen receptor
complex alone or in combination with the accessory molecule CD28
releases soluble CD27 from activated T cells (Hintzen et al., 1991,
J Immunol 147:29-35). Naive B cells do not express CD27, but its
expression is induced and, in contrast to CD70, sustained after
antigenic triggering of B cells (Jacquot S et al., 1997 J Immunol
159:2652-57; Kobata T et al., 1995, Proc Natl Acad Sci USA
92:11249-53).
[0007] In marked contrast to the restricted expression of CD27 and
CD70 in normal B lineage cells, both CD27 and CD70 are frequently
co-expressed in many B cell non-Hodgkin's lymphomas and leukemias.
This could potentially lead to functional CD27-CD70 interactions on
these cells in the form of an autocrine loop, resulting in CD27
signaling and in CD70-induced proliferation, thereby providing a
growth advantage to malignant cells (Lens et al., 1999, supra).
[0008] The available data supports a model in which ligation of
CD27 on activated lymphocytes by CD70 delivers signals to the
CD27-expressing cells, including co-stimulatory signals in T, B,
and NK cells. (See, e.g., Goodwin et al., supra; Hintzen et al.,
1995, J Immunol 154:2612-23; Oshima et al., 1998, Int Immunol
10:517-26; Smith et al., supra; Van Lier et al., 1987, J Immunol
139:1589-96; Gravestein et al., 1995, Int Immunol 7:551-7;
Tesselaar et al., 1997, J Immunol 159:4959-65; Jacquot et al.,
supra; Agematsu et al., 1998, Blood 91:173-80; Kobata et al.,
supra; Agematsu et al., 1997, Eur J Immunol 27:2073-79; Sugita et
al., 1992, J Immunol 149:1199-1203; Orengo et al., 1997, Clin Exp
Immunol 107:608-13). Antibodies against both murine and human CD70
have been demonstrated to inhibit such activities, presumably by
blocking the CD70/CD27 interaction (Hintzen et al., 1994, supra;
Hintzen et al., 1995, supra; Oshima et al., supra).
[0009] Limited information is available on the modulation of
cellular functions through CD70 signaling upon CD70/CD27
interaction, i.e., `reverse signaling`. Some CD70 antibodies have
the ability to enhance T cell proliferation when they are presented
to CD70-expressing T cells either cross-linked with a secondary
antibody or immobilized on tissue culture plates (Bowman et al.,
1994, J Immunol 152:1756-61; Brugnoni, 1997, Immunol Lett
55:99-104). Such `reverse signaling` has also been described in a
subset of B chronic lymphocytic leukemia (B-CLL) cells, and CD70
can function as a receptor to transduce signals to facilitate
proliferation of PMA-stimulated purified B-CLL cells (Lens et al.,
1999, supra). These observations suggest situations in which
engagement of CD27 and CD70 can result in the delivery of agonistic
signals to both the CD27 and CD70 expressing cells.
[0010] The role of CD70/CD27 co-stimulation in cell-mediated
autoimmune diseases has been investigated in a model of
experimental autoimmune encephalomyelitis (EAE) (Nakajima et al.,
2000, J Neuroimmunol 109:188-96). In vivo administration of a
particular anti-mouse CD70 mAb (clone FR-70) markedly suppressed
the onset of EAE by inhibiting antigen-induced TNF-alpha production
without affecting T cell priming, Ig production or
T.sub.H1/T.sub.H2 cell balance. However, such treatment had little
efficacy in established disease. It has been reported that
expression of CD70 on T cells was enhanced by TNF-alpha and IL-12
and down regulated by IL4 (Lens et al., 1998, supra). Thus, the
CD70/CD27 mediated T cell-T cell interactions may play a role in
enhancing T.sub.H1-mediated immune responses rather than
T.sub.H2-mediated responses. Supporting this hypothesis, the
anti-mouse CD70 mAb FR-70 is also effective in inhibiting
T.sub.H1-mediated collagen-induced arthritis (Nakajima et al.,
2000, supra). In contrast, the same anti-mouse CD70 mAb did not
show any efficacy in modulating lupus in NZB/NZW F1 mice and
experimental Leishmania major infection in susceptible BALB/c mice,
both of which are predominantly T.sub.H2-mediated autoimmue
response (Nakajima et al., 1997, J Immunol 158, 1466-72; Akiba et
al., 2000, J Exp Med 191:375-380).
[0011] The role of CD70 has not yet been investigated in acute
graft versus host disease (aGVHD), another T.sub.H1-mediated immune
response. GVHD is a major and often lethal consequence of
allogeneic bone marrow transplantation (BMT) therapy that occurs
when histocompatibility antigen differences between the BM donor
and the recipient of the transplant are present (den Haan et al.,
1995, Science 268:1476). GVHD is caused by mature T cells present
in the transplanted marrow, as well as other minor cell populations
(Giralt and Champlin, 1994, Blood 84:3603). It is noteworthy that
CD70 has been detected in vivo on CD4+ cells in conditions
characterized by allogeneic reaction, as in cases of maternal T
cell engraftment in severe combined immune deficiency patients
(Brugnoni et al., Immunol Lett 55:99-104). Prophylaxis of GVHD is
achieved by pan-T cell immunosuppressive agents such as
cyclosporine, corticosteroids, or methotrexate. In addition to the
lack of specificity, these agents are also associated with
significant adverse side effects. To limit these undesirable
effects and the disruption of normal T cell functions, other
therapeutic interventions based on selective targeting of T cells
directly participating in allo-recognition and graft rejection are
much needed.
[0012] CD70 is a potentially useful target for antibody-directed
immunotherapy. As indicated supra, CD70 has a restricted expression
pattern in normal cells: CD70 expression is mostly restricted to
recently antigen-activated T and B cells under physiological
conditions, and its expression is down-regulated when antigenic
stimulation ceases. The key role of CD70 is believed to be
facilitating plasma cell differentiation and contributing to the
generation and maintenance of long-term T cell memory. Further,
evidence from animal models suggests that unregulated CD70/CD27
interaction may contribute to immunological disorders, and, in
humans, experimental data have also pointed towards potential
abnormal regulation of the CD70/CD27 pathway in T.sub.H1-mediated
immune disorders such as, e.g., rheumatoid arthritis, psoriasis,
and multiple sclerosis. It is of particular interest that CD70 is
expressed on a variety of transformed cells including lymphoma B
cells, Hodgkin and Reed-Sternberg cells, malignant cells of neural
origin, and a number of carcinomas.
[0013] Several groups have demonstrated the inhibitory effect of
anti-CD70 mAb in both in vitro models of lymphocyte activation and
animal models of T.sub.H1-mediated responses. The focus has been on
the use of antibodies to block the CD70/CD27 co-stimulation pathway
to achieve therapeutic efficacy. However, one main shortcoming of
such an approach is the large number of signaling receptors, e.g.,
the CD28/CD80/CD86 co-stimulatory pathway, known to participate in
immunological diseases. Consequently, blocking one specific
signaling pathway may only have minimal impact on disease
development. This is supported by the observations that anti-CD70
mAb can only partially inhibit in vitro T cell activation induced
by allogeneic stimulator cells (Hintzen et al., 1995, supra) and an
anti-CD70 mAb showed no therapeutic efficacy in EAE once the
disease is established (Nakajima et al., 2000, supra).
[0014] Thus, there is a need in the art for developing an approach
for depleting or inhibiting the growth of CD70-expressing cells
involved in cancers and/or immunological diseases by means other
than or in addition to blocking the CD70/CD27 interaction. As CD70
is expressed on the surface of mature antigen presenting dendritic
cells, activated T cells, and activated B cells, agents that can
target and inhibit or deplete CD70.sup.+ cells may prove to be
effective in the removal of antigen presenting cells presenting
autoantigens and offending autoreactive activated T or B cells, as
wells as CD70-expressing tumor cells.
[0015] Approaches that have been used for increasing the
therapeutic efficacy of antibodies are radiolabeling and
combination with chemotherapy; however, these approaches include
associated with undesirable side effects. For example, isotope
therapy is associated with myelosuppression (Witzig, 2001, Cancer
Chemother Pharmacol 48 (Suppl 1):S91-5), and combining therapy with
antibodies and chemotherapeutics is associated with
immunosuppression. Further, isotopically labeled substances are
difficult to produce, and patients often experience relapse after
initial treatment with isotopically labeled substances.
[0016] Accordingly, there is a need for anti-CD70 antibody-drug
conjugates (ADCs) that are constructed in such a manner so as to be
capable exerting a clinically useful cytotoxic, cytostatic, or
immunosuppressive effect on CD70-expressing cells, particularly
without exerting undesirable effects on non-CD70-expressing cells.
Such compounds would be useful therapeutic agents against cancers
that express CD70 or immune disorders that are mediated by
CD70-expressing cells. (The recitation of any reference in this
application is not an admission that the reference is prior art to
this application.)
BRIEF SUMMARY OF THE INVENTION
[0017] The present invention provides antibody-drug conjugates
(ADCs) and ADC derivatives and methods relating to the use of such
conjugates to treat CD70-expressing cancers and immunological
disorders. The antibody, or other targeting moiety in the ADC,
binds to CD70. A drug conjugated to the antibody or targeting
moiety exerts a cytotoxic, cytostatic, or immunosuppressive effect
on CD70-expressing cells to treat or prevent recurrence of
CD70-expressing cancers or immunological disorders.
[0018] In one aspect, methods are provided for the treatment of a
CD70-expressing cancer in a subject. The methods generally include
administering to the subject an effective amount of an
antibody-drug conjugate. The antibody-drug conjugate includes an
antibody that binds to CD70 ("CD70 antibody"). The antibody is
conjugated to a drug that is a cytotoxic or cytostatic agent. In
certain embodiments, the antibody is the monoclonal antibody 1F6 or
2F2.
[0019] In other embodiments, the antibody binds to CD70 and
competes for binding to CD70 with monoclonal antibody 1F6 or 2F2.
Such a CD70 antibody can include at least one polypeptide region
selected from (a) an H1 region having at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:6; (b)
an H2 region having at least 80% sequence identity to the amino
acid sequence set forth in SEQ ID NO:8; an H3 region having at
least 80% sequence identity to the amino acid sequence set forth in
SEQ ID NO:10; (d) an L1 region having at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:16; (e)
an L2 region having at least 80% sequence identity to the amino
acid sequence set forth in SEQ ID NO:18; (f) an L3 region having at
least 80% sequence identity to the amino acid sequence set forth in
SEQ ID NO:20; (g) an H1 region having at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:26; (h)
an H2 region having at least 80% sequence identity to the amino
acid sequence set forth in SEQ ID NO:28; (i) an H3 region having at
least 80% sequence identity to the amino acid sequence set forth in
SEQ ID NO:30; (j) an L1 region having at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:36; (k)
an L2 region having at least 80% sequence identity to the amino
acid sequence set forth in SEQ ID NO:38; and (l) an L3 region
having at least 80% sequence identity to the amino acid sequence
set forth in SEQ ID NO:40.
[0020] In certain embodiments, the CD70 antibody includes the H1,
H2, and H3 regions of (a), (b), and (c) (supra); or the H1, H2, and
H3 regions of (g), (h), and (i) (supra). In additional embodiments,
the H1, H2, H3, L1, L2 and L3 regions of the CD70 antibody include
polypeptide regions have the amino acid sequences set forth in SEQ
ID NO:6, SEQ ID NO:8, SEQ ID NO:10; SEQ ID NO:16, SEQ ID NO:18, and
SEQ ID NO:20, respectively.
[0021] In additional embodiments, the H1, H2, and H3 regions
correspond to (a), (b), and (c) (supra) and the L1, L2, and L3
regions correspond to (d), (e), and (f) (supra), respectively; or
the H1, H2, and H3 regions correspond to (g), (h), and (i) (supra)
and the L1, L2, and L3 regions correspond to (j), (k), and (l)
(supra). In further embodiments, the CD70 antibody comprises a
heavy chain variable region having at least 80% sequence identity
to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO
22. The CD70 heavy chain variable region also can have the amino
acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:22.
[0022] In yet other embodiments, the heavy chain variable region
has at least 80% sequence identity to SEQ ID NO:2 or SEQ ID NO:22,
and the light chain variable region has at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:12 or
SEQ ID NO:32. The light chain variable region also can have the
amino acid sequence set forth in SEQ ID NO:12 or SEQ ID NO:32.
[0023] In exemplary embodiments, the antibody-drug conjugate is
1F6-val-cit-AFP, 1F6-val-cit-MMAF, 1F6-val-cit-MMAE,
2F2-val-cit-AFP, 2F2-val-cit-MMAF, and 2F2-val-cit-MMAE.
[0024] The CD70 antibody can be, for example, a chimeric,
humanized, or human antibody. In certain embodiments, the CD70
antibody is a chimeric antibody having a human constant region. The
CD70 antibody can be monovalent or polyvalent.
[0025] Suitable cytotoxic agents can be, for example, an
auristatin, a DNA minor groove binding agent, a DNA minor groove
alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a
taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca
alkaloid. In specific embodiments, the cytotoxic agent is AFP,
MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel,
CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin,
cyanomorpholino-doxorubicin, dolastatin-10, echinomycin,
combretatstatin, chalicheamicin, maytansine, DM-1, or netropsin.
Other suitable cytotoxic agents include anti-tubulin agents, such
as an auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a
baccatin derivative, a cryptophysin, a maytansinoid, a
combretastatin, or a dolastatin. In specific embodiments, the
antitubulin agent is AFP, MMAF, MMAE, AEB, AEVB, auristatin E,
vincristine, vinblastine, vindesine, vinorelbine, VP-16,
camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B,
nocodazole, colchicines, colcimid, estramustine, cemadotin,
discodermolide, maytansine, DM-1, or eleutherobin.
[0026] In antibody drug conjugates, the antibody can be conjugated
directly to the cytotoxic agent or via a linker. Suitable linkers
include, for example, cleavable and non-cleavable linkers. A
cleavable linker is typically susceptible to cleavage under
intracellular conditions. Suitable cleavable linkers include, for
example, a peptide linker cleavable by an intracellular protease,
such as lysosomal protease or an endosomal protease. In exemplary
embodiments, the linker can be a dipeptide linker, such as a
valine-citrulline (val-cit) or a phenylalanine-lysine (phe-lys)
linker. Other suitable linkers include linkers hydrolyzable at a pH
of less than 5.5, such as a hydrazone linker. Additional suitable
cleavable linkers include disulfide linkers.
[0027] An antibody-drug conjugate can be targeted to
CD70-expressing cells, such as for example, Hodgkin's disease
(e.g., Reed-Sternberg cells); cancers of the B-cell lineage,
including, e.g., diffuse large B-cell lymphomas, follicular
lymphomas, Burkitt's lymphoma, mantle cell lymphomas, B-cell
lymphocytic leukemias (e.g., acute lymphocytic leukemia, chronic
lymphocytic leukemia); Epstein Barr Virus positive B cell
lymphomas; gliomas; glioblastomas; neuroblastomas; astrocytomas;
meningiomas; Waldenstrom macroglobulinemia; multiple myelomas; and
colon, stomach, and rectal carcinomas. The antibody-drug conjugate
also can be targeted to CD70-expressing immune cells, such as an
activated T cell, an activated B cell, or a mature dendritic cell.
The antibody-drug conjugates can be administered to humans or to
non-human animals.
[0028] In another aspect, methods are provided for treating an
immunological disorder. The methods generally include administering
to a subject an amount effective of an antibody-drug conjugate
comprising an antibody that binds CD70. The antibody is conjugated
to a cytotoxic agent or an immunosuppressive agent. In certain
embodiments, the antibody is the monoclonal antibody 1F6 or
2F2.
[0029] In other embodiments, the antibody binds to CD70 and
competes for binding to CD70 with monoclonal antibody 1F6 or 2F2.
The CD70 antibody can include at least one polypeptide region
selected from (a) an H1 region having at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:6; (b)
an H2 region having at least 80% sequence identity to the amino
acid sequence set forth in SEQ ID NO:8; an H3 region having at
least 80% sequence identity to the amino acid sequence set forth in
SEQ ID NO:10; (d) an L1 region having at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:16; (e)
an L2 region having at least 80% sequence identity to the amino
acid sequence set forth in SEQ ID NO:18; (f) an L3 region having at
least 80% sequence identity to the amino acid sequence set forth in
SEQ ID NO:20; (g) an H1 region having at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:26; (h)
an H2 region having at least 80% sequence identity to the amino
acid sequence set forth in SEQ ID NO:28; (i) an H3 region having at
least 80% sequence identity to the amino acid sequence set forth in
SEQ ID NO:30; (j) an L1 region having at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:36; (k)
an L2 region having at least 80% sequence identity to the amino
acid sequence set forth in SEQ ID NO:38; and (l) an L3 region
having at least 80% sequence identity to the amino acid sequence
set forth in SEQ ID NO:40.
[0030] In certain embodiments, the CD70 antibody includes the H1,
H2, and H3 regions of (a), (b), and (c) (supra); or the H1, H2, and
H3 regions of (g), (h), and (i) (supra), respectively. In
additional embodiments, the H1, H2, H3, L1, L2 and L3 regions of
the CD70 antibody includes polypeptide regions have the amino acid
sequences set forth in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10; SEQ
ID NO:16, SEQ ID NO:18, and SEQ ID NO:20, respectively.
[0031] In additional embodiments, the H1, H2, and H3 regions
correspond to (a), (b), and (c) (supra) and the L1, L2, and L3
regions correspond to (d), (e), and (f) (supra), respectively; or
the H1, H2, and H3 regions correspond to (g), (h), and (i) (supra)
and the L1, L2, and L3 regions correspond to (j), (k), and (l)
(supra), respectively. In further embodiments, the CD70 antibody
comprises a heavy chain variable region having at least 80%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:2 or SEQ ID NO 22. The CD70 heavy chain variable region also can
have the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID
NO:22.
[0032] In yet other embodiments, the heavy chain variable region
has at least 80% sequence identity to SEQ ID NO:2 or SEQ ID NO:22,
and the light chain variable region has at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:12 or
SEQ ID NO:32. The light chain variable region also can have the
amino acid sequence set forth in SEQ ID NO:12 or SEQ ID NO:32.
[0033] In exemplary embodiments, the antibody-drug conjugate is
1F6-val-cit-AFP, 1F6-val-cit-MMAF, 1F6-val-cit-MMAE,
2F2-val-cit-AFP, 2F2-val-cit-MMAF, and 2F2-val-cit-MMAE.
[0034] The CD70 antibody can be, for example, a chimeric,
humanized, or human antibody. In certain embodiments, the CD70
antibody is a chimeric antibody having a human constant region. The
CD70 antibody can be monovalent or polyvalent.
[0035] Suitable cytotoxic agents can be, for example, an
auristatin, a DNA minor groove binding agent, a DNA minor groove
alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a
taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca
alkaloid. In specific embodiments, the drug is cytotoxic agent is
AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel,
CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin,
cyanomorpholino-doxorubicin, dolastatin-10, echinomycin,
combretatstatin, chalicheamicin, maytansine, DM-1, or netropsin.
Other suitable cytotoxic agents include anti-tubulin agents, such
as an auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a
baccatin derivative, a cryptophysin, a maytansinoid, a
combretastatin, or a dolastatin. In specific embodiments, the
antitubulin agent is AFP, MMAF, MMAE, AEB, AEVB, auristatin E,
vincristine, vinblastine, vindesine, vinorelbine, VP-16,
camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B,
nocodazole, colchicines, colcimid, estramustine, cemadotin,
discodermolide, maytansine, DM-1, or eleutherobin.
[0036] Suitable immunosuppressive agents include, for example,
gancyclovir, etanercept, cyclosporine, tacrolimus, rapamycin,
cyclophosphamide, azathioprine, mycophenolate mofetil,
methotrexate, cortisol, aldosterone, dexamethasone, a
cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, or a
leukotriene receptor antagonist.
[0037] In antibody drug conjugates, the antibody can be conjugated
directly to the cytotoxic agent or immunosuppressive agent, or via
a linker. Suitable linkers include, for example, cleavable and
non-cleavable linkers. A cleavable linker is typically susceptible
to cleavage under intracellular conditions. Suitable cleavable
linkers include, for example, a peptide linker cleavable by an
intracellular protease, such as lysosomal protease or an endosomal
protease.
[0038] In exemplary embodiments, the linker can be a dipeptide
linker, such as a valine-citrulline (val-cit) or a
phenylalanine-lysine (phe-lys) linker. Other suitable linkers
include cleavable linker hydrolyzable at a pH of less than 5.5,
such as a hydrazone linker. Further suitable cleavable linkers
include disulfide linkers.
[0039] The ADC can be targeted to cells of an immunological
disorder, such as, for example, Th1-mediated immunological
disorders. Th1-mediated immunological disorders include, for
example, rheumatoid arthritis, multiple sclerosis, psoriasis,
Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease,
primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis,
or acute graft versus host disease. Other immunological disorders
include, for example, an activated B-lymphocyte disorder. The
subject can be a human or a non-human animal.
[0040] In yet another aspect, antibody-drug conjugates are
provided. The antibody-drug conjugates include an antibody that
binds to CD70. The antibody is conjugated to a cytotoxic agent or
an immunosuppressive agent. The antibody-drug conjugate can exerts
a cytotoxic or cytostatic effect on a CD70-expressing cancer cell
line, and/or a cytotoxic, cytostatic, or immunosuppressive effect
on a CD70-expressing immune cell.
[0041] In certain embodiments, the antibody is the monoclonal
antibody 1F6 or 2F2. In other embodiments, the antibody binds to
CD70 and competes for binding to CD70 with monoclonal antibody 1F6
or 2F2. The CD70 antibody can include at least one polypeptide
region selected from (a) an H1 region having at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:6; (b)
an H2 region having at least 80% sequence identity to the amino
acid sequence set forth in SEQ ID NO:8; (c) an H3 region having at
least 80% sequence identity to the amino acid sequence set forth in
SEQ ID NO:10; (d) an L1 region having at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:16; (e)
an L2 region having at least 80% sequence identity to the amino
acid sequence set forth in SEQ ID NO:18; (f) an L3 region having at
least 80% sequence identity to the amino acid sequence set forth in
SEQ ID NO:20; (g) an H1 region having at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:26; (h)
an H2 region having at least 80% sequence identity to the amino
acid sequence set forth in SEQ ID NO:28; (i) an H3 region having at
least 80% sequence identity to the amino acid sequence set forth in
SEQ ID NO:30; (j) an L1 region having at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:36; (k)
an L2 region having at least 80% sequence identity to the amino
acid sequence set forth in SEQ ID NO:38; and (l) an L3 region
having at least 80% sequence identity to the amino acid sequence
set forth in SEQ ID NO:40.
[0042] In certain embodiments, the CD70 antibody includes the H1,
H2, and H3 regions of (a), (b), and (c) (supra); or the H1, H2, and
H3 regions of (g), (h), and (i) (supra), respectively. In
additional embodiments, the H1, H2, H3, L1, L2 and L3 regions of
the CD70 antibody includes polypeptide regions have the amino acid
sequences set forth in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10; SEQ
ID NO:16, SEQ ID NO:18, and SEQ ID NO:20, respectively.
[0043] In additional embodiments, the H1, H2, and H3 regions
correspond to (a), (b), and (c) (supra) and the L1, L2, and L3
regions correspond to (d), (e), and (f) (supra), respectively; or
the H1, H2, and H3 regions correspond to (g), (h), and (i) (supra)
and the L1, L2, and L3 regions correspond to (j), (k), and (l)
(supra), respectively. In further embodiments, the CD70 antibody
comprises a heavy chain variable region having at least 80%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:2 or SEQ ID NO 22. The CD70 heavy chain variable region also can
have the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID
NO:22.
[0044] In yet other embodiments, the heavy chain variable region
has at least 80% sequence identity to SEQ ID NO:2 or SEQ ID NO:22,
and the light chain variable region has at least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO:12 or
SEQ ID NO:32. The light chain variable region also can have the
amino acid sequence set forth in SEQ ID NO:12 or SEQ ID NO:32.
[0045] In exemplary embodiments, the antibody-drug conjugate is
1F6-val-cit-AFP, 1F6-val-cit-MMAF, 1F6-val-cit-MMAE,
2F2-val-cit-AFP, 2F2-val-cit-MMAF, and 2F2-val-cit-MMAE.
[0046] The CD70 antibody can be, for example, a chimeric,
humanized, or human antibody. In certain embodiments, the CD70
antibody is a chimeric antibody having a human constant region. The
CD70 antibody can be monovalent or polyvalent.
[0047] Suitable cytotoxic agents can be, for example, an
auristatin, a DNA minor groove binding agent, a DNA minor groove
alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a
taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca
alkaloid. In specific embodiments, the drug is cytotoxic agent is
AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel,
CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin,
cyanomorpholino-doxorubicin, dolastatin-10, echinomycin,
combretatstatin, chalicheamicin, maytansine, DM-1, or netropsin.
Other suitable cytotoxic agents include anti-tubulin agents, such
as an auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a
baccatin derivative, a cryptophysin, a maytansinoid, a
combretastatin, or a dolastatin. In specific embodiments, the
antitubulin agent is AFP, MMAF, MMAE, AEB, AEVB, auristatin E,
vincristine, vinblastine, vindesine, vinorelbine, VP-16,
camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B,
nocodazole, colchicines, colcimid, estramustine, cemadotin,
discodermolide, maytansine, DM-1, or eleutherobin.
[0048] Suitable immunosuppressive agents include, for example,
gancyclovir, etanercept, cyclosporine, tacrolimus, rapamycin,
cyclophosphamide, azathioprine, mycophenolate mofetil,
methotrexate, cortisol, aldosterone, dexamethasone, a
cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, or a
leukotriene receptor antagonist.
[0049] In antibody drug conjugates, the antibody can be conjugated
directly to the cytotoxic agent or immunosuppressive agent, or via
a linker. Suitable linkers include, for example, cleavable and
non-cleavable linkers. A cleavable linker is typically susceptible
to cleavage under intracellular conditions. Suitable cleavable
linkers include, for example, a peptide linker cleavable by an
intracellular protease, such as a lysosomal protease or an
endosomal protease.
[0050] Antibody drug conjugates can be targeted to CD70-expressing
cancer cells, such as for example, Hodgkin's disease (e.g.,
Reed-Sternberg cells); cancers of the B-cell lineage, including,
e.g., diffuse large B-cell lymphomas, follicular lymphomas,
Burkitt's lymphoma, mantle cell lymphomas, B-cell lymphocytic
leukemias (e.g., acute lymphocytic leukemia, chronic lymphocytic
leukemia); Epstein Barr Virus positive B cell lymphomas; gliomas;
glioblastomas; neuroblastomas; astrocytomas; meningiomas;
Waldenstrom macroglobulinemia; multiple myelomas; and colon,
stomach, and rectal carcinomas. The antibody-drug conjugate also
can be targeted to CD70-expressing immune cells, such as an
activated T cell, an activated B cell, or a mature dendritic
cell.
[0051] In yet additional aspects, pharmaceutical compositions are
provided for the treatment of a CD70-expressing cancer or an
immunological disorder. The pharmaceutical compositions include an
antibody-drug conjugate and at least one pharmaceutically
compatible ingredient. In a related aspect, pharmaceutical kits are
provided. The kits include a container containing an antibody-drug
conjugate. The antibody-drug conjugate is typically lyophilized.
The kits can further include a second container containing a
pharmaceutically acceptable diluent.
[0052] The present invention may be more fully understood by
reference to the following detailed description of the invention,
non-limiting examples of specific embodiments of the invention and
the appended figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1: The 1F6 V.sub.L and V.sub.H cDNA and amino acid
sequences. The coding and amino acid sequences for the light
(V.sub.L, upper 2 panels) and heavy chain (V.sub.H, lower 2 panels)
variable regions of 1F6 were determined. The complementarity
determining regions (CDRs) for the V.sub.L and V.sub.H were
identified according to criteria described in Kabat et al., 1991,
Sequences of proteins of Immunogical Interest, Washington, D.C., US
Department of Health and Public Services; Chothia and Lesk, 1987, J
Mol Biol 196: 901-917. Amino acid residues corresponding to the
CDRs are underlined. The signal peptide for the V.sub.L and V.sub.H
are identified to be amino residues -20 to 0 and -19 to 0,
respectively.
[0054] FIG. 2: The 2F2 V.sub.L and V.sub.H cDNA and amino acid
sequences. The coding and amino acid sequences for the light
(V.sub.L, upper 2 panels) and heavy chain (V.sub.H, lower 2 panels)
variable regions of 2F2 were determined. The complementarity
determining regions (CDRs) for the V.sub.L and V.sub.H were
identified according to criteria described in Kabat et al., 1991,
Sequences of Proteins of Immunogical Interest, Washington, D.C., US
Department of Health and Public Services; Chothia and Lesk, 1987,
J. Mol. Biol. 196:901-917. Amino acid residues corresponding to the
CDRs are underlined. The signal peptides for the V.sub.L and
V.sub.H were identified to be amino residues -20 to 0 and -19 to 0,
respectively.
[0055] FIG. 3: Amino acid sequence comparison between the 1F6 and
2F2 CDRs. The amino acid sequences of 1F6 and 2F2 CDRs are aligned.
Underlined residues represent conservative substitutions and boxed
and italic residues represent divergent substitutions.
[0056] FIG. 4: Expression of CD70 on hematologic cell lines. CD70
expression on the surface of a panel of hematologic cell lines of
different tissue origins and sources was determined by flow
cytometry. CD70 expression was expressed as binding ratios between
the anti-CD70 mAb and control IgG.
[0057] FIG. 5: Effects of anti-CD70 ADCs on the proliferation of
transformed hematologic cell lines. A subset of the CD70.sup.+ cell
lines shown in FIG. 4 and the CD70.sup.- Jurkat cell line were
tested for their sensitivity toward 1F6 ADCs. Graded doses of the
1F6-vcMMAE, 1F6-vcAFP, or the corresponding non-binding control IgG
(IgG) ADCs were added to cells at the initiation of culture. Cells
were exposed to the ADCs continuously for a total of 96 hours.
Proliferation was determined by pulsing with .sup.3H-TdR during the
last 16 hours of incubation. The upper panels show the response of
the WSU-NHL and Jurkat lines. The lower panel summaries the
responses by the tested cell lines to 1F6-vcMMAE and 1F6-vcAFP in
form of IC.sub.50s.
[0058] FIG. 6: Cancer Profiling Array (CPA) analysis of the
expression of CD70 message in multiple cancer types. Expression of
CD70 in 241 tumor isolates and the corresponding adjacent normal
tissues on a CPA was determined by hybridization with a CD70 cDNA
probe. Thirteen tumor types are present on the array as indicated
in the figure. Each pair of dots represents a tumor sample (right
side, T) and the corresponding normal tissue (left side, N). For
some cDNA sets, as exemplified by the boxed sample set, the cDNA
sample on the upper left spot is derived from the normal tissue,
the upper right from the metastatic tumor, and the lower right from
the primary tumor. Asterisks denote cDNA pairs in which CD70
transcript over-expression was detected in the tumor cDNA
(tumor:normal ratio of >2-fold, based on hybridization
intensity).
[0059] FIG. 7: Over-expression of CD70 transcripts in renal cell
carcinoma (RCC) detected by quantitative PCR. CD70 expression in
RCC cDNA samples was compared to that detected in a pool of 4
normal kidneys by quantitative PCR using an ABI PRISM 7000 Sequence
Detection System and the .DELTA.Ct method. Numbers represent the
fold of CD70 transcript expression in RCC compared to that in
normal kidneys.
[0060] FIGS. 8A and B: CD70 protein expression in frozen tumor
sections of RCC. (A) Binding of the anti-CD70 mAb 2F2 (right
column) and a control IgG (left column) to serial frozen tumor
sections derived two RCC donors was determined by
immunohistochemistry staining. Photomicrographs were taken under
40.times. magnification. (B) The same pair of antibodies was used
to stain serial frozen normal tissue sections adjacent to the tumor
obtained from the same RCC donors used in (A).
[0061] FIGS. 9A and B: Expression of CD70 on renal cell carcinoma
cell lines (RCC). CD70 expression on the surface of a panel of RCC
was determined by flow cytometry. (A) The staining profiles of an
anti-CD70 (open curves) and a control IgG (closed curves) on four
representative RCC cell lines 786-O, Caki-1, Caki-2, and Hs 835.T
and two normal renal tubule epithelial lines RPTEC and HRCE are
shown. (B) CD70 expression on additional RCC cell lines (CAL54,
SK-RC-6, SK-RC-7, 786-O, A498, and ACHN) is compared to cell lines
shown in (A). Mean fluorescence intensity of anti-CD70 staining and
background fluorescence are plotted. Numbers on top of each bar
indicate either mean fluorescence intensity obtained from anti-CD70
binding or background fluorescence.
[0062] FIG. 10: Growth inhibitory and cytotoxicity activities of
1F6-vcAFP. 786-O or Caki-1 cells were incubated with graded doses
of 1F6-vcAFP or the non-binding control IgG-vcAFP. Cells were
incubated for a total of 96 hours. .sup.3H-thymidine incorporation
(upper panel) and reduction of alamarBlue (lower panel) were used
to determine proliferation and cell viability, respectively. Data
points are expressed as percentages of the untreated control. Error
bars represent the standard deviations of values obtained from
quadruplicate wells.
[0063] FIG. 11: Effects of anti-CD70 ADCs on the proliferation of
RCC cell lines. Dose-response curves on growth inhibitory or
cytotoxicity of 1F6 ADCs were determined as described in FIG. 10 to
obtain IC.sub.50 values. The IC.sub.50s of 1F6-vcMMAE, 1F6-vcMMAF,
1F6-vcAFP, or two control non-binding IgG (IgG) ADCs on the
proliferation of CD70.sup.+ RCC lines (Caki-1, Caki-2, 786-O,
769-P, SK-RC-6, SK-RC-7, ACHN, CAL54, and A498), a CD70.sup.- RCC
line (Hs 835.T), and normal kidney tubule epithelial cell lines
(RPTEC and HRCE) are summarized. Effects of 1F6-vcMMAF and
1F6-vcAFP on the viability of a subset of the cell lines are also
summarized.
[0064] FIG. 12: In vivo efficacy of 1F6-vcAFP in a RCC model.
Tumors were initiated in nude mice by implanting tissue tumor
blocks (30 mm.sup.3 in size) containing Caki-1 cells. Treatment
began when average tumor size within a treatment group was around
100 mm.sup.3. In the upper panel, treatment for the IgG and 1F6
groups began 17 days post implantation (filled arrow), while
treatment for the 1F6-vcAFP and IgG-vcAFP groups began 25 days post
implantation (open arrow). Indicated doses of either unconjugated
mAbs or their ADCs were given for one course on a q4d.times.4
schedule. The lower panel shows results from a repeat experiment
when treatment began 14 days (filled arrow) after tumor
implantation with 3 mg/kg and a q4d.times.4 schedule of 1F6-vcAFP
or IgG-vcAFP.
[0065] FIG. 13: Activation-induced CD70 expression on a T cell
clone. A representative T cell clone C1A was activated by
phytohemaggutinin A-L (PHA-L), irradiated feeder cells, IL-2, and
IL-4. CD25 (left panel) and CD70 (right panel) expression on days
0, 2, 4, and 8 were determined by flow cytometric analysis.
[0066] FIG. 14: Effects of anti-CD70 ADCs on the proliferation of
activated T cell clones. Resting T cell clones C2A, 40D8, and
4.01.1 were induced to express CD70 as described. At the peak of
CD70 expression (2 days after activation), cells were harvested and
treated with graded doses of 1F6-vcMMAE, 1F6-vcAFP, or the
corresponding non-binding control IgG (IgG) ADCs as indicated in
the figure in the presence of rhIL-2 and rhIL-4. Cells were exposed
to the ADCs continuously for an additional 72 hours. T cell
proliferation was determined by pulsing with .sup.3H-TdR during the
last 16 hours of incubation.
[0067] FIG. 15: Effects of anti-CD70 ADCs on mixed lymphocyte
reaction. An MLR was set up between PBMCs and irradiated allogeneic
CESS cells. Expression of CD70 on activated allo-reactive T cells
is shown on the upper panel. Binding of anti-CD70 mAb and control
IgG is indicated by the open and closed curves, respectively.
Graded doses of either 1F6-vcMMAE or 1F6-vcAFP were added to the
culture at initiation. Cultures were exposed to the ADCs for a
total of 120 hours. T cell proliferation was determined by pulsing
with .sup.3H-thymidine during the last 16 hours of incubation
(lower panel).
[0068] FIG. 16: Effects of anti-CD70 or anti-CD70 ADC on
antigen-induced T cell proliferation. T cells enriched for tetanus
toxoid reactivity were stimulated with autologous dendritic cells
and tetanus toxoid in the presence of graded doses of 1F6,
1F6-vcMMAE, or 1F6-vcAFP for a total of 96 hours. T cell
proliferation was determined by pulsing with .sup.3H-thymidine
during the last 16 hours of incubation.
[0069] FIG. 17: CD70 induction during antigen-specific T cell
expansion. PBMCs from a normal HLA-A0201 donor were stimulated with
the M1 peptide derived from the influenza virus matrix protein. (A)
Expansion of M1 peptide-specific CD8.sup.+ cells expressing TCR
V.beta.17 chain was followed by flow cytometry (upper panels). The
lower left panel shows the percentage of CD8.sup.+/V.beta.17.sup.+
cells expressing CD70 and the lower right panel shows the intensity
of CD70 expression on the expanding CD8.sup.+/V.beta.17.sup.+
cells. (B) A representative example of specific CD70 induction on
the expanding CD8.sup.+/V.beta.17.sup.+ after stimulation with the
M1 peptide for five days. Binding of the control IgG (open curves)
and anti-CD70 mAb (closed curves) on the CD8.sup.+/V.beta.17.sup.-
or CD8.sup.+/V.beta.17.sup.+ cells are shown.
[0070] FIG. 18: Specific deletion of antigen-specific
CD8.sup.+/V.beta.17.sup.+ T cells by anti-CD70 ADCs. PBMCs from a
normal HLA-A0201 donor were stimulated with the M1 peptide for 5
days. 1F6 ADCs or control non-binding IgG (cIgG) ADCs were added to
cells to a final concentration of 1 .mu.g/ml. Total viable cell
counts were conducted at 24, 48, and 76 hours post drug addition.
The percentage of CD8.sup.+/V.beta.17.sup.+ in each culture
condition was determined by flow cytometry as shown in FIG. 17.
Total numbers of CD8.sup.+/V.beta.17.sup.+ for each culture
condition are calculated (upper panel). The numbers of
CD8.sup.+/V.beta.17.sup.+ and CD8.sup.+/V.beta.17.sup.- cells in
the 1F6-ADC treated cultures are expressed as percentages of the
untreated control cultures (lower panel).
[0071] FIG. 19: Dose-response comparison of anti-CD70 ADCs on
deletion of antigen-specific CD8.sup.+/V.beta.17.sup.+ cells. PBMCs
from a normal HLA-A0201 donor were stimulated with the M1 peptide
for 5 days. 1F6 ADCs or control non-binding IgG (cIgG) ADCs were
given to cells to the specified final concentrations. The
percentages of CD8.sup.+/V.beta.17.sup.+ cells 4 days post ADC
addition was determined by flow cytometry as in FIG. 17.
[0072] FIG. 20: Limited effects of 1F6 ADCs on the proliferation
capacity of the CD70.sup.- bystander T cells. PBMCs from a normal
HLA-A0201 donor were stimulated with the M1 peptide for 5 days.
Cells were either allowed to expand or treated with 1 .mu.g/ml
1F6-vcMMAF for an additional 4 days. Percentages of
CD8.sup.+/V.beta.17.sup.+ cells were determined by flow cytometry
(left column). In order to assess the proliferation capacity of the
CD70.sup.- bystander, antigen non-specific T cells, V.beta.17.sup.+
T cells were eliminated from the cultures by immuno-depletion
(center column). The resulting V.beta.17.sup.- cells were then
stimulated with graded doses of immobilized anti-CD3 and anti-CD28
mAb for a total of 96 hours, and DNA synthesis was detected by
pulsing with .sup.3H-TdR during the last 18 hours of
incubation.
[0073] FIG. 21: Mouse Xenograft model of Renal Cell Carcinoma. (A)
Subcutaneous 786-O tumors were initiated in nude mice by implanting
tumor fragments (N=5 or 6/group) of approximately 30 mm.sup.3.
Tumor growth was allowed to establish and treatment began when
average tumor size within each group was approximately 100
mm.sup.3. h1F6-mcMMAF4 or h1F6-vcMMAE4 at the indicated doses was
administered at a q4d.times.4 schedule beginning on day 17 after
tumor implantation, as indicated by the arrows. Cross-strikes
indicate when animals with tumors >1000 mm.sup.3 were
euthanized. (B) 786-O tumor implantation and treatment initiation
are the same as given in (A). Groups of mice (N=5-7) were
administered with h1F6-mcMMAF4 or h1F6-vcMMAE4 at 0.17 mg/kg at a
q4d.times.4 or q4d.times.10 schedule beginning on day 13 after
tumor implantation. Tumor growth is represented by Kaplan-Meier
plots. An event was registered when a mouse with a tumor quadrupled
in size compared to day 13 when treatment began. Mice with tumor
that did not quadruple in size at the end of the experiment on day
43 were censored. The log-rank test was used to generate p values
between treatment groups and the untreated group.
[0074] FIG. 22: Mouse Xenograft Model of Multiple Myeloma. (A) Ten
million MM-1S cells were injected intravenously into each SCID
mouse. Groups of mice (N=8-10) were left untreated, received
IgG-vcMMAF4, IgG-mcMMAF4, h1F6-vcMMAF4, or h1F6-mcMMAF4 at the
specified doses on a q7d.times.5 schedule, as indicated by the
arrows. Mice showing symptoms of hind limb paralysis, hunched
posture, cranial swelling, and/or scruffy coat were euthanized, and
the percent survival of each group was plotted. The log-rank test
was used to generate p values between treatment groups and the
control groups. (B) Bone marrow cells were recovered from the
femurs of euthanized mice due to the above disease symptoms or on
day 122 post tumor cell implantation when the experiment was
terminated. The percentage of CD138-expressing MM-1S cells in the
femers of each mouse was determined by flow cytometry. The
Mann-Whitney test was used to derived p values between the
indicated groups.
[0075] FIG. 23: Mouse Xenograft model of Multiple Myeloma. (A) Ten
million L363 cells were injected intravenously into each SCID
mouse. Groups of mice (N=7) were left untreated, received
IgG-vcMMAF4, or h1F6-vcMMAF4 at the specified doses on a
q7d.times.5 schedule as indicated by the arrows. Mice showing
palpable tumor masses were euthanized, and the percent survival of
each group was plotted. The log-rank test was used to generate the
p value between the treated group and the untreated group. (B)
Serum samples were obtained from mice 40 days after tumor implant.
The concentration of human .lamda. light chain in the serum of each
mice was determined by ELISA. The Mann-Whitney test was used to
derived p values between the indicated groups.
DETAILED DESCRIPTION OF THE INVENTION
[0076] Provided are methods and compositions relating to the use of
antibody-drug conjugates (ADCs) and ADC derivatives that bind to
CD70. A drug conjugated to the antibody exerts a cytotoxic,
cytostatic, or immunosuppressive effect on CD70-expressing cells to
treat or immunological disorders or CD70-expressing cancers. In one
aspect, the methods and compositions relate to antibodies and
derivatives thereof that compete with monoclonal antibody 1F6 or
2F2 for binding to CD70 and that, when conjugated to a cytotoxic
agent, exert a cytotoxic or cytostatic effect to deplete or inhibit
the proliferation of CD70-expressing cells. Antibodies to CD70
which can be used in accordance with the methods and compositions
described herein include monoclonal antibodies as well as chimeric,
humanized, or human antibodies (e.g., a humanized or chimeric form
of 1F6 or 2F2), and such antibodies conjugated to cytotoxic or
immunosuppressive agents such as, for example, chemotherapeutic
drugs.
[0077] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections which follow.
I. Definitions and Abbreviations
[0078] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art pertinent to the methods and compositions
described. As used herein, the following terms and phrases have the
meanings ascribed to them unless specified otherwise.
[0079] The term "inhibit" or "inhibition of" as used herein means
to a reduce by a measurable amount, or to prevent entirely.
[0080] The term "agent" as used herein means an element, compound,
or molecular entity, including, e.g., a pharmaceutical,
therapeutic, or pharmacologic compound. Agents can be natural or
synthetic or a combination thereof. A "therapeutic agent" is an
agent that exerts a therapeutic (e.g., beneficial) effect on cancer
cells or activated immune cells, either alone or in combination
with another agent (e.g., a prodrug converting enzyme in
combination with a prodrug). Typically, therapeutic agents useful
in accordance with the methods and compositions described herein
are those that exert a cytotoxic, cytostatic, or immunosuppressive
effect.
[0081] "Cytotoxic effect," in reference to the effect of an agent
on a cell, means killing of the cell. "Cytostatic effect" means an
inhibition of cell proliferation. A "cytotoxic agent" means an
agent that has a cytotoxic or cytostatic effect on a cell, thereby
depleting or inhibiting the growth of, respectively, cells within a
cell population.
[0082] The term "deplete," in the context of the effect of a
CD70-targeting moiety-drug conjugate on CD70-expressing cells,
refers to a reduction or elimination of the CD70-expressing
cells.
[0083] The term "immunosuppressive agent" as used herein means an
agent that inhibits the development or maintenance of an
immunologic response. Such inhibition by an immunosuppressive agent
can be effected by, for example, elimination of immune cells (e.g.,
T or B lymphocytes); induction or generation of immune cells that
can modulate (e.g., down-regulate) the functional capacity of other
cells; induction of an unresponsive state in immune cells (e.g.,
anergy); or increasing, decreasing or changing the activity or
function of immune cells, including, for example, altering the
pattern of proteins expressed by these cells (e.g., altered
production and/or secretion of certain clases of molecules such as
cytokines, chemokines, growth factors, transcription factors,
kinases, costimulatory molecules or other cell surface receptors,
and the like). In typical embodiments, an immunosuppressive agent
has a cytotoxic or cytostatic effect on an immune cell that
promotes an immune response.
[0084] "Immune cell" as used herein means any cell of hematopoietic
lineage involved in regulating an immune response against an
antigen (e.g., an autoantigen). In typical embodiments, an immune
cell is a T lymphocyte, a B lymphocyte, or a dendritic cell.
[0085] The term "polypeptide" refers to a polymer of amino acids
and its equivalent and does not refer to a specific length of a
product; thus, "peptides" and "proteins" are included within the
definition of a polypeptide. Also included within the definition of
polypeptides are "antibodies" as defined herein. A "polypeptide
region" refers to a segment of a polypeptide, which segment may
contain, for example, one or more domains or motifs (e.g., a
polypeptide region of an antibody can contain, for example, one or
more CDRs). The term "fragment" refers to a portion of a
polypeptide typically having at least 20 contiguous or at least 50
contiguous amino acids of the polypeptide. A "derivative" includes
a polypeptide or fragment thereof having conservative amino acid
substitutions relative to a second polypeptide; or a polypeptide or
fragment thereof that is modified by covalent attachment of a
second molecule such as, e.g., by attachment of a heterologous
polypeptide, or by glycosylation, acetylation, phosphorylation, and
the like. Further included within the definition of "polypeptide"
are, for example, polypeptides containing one or more analogs of an
amino acid (e.g., unnatural amino acids and the like), polypeptides
with unsubstituted linkages, as well as other modifications known
in the art, both naturally and non-naturally occurring.
[0086] The term "antibody" as used herein refers to (a)
immunoglobulin polypeptides and immunologically active portions of
immunoglobulin polypeptides, i.e., polypeptides of the
immunoglobulin family, or fragments thereof, that contain an
antigen binding site that immunospecifically binds to a specific
antigen (e.g., CD70), or (b) conservatively substituted derivatives
of such immunoglobulin polypeptides or fragments that
immunospecifically bind to the antigen (e.g., CD70). Antibodies are
generally described in, for example, Harlow & Lane, Antibodies:
A Laboratory Manual (Cold Spring Harbor Laboratory Press,
1988).
[0087] In the context of immunoglobulin polypeptides or fragments
thereof as defined above, "conservative substitution" means one or
more amino acid substitutions that do not substantially reduce
specific binding (e.g., as measured by the K.sub.D) of the
immunoglobulin polypeptide or fragment thereof to an antigen (i.e.,
substitutions that increase binding, that do not significantly
alter binding, or that reduce binding by no more than about 40%,
typically no more than about 30%, more typically no more than about
20%, even more typically no more than about 10%, or most typically
no more than about 5%, as determined by standard binding assays
such as, e.g., ELISA).
[0088] An "antibody derivative" as used herein means an antibody,
as defined above, that is modified by covalent attachment of a
heterologous molecule such as, e.g., by attachment of a
heterologous polypeptide, or by glycosylation, acetylation or
phosphorylation not normally associated with the antibody, and the
like.
[0089] The term "monoclonal antibody" refers to an antibody that is
derived from a single cell clone, including any eukaryotic or
prokaryotic cell clone, or a phage clone, and not the method by
which it is produced. Thus, the term "monoclonal antibody" as used
herein is not limited to antibodies produced through hybridoma
technology.
[0090] The term "heterologous," in the context of a polypeptide,
means from a different source (e.g., a cell, tissue, organism, or
species) as compared with another polypeptide, so that the two
polypeptides are different. Typically, a heterologous polypeptide
is from a different species.
[0091] As used herein, the term "functional," in the context of an
anti-CD70 antibody or derivative thereof to be used in accordance
with the methods described herein, indicates that the antibody or
derivative thereof is (1) capable of binding to CD70 and (2)
depletes or inhibits the proliferation of CD70-expressing cells
when conjugated to a cytotoxic agent, or has an immunosuppressive
effect on an immune cell when conjugated to an immunosuppressive
agent.
[0092] The terms "identical" or "percent identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of nucleotides or amino acid residues that are
the same, when compared and aligned for maximum correspondence. To
determine the percent identity, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % identity=# of
identical positions/total # of positions (e.g., overlapping
positions).times.100). In certain embodiments, the two sequences
are the same length.
[0093] The term "substantially identical," in the context of two
nucleic acids or polypeptides, refers to two or more sequences or
subsequences that have at least 50%, at least 55%, at least 60%, or
at least 65% identity; typically at least 70% or at least 75%
identity; more typically at least 80% or at least 85% identity; and
even more typically at least 90%, at least 95%, or at least 98%
identity (as determined using one of the methods set forth
infra).
[0094] "Similarity" or "percent similarity" in the context of two
or more polypeptide sequences, refer to two or more sequences or
subsequences that have a specified percentage of amino acid
residues that are the same or conservatively substituted when
compared and aligned for maximum correspondence, as measured using
one of the methods set forth infra. By way of example, a first
amino acid sequence can be considered similar to a second amino
acid sequence when the first amino acid sequence is at least 50%,
60%, 70%, 75%, 80%, 90%, or even 95% identical, or conservatively
substituted, to the second amino acid sequence when compared to an
equal number of amino acids as the number contained in the first
sequence, or when compared to an alignment of polypeptides that has
been aligned by a computer similarity program known in the art (see
infra).
[0095] The terms "substantial similarity" or "substantial
similarity," in the context of polypeptide sequences, indicates
that a polypeptide region has a sequence with at least 70%,
typically at least 80%, more typically at least 85%, and even more
typically at least 90% or at least 95% sequence similarity to a
reference sequence. For example, a polypeptide is substantially
similar to a second polypeptide, for example, where the two
peptides differ by one or more conservative substitutions.
[0096] In the context of anti-CD70 antibodies or derivatives
thereof, a protein that has one or more polypeptide regions
substantially identical or substantially similar to one or more
antigen-binding regions (e.g., a heavy or light chain variable
region, or a heavy or light chain CDR) of an anti-CD70 antibody
retains specific binding to an epitope of CD70 recognized by the
anti-CD70 antibody, as determined using any of various standard
immunoassays known in the art or as referred to herein.
[0097] The determination of percent identity or percent similarity
between two sequences can be accomplished using a mathematical
algorithm. A preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of two sequences is the
algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA
87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl.
Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into
the NBLAST and XBLAST programs of Altschul, et al., 1990, J. Mol.
Biol. 215:403-410. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to a nucleic acid encoding a protein of
interest. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to protein of interest. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.
Alternatively, PSI-Blast can be used to perform an iterated search
which detects distant relationships between molecules (Id.). When
utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can
be used. (See, e.g., the NCBI internet web site.) Another
preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algorithm of Myers
and Miller, CABIOS (1989). Such an algorithm is incorporated into
the ALIGN program (version 2.0) which is part of the GCG sequence
alignment software package. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4 can be used.
Additional algorithms for sequence analysis are known in the art
and include ADVANCE and ADAM as described in Torellis and Robotti,
1994, Comput. Appl. Biosci. 10:3-5; and FASTA described in Pearson
and Lipman, 1988, Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA,
ktup is a control option that sets the sensitivity and speed of the
search. If ktup=2, similar regions in the two sequences being
compared are found by looking at pairs of aligned residues; if
ktup=1, single aligned amino acids are examined. ktup can be set to
2 or 1 for protein sequences, or from 1 to 6 for DNA sequences. The
default if ktup is not specified is 2 for proteins and 6 for DNA.
For a further description of FASTA parameters, see, e.g., the
documentation at the Pasteur Institute web site, the contents of
which are incorporated herein by reference.
[0098] Alternatively, protein sequence alignment may be carried out
using the CLUSTAL W algorithm, as described by Higgins et al.,
1996, Methods Enzymol. 266:383-402.
[0099] As used herein, the terms "prevention" and "prevent" refer
to administration of an anti-CD70 antibody-drug conjugate (ADC) or
ADC derivative to a subject before the onset of a clinical or
diagnostic symptom of a CD70-expressing cancer or immunological
disorder (e.g., administration to an individual with a
predisposition or at a high risk of acquiring the CD70-expressing
cancer or immunological disorder) to (a) block the occurrence or
onset of the CD70-expressing cancer or immunological disorder, or
one or more of clinical or diagnostic symptoms thereof, (b) inhibit
the severity of onset of the CD70-expressing cancer or
immunological disorder, or (c) to lessen the likelihood of the
onset of the CD70-expressing cancer or immunological disorder.
[0100] As used herein, the terms "treatment" or "treat" refer to
slowing, stopping, or reversing the progression of a
CD70-expressing cancer or immunological disorder in a subject, as
evidenced by a decrease or elimination of a clinical or diagnostic
symptom of the disease, by administration of an anti-CD70 ADC or
ADC derivative to the subject after the onset of the clinical or
diagnostic symptom of the CD70-expressing cancer or immunological
disorder at any clinical stage. Treatment can include, for example,
a decrease in the severity of a symptom, the number of symptoms, or
frequency of relapse.
[0101] The term "pharmaceutically acceptable" as used herein means
approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, and more particularly
in humans. The term "pharmaceutically compatible ingredient" refers
to a pharmaceutically acceptable diluent, adjuvant, excipient, or
vehicle with which a CD70-targeting moiety-drug conjugate is
administered.
[0102] The term "effective amount," in the context of the
administration of an anti-CD70 ADC or ADC derivative, refers to the
amount of the ADC or ADC derivative that is sufficient to inhibit
the occurrence or ameliorate one or more clinical or diagnostic
symptoms of a CD70-expressing cancer or immunological disorder in a
subject. An effective amount of an agent is administered according
to the methods described herein in an "effective regime." The term
"effective regime" refers to a combination of amount of the agent
and dosage frequency adequate to accomplish treatment or prevention
of a CD70-expressing cancer or immunological disorder.
[0103] The abbreviation "AFP" refers to
dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylened-
iamine (see Formula XVI infra).
[0104] The abbreviation "MMAE" refers to monomethyl auristatin E
(see Formula XI infra).
[0105] The abbreviation "AEB" refers to an ester produced by
reacting auristatin E with paraacetyl benzoic acid (see Formula XX
infra)
[0106] The abbreviation "AEVB" refers to an ester produced by
reacting auristatin E with benzoylvaleric acid (see Formula XXI
infra).
[0107] The abbreviation "MMAF" refers to
dovaline-valine-dolaisoleuine-dolaproine-phenylalanine (see Formula
IVIV infra).
[0108] The abbreviations "fk" and "phe-lys" refer to the linker
phenylalanine-lysine.
[0109] The abbreviations "vc" and "val-cit" refer to the linker
valine-citrulline.
II. Anti-CD70 Antibodies and Derivatives Thereof
[0110] The methods and compositions described herein encompass the
use of antibodies, or a derivative thereof, as ADCs that (a)
specifically bind to CD70 and (b) when conjugated to a therapeutic
agent, the therapeutic agent exerts a cytotoxic, cytostatic or
immunosuppressive effect on CD70-expressing cancer cells or
activated immune cells. As used herein, the term "derivative," in
the context of an anti-CD70 antibody, refers to a molecule that (i)
has an antigen-binding region of an anti-CD70 antibody, or a region
derived therefrom (e.g., by conservative substitution), and at
least one polypeptide region or other moiety heterologous to the
anti-CD70 antibody, and (ii) specifically binds to CD70 via the
antigen-binding region or region derived therefrom. In specific
embodiments, the anti-CD70 antibody is mAb 1F6 or 2F2 or a
derivative thereof. In certain aspects, the anti-CD70 antibody or
derivative thereof competes with monoclonal antibody 1F6 or 2F2 for
binding to CD70.
[0111] In typical embodiments, the anti-CD70 antibody or derivative
thereof, when conjugated to a cytotoxic agent, exerts a cytotoxic
or cytostatic effect on CD70-expressing cancer cells, or, when
conjugated to a cytotoxic or immunosuppressive agent, exerts a
cytotoxic, cytostatic, or immunosuppressive effect on activated
lymphocytes or dendritic cells, for the treatment of a
CD70-expressing cancer or an immunological disorder, respectively,
in a subject (e.g., the extracellular domain of human CD70).
[0112] Anti-CD70 antibodies suitable for use in accordance with the
present compositions and methods are typically monoclonal and can
include, for example, chimeric (e.g., having a human constant
region and mouse variable region), humanized, or human antibodies;
single chain antibodies; or the like. The immunoglobulin molecules
can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class
(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of
immunoglobulin molecule.
[0113] In certain embodiments, the antibody is an antigen-binding
antibody fragment such as, for example, a Fab, a F(ab'), a
F(ab').sub.2, a Fd chain, a single-chain Fv (scFv), a single-chain
antibody, a disulfide-linked Fv (sdFv), a fragment comprising
either a V.sub.L or V.sub.H domain, or fragments produced by a Fab
expression library, or a CD70-binding fragments of any of the above
antibodies described supra. Antigen-binding antibody fragments,
including single-chain antibodies, can comprise the variable
region(s) alone or in combination with the entirety or a portion of
the following: hinge region, C.sub.H1, C.sub.H2, C.sub.H3 and
C.sub.L domains. Also, antigen-binding fragments can comprise any
combination of variable region(s) with a hinge region, C.sub.H1,
C.sub.H2, C.sub.H3 and C.sub.L domains. Typically, the antibodies
are human, rodent (e.g., mouse and rat), donkey, sheep, rabbit,
goat, guinea pig, camelid, horse, or chicken. As used herein,
"human" antibodies include antibodies having the amino acid
sequence of a human immunoglobulin and include antibodies isolated
from human immunoglobulin libraries, from human B cells, or from
animals transgenic for one or more human immunoglobulin, as
described infra and, for example in U.S. Pat. Nos. 5,939,598 and
6,111,166.
[0114] The antibodies may be monospecific, bispecific, trispecific,
or of greater multispecificity. Multispecific antibodies may be
specific for different epitopes of CD70 or may be specific for both
CD70 as well as for a heterologous protein. (See, e.g., PCT
publications WO 93/17715; WO 92/08802; WO 91/00360; and WO
92/05793; Tutt et al., 1991, J Immunol 147:60-69; U.S. Pat. Nos.
4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819; Kostelny
et al., 1992, J Immunol 148:1547-1553.) Multispecific antibodies,
including bispecific and trispecific antibodies, useful for
practicing the methods described herein are antibodies that
immunospecifically bind to both CD70 (including but not limited to
antibodies that have the CDRs and/or heavy chains of the monoclonal
antibodies 2F2 and 1F6) and a second cell surface receptor or
receptor complex, such as an immunoglobulin gene superfamily
member, a TNF receptor superfamily member, an integrin, a cytokine
receptor, a chemokine receptor, a major histocompatibility protein,
a lectin (C-type, S-type, or I-type), or a complement control
protein. In a typical embodiment, the binding of the portion of the
multispecific antibody to the second cell surface molecule or
receptor complex enhances the cytotoxic or cytostatic effect of an
anti-CD70 antibody-drug conjugate.
[0115] In certain specific embodiments, the anti-CD70 antibody is
agonistic, non-agonistic or antagonistic with respective to CD70.
In another specific embodiment, the anti-CD70 antibody does not
block binding of CD70 ligand to CD70. In yet another embodiment,
the anti-CD70 antibody or derivative thereof is a blocking antibody
(i.e., an antibody that blocks the binding of CD27 to CD70).
[0116] In one aspect, an anti-CD70 antibody comprises one or more
complementarity determining regions (CDRs) substantially identical
or substantially similar to one or more CDR(s) of monoclonal
antibody 1F6 (see Table 1). For example, the antibody can include a
heavy chain CDR and/or a light chain CDR that is substantially
identical or substantially similar to a corresponding heavy chain
CDR (H1, H2, or H3 regions) or corresponding light chain CDR (L1,
L2, or L3 regions) of mAb 1F6 (SEQ ID NO:6; SEQ ID NO:8; SEQ ID
NO:10; SEQ ID NO:16; SEQ ID NO:18; or SEQ ID NO:20, respectively).
In typical embodiments, the anti-CD70 antibody has two or three
heavy chain CDRs and/or two or three light chain CDRs that are
substantially identical or substantially similar to corresponding
heavy and/or light chain CDRs of mAb 1F6. In specific embodiments,
a CDR substantially identical or substantially similar to a heavy
or light chain CDR of 1F6 has the amino acid sequence set forth in
SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:18,
or SEQ ID NO:20.
[0117] For example, in certain embodiments, where an anti-CD70
antibody has at least one heavy chain CDR substantially identical
or substantially similar to a heavy chain CDR of mAb 1F6, the
antibody or derivative thereof further includes at least one light
chain CDR that is substantially identical or substantially similar
to a light chain CDR of mAb 1F6.
[0118] In certain typical embodiments, an anti-CD70 antibody
includes a heavy or light chain variable domain, the variable
domain having (a) a set of three CDRs substantially identical or
substantially similar to corresponding CDRs of mAb 1F6, and (b) a
set of four framework regions. For example, an anti-CD70 antibody
can include a heavy or light chain variable domain, the variable
domain having (a) a set of three CDRs, in which the set of CDRs are
from monoclonal antibody 1F6, and (b) a set of four framework
regions, in which the set of framework regions are identical to or
different from the set of framework regions in mAb 1F6.
[0119] In a specific embodiment, the anti-CD70 antibody includes a
heavy chain variable region that is substantially identical or
substantially similar to the heavy chain variable region of mAb 1F6
(i.e., substantially identical or substantially similar to the
amino acid sequences set forth in SEQ ID NO:2, see Table 1) and/or
a light chain variable region that is substantially identical or
substantially similar to the light chain variable regions of mAb
1F6 (i.e., substantially identical or substantially similar to the
amino acid sequences set forth in SEQ ID NO:12, see Table 1). For
example, the antibody can include a heavy chain variable region
having the amino acid sequence set forth in SEQ ID NO:2 and,
optionally, can further include a light chain variable region
having the amino acid sequence set forth in SEQ ID NO:12. In one
exemplary embodiment, the anti-CD70 antibody is mAb 1F6.
[0120] In another aspect, an anti-CD70 antibody comprises one or
more CDRs substantially identical or substantially similar to one
or more CDR(s) of monoclonal antibody 2F2 (see Table 1). For
example, the antibody can include a heavy chain CDR and/or a light
chain CDR that is substantially identical or substantially similar
to a corresponding heavy chain CDR (H1, H2, or H3 regions) or
corresponding light chain CDR (L1, L2, or L3 regions) of mAb 2F2
(SEQ ID NO:26, SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:36, SEQ ID
NO:38 or SEQ ID NO:40). In typical embodiments, the anti-CD70
antibody has two or three heavy chain CDRs and/or two or three
light chain CDRs that are substantially identical or substantially
similar to corresponding heavy and/or light chain CDRs of mAb 2F2.
In specific embodiments, a CDR substantially identical or
substantially similar to a heavy or light chain CDR of 2F2 has the
amino acid sequence set forth in SEQ ID NO:26, SEQ ID NO:28, SEQ ID
NO:30; SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40.
[0121] For example, in certain embodiments, where an anti-CD70
antibody has at least one heavy chain CDR substantially identical
or substantially similar to a heavy chain CDR of mAb 2F2, the
antibody or derivative thereof further includes at least one light
chain CDR that is substantially identical or substantially similar
to a light chain CDR of mAb 2F2.
[0122] In certain typical embodiments, an anti-CD70 antibody
includes a heavy or light chain variable domain, the variable
domain having (a) a set of three CDRs substantially identical or
substantially similar to corresponding CDRs of mAb 2F2, and (b) a
set of four framework regions. For example, an anti-CD70 antibody
can include a heavy or light chain variable domain, the variable
domain having (a) a set of three CDRs, in which the set of CDRs are
from monoclonal antibody 2F2, and (b) a set of four framework
regions, in which the set of framework regions is identical to or
different from the set of framework regions in mAb 2F2.
[0123] In a specific embodiment, the anti-CD70 antibody includes a
heavy chain variable region that is substantially identical or
substantially similar to the heavy chain variable region of mAb 2F2
(i.e., substantially identical or substantially similar to the
amino acid sequences set forth in SEQ ID NO:22, see Table 1) and/or
a light chain variable region that is substantially identical or
substantially similar to the light chain variable regions of mAb
2F2 (i.e., substantially identical or substantially similar to the
amino acid sequences set forth in SEQ ID NO:32, see Table 1). For
example, the antibody can include a heavy chain variable region
having the amino acid sequence set forth in SEQ ID NO:22 and,
optionally, can further include a light chain variable region
having the amino acid sequence set forth in SEQ ID NO:32. In one
exemplary embodiment, the anti-CD70 antibody is mAb 2F2.
[0124] A table indicating the region of 1F6 or 2F2 to which each
SEQ ID NO. corresponds is provided below: TABLE-US-00001 TABLE 1
NUCLEOTIDE MOLECULE OR AMINO ACID SEQ ID NO 1F6 Heavy Chain
Variable Region Nucleotide 1 1F6 Heavy Chain Variable Region Amino
Acid 2 1F6 Heavy Chain Signal Peptide Nucleotide 3 1F6 Heavy Chain
Signal Peptide Amino Acid 4 1F6 Heavy Chain-CDR1(H1) Nucleotide 5
1F6 Heavy Chain-CDR1(H1) Amino Acid 6 1F6 Heavy Chain-CDR2(H2)
Nucleotide 7 1F6 Heavy Chain-CDR2(H2) Amino Acid 8 1F6 Heavy
Chain-CDR3(H3) Nucleotide 9 1F6 Heavy Chain-CDR3(H3) Amino Acid 10
1F6 Light Chain Variable Region Nucleotide 11 1F6 Light Chain
Variable Region Amino Acid 12 1F6 Light Chain Signal Peptide
Nucleotide 13 1F6 Light Chain Signal Peptide Amino Acid 14 1F6
Light Chain-CDR1(L1) Nucleotide 15 1F6 Light Chain-CDR1(L1) Amino
Acid 16 1F6 Light Chain-CDR2(L2) Nucleotide 17 1F6 Light
Chain-CDR2(L2) Amino Acid 18 1F6 Light Chain-CDR3(L3) Nucleotide 19
1F6 Light Chain-CDR3(L3) Amino Acid 20 2F2 Heavy Chain Variable
Region Nucleotide 21 2F2 Heavy Chain Variable Region Amino Acid 22
2F2 Heavy Chain Signal Peptide Nucleotide 23 2F2 Heavy Chain Signal
Peptide Amino Acid 24 2F2 Heavy Chain-CDR1(H1) Nucleotide 25 2F2
Heavy Chain-CDR1(H1) Amino Acid 26 2F2 Heavy Chain-CDR2(H2)
Nucleotide 27 2F2 Heavy Chain-CDR2(H2) Amino Acid 28 2F2 Heavy
Chain-CDR3(H3) Nucleotide 29 2F2 Heavy Chain-CDR3(H3) Amino Acid 30
2F2 Light Chain Variable Region Nucleotide 31 2F2 Light Chain
Variable Region Amino Acid 32 2F2 Light Chain Signal Peptide
Nucleotide 33 2F2 Light Chain Signal Peptide Amino Acid 34 2F2
Light Chain-CDR1(L1) Nucleotide 35 2F2 Light Chain-CDR1(L1) Amino
Acid 36 2F2 Light Chain-CDR2(L2) Nucleotide 37 2F2 Light
Chain-CDR2(L2) Amino Acid 38 2F2 Light Chain-CDR3(L3) Nucleotide 39
2F2 Light Chain-CDR3(L3) Amino Acid 40
[0125] Anti-CD70 antibodies useful in the methods set forth herein
may also be described or specified in terms of their binding
affinity to CD70. Typical binding affinities include those with a
dissociation constant or Kd less than 5.times.10.sup.-2 M,
10.sup.-2 M, 5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M,
10.sup.-4 M, 5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M,
10.sup.-6 M, 5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M,
10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10
M, 10.sup.-10 M, 5.times.10.sup.-11 M, 10.sup.-11 M,
5.times.10.sup.-12 M, 10.sup.-12 M, 5.times.10.sup.-13 M,
10.sup.-13 M, 5.times.10.sup.-14 M, 10.sup.-14 M,
5.times.10.sup.-15 M, or 10.sup.-15 M.
[0126] The antibodies that may be used in the treatment of
immunological disorders or CD70-expressing cancers can be generated
by any suitable method known in the art. For example, monoclonal
antibodies can be prepared using a wide variety of techniques
including, e.g., the use of hybridoma, recombinant, and phage
display technologies, or a combination thereof. Hybridoma
techniques are generally discussed in, for example, Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed., 1988); and Hammerling, et al., In Monoclonal
Antibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier, N.Y.,
1981). Examples of phage display methods that can be used to make
the anti-CD70 antibodies include, e.g., those disclosed in Brinkman
et al., 1995, J Immunol Methods 182:41-50; Ames et al., 1995, J
Immunol Methods 184:177-186; Kettleborough et al., 1994, Eur J
Immunol 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et
al., 1994, Advances in Immunology 57:191-280; PCT Application No.
PCT/GB91/01134; PCT Publications WO 90/02809; WO 91/10737; WO
92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and
U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108 (the disclosures of
which are incorporated by reference herein).
[0127] Techniques for generating antibody fragments that recognize
specific epitopes are also generally known in the art. For example,
Fab and F(ab').sub.2 fragments can be produced by proteolytic
cleavage of immunoglobulin molecules, using enzymes such as papain
(to produce Fab fragments) or pepsin (to produce F(ab')2
fragments). F(ab')2 fragments contain the variable region, the
light chain constant region and the C.sub.H1 domain of the heavy
chain. Techniques to recombinantly produce Fab, Fab' and
F(ab').sub.2 fragments can also be employed using, e.g., methods
disclosed in PCT publication WO 92/22324; Mullinax et al., 1992,
BioTechniques 12(6):864-869; and Sawai et al., 1995, AJRI 34:26-34;
and Better et al., 1988, Science 240:1041-1043 (the disclosures of
which are incorporated by reference herein).
[0128] Examples of techniques that can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in
Enzymology 203:46-88; Shu et al., 1993, Proc Natl Acad Sci USA
90:7995-7999; and Skerra et al., 1988, Science 240:1038-1040.
[0129] In certain embodiments, the anti-CD70 antibody is a chimeric
antibody. A chimeric antibody is a molecule in which different
portions of the antibody are derived from different animal species,
such as for example antibodies having a variable region derived
from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are
known in the art. (See e.g., Morrison, Science, 1985, 229:1202; Oi
et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J.
Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567;
and 4,816,397.)
[0130] An anti-CD70 antibody can also be a humanized antibody.
Humanized antibodies are antibody molecules that bind the desired
antigen and have one or more CDRs from a non-human species, and
framework and constant regions from a human immunoglobulin
molecule. Often, framework residues in the human framework regions
will be substituted with the corresponding residue from the CDR
donor antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; Riechmann et al., 1988, Nature 332:323).
Antibodies can be humanized using a variety of techniques known in
the art including, for example, CDR-grafting (EP 0 239 400; PCT
publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and
5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596;
Padlan, Molecular Immunology, 1991, 28(4/5):489-498; Studnicka et
al., 1994, Protein Engineering 7(6):805-814; Roguska et al., 1994,
Proc. Natl. Acad. Sci. 91:969-973), and chain shuffling (U.S. Pat.
No. 5,565,332) (all of these references are incorporated by
reference herein).
[0131] In some embodiments, the antibody is a humanized 1F6 or 2F2
antibody, as disclosed in U.S. Provisional Patent Application No.
60/673,070, filed Apr. 19, 2005; and in PCT International
Publication No. WO 2006/113909; the disclosures of which are
incorporated by reference herein.
[0132] In yet other embodiments, the anti-CD70 antibody is a human
antibody. Human antibodies can be made by a variety of methods
known in the art including, e.g., phage display methods (see supra)
using antibody libraries derived from human immunoglobulin
sequences. See also, e.g., U.S. Pat. Nos. 4,444,887 and 4,716,111;
and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO
98/16654, WO 96/34096, WO 96/33735, and WO 91/10741. In addition, a
human antibody recognizing a selected epitope can be generated
using a technique referred to as "guided selection," in which a
selected non-human monoclonal antibody, e.g., a mouse antibody, is
used to guide the selection of a completely human antibody
recognizing the same epitope (see, e.g., Jespers et al., 1994,
Bio/technology 12:899-903). Human antibodies can also be produced
using transgenic mice that express human immunoglobulin genes.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. For an overview of this technology for producing human
antibodies, see Lonberg and Huszar, 1995, Int. Rev. Immunol.
13:65-93. For a detailed discussion of this technology for
producing human antibodies and human monoclonal antibodies and
protocols for producing such antibodies, see, e.g., PCT
publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;
European Patent No. 0 598, 877; U.S. Pat. Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;
5,885,793; 5,916,771; and 5,939,598. In addition, companies such as
Abgenix, Inc. (now Amgen, Fremont, Calif.) and Medarex (Princeton,
N.J.) can be engaged to provide human antibodies directed against a
selected antigen using technology similar to that described
above.
[0133] As set forth supra, a derivative of an anti-CD70 antibody
can also be used in the practice of present methods. Generally, an
anti-CD70 antibody derivative comprises an anti-CD70 antibody
(including, e.g., an antigen-binding fragment or conservatively
substituted polypeptides) and at least one polypeptide region or
other moiety heterologous to the anti-CD70 antibody. For example,
an anti-CD70 antibody can be modified, e.g., by the covalent
attachment of any type of molecule, such that covalent attachment
does not prevent the antibody derivative from specifically binding
to CD70 via the antigen-binding region or region derived therefrom,
or the conjugated drug from exerting (a) a cytostatic or cytotoxic
effect on CD70-expressing cancer cells, or (b) a cytostatic,
cytotoxic, or immunosuppressive effect on activated lymphocytes.
Typical modifications include, e.g., glycosylation, acetylation,
pegylation, phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, and the like. Any of numerous
chemical modifications may be carried out by known techniques,
including, but not limited to specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical
amino acids.
[0134] In certain embodiments, the antibody derivative is a
multimer, such as, for example, a dimer, comprising one or more
monomers, where each monomer includes (i) an antigen-binding region
of an anti-CD70 antibody, or a polypeptide region derived therefrom
(such as, e.g., by conservative substitution of one or more amino
acids), and (ii) a multimerizing (e.g., dimerizing) polypeptide
region, such that the antibody derivative forms multimers (e.g.,
homodimers) that specifically bind to CD70. In typical embodiments,
an antigen binding region of an anti-CD70 antibody, or a
polypeptide region derived therefrom, is recombinantly or
chemically fused with a heterologous protein, wherein the
heterologous protein comprises a dimerization or multimerization
domain. Prior to administration of the antibody derivative to a
subject for the purpose of treating or preventing immunological
disorders or CD70-expressing cancers, the derivative is subjected
to conditions that allow formation of a homodimer or heterodimer. A
heterodimer, as used herein, may comprise identical dimerization
domains but different CD70 antigen-binding regions, identical CD70
antigen-binding regions but different dimerization domains, or
different CD70 antigen-binding regions and dimerization
domains.
[0135] Typical dimerization domains are those that originate from
transcription factors. In one embodiment, the dimerization domain
is that of a basic region leucine zipper ("bZIP"). (See C. Vinson
et al., 1989, Science 246:911-916). Useful leucine zipper domains
include, for example, those of the yeast transcription factor GCN4,
the mammalian transcription factor CCAAT/enhancer-binding protein
C/EBP, and the nuclear transform in oncogene products, Fos and Jun.
(See Landschultz et al., 1988, Science 240:1759-1764; Baxevanis and
Vinson, 1993, Curr. Op. Gen. Devel. 3:278-285; O'Shea et al., 1989,
Science 243:538-542.) In another embodiment, the dimerization
domain is that of a basic-region helix-loop-helix ("bHLH") protein.
(See Murre et al., 1989, Cell 56:777-783. See also Davis et al.,
1990, Cell 60:733-746; Voronova and Baltimore, 1990, Proc Natl Acad
Sci USA 87:4722-4726.) Particularly useful hHLH proteins are myc,
max, and mac.
[0136] In yet other embodiments, the dimerization domain is an
immunoglobulin constant region such as, for example, a heavy chain
constant region or a domain thereof (e.g., a C.sub.H1 domain, a
C.sub.H2 domain, or a C.sub.H3 domain). (See, e.g., U.S. Pat. Nos.
5,155,027; 5,336,603; 5,359,046; and 5,349,053; EP 0 367 166; WO
96/04388.)
[0137] Heterodimers are known to form between Fos and Jun (Bohmann
et al., 1987, Science 238:1386-1392), among members of the ATF/CREB
family (Hai et al., 1989, Genes Dev. 3:2083-2090), among members of
the C/EBP family (Cao et al., 1991, Genes Dev. 5:1538-1552;
Williams et al., 1991, Genes Dev. 5:1553-1567; Roman et al., 1990,
Genes Dev. 4:1404-1415), and between members of the ATF/CREB and
Fos/Jun families Hai and Curran, 1991, Proc Natl Acad Sci USA
88:3720-3724). Therefore, when a CD70-binding protein-drug
conjugate is administered to a subject as a heterodimer comprising
different dimerization domains, any combination of the foregoing
may be used.
[0138] In other embodiments, an anti-CD70 antibody derivative is an
anti-CD70 antibody conjugated to a second antibody (an "antibody
heteroconjugate") (see U.S. Pat. No. 4,676,980). Heteroconjugates
useful for practicing the present methods comprise an antibody that
binds to CD70 (e.g., an antibody that has the CDRs and/or heavy
chains of the monoclonal antibodies 2F2 or 1F6) and an antibody
that binds to a surface receptor or receptor complex, such as an
immunoglobulin gene superfamily member, a TNF receptor superfamily
member, an integrin, a cytokine receptor, a chemokine receptor, a
major histocompatibility protein, a lectin (C-type, S-type, or
I-type), or a complement control protein.
[0139] In certain embodiments, the anti-CD70 antibody or derivative
thereof competitively inhibits binding of mAb 1F6 or 2F2 to CD70,
as determined by any method known in the art for determining
competitive binding (such as, e.g., the immunoassays described
herein). In typical embodiments, the antibody competitively
inhibits binding of 1F6 or 2F2 to CD70 by at least 50%, more
typically at least 60%, yet more typically at least 70%, and most
typically at least 75%. In other embodiments, the antibody
competitively inhibits binding of 1F6 or 2F2 to CD70 by at least
80%, at least 85%, at least 90%, or at least 95%.
[0140] Antibodies can be assayed for specific binding to CD70 by
any of various known methods. Immunoassays which can be used
include, for example, competitive and non-competitive assay systems
using techniques such as Western blots, radioimmunoassays, ELISA
(enzyme linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation assays, precipitin reactions, gel diffusion
precipitin reactions, immunodiffusion assays, agglutination assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays, protein A immunoassays, to name but a few. Such
assays are routine and well-known in the art. (See, e.g., Ausubel
et al., eds., Short Protocols in Molecular Biology (John Wiley
& Sons, Inc., New York, 4th ed. 1999); Harlow & Lane, Using
Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1999.)
[0141] Further, the binding affinity of an antibody to CD70 and the
off-rate of an antibody CD70 interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
CD70 (e.g., .sup.3H or .sup.125I) with the antibody of interest in
the presence of increasing amounts of unlabeled CD70, and the
detection of the antibody bound to the labeled CD70. The affinity
of the antibody for CD70 and the binding off-rates can then be
determined from the data by Scatchard plot analysis. Competition
with a second antibody (such as, e.g., mAb 1F6 or 2F2) can also be
determined using radioimmunoassays. In this case, CD70 is incubated
with the antibody of interest conjugated to a labeled compound
(e.g., .sup.3H or .sup.125I) in the presence of increasing amounts
of an unlabeled second antibody. Alternatively, the binding
affinity of an antibody to CD70 and the on- and off-rates of an
antibody-CD70 interaction can be determined by surface plasmon
resonance.
[0142] In accordance with the methods described herein, anti-CD70
antibodies or derivatives thereof, when conjugated to a therapeutic
agent, can be internalized and accumulate within a CD70-expressing
cell, where the therapeutic agent exerts an effect (e.g., a
cytotoxic, cytostatic, or immunosuppressive effect). In additional
embodiments, anti-CD70 antibodies or derivatives thereof, when
conjugated to a therapeutic agent, can be targeted to and
accumulate on the membrane of a CD70-expressing cell, where the
therapeutic agent exerts an effect (e.g., a cytotoxic, cytostatic,
or immunosuppressive effect). In yet other embodiments, anti-CD70
antibodies or derivatives thereof, when conjugated to a therapeutic
agent, can be targeted to a biological molecules in a cell (e.g.,
an inflammatory agent) and accumulate at or adjacent cells
secreting or binding the biological molecule, where the therapeutic
agent exerts an effect (e.g., a cytotoxic, cytostatic, or
immunosuppressive effect).
[0143] Whether a given anti-CD70 antibody or derivative, when
conjugated to a therapeutic agent, exerts a corresponding
therapeutic effect upon binding a CD70-expressing cell can be
readily determined, e.g., by (1) incubating CD70-expressing cells
independently with the anti-CD70 antibody or derivative thereof,
(2) incubating the cells with a secondary reagent that is
conjugated to the therapeutic agent and that specifically binds to
the antibody or derivative thereof, and (3) assaying the cells for
the corresponding therapeutic effect. Multiple antibodies or
antibody derivatives can be readily evaluated via such assays using
a secondary reagent that specifically binds a polypeptide region
shared by each antibody or derivative thereof (e.g., an anti-Ig
antibody). For example, an anti-CD70 mAb that binds CD70 and exerts
a cytotoxic effect when conjugated to a cytotoxic agent (e.g., an
auristatin such as, for example, AFP, MMAF, or MMAE) can be
identified by an indirect immunotoxicity assay such as, for
example, described by Chun et al., 2003, Supplement to Clinical
Cancer Research, Vol. 9. Briefly, the cytotoxic agent is conjugated
to a secondary antibody (e.g., for murine mAbs, a polyclonal
anti-mouse IgG); CD70-expressing cells are incubated with both the
primary and cytotoxic agent-conjugated secondary antibody (e.g., in
96-well plates, using hybridoma supernatant for the primary
antibody); and primary antibody-dependent cytotoxicity is assessed
in a standard cytotoxicity assay (e.g., an MTT cell viability
assay). (See id.)
[0144] The anti-CD70 antibodies and derivatives thereof that are
useful in the present methods can be produced by any method known
in the art for the synthesis of proteins, typically, e.g., by
recombinant expression techniques. Recombinant expression of an
antibody or derivative thereof that binds to CD70 and depletes or
inhibits the proliferation of CD70-expressing cells requires
construction of an expression vector containing a nucleic acid that
encodes the antibody or derivative thereof. Once a nucleic acid
encoding such a protein has been obtained, the vector for the
production of the protein molecule may be produced by recombinant
DNA technology using techniques well known in the art. Standard
techniques such as, for example, those described in Sambrook and
Russell, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 3rd ed., 2001);
Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2nd ed.,
1989); Short Protocols in Molecular Biology (Ausubel et al., John
Wiley & Sons, New York, 4th ed., 1999); and Glick &
Pasternak, Molecular Biotechnology: Principles and Applications of
Recombinant DNA (ASM Press, Washington, D.C., 2nd ed., 1998) can be
used for recombinant nucleic acid methods, nucleic acid synthesis,
cell culture, transgene incorporation, and recombinant protein
expression.
[0145] For example, for recombinant expression of an anti-CD70
antibody, an expression vector may encode a heavy or light chain
thereof, or a heavy or light chain variable domain, operably linked
to a promoter. An expression vector may include, for example, the
nucleotide sequence encoding the constant region of the antibody
molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication
WO 89/01036; and U.S. Pat. No. 5,122,464), and the variable domain
of the antibody may be cloned into such a vector for expression of
the entire heavy or light chain. The expression vector is
transferred to a host cell by conventional techniques, and the
transfected cells are then cultured by conventional techniques to
produce the anti-CD70 antibody. In typical embodiments for the
expression of double-chained antibodies, vectors encoding both the
heavy and light chains can be co-expressed in the host cell for
expression of the entire immunoglobulin molecule.
[0146] A variety of prokaryotic and eukaryotic host-expression
vector systems can be utilized to express an anti-CD70 antibody or
derivative thereof. Typically, eukaryotic cells, particularly for
whole recombinant anti-CD70 antibody molecules, are used for the
expression of the recombinant protein. For example, mammalian cells
such as Chinese hamster ovary cells (CHO), in conjunction with a
vector such as the major intermediate early gene promoter element
from human cytomegalovirus, is an effective expression system for
the production of anti-CD70 antiobies and derivatives thereof (see,
e.g., Foecking et al., 1986, Gene 45:101; Cockett et al., 1990,
Bio/Technology 8:2).
[0147] Other host-expression systems include, for example,
plasmid-based expression systems in bacterial cells (see, e.g.,
Ruther et al., 1983, EMBO 1, 2:1791; Inouye & Inouye, 1985,
Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J.
Biol. Chem. 24:5503-5509); insect systems such as, e.g., the use of
Autographa californica nuclear polyhedrosis virus (AcNPV)
expression vector in Spodoptera frugiperda cells; and viral-based
expression systems in mammalian cells, such as, e.g.,
adenoviral-based systems (see, e.g., Logan & Shenk, 1984, Proc.
Natl. Acad. Sci. USA 81:355-359; Bittner et al., 1987, Methods in
Enzymol. 153:51-544).
[0148] In addition, a host cell strain can be chosen that modulates
the expression of the inserted sequences, or modifies and processes
the gene product in the specific fashion desired. Appropriate cell
lines or host systems can be chosen to ensure the correct
modification and processing (e.g., glycosylation, phosphorylation,
and cleavage) of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript and gene product can be
used. Such mammalian host cells include, for example, CHO, VERO,
BHK, HeLa, COS, MDCK, 293, 3T3, and W138.
[0149] A stable expression system is typically used for long-term,
high-yield production of recombinant anti-CD70 antibody or
derivative thereof. For example, cell lines that stably express the
anti-CD70 antibody or derivative thereof can be engineered by
transformation of host cells with DNA controlled by appropriate
expression control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites) and a selectable
marker, followed by growth of the transformed cells in a selective
media. The selectable marker confers resistance to the selection
and allows cells to stably integrate the DNA into their chromosomes
and grow to form foci which in turn can be cloned and expanded into
cell lines. A number of selection systems can be used, including,
for example, the herpes simplex virus thymidine kinase,
hypoxanthineguanine phosphoribosyltransferase, and adenine
phosphoribosyltransferase genes, which can be employed in tk.sup.-,
hgprt.sup.- or aprt.sup.- cells, respectively. Also, antimetabolite
resistance can be used as the basis of selection for the following
genes: dhfr, which confers resistance to methotrexate; gpt, which
confers resistance to mycophenolic acid; neo, which confers
resistance to the aminoglycoside G-418; and hygro, which confers
resistance to hygromycin. Methods commonly known in the art of
recombinant DNA technology can be routinely applied to select the
desired recombinant clone, and such methods are described, for
example, in Current Protocols in Molecular Biology (Ausubel et al.
eds., John Wiley & Sons, N.Y., 1993); Kriegler, Gene Transfer
and Expression, A Laboratory Manual (Stockton Press, N.Y., 1990);
Current Protocols in Human Genetics (Dracopoli et al. eds., John
Wiley & Sons, N.Y., 1994, Chapters 12 and 13); and
Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1.
[0150] The expression levels of an antibody or derivative can be
increased by vector amplification. (See generally, e.g., Bebbington
& Hentschel, The Use of Vectors Based on Gene Amplification for
the Expression of Cloned Genes in Mammalian Cells in DNA Cloning,
Vol. 3 (Academic Press, New York, 1987).) When a marker in the
vector system expressing an anti-CD70 antibody or derivative
thereof is amplifiable, an increase in the level of inhibitor
present in host cell culture media will select host cells that have
increased copy number of a marker gene conferring resistance to the
inhibitor. The copy number of an associated antibody gene will also
be increased, thereby increasing expression of the antibody or
derivative thereof (see Crouse et al., 1983, Mol. Cell. Biol.
3:257). Expression levels can also be increased by optimizating the
vector, and in particular the nucleic acids encoding the antibody
or derivative, for the host organism (e.g., by modifying the codon
usage, CpG content, and the like).
[0151] Where the anti-CD70 antibody comprises both a heavy and a
light chain or derivatives thereof, the host cell may be
co-transfected with two expression vectors, the first vector
encoding the heavy chain protein and the second vector encoding the
light chain protein. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain proteins. Alternatively, a single vector may be used which
encodes, and is capable of expressing, both heavy and light chain
proteins. In such situations, the light chain is typically placed
before the heavy chain to avoid an excess of toxic free heavy chain
(see Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl.
Acad. Sci. USA 77:2197). The coding sequences for the heavy and
light chains may comprise cDNA or genomic DNA.
[0152] Once an anti-CD70 antibody or derivative thereof has been
produced (e.g., by an animal, chemical synthesis, or recombinant
expression), it can be purified by any suitable method for
purification of proteins, including, for example, by chromatography
(e.g., ion exchange or affinity chromatography (such as, for
example, Protein A chromatography for purification of antibodies
having an intact Fc region)), centrifugation, differential
solubility, or by any other standard technique for the purification
of proteins. An anti-CD70 antibody or derivative thereof can, for
example, be fused to a marker sequence, such as a peptide, to
facilitate purification by affinity chromatography. Suitable marker
amino acid sequences include, e.g., a hexa-histidine peptide, such
as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton
Avenue, Chatsworth, Calif., 91311), and the "HA" tag, which
corresponds to an epitope derived from the influenza hemagglutinin
protein (Wilson et al., 1984, Cell 37:767), and the "flag" tag.
[0153] Once an anti-CD70 antibody or derivative thereof is
produced, its ability to exert a cytostatic or cytotoxic effect on
CD70-expressing cancer cells (when conjugated to a cytotoxic agent)
or an immunosuppressive effect on a CD70-expressing immune cell
(when conjugated to an immunosuppressive agent) is determined by
the methods described infra or as known in the art.
III. Anti-CD70 Antibody-Drug Conjugates
[0154] Compositions useful in the treatment of a CD70-expressing
cancer or an immunological disorder comprise anti-CD70
antibody-drug conjugates (ADCs) or anti-CD70 ADC derivatives. An
"anti-CD70 ADC" as used herein refers to an anti-CD70 antibody (as
described in Section II, supra) conjugated to a therapeutic agent.
An "anti-CD70 derivative" as used herein refers to a derivative of
an anti-CD70 antibody (as described in Section II, supra)
conjugated to a therapeutic agent. In certain embodiments, the ADC
comprises an anti-CD70 antibody (e.g., mAb 1F6 or 2F2 or a fragment
or derivative thereof, including, for example, a chimeric or
humanized form thereof). The ADCs or ADC derivatives as described
herein produce clinically beneficial effects on CD70-expressing
cells when administered to a subject with a CD70-expressing cancer
or an immunological disorder, typically when administered alone but
also in combination with other therapeutic agents.
[0155] In typical embodiments, the anti-CD70 antibody or derivative
thereof is conjugated to a cytotoxic or immunosuppressive agent,
such that the resulting ADC or ADC derivative exerts a cytotoxic or
cytostatic effect on a CD70-expressing cancer cell, or a cytotoxic,
cytostatic, or immunosuppressive effect on an immune cell (e.g., an
activated lymphocyte or dendritic cell) when taken up or
internalized by the cell. Particularly suitable moieties for
conjugation to antibodies or antibody derivatives are
chemotherapeutic agents, prodrug converting enzymes, radioactive
isotopes or compounds, or toxins. For example, an anti-CD70
antibody or derivative thereof can be conjugated to a cytotoxic
agent such as a chemotherapeutic agent (see infra), or a toxin
(e.g., a cytostatic or cytocidal agent such as, e.g., abrin, ricin
A, pseudomonas exotoxin, or diphtheria toxin). Examples of
additional agents that are useful for conjugation to the anti-CD70
molecules are provided infra.
[0156] In other embodiments, the anti-CD70 antibody or derivative
thereof is conjugated to a pro-drug converting enzyme. The pro-drug
converting enzyme can be recombinantly fused to the antibody or
derivative thereof or chemically conjugated thereto using known
methods. Exemplary pro-drug converting enzymes are carboxypeptidase
G2, beta-glucuronidase, penicillin-V-amidase, penicillin-G-amidase,
.beta.-lactamase, .beta.-glucosidase, nitroreductase and
carboxypeptidase A.
[0157] Techniques for conjugating therapeutic agents to proteins,
and in particular to antibodies, are well-known. (See, e.g., Arnon
et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In
Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy
(Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al.,
"Antibodies For Drug Delivery," in Controlled Drug Delivery
(Robinson et al. eds., Marcel Dekker, Inc., 2nd ed. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review," in Monoclonal Antibodies '84: Biological And Clinical
Applications (Pinchera et al. eds., 1985); "Analysis, Results, and
Future Prospective of the Therapeutic Use of Radiolabeled Antibody
In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection
And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe
et al., 1982, Immunol. Rev. 62:119-58. See also, e.g., PCT
publication WO 89/12624.)
[0158] In accordance with the methods described herein, the
anti-CD70 ADC or ADC derivative is internalized and accumulates
within a CD70-expressing cell, where the ADC or ADC derivative
exerts a therapeutic effect (e.g., a cytotoxic, cytostatic, or
immunosuppressive effect). Methods for determining accumulation and
rates of accumulation are found in U.S. Provisional Application
Ser. No. 60/400,404, filed Jul. 31, 2002; the disclosure of which
is incorporated by reference herein.
[0159] Typically, when using an anti-CD70 antibody or derivative
thereof conjugated to a therapeutic agent (e.g., a drug or a
prodrug converting enzyme), the agent is preferentially active when
internalized by cells of the cancer to be treated or by activated
immune cells (e.g., activated lymphocytes or dendritic cells). In
other embodiments, the anti-CD70 ADC or ADC derivative is not
internalized, and the drug is effective to deplete or inhibit
CD70-expressing cells by binding to the cell membrane. In yet other
embodiments, anti-CD70 antibodies ADC or ADC derivatives thereof
can be targeted to a biological molecules in a cell (e.g., an
inflammatory agent) and accumulate at or adjacent cells secreting
or binding the biological molecule, where the therapeutic agent
exerts an effect (e.g., a cytotoxic, cytostatic, or
immunosuppressive effect).
[0160] To minimize activity of the therapeutic agent outside the
activated immune cells or CD70-expressing cancer cells, an antibody
that specifically binds to cell membrane-bound CD70, but not to
soluble CD70, can be used, so that the therapeutic agent is
concentrated at the cell surface of the activated immune cell or
CD70-expressing cancer cell. Alternatively, in a more typical
embodiment, the therapeutic agent is conjugated in a manner that
reduces its activity unless it is cleaved off the antibody (e.g.,
by hydrolysis or by a cleaving agent). In such embodiments, the
therapeutic agent is attached to the antibody or derivative thereof
with a cleavable linker that is sensitive to cleavage in the
intracellular environment of the activated immune cell or
CD70-expressing cancer cell but is not substantially sensitive to
the extracellular environment, such that the conjugate is cleaved
from the antibody or derivative thereof when it is internalized by
the activated immune cell or CD70-expressing cancer cell (e.g., in
the endosomal or, for example by virtue of pH sensitivity or
protease sensitivity, in the lysosomal environment or in a
caveolea). (See Section III(A), infra.)
[0161] Further, in certain embodiments, the ADC or ADC derivative
comprises a therapeutic agent that is charged relative to the
plasma membrane, thereby further minimizing the ability of the
agent to cross the plasma membrane once internalized by a cell. As
used herein, a "charged agent" means an agent that (a) is
polarized, such that one region of the agent has a charge relative
to the plasma membrane, or (b) has a net charge relative to the
plasma membrane.
[0162] Typically, the anti-CD70 antibody-drug conjugate (ADC) or
ADC derivative is substantially purified (e.g., substantially free
from substances that limit its effect or produce undesired
side-effects). In certain specific embodiments, the anti-CD70 ADC
or ADC derivative is 40% pure, more typically about 50% pure, and
most typically about 60% pure. In other specific embodiments, the
anti-CD70 ADC or ADC derivative is at least approximately 60-65%,
65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-98% pure. In
another specific embodiment, the anti-CD70 ADC or ADC derivative is
approximately 99% pure.
[0163] A. Linkers
[0164] Typically, the ADC or ADC derivative comprises a linker
region between the therapeutic agent and the anti-CD70 antibody or
derivative thereof. As noted supra, in typical embodiments, the
linker is cleavable under intracellular conditions, such that
cleavage of the linker releases the therapeutic agent from the
antibody in the intracellular environment.
[0165] For example, in some embodiments, the linker is cleavable by
a cleaving agent that is present in the intracellular environment
(e.g., within a lysosome or endosome or caveolea). The linker can
be, e.g., a peptidyl linker that is cleaved by an intracellular
peptidase or protease enzyme, including, but not limited to, a
lysosomal or endosomal protease. Typically, the peptidyl linker is
at least two amino acids long or at least three amino acids long.
Cleaving agents can include cathepsins B and D and plasmin, all of
which are known to hydrolyze dipeptide drug derivatives resulting
in the release of active drug inside target cells (see, e.g.,
Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most
typical are peptidyl linkers that are cleavable by enzymes that are
present in CD70-expressing cells. For example, a peptidyl linker
that is cleavable by the thiol-dependent protease cathepsin-B,
which is highly expressed in cancerous tissue, can be used (e.g., a
Phe-Leu or a Gly-Phe-Leu-Gly linker). Other such linkers are
described, e.g., in U.S. Pat. No. 6,214,345. In specific
embodiments, the peptidyl linker cleavable by an intracellular
protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S.
Pat. No. 6,214,345, which describes the synthesis of doxorubicin
with the val-cit linker). One advantage of using intracellular
proteolytic release of the therapeutic agent is that the agent is
typically attenuated when conjugated and the serum stabilities of
the conjugates are typically high.
[0166] In other embodiments, the cleavable linker is pH-sensitive,
i.e., sensitive to hydrolysis at certain pH values. Typically, the
pH-sensitive linker hydrolyzable under acidic conditions. For
example, an acid-labile linker that is hydrolyzable in the lysosome
(e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic
amide, orthoester, acetal, ketal, or the like) can be used. (See,
e.g. U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and
Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989,
Biol. Chem. 264:14653-14661.) Such linkers are relatively stable
under neutral pH conditions, such as those in the blood, but are
unstable at below pH 5.5 or 5.0, the approximate pH of the
lysosome. In certain embodiments, the hydrolyzable linker is a
thioether linker (such as, e.g., a thioether attached to the
therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat.
No. 5,622,929)).
[0167] In yet other embodiments, the linker is cleavable under
reducing conditions (e.g., a disulfide linker). A variety of
disulfide linkers are known in the art, including, for example,
those that can be formed using SATA
(N-succinimidyl-5-acetylthioacetate), SPDP
(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB
(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT
(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-
, SPDB and SMPT (See, e.g., Thorpe et al., 1987, Cancer Res.
47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody
Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,
Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)
[0168] In yet other specific embodiments, the linker is a malonate
linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a
maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem.
3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995,
Bioorg-Med-Chem. 3(10):1305-12).
[0169] Typically, the linker is not substantially sensitive to the
extracellular environment. As used herein, "not substantially
sensitive to the extracellular environment," in the context of a
linker, means that no more than about 20%, typically no more than
about 15%, more typically no more than about 10%, and even more
typically no more than about 5%, no more than about 3%, or no more
than about 1% of the linkers, in a sample of ADC or ADC derivative,
are cleaved when the ADC or ADC derivative present in an
extracellular environment (e.g., in plasma). Whether a linker is
not substantially sensitive to the extracellular environment can be
determined, for example, by incubating independently with plasma
both (a) the ADC or ADC derivative (the "ADC sample") and (b) an
equal molar amount of unconjugated antibody or therapeutic agent
(the "control sample") for a predetermined time period (e.g., 2, 4,
8, 16, or 24 hours) and then comparing the amount of unconjugated
antibody or therapeutic agent present in the ADC sample with that
present in control sample, as measured, for example, by high
performance liquid chromatography.
[0170] In other, non-mutually exclusive embodiments, the linker
promotes cellular internalization. In certain embodiments, the
linker promotes cellular internalization when conjugated to the
therapeutic agent (i.e., in the milieu of the linker-therapeutic
agent moiety of the ADC or ADC derivate as described herein). In
yet other embodiments, the linker promotes cellular internalization
when conjugated to both the therapeutic agent and the anti-CD70
antibody or derivative thereof (i.e., in the milieu of the ADC or
ADC derivative as described herein).
[0171] A variety of linkers that can be used with the present
compositions and methods are described in WO 2004010957 entitled
"Drug Conjugates and Their Use for Treating Cancer, An Autoimmune
Disease or an Infectious Disease" filed Jul. 31, 2003, and U.S.
Provisional Application No. 60/400,403, entitled "Drug Conjugates
and their use for treating cancer, an autoimmune disease or an
infectious disease", filed Jul. 31, 2002 (the disclosure of which
is incorporated by reference herein).
[0172] In certain embodiments, the linker unit has the following
general formula: -T.sub.a-W.sub.w--Y.sub.y--
[0173] wherein:
[0174] -T- is a stretcher unit;
[0175] a is 0 or 1;
[0176] each --W-- is independently an amino acid unit;
[0177] w is independently an integer ranging from 2 to 12;
[0178] --Y-- is a spacer unit; and
[0179] y is 0, 1 or 2.
[0180] 1. The Stretcher Unit
[0181] The stretcher unit (-T-), when present, links the anti-CD70
antibody unit to an amino acid unit (--W--). Useful functional
groups that can be present on an anti-CD70 antibody, either
naturally or via chemical manipulation include, but are not limited
to, sulfhydryl, amino, hydroxyl, the anomeric hydroxyl group of a
carbohydrate, and carboxyl. Suitable functional groups are
sulfhydryl and amino. Sulfhydryl groups can be generated by
reduction of the intramolecular disulfide bonds of an anti-CD70
antibody. Alternatively, sulfhydryl groups can be generated by
reaction of an amino group of a lysine moiety of an anti-CD70
antibody with 2-iminothiolane (Traut's reagent) or other sulfhydryl
generating reagents. In specific embodiments, the anti-CD70
antibody is a recombinant antibody and is engineered to carry one
or more lysines. In other embodiments, the recombinant anti-CD70
antibody is engineered to carry additional sulfhydryl groups, e.g.,
additional cysteines.
[0182] In certain specific embodiments, the stretcher unit forms a
bond with a sulfur atom of the anti-CD70 antibody unit. The sulfur
atom can be derived from a sulfhydryl (--SH) group of a reduced
anti-CD70 antibody (A). Representative stretcher units of these
embodiments are depicted within the square brackets of Formulas
(Ia) and (Ib; see infra), wherein A-, --W--, --Y--, -D, w and y are
as defined above and R.sup.1 is selected from --C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 carbocyclo-, --O--(C.sub.1-C.sub.8
alkyl)-, -arylene-, --C.sub.1-C.sub.10 alkylene-arylene-,
-arylene-C.sub.1-C.sub.10 alkylene-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 carbocyclo)-, --(C.sub.3-C.sub.8
carbocyclo)-C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
heterocyclo-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
heterocyclo)-, --(C.sub.3-C.sub.8 heterocyclo)-C.sub.1-C.sub.10
alkylene-, --(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r is an integer
ranging from 1-10. ##STR1##
[0183] An illustrative stretcher unit is that of formula (Ia) where
R.sup.1 is --(CH.sub.2).sub.5--: ##STR2##
[0184] Another illustrative stretcher unit is that of formula (Ia)
where R.sup.1 is --(CH.sub.2CH.sub.2O).sub.r--CH.sub.2-- and r is
2: ##STR3##
[0185] Still another illustrative stretcher unit is that of formula
(Ib) where R.sup.1 is --(CH.sub.2).sub.5--: ##STR4##
[0186] In certain other specific embodiments, the stretcher unit is
linked to the anti-CD70 antibody unit (A) via a disulfide bond
between a sulfur atom of the anti-CD70 antibody unit and a sulfur
atom of the stretcher unit. A representative stretcher unit of this
embodiment is depicted within the square brackets of Formula (II),
wherein R.sup.1, A-, --W--, --Y--, -D, w and y are as defined
above. AS--R.sup.1--C(O)W.sub.w--Y.sub.y-D (II)
[0187] In other specific embodiments, the reactive group of the
stretcher contains a reactive site that can be reactive to an amino
group of an anti-CD70 antibody. The amino group can be that of an
arginine or a lysine. Suitable amine reactive sites include, but
are not limited to, activated esters such as succinimide esters,
4-nitrophenyl esters, pentafluorophenyl esters, anhydrides, acid
chlorides, sulfonyl chlorides, isocyanates and isothiocyanates.
Representative stretcher units of these embodiments are depicted
within the square brackets of Formulas (IIIa) and (IIIb), wherein
R.sup.1, A-, --W--, --Y--, -D, w and y are as defined above;
##STR5##
[0188] In yet another aspect, the reactive function of the
stretcher contains a reactive site that is reactive to a modified
carbohydrate group that can be present on an anti-CD70 antibody. In
a specific embodiment, the anti-CD70 antibody is glycosylated
enzymatically to provide a carbohydrate moiety. The carbohydrate
may be mildly oxidized with a reagent such as sodium periodate and
the resulting carbonyl unit of the oxidized carbohydrate can be
condensed with a stretcher that contains a functionality such as a
hydrazide, an oxime, a reactive amine, a hydrazine, a
thiosemicarbazide, a hydrazine carboxylate, or an arylhydrazide
such as those described by Kaneko et al., 1991, Bioconju gate Chem
2:133-41. Representative stretcher units of this embodiment are
depicted within the square brackets of Formulas (IVa)-(IVc),
wherein R.sup.1, A-, --W--, --Y--, -D, w and y are as defined
above. ##STR6##
[0189] 2. The Amino Acid Unit
[0190] The amino acid unit (--W--) links the stretcher unit (-T-)
to the Spacer unit (--Y--) if the Spacer unit is present, and links
the stretcher unit to the cytotoxic or cytostatic agent (Drug unit;
D) if the spacer unit is absent.
[0191] --W.sub.w-- is a dipeptide, tripeptide, tetrapeptide,
pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide,
decapeptide, undecapeptide or dodecapeptide unit. Each --W-- unit
independently has the formula denoted below in the square brackets,
and w is an integer ranging from 2 to 12: ##STR7## wherein R.sup.2
is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,
p-hydroxybenzyl, --CH.sub.2OH, --CH(OH)CH.sub.3,
--CH.sub.2CH.sub.2SCH.sub.3, --CH.sub.2CONH.sub.2, --CH.sub.2COOH,
--CH.sub.2CH.sub.2CONH.sub.2, --CH.sub.2CH.sub.2COOH,
--(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2, --(CH.sub.2).sub.3NH.sub.2,
--(CH.sub.2).sub.3NHCOCH.sub.3, --(CH.sub.2).sub.3NHCHO,
--(CH.sub.2).sub.4NHC(.dbd.NH)NH.sub.2, --(CH.sub.2).sub.4NH.sub.2,
--(CH.sub.2).sub.4NHCOCH.sub.3, --(CH.sub.2).sub.4NHCHO,
--(CH.sub.2).sub.3NHCONH.sub.2, --(CH.sub.2).sub.4NHCONH.sub.2,
--CH.sub.2CH.sub.2CH(OH)CH.sub.2NH.sub.2, 2-pyridylmethyl-,
3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,
##STR8##
[0192] The amino acid unit of the linker unit can be enzymatically
cleaved by an enzyme including, but not limited to, a
tumor-associated protease to liberate the drug unit (-D) which is
protonated in vivo upon release to provide a cytotoxic drug
(D).
[0193] Illustrative W.sub.w units are represented by formulas
(V)-(VII): ##STR9##
[0194] wherein R.sup.3 and R.sup.4 are as follows: TABLE-US-00002
R.sup.3 R.sup.4 Benzyl (CH.sub.2).sub.4NH.sub.2; Methyl
(CH.sub.2).sub.4NH.sub.2; Isopropyl (CH.sub.2).sub.4NH.sub.2;
Isopropyl (CH.sub.2).sub.3NHCONH.sub.2; Benzyl
(CH.sub.2).sub.3NHCONH.sub.2; Isobutyl
(CH.sub.2).sub.3NHCONH.sub.2; sec-butyl
(CH.sub.2).sub.3NHCONH.sub.2; ##STR10##
(CH.sub.2).sub.3NHCONH.sub.2; Benzyl methyl; and Benzyl
(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2;
[0195] ##STR11##
[0196] wherein R.sup.3, R.sup.4 and R.sup.5 are as follows:
TABLE-US-00003 R.sup.3 R.sup.4 R.sup.5 Benzyl benzyl
(CH.sub.2).sub.4NH.sub.2; Isopropyl benzyl
(CH.sub.2).sub.4NH.sub.2; and H benzyl
(CH.sub.2).sub.4NH.sub.2;
or ##STR12##
[0197] wherein R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are as
follows: TABLE-US-00004 R.sup.3 R.sup.4 R.sup.5 R.sup.6 H Benzyl
isobutyl H; and methyl Isobutyl methyl isobutyl.
[0198] Suitable amino acid units include, but are not limited to,
units of formula (V) where: R.sup.3 is benzyl and R.sup.4 is
--(CH.sub.2).sub.4NH.sub.2; R.sup.3 is isopropyl and R.sup.4 is
--(CH.sub.2).sub.4NH.sub.2; or R.sup.3 is isopropyl and R.sup.4 is
--(CH.sub.2).sub.3NHCONH.sub.2. Another suitable amino acid unit is
a unit of formula (VI), where: R.sup.3 is benzyl, R.sup.4 is
benzyl, and R.sup.5 is --(CH.sub.2).sub.4NH.sub.2.
[0199] --W.sub.w-- units can be designed and optimized in their
selectivity for enzymatic cleavage by a particular tumor-associated
protease. The suitable -Ww- units are those whose cleavage is
catalyzed by the proteases, cathepsin B, C and D, and plasmin.
[0200] In one embodiment, --W.sub.w-- is a dipeptide, tripeptide or
tetrapeptide unit.
[0201] Where R.sup.2, R.sup.3, R.sup.4, R.sup.5 or R.sup.6 is other
than hydrogen, the carbon atom to which R.sup.2, R.sup.3, R.sup.4,
R.sup.5 or R.sup.6 is attached is chiral.
[0202] Each carbon atom to which R.sup.2, R.sup.3, R.sup.4, R.sup.5
or R.sup.6 is attached is independently in the (S) or (R)
configuration.
[0203] In a certain embodiment, the amino acid unit is a
phenylalanine-lysine dipeptide (Phe-Lys or FK linker). In another
embodiment, the amino acid unit is a valine-citrulline dipeptide
(Val-Cit or VC linker).
[0204] 3. The Spacer Unit
[0205] The spacer unit (--Y--), when present, links an amino acid
unit to the drug unit. Spacer units are of two general types:
self-immolative and non self-immolative. A non self-immolative
spacer unit is one in which part or all of the spacer unit remains
bound to the drug unit after enzymatic cleavage of an amino acid
unit from the anti-CD70 antibody-linker-drug conjugate or the
drug-linker compound. Examples of a non self-immolative spacer unit
include, but are not limited to a (glycine-glycine) spacer unit and
a glycine spacer unit (both depicted in Scheme 1). When an
anti-CD70 antibody-linker-drug conjugate containing a
glycine-glycine spacer unit or a glycine spacer unit undergoes
enzymatic cleavage via a tumor-cell associated-protease, a
cancer-cell-associated protease or a lymphocyte-associated
protease, a glycine-glycine-drug moiety or a glycine-drug moiety is
cleaved from A-T-W.sub.w--. To liberate the drug, an independent
hydrolysis reaction should take place within the target cell to
cleave the glycine-drug unit bond.
[0206] In a typical embodiment, --Y.sub.y-- is a p-aminobenzyl
ether which can be substituted with Q.sub.m where Q is
--C.sub.1-C.sub.8 alkyl, --C.sub.1-C.sub.8 alkoxy, -halogen, -nitro
or -cyano; and m is an integer ranging from 0-4.
Scheme 1
[0207] In one embodiment, a non self-immolative spacer unit (--Y--)
is -Gly-Gly-. ##STR13##
[0208] In another embodiment, a non self-immolative the spacer unit
(--Y--) is -Gly-.
[0209] In one embodiment, the drug-linker compound or an anti-CD70
antibody-linker-drug conjugate lacks a spacer unit (y=0).
[0210] Alternatively, an anti-CD70 antibody-linker-drug conjugate
containing a self-immolative spacer unit can release the drug (D)
without the need for a separate hydrolysis step. In these
embodiments, --Y-- is a p-aminobenzyl alcohol (PAB) unit that is
linked to --W.sub.w-- via the nitrogen atom of the PAB group, and
connected directly to -D via a carbonate, carbamate or ether group
(Scheme 2 and Scheme 3). ##STR14## where Q is --C.sub.1-C.sub.8
alkyl, --C.sub.1-C.sub.8 alkoxy, -halogen, -nitro or -cyano; m is
an integer ranging from 0-4; and p is an integer ranging from 1-20.
##STR15## where Q is --C.sub.1-C.sub.8 alkyl, --C.sub.1-C.sub.8
alkoxy, -halogen, -nitro or -cyano; m is an integer ranging from
0-4; and p is an integer ranging from 1-20.
[0211] Other examples of self-immolative spacers include, but are
not limited to, aromatic compounds that are electronically
equivalent to the PAB group such as 2-aminoimidazol-5-methanol
derivatives (see Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237
for examples) and ortho or para-aminobenzylacetals. Spacers can be
used that undergo facile cyclization upon amide bond hydrolysis,
such as substituted and unsubstituted 4-aminobutyric acid amides
(Rodrigues et al., 1995, Chemistry Biology 2:223), appropriately
substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm
et al., 1972, J. Amer. Chem. Soc. 94:5815) and
2-aminophenylpropionic acid amides (Amsberry et al., 1990, J. Org.
Chem. 55:5867). Elimination of amine-containing drugs that are
substituted at the .alpha.-position of glycine (Kingsbury et al.,
1984, J. Med. Chem. 27:1447) are also examples of self-immolative
spacer strategies that can be applied to the anti-CD70
antibody-linker-drug conjugates.
[0212] In an alternate embodiment, the spacer unit is a branched
bis(hydroxymethyl)styrene (BHMS) unit (Scheme 4), which can be used
to incorporate additional drugs. ##STR16## where Q is
--C.sub.1-C.sub.8 alkyl, --C.sub.1-C.sub.8 alkoxy, -halogen, -nitro
or -cyano; m is an integer ranging from 0-4; n is 0 or 1; and p is
an integer raging from 1-20.
[0213] In one embodiment, the two -D moieties are the same.
[0214] In another embodiment, the two -D moieties are
different.
[0215] Typical spacer units (--Y.sub.y--) are represented by
Formulas (VIII)-(X): ##STR17## where Q is C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxy, halogen, nitro or cyano; and m is an
integer ranging from 0-4; ##STR18##
[0216] B. Therapeutic Agents
[0217] In accordance with the methods described herein, any agent
that exerts a therapeutic effect on cancer cells or activated
immune cells can be used as the therapeutic agent for conjugation
to an anti-CD70 antibody or derivative thereof. (See, e.g., WO
2004/010957, "Drug Conjugates and Their Use for Treating Cancer, An
Autoimmune Disease or an Infectious Disease" (supra) and U.S.
Provisional Application No. 60/400,403 (supra)). Typically, the
therapeutic agent is a cytotoxic or immunosuppressive agent. In
some embodiments, an anti-CD70 drug conjugate or derivative
conjugate comprises from about 1 to about 20 therapeutic agents per
conjugate. In some embodiments, an anti-CD70 drug conjugate or
derivative conjugate comprises from about 2 to about 10, from about
2 to about 8, about 4 or about 6 therapeutic agents per
conjugate.
[0218] Useful classes of cytotoxic or immunosuppressive agents
include, for example, antitubulin agents, auristatins, DNA minor
groove binders, DNA replication inhibitors, alkylating agents
(e.g., platinum complexes such as cis-platin, mono(platinum),
bis(platinum) and tri-nuclear platinum complexes and carboplatin),
anthracyclines, antibiotics, antifolates, antimetabolites,
chemotherapy sensitizers, duocarmycins, etoposides, fluorinated
pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols,
pre-forming compounds, purine antimetabolites, puromycins,
radiation sensitizers, steroids, taxanes, topoisomerase inhibitors,
vinca alkaloids, or the like.
[0219] Individual cytotoxic or immunosuppressive agents include,
for example, an androgen, anthramycin (AMC), asparaginase,
5-azacytidine, azathioprine, bleomycin, busulfan, buthionine
sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC-1065,
chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine,
cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin
(formerly actinomycin), daunorubicin, decarbazine, docetaxel,
doxorubicin, an estrogen, 5-fluordeoxyuridine, 5-fluorouracil,
gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan,
lomustine (CCNU), mechlorethamine, melphalan, 6-mercaptopurine,
methotrexate, mithramycin, mitomycin C, mitoxantrone,
nitroimidazole, paclitaxel, plicamycin, procarbizine,
streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan,
vinblastine, vincristine, vinorelbine, VP-16 and VM-26.
[0220] In some typical embodiments, the therapeutic agent is a
cytotoxic agent. Suitable cytotoxic agents include, for example,
dolastatins (e.g., auristatin E, AFP, MMAF, MMAE), DNA minor groove
binders (e.g., enediynes and lexitropsins), duocarmycins, taxanes
(e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids,
CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin,
cyanomorpholino-doxorubicin, echinomycin, combretastatin,
netropsin, epothilone A and B, estramustine, cryptophysins,
cemadotin, maytansinoids, discodermolide, eleutherobin, and
mitoxantrone.
[0221] In certain embodiments, the cytotoxic agent is a
conventional chemotherapeutic such as, for example, doxorubicin,
paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C
or etoposide. In addition, potent agents such as CC-1065 analogues,
calicheamicin, maytansine, analogues of dolastatin 10, rhizoxin,
and palytoxin can be linked to the anti-CD70 antibodies or
derivatives thereof.
[0222] In specific embodiments, the cytotoxic or cytostatic agent
is auristatin E (a derivative of dolastatin-10) or a derivative
thereof. Typically, the auristatin E derivative is, e.g., an ester
formed between auristatin E and a keto acid. For example,
auristatin E can be reacted with paraacetyl benzoic acid or
benzoylvaleric acid to produce AEB and AEVB, respectively. Other
typical auristatin derivatives include AFP, MMAF, and MMAE. The
synthesis and structure of auristatin E and its derivatives are
described in U.S. patent application Ser. Nos. 09/845,786 (U.S.
Patent Application Publication No. 2003-0083263) and 10/001,191
(U.S. Patent Application Publication No. 2005-0009751);
International Patent Application No. PCT/US03/24209, International
Patent Application No. PCT/US02/13435, and U.S. Pat. Nos.
6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149;
5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191;
5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278;
4,816,444; and 4,486,414.
[0223] In specific embodiments, the cytotoxic agent is a DNA minor
groove binding agent. (See, e.g., U.S. Pat. No. 6,130,237.) For
example, in certain embodiments, the minor groove binding agent is
a CBI compound. In other embodiments, the minor groove binding
agent is an enediyne (e.g., calicheamicin).
[0224] In certain embodiments, the ADC or ADC derivative comprises
an anti-tubulin agent. Examples of anti-tubulin agents include, but
are not limited to, taxanes (e.g., Taxol.RTM. (paclitaxel),
Taxotere.RTM. (docetaxel)), T67 (Tularik), vinca alkyloids (e.g.,
vincristine, vinblastine, vindesine, and vinorelbine), and
dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Other
antitubulin agents include, for example, baccatin derivatives,
taxane analogs (e.g., epothilone A and B), nocodazole, colchicine
and colcimid, estramustine, cryptophysins, cemadotin,
maytansinoids, combretastatins, discodermolide, and
eleutherobin.
[0225] In certain embodiments, the cytotoxic agent is a
maytansinoid, another group of anti-tubulin agents. For example, in
specific embodiments, the maytansinoid is maytansine or DM-1
(ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res.
52:127-131).
[0226] In certain embodiments, the therapeutic agent is not a
radioisotope. In certain embodiments, the therapeutic agent is not
a peptide toxin.
[0227] In certain embodiments, the cytotoxic or immunosuppressive
agent is an antimetabolite. The antimetabolite can be, for example,
a purine antagonist (e.g. azothioprine or mycophenolate mofetil), a
dihydrofolate reductase inhibitor (e.g., methotrexate), acyclovir,
gangcyclovir, zidovudine, vidarabine, ribavarin, azidothymidine,
cytidine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine,
poscarnet, or trifluridine.
[0228] In other embodiments, the cytotoxic or immunosuppressive
agent is tacrolimus, cyclosporine or rapamycin. In further
embodiments, the cytoxic agent is aldesleukin, alemtuzumab,
alitretinoin, allopurinol, altretamine, amifostine, anastrozole,
arsenic trioxide, bexarotene, bexarotene, calusterone,
capecitabine, celecoxib, cladribine, Darbepoetin alfa, Denileukin
diftitox, dexrazoxane, dromostanolone propionate, epirubicin,
Epoetin alfa, estramustine, exemestane, Filgrastim, floxuridine,
fludarabine, fulvestrant, gemcitabine, gemtuzumab ozogamicin,
goserelin, idarubicin, ifosfamide, imatinib mesylate, Interferon
alfa-2a, irinotecan, letrozole, leucovorin, levamisole,
meclorethamine or nitrogen mustard, megestrol, mesna, methotrexate,
methoxsalen, mitomycin C, mitotane, nandrolone phenpropionate,
oprelvekin, oxaliplatin, pamidronate, pegademase, pegaspargase,
pegfilgrastim, pentostatin, pipobroman, plicamycin, porfimer
sodium, procarbazine, quinacrine, rasburicase, Rituximab,
Sargramostim, streptozocin, tamoxifen, temozolomide, teniposide,
testolactone, thioguanine, toremifene, Tositumomab, Trastuzumab,
tretinoin, uracil mustard, valrubicin, vinblastine, vincristine,
vinorelbine or zoledronate.
[0229] In additional embodiments, the drug is a humanized anti HER2
monoclonal antibody, RITUXAN (rituximab; Genentech; a chimeric anti
CD20 monoclonal antibody); OVAREX (AltaRex Corporation, MA);
PANOREX (Glaxo Wellcome, NC; a murine IgG2a antibody); Cetuximab
Erbitux (Imclone Systems Inc., NY; an anti-EGFR IgG chimeric
antibody); Vitaxin (MedImmune, Inc., MD; Campath I/H (Leukosite,
MA; a humanized IgG1 antibody); lintuzumab (Protein Design Labs,
Inc. and Seattle Genetics, Inc.; a humanized anti-CD33 IgG
antibody); LymphoCide.TM. (Immunomedics, Inc., NJ; a humanized
anti-CD22 IgG antibody); Smart ID10 (Protein Design Labs, Inc., CA;
a humanized anti-HLA-DR antibody); Oncolym.TM. (Techniclone, Inc.,
CA; a radiolabeled murine anti-HLA-Dr10 antibody); Allomune.TM.
(BioTransplant, CA; a humanized anti-CD2 mAb); Avastin.TM.
(Genentech, Inc., CA; an anti-VEGF humanized antibody); Epratuzamab
(Immunomedics, Inc., NJ and Amgen, CA; an anti-CD22 antibody);
CEAcide.TM. (Immunomedics, NJ; a humanized anti-CEA antibody), or
an anti-CD40 antibody (e.g., as disclosed in U.S. Pat. No.
6,838,261).
[0230] Other suitable antibodies include, but are not limited to,
antibodies against the following antigens: CA125, CA15-3, CA19-9,
L6, Lewis Y, Lewis X, alpha fetoprotein, CA 242, placental alkaline
phosphatase, prostate specific antigen, prostatic acid phosphatase,
epidermal growth factor, MAGE-1, MAGE-2, MAGE-3, MAGE-4, anti
transferrin receptor, p97, MUC1-KLH, CEA, gp100, MART1, Prostate
Specific Antigen, IL-2 receptor, CD20, CD52, CD33, CD22, human
chorionic gonadotropin, CD38, CD40, mucin, P21, MPG, and Neu
oncogene product.
[0231] In certain embodiments, the therapeutic agent is an
immunosuppressive agent. The immunosuppressive agent can be, for
example, gancyclovir, etanercept, tacrolimus, cyclosporine,
rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil or
methotrexate. Alternatively, the immunosuppressive agent can be,
for example, a glucocorticoid (e.g., cortisol or aldosterone) or a
glucocorticoid analogue (e.g. prednisone or dexamethasone).
[0232] In certain typical embodiments, the immunosuppressive agent
is an anti-inflammatory agent, such as arylcarboxylic derivatives,
pyrazole-containing derivatives, oxicam derivatives and nicotinic
acid derivatives. Classes of anti-inflammatory agents include, for
example, cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, or
leukotriene receptor antagonists.
[0233] Suitable cyclooxygenase inhibitors include meclofenamic
acid, mefenamic acid, carprofen, diclofenac, diflunisal, fenbufen,
fenoprofen, ibuprofen, indomethacin, ketoprofen, nabumetone,
naproxen, sulindac, tenoxicam, tolmetin, and acetylsalicylic
acid.
[0234] Suitable lipoxygenase inhibitors include redox inhibitors
(e.g., catechol butane derivatives, nordihydroguaiaretic acid
(NDGA), masoprocol, phenidone, Ianopalen, indazolinones,
naphazatrom, benzofuranol, alkylhydroxylamine), and non-redox
inhibitors (e.g., hydroxythiazoles, methoxyalkylthiazoles,
benzopyrans and derivatives thereof, methoxytetrahydropyran,
boswellic acids and acetylated derivatives of boswellic acids, and
quinolinemethoxyphenylacetic acids substituted with cycloalkyl
radicals), and precursors of redox inhibitors.
[0235] Other suitable lipoxygenase inhibitors include antioxidants
(e.g., phenols, propyl gallate, flavonoids and/or naturally
occurring substrates containing flavonoids, hydroxylated
derivatives of the flavones, flavonol, dihydroquercetin, luteolin,
galangin, orobol, derivatives of chalcone,
4,2',4'-trihydroxychalcone, ortho-aminophenols, N-hydroxyureas,
benzofuranols, ebselen and species that increase the activity of
the reducing selenoenzymes), iron chelating agents (e.g.,
hydroxamic acids and derivatives thereof, N-hydroxyureas,
2-benzyl-1-naphthol, catechols, hydroxylamines, carnosol trolox C,
catechol, naphthol, sulfasalazine, zyleuton, 5-hydroxyanthranilic
acid and 4-(omega-arylalkyl)phenylalkanoic acids),
imidazole-containing compounds (e.g., ketoconazole and
itraconazole), phenothiazines, and benzopyran derivatives.
[0236] Yet other suitable lipoxygenase inhibitors include
inhibitors of eicosanoids (e.g., octadecatetraenoic,
eicosatetraenoic, docosapentaenoic, eicosahexaenoic and
docosahexaenoic acids and esters thereof, PGE1 (prostaglandin E1),
PGA2 (prostaglandin A2), viprostol, 15-monohydroxyeicosatetraenoic,
15-monohydroxy-eicosatrienoic and 15-monohydroxyeicosapentaenoic
acids, and leukotrienes B5, C5 and D5), compounds interfering with
calcium flows, phenothiazines, diphenylbutylamines, verapamil,
fuscoside, curcumin, chlorogenic acid, caffeic acid,
5,8,11,14-eicosatetrayenoic acid (ETYA), hydroxyphenylretinamide,
Ionapalen, esculin, diethylcarbamazine, phenantroline, baicalein,
proxicromil, thioethers, diallyl sulfide and di-(1-propenyl)
sulfide.
[0237] Leukotriene receptor antagonists include calcitriol,
ontazolast, Bayer Bay-x-1005, Ciba-Geigy CGS-25019C, ebselen, Leo
Denmark ETH-615, Lilly LY-293111, Ono ONO-4057, Terumo TMK-688,
Boehringer Ingleheim BI-RM-270, Lilly LY 213024, Lilly LY 264086,
Lilly LY 292728, Ono ONO LB457, Pfizer 105696, Perdue Frederick PF
10042, Rhone-Poulenc Rorer RP 66153, SmithKline Beecham SB-201146,
SmithKline Beecham SB-201993, SmithKline Beecham SB-209247, Searle
SC-53228, Sumitamo SM 15178, American Home Products WAY 121006,
Bayer Bay-o-8276, Warner-Lambert CI-987, Warner-Lambert
CI-987BPC-15LY 223982, Lilly LY 233569, Lilly LY-255283, MacroNex
MNX-160, Merck and Co. MK-591, Merck and Co. MK-886, Ono
ONO-LB-448, Purdue Frederick PF-5901, Rhone-Poulenc Rorer RG 14893,
Rhone-Poulenc Rorer RP 66364, Rhone-Poulenc Rorer RP 69698,
Shionoogi S-2474, Searle SC-41930, Searle SC-50505, Searle
SC-51146, Searle SC-52798, SmithKline Beecham SK&F-104493, Leo
Denmark SR-2566, Tanabe T-757 and Teijin TEI-1338.
[0238] 1. Dolastatin Drugs
[0239] In certain embodiments, the cytotoxic or cytostatic agent is
a dolastatin. In more specific embodiments, the dolastatin is of
the auristatin class. As used herein, the term dolastatin
encompasses naturally occurring auristatins and non-naturally
occurring derivatives, for example MMAE. Thus, in a specific
embodiment, the cytotoxic or cytostatic agent is MMAE (Formula XI).
In another specific embodiment, the cytotoxic or cytostatic agent
is AFP (Formula XVI). ##STR19##
[0240] In certain embodiments, the cytotoxic or cytostatic agent is
a dolastatin of formulas XII-XXI. ##STR20## ##STR21##
[0241] C. Formation of Anti-CD70 ADCs and ADC Derivatives
[0242] The generation of anti-CD70 ADCs and ADC derivatives can be
accomplished by any technique known to the skilled artisan.
Briefly, the anti-CD70 ADCs comprise an anti-CD70 antibody, a drug,
and optionally a linker that joins the drug and the antibody. A
number of different reactions are available for covalent attachment
of drugs to antibodies. This is often accomplished by reaction of
the amino acid residues of the antibody molecule, including the
amine groups of lysine, the free carboxylic acid groups of glutamic
and aspartic acid, the sulfhydryl groups of cysteine and the
various moieties of the aromatic amino acids. One of the most
commonly used non-specific methods of covalent attachment is the
carbodiimide reaction to link a carboxy (or amino) group of a
compound to amino (or carboxy) groups of the antibody.
Additionally, bifunctional agents such as dialdehydes or
imidoesters have been used to link the amino group of a compound to
amino groups of the antibody molecule. Also available for
attachment of drugs to antibodies is the Schiff base reaction. This
method involves the periodate oxidation of a drug that contains
glycol or hydroxy groups, thus forming an aldehyde which is then
reacted with the antibody molecule. Attachment occurs via formation
of a Schiff base with amino groups of the antibody molecule.
Isothiocyanates can also be used as coupling agents for covalently
attaching drugs to antibodies. Other techniques are known to the
skilled artisan and within the scope of the present invention.
Non-limiting examples of such techniques are described in, e.g.,
U.S. Pat. Nos. 5,665,358; 5,643,573; and 5,556,623, which are
incorporated by reference in their entireties herein.
[0243] In certain embodiments, an intermediate, which is the
precursor of the linker, is reacted with the drug under appropriate
conditions. In certain embodiments, reactive groups are used on the
drug and/or the intermediate. The product of the reaction between
the drug and the intermediate, or the derivatized drug, is
subsequently reacted with the anti-CD70 antibody under appropriate
conditions.
IV. Other CD70-Targeting Moieties and Protein-Drug Conjugates
[0244] As indicated supra, in other embodiments, the CD70-targeting
moiety need not be an antibody to be useful in accordance with the
methods described herein. Accordingly, a CD70-targeting moiety can
include one or more CDRs from an antibody that binds to CD70 and
depletes or inhibits the proliferation of CD70-expressing cells
when conjugated to a cytotoxic agent, or from an antibody that
binds to CD70 and exerts an immunosuppressive effect when
conjugated to an immunosuppressive agent. Typically, the protein is
a multimer, most typically a dimer.
[0245] Further, CD70-binding proteins useful in accordance with the
methods provided herein include fusion proteins, i.e., proteins
that are recombinantly fused or chemically conjugated (including
both covalent and non-covalent conjugation) to heterologous
proteins (of typically at least 10, 20, 30, 40, 50, 60, 70, 80, 90
or at least 100 amino acids). The fusion does not necessarily need
to be direct, but may occur through linker sequences.
[0246] For example, CD70-targeting moieties useful in the present
methods can be produced recombinantly by fusing the coding region
of one or more of the CDRs of an anti-CD70 antibody in frame with a
sequence coding for a heterologous protein. The heterologous
protein may provide one or more of the following characteristics:
added therapeutic benefits; promote stable expression; provide a
means of facilitating high yield recombinant expression; and/or
provide a multimerization domain.
[0247] In other aspects, CD70-targeting moieties can include CD27
and variants or fragments thereof that bind to CD70. CD70-targeting
moieties can further include peptides, ligands and other molecules
that specifically bind to CD70.
[0248] CD70-targeting moieties useful in the methods described
herein can be identified using any method suitable for screening
for protein-protein interactions. Typically, proteins are initially
identified by their ability to specifically bind to CD70, then
their ability to exert a cytostatic or cytotoxic effect on
activated lymphocytes or CD70-expressing cancer cells when
conjugated to a cytotoxic or cytostatic agent, or an
immunosuppressive effect on an immune cell when conjugated to an
immunosuppressive agent. Among the traditional methods which can be
employed are "interaction cloning" techniques which entail probing
expression libraries with labeled CD70 in a manner similar to the
technique of antibody probing of .lamda.gt11 libraries. By way of
example and not limitation, this can be achieved as follows: a cDNA
clone encoding CD70 (or a 1F6 or 2F2 binding domain thereof) is
modified at the terminus by inserting the phosphorylation site for
the heart muscle kinase (HMK) (see, e.g., Blanar and Rutter, 1992,
Science 256:1014-1018). The recombinant protein is expressed in E.
coli and purified on a GDP-affinity column to homogeneity (Edery et
al., 1988, Gene 74:517-525) and labeled using .gamma..sup.32P-ATP
and bovine heart muscle kinase (Sigma) to a specific activity of
1.times.10.sup.8 cpm/.mu.g, and used to screen a human placenta
.lamda.gt11 cDNA library in a "far-Western assay" (Blanar and
Rutter, 1992, Science 256:1014-1018). Plaques which interact with
the CD70 probe are isolated. The cDNA inserts of positive .lamda.
plaques are released and subcloned into a vector suitable for
sequencing, such as pBluescript KS (Stratagene).
[0249] One method which detects protein interactions in vivo, the
two-hybrid system, is described in detail for illustration purposes
only and not by way of limitation. One version of this system has
been described (Chien et al., 1991, Proc. Natl. Acad. Sci. USA
88:9578-9582) and is commercially available from Clontech (Palo
Alto, Calif.).
[0250] Once a CD70-binding protein is identified, its ability
(alone or when multimerized or fused to a dimerization or
multimerization domain) to exert a cytostatic or cytotoxic effect
on CD70-expressing cancer cells (when conjugated to a cytotoxic
agent) or an immunosuppressive effect on a CD70-expressing immune
cell (when conjugated to an immunosuppressive agent) is determined
by the methods described infra.
V. Assays for Cytotoxic Cytostatic, and Immunosuppressive
Activities
[0251] In accordance with the methods described herein, an
anti-CD70 ADC or ADC derivative exerts a cytotoxic or cytostatic
effect on a CD70-expressing cancer cell, or a cytotoxic,
cytostatic, or immunosuppressive effect on activated
CD70-expressing immune cell (e.g., activated lymphocyte or
dendritic cell). Activated lymphocytes that can be assayed for a
cytotoxic, cytostatic, or immunosuppressive effect of an anti-CD70
ADC or ADC derivative can be cultured cell lines (e.g., CESS, which
is available from the ATCC; or KMH2 and L428, both of which are
available from Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH), or from lymphocytes prepared from a fresh blood
sample or other sources. Lymphocytes can be activated by the
appropriate cocktails of antibodies and cytokines. For example, T
lymphocytes can be activated using allogeneic stimulator cells or
antigen-pulsed autologous antigen presenting cells and T cell
clones can be activated by PHA and cytokines in the presence of
feeder cells, as described in Example 9, infra. CD70-expressing
cancer cells that can be assayed for a cytotoxic or cytotoxic
effect can be tissue culture cell lines as described in Examples 7,
infra, or from CD70-expressing cancer cells prepared from a
subject.
[0252] Once an anti-CD70 ADC or ADC derivative is confirmed as
exerting a cytotoxic or cytostatic on CD70-expressing cancer cells
or a cytotoxic, cytostatic, or immunosuppressive effect on
activated immune cells, its therapeutic value can be validated in
an animal model. Exemplary animal models of immunological disorders
or CD70-expressing cancers are described in Section VI, infra.
[0253] Methods of determining whether an agent exerts a cytostatic
or cytotoxic effect on a cell are known. Illustrative examples of
such methods are described infra.
[0254] For determining whether an anti-CD70 ADC or ADC derivative
exerts a cytostatic effect on activated immune cells or
CD70-expressing cancer cells, a thymidine incorporation assay may
be used. For example, activated immune cells (e.g., activated
lymphocytes) or CD70-expressing cancer cells at a density of 5,000
cells/well of a 96-well plated can be cultured for a 72-hour period
and exposed to 0.5 .mu.Ci of .sup.3H-thymidine during the final 8
hours of the 72-hour period, and the incorporation of
.sup.3H-thymidine into cells of the culture is measured in the
presence and absence of the ADC or ADC derivative.
[0255] For determining cytotoxicity, necrosis or apoptosis
(programmed cell death) can be measured. Necrosis is typically
accompanied by increased permeability of the plasma membrane;
swelling of the cell, and rupture of the plasma membrane. Apoptosis
is typically characterized by membrane blebbing, condensation of
cytoplasm, and the activation of endogenous endonucleases.
Determination of any of these effects on activated immune cells or
CD70 expressing cancer cells indicates that an anti-CD70 ADC or ADC
derivative is useful in the treatment or prevention of
immunological disorders and CD70-expressing cancers.
[0256] Cell viability can be measured by determining in a cell the
uptake of a dye such as neutral red, typan blue, or ALAMAR.TM. blue
(see, e.g., Page et al., 1993, Intl. J. of Oncology 3:473-476). In
such an assay, the cells are incubated in media containing the dye,
the cells are washed, and the remaining dye, reflecting cellular
uptake of the dye, is measured spectrophotometrically. The
protein-binding dye sulforhodamine B (SRB) can also be used to
measure cytoxicity (Skehan et al., 1990, J. Nat'l Cancer Inst.
82:1107-12).
[0257] Alternatively, a tetrazolium salt, such as MTT, is used in a
quantitative colorimetric assay for mammalian cell survival and
proliferation by detecting living, but not dead, cells (see, e.g.,
Mosmann, 1983, J. Immunol. Methods 65:55-63).
[0258] Apoptosis can be quantitated by measuring, for example, DNA
fragmentation. Commercial photometric methods for the quantitative
in vitro determination of DNA fragmentation are available. Examples
of such assays, including TUNEL (which detects incorporation of
labeled nucleotides in fragmented DNA) and ELISA-based assays, are
described in Biochemica, 1999, no. 2, pp. 34-37 (Roche Molecular
Biochemicals).
[0259] Apoptosis can also be determined by measuring morphological
changes in a cell. For example, as with necrosis, loss of plasma
membrane integrity can be determined by measuring uptake of certain
dyes (e.g., a fluorescent dye such as, for example, acridine orange
or ethidium bromide). A method for measuring apoptotic cell number
has previously been described by Duke and Cohen, Current Protocols
In Immunology (Coligan et al. eds., 1992, pp. 3.17.1-3.17.16).
Cells can be also labeled with a DNA dye (e.g., acridine orange,
ethidium bromide, or propidium iodide) and the cells observed for
chromatin condensation and margination along the inner nuclear
membrane. Other morphological changes that can be measured to
determine apoptosis include, e.g., cytoplasmic condensation,
increased membrane blebbing, and cellular shrinkage.
[0260] The presence of apoptotic cells can be measured in both the
attached and "floating" compartments of the cultures. For example,
both compartments can be collected by removing the supernatant,
trypsining the attached cells, combining the preparations following
a centrifugation wash step (e.g., 10 minutes, 2000 rpm), and
detecting apoptosis (e.g., by measuring DNA fragmentation). (See,
e.g., Piazza et al., 1995, Cancer Research 55:3110-16).
[0261] Immunosuppressive effects can be measured, for example, by
assaying for a cytotoxic or cytostatic effect (as described supra)
on an activated immune cell involved in promoting immune responses
(e.g., CD8.sup.+ cytotoxic T cells, CD4.sup.+ Th1 cells, B cells,
or mature dendritic cells). In addition or alternatively, an
immunosuppressive effect can be determined by examination of the
presence or absence of specific immune cell populations (using,
e.g., flow cytometry) such as, for example, the presence of T
regulatory cells involved in suppression of an immune response;
measurement of the functional capacity of immune cells, including,
e.g., the cytolytic capacity of cytotoxic T cells; measurements of
the cytokines, chemokines, cell surface molecules, antibodies or
other secreted or cell-surface molecules of the cells (e.g., by
flow cytometry, enzyme-linked immunosorbent assays, Western blot
analysis, protein microarray analysis, immunoprecipitation
analysis); or measurement of biochemical markers of activation of
immune cells or signaling pathways within immune cells (e.g.,
Western blot and immunoprecipitation analysis of tyrosine, serine
or threonine phosphorylation, polypeptide cleavage, and formation
or dissociation of protein complexes; protein array analysis; DNA
transcriptional profiling using DNA arrays or subtractive
hybridization).
VI. Animal Models of Immunological Disorders or CD70-Expressing
Cancers
[0262] The anti-CD70 ADCs or ADC derivatives can be tested or
validated in animal models of immunological disorders or
CD70-expressing cancers. A number of established animal models of
immunological disorders or CD70-expressing cancers are known to the
skilled artisan, any of which can be used to assay the efficacy of
the ADC or ADC derivative. Non-limiting examples of such models are
described infra.
[0263] Some examples for animal models of systemic and
organ-specific autoimmune diseases including diabetes, systemic
lupus erythematosus, systemic sclerosis, Sjogren's Syndrome,
experimental autoimmune encephalomyelitis (multiple sclerosis),
thyroiditis, myasthenia gravis, arthritis, uveitis, inflammatory
bowel disease have been described by Bigazzi, "Animal Models of
Autoimmunity: Spontaneous and Induced," in The Autoimmune Diseases
(Rose & Mackay eds., Academic Press, 1998) and in "Animal
Models for Autoimmune and Inflammatory Disease," in Current
Protocols in Immunology (Coligan et al. eds., Wiley & Sons,
1997).
[0264] Allergic conditions, e.g., asthma and dermatitis, can also
be modeled in rodents. Airway hypersensitivity can be induced in
mice by ovalbumin (Tomkinson et al., 2001, J. Immunol.
166:5792-5800) or Schistosoma mansoni egg antigen (Tesciuba et al.,
2001, J. Immunol. 167:1996-2003). The Nc/Nga strain of mice show
marked increase in serum IgE and spontaneously develop atopic
dermatitis-like leisons (Vestergaard et al., 2000, Mol. Med. Today
6:209-210; Watanabe et al., 1997, Int. Immunol. 9:461-466; Saskawa
et al., 2001, Int. Arch. Allergy Immunol. 126:239-247).
[0265] Injection of immuno-competent donor lymphocytes into a
lethally irradiated histo-incompatible host is a classical approach
to induce acute GVHD in mice. Alternatively, the parent B6D2F1
murine model provides a system to induce both acute and chronic
GVHD. In this model the B6D2F1 mice are F1 progeny from a cross
between the parental strains of C57BL/6 and DBA/2 mice. Transfer of
DBA/2 lymphoid cells into non-irradiated B6D2F1 mice causes chronic
GVHD, whereas transfer of C57BL/6, C57BL/10 or B10.D2 lymphoid
cells causes acute GVHD (Slayback et al., 2000, Bone Marrow
Transpl. 26:931-938; Kataoka et al., 2001, Immunology
103:310-318).
[0266] Additionally, both human hematopoietic stem cells and mature
peripheral blood lymphoid cells can be engrafted into SCID mice,
and these human lympho-hematopoietic cells remain functional in the
SCID mice (McCune et al., 1988, Science 241:1632-1639; Kamel-Reid
and Dick, 1988, Science 242:1706-1709; Mosier et al., 1988, Nature
335:256-259). This has provided a small animal model system for the
direct testing of potential therapeutic agents on human lymphoid
cells. (See, e.g., Tournoy et al., 2001, J. Immunol.
166:6982-6991).
[0267] Moreover, small animal models to examine the in vivo
efficacies of the anti-CD70 ADCs or ADC derivatives can be created
by implanting CD70-expressing human tumor cell lines into
appropriate immunodeficient rodent strains, e.g., athymic nude mice
or SCID mice. Examples of CD70-expressing human lymphoma cell lines
include, for example, Daudi (Ghetie et al., 1994, Blood 83:1329-36;
Ghetie et al., 1990, Int J Cancer 15:481-5; de Mont et al., 2001,
Cancer Res 61:7654-59), HS-Sultan (Cattan & Maung, 1996, Cancer
Chemother Pharmacol 38:548-52; Cattan and Douglas, 1994, LeukRes
18:513-22), and Raji (Ochakovskaya et al., 2001, Clin Cancer Res
7:1505-10; Breisto et al., 1999, Cancer Res 59:2944-49). A
non-limiting example of a CD70-expressing Hodgkin's lymphoma line
is L428 (Drexler, 1993, Leukemia and Lymphoma 9:1-25; Dewan et al.,
2005, Cancer Sci. 96:466-473). Non-limiting examples of CD70
expressing human renal cell carcinoma cell lines include 786-O
(Ananth et al., 1999, Cancer Res 59:2210-6; Datta et al., 2001,
Cancer Res 61:1768-75), ACHN (Hara et al., 2001, J Urol.
166:2491-4; Miyake et al., 2002, J Urol. 167:2203-8), Caki-1
(Prewett et al., 1998, Clin. Cancer Res. 4:2957-66; Shi and
Siemann, 2002, Br. J. Cancer 87:119-26), and Caki-2 (Zellweger et
al., 2001, Neoplasia 3:360-7). Non-limiting examples of
CD70-expressing nasopharyngeal carcinoma cell lines include C15 and
C17 (Busson et al., 1988, Int. J. Cancer 42:599-606; Bernheim et
al., 1993, Cancer Genet. Cytogenet. 66:11-5). Non-limiting examples
of CD70-expressing human glioma cell lines include U373 (Palma et
al., 2000, Br. J. Cancer 82:480-7) and U87MG (Johns et al., 2002,
Int. J. Cancer 98:398-408). Non-limiting examples of multiple
myeloma cell lines include MM.1S (Greenstein et al., 2003,
Experimental Hematology 31:271-282) and L363 (Diehl et al., 1978,
Blut 36:331-338). (See also Drexler and Matsuo, 2000, Leukemia
Research 24:681-703). These tumor cell lines can be established in
immunodeficient rodent hosts either as solid tumor by subcutaneous
injections or as disseminated tumors by intravenous injections.
Once established within a host, these tumor models can be applied
to evaluate the therapeutic efficacies of the anti-CD70 ADCs or ADC
derivatives as described herein on modulating in vivo tumor
growth.
VII. Immune Disorders and CD70-Expressing Cancers
[0268] The anti-CD70 ADCs and ADC derivatives as described herein
are useful for treating or preventing an immunological disorder
characterized by inappropriate activation of immune cells (e.g.,
lymphocytes or dendritic cells). Treatment or prevention of the
immunological disorder, according to the methods described herein,
is achieved by administering to a subject in need of such treatment
or prevention an effective amount of the anti-CD70 ADC or ADC
derivative, whereby the ADC or ADC derivative (i) binds to
activated immune cells that express CD70 and that are associated
with the disease state and (ii) exerts a cytotoxic, cytostatic, or
immunosuppressive effect on the activated immune cells.
[0269] Immunological diseases that are characterized by
inappropriate activation of immune cells and that can be treated or
prevented by the methods described herein can be classified, for
example, by the type(s) of hypersensitivity reaction(s) that
underlie the disorder. These reactions are typically classified
into four types: anaphylactic reactions, cytotoxic (cytolytic)
reactions, immune complex reactions, or cell-mediated immunity
(CMI) reactions (also referred to as delayed-type hypersensitivity
(DTH) reactions). (See, e.g., Fundamental Immunology (William E.
Paul ed., Raven Press, N.Y., 3rd ed. 1993).)
[0270] Specific examples of such immunological diseases include the
following: rheumatoid arthritis, psoriatic arthritis, autoimmune
demyelinative diseases (e.g., multiple sclerosis, allergic
encephalomyelitis), endocrine opthalmopathy, uveoretinitis,
systemic lupus erythematosus, myasthenia gravis, Grave's disease,
glomerulonephritis, autoimmune hepatological disorder, inflammatory
bowel disease (e.g., Crohn's disease), anaphylaxis, allergic
reaction, Sjogren's syndrome, type I diabetes mellitus, primary
biliary cirrhosis, Wegener's granulomatosis, fibromyalgia,
polymyositis, dermatomyositis, multiple endocrine failure,
Schmidt's syndrome, autoimmune uveitis, Addison's disease,
adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune
thyroid disease, pernicious anemia, gastric atrophy, chronic
hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous
lupus erythematosus, hypoparathyroidism, Dressler's syndrome,
autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura,
hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis
herpetiformis, alopecia arcata, pemphigoid, psoriasis, scleroderma,
progressive systemic sclerosis, CREST syndrome (calcinosis,
Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and
telangiectasia), male and female autoimmune infertility, ankylosing
spondolytis, ulcerative colitis, mixed connective tissue disease,
polyarteritis nedosa, systemic necrotizing vasculitis, atopic
dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas'
disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion,
anti-phospholipid syndrome, farmer's lung, erythema multiforme,
post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic
active hepatitis, bird-fancier's lung, toxic epidermal necrolysis,
Alport's syndrome, alveolitis, allergic alveolitis, fibrosing
alveolitis, interstitial lung disease, erythema nodosum, pyoderma
gangrenosum, transfusion reaction, Takayasu's arteritis,
polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant
cell arteritis, ascariasis, aspergillosis, Sampter's syndrome,
eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's
syndrome, Kawasaki's disease, dengue, encephalomyelitis,
endocarditis, endomyocardial fibrosis, endophthalmitis, erythema
elevatum et diutinum, psoriasis, erythroblastosis fetalis,
eosinophilic faciitis, Shulman's syndrome, Felty's syndrome,
filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis,
Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft
versus host disease (e.g., following hematopoeitic stem cell
transplant), transplantation rejection, cardiomyopathy,
Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia,
Waldenstrom's macroglobulemia, Evan's syndrome, and autoimmune
gonadal failure.
[0271] Accordingly, the methods described herein encompass
treatment of disorders of B lymphocytes (e.g., systemic lupus
erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and
type I diabetes), Th.sub.1-lymphocytes (e.g., rheumatoid arthritis,
multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's
thyroiditis, Grave's disease, primary biliary cirrhosis, Wegener's
granulomatosis, tuberculosis, or acute graft versus host disease),
or Th.sub.2-lymphocytes (e.g., atopic dermatitis, systemic lupus
erythematosus, atopic asthma, rhinoconjunctivitis, allergic
rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft
versus host disease). Generally, disorders involving dendritic
cells involve disorders of Th.sub.1-lymphocytes or
Th.sub.2-lymphocytes.
[0272] In certain embodiments, the immunological disorder is a T
cell-mediated immunological disorder, such as a T cell disorder in
which activated T cell associated with the disorder express CD70.
ADCs or ADC derivatives can be administered to deplete such
CD70-expressing activated T cells. In a specific embodiment,
administration of ADCs or ADC derivatives can deplete
CD70-expressing activated T cells, while resting T cells are not
substantially depleted by the ADC or ADC derivative. In this
context, "not substantially depleted" means that less than about
60%, or less than about 70% or less than about 80% of resting T
cells are not depleted.
[0273] The anti-CD70 ADCs and ADC derivatives as described herein
are also useful for treating or preventing a CD70-expressing
cancer. Treatment or prevention of a CD70-expressing cancer,
according to the methods described herein, is achieved by
administering to a subject in need of such treatment or prevention
an effective amount of the anti-CD70 ADC or ADC derivative, whereby
the ADC or ADC derivative (i) binds to CD70-expressing cancer cells
and (ii) exerts a cytotoxic or cytostatic effect to deplete or
inhibit the proliferation of the CD70-expressing cancer cells.
[0274] CD70-expressing cancers that can be treated or prevented by
the methods described herein include, for example, different
subtypes of Non-Hodgkin's Lymphoma (indolent NHLs, follicular NHLs,
small lymphocytic lymphomas, lymphoplasmacytic NHLs, or marginal
zone NHLs); Hodgkin's disease (e.g., Reed-Sternberg cells); cancers
of the B-cell lineage, including, e.g., diffuse large B-cell
lymphomas, follicular lymphomas, Burkitt's lymphoma, mantle cell
lymphomas, B-cell lymphocytic leukemias (e.g., acute lymphocytic
leukemia, chronic lymphocytic leukemia); Epstein Barr Virus
positive B cell lymphomas; renal cell carcinomas (e.g., clear cell
and papillary); nasopharyngeal carcinomas; thymic carcinomas;
gliomas; glioblastomas; neuroblastomas; astrocytomas; meningiomas;
Waldenstrom macroglobulinemia; multiple myelomas; and colon,
stomach, and rectal carcinomas.
[0275] In some embodiments, a CD70-expressing cancer has at least
about 15,000, at least about 10,000 or at least about 5,000 CD70
molecules/cell.
VIII. Pharmaceutical Compositions Comprising Anti-CD70 ADCs and ADC
Derivatives and Administration Thereof
[0276] In accordance with the present methods, a composition
comprising an anti-CD70 ADC or ADC derivative as described herein
is administered to a subject having or at risk of having an
immunological disorder or a CD70-expressing cancer. The term
"subject" as used herein means any mammalian patient to which a
CD70-binding protein-drug conjugate may be administered, including,
e.g., humans and non-human mammals, such as primates, rodents, and
dogs. Subjects specifically intended for treatment using the
methods described herein include humans. The ADCs or ADC
derivatives can be administered either alone or in combination with
other compositions in the prevention or treatment of the
immunological disorder or CD70-expressing cancer.
[0277] Various delivery systems are known and can be used to
administer the anti-CD70 ADC or ADC derivative. Methods of
introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The ADCs or ADC derivatives
can be administered, for example by infusion or bolus injection, by
absorption through epithelial or mucocutaneous linings (e.g., oral
mucosa, rectal and intestinal mucosa, and the like) and can be
administered together with other biologically active agents such as
chemotherapeutic agents. Administration can be systemic or
local.
[0278] In specific embodiments, the anti-CD70 ADC or ADC derivative
composition is administered by injection, by means of a catheter,
by means of a suppository, or by means of an implant, the implant
being of a porous, non-porous, or gelatinous material, including a
membrane, such as a sialastic membrane, or a fiber. Typically, when
administering the composition, materials to which the ADC or ADC
derivative does not absorb are used.
[0279] In other embodiments, the ADC or ADC derivative is delivered
in a controlled release system. In one embodiment, a pump may be
used (see Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC
Crit. Ref Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery
88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another
embodiment, polymeric materials can be used. (See Medical
Applications of Controlled Release (Langer & Wise eds., CRC
Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability,
Drug Product Design and Performance (Smolen & Ball eds., Wiley,
New York, 1984); Ranger & Peppas, 1983, Macromol. Sci. Rev.
Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228:190;
During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J.
Neurosurg. 71:105.) Other controlled release systems are discussed,
for example, in Langer, supra.
[0280] The anti-CD70 ADCs or ADC derivatives are administered as
pharmaceutical compositions comprising a therapeutically effective
amount of the ADC or ADC derivative and one or more
pharmaceutically compatible ingredients. For example, the
pharmaceutical composition typically includes one or more
pharmaceutical carriers (e.g., sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like). Water is a more typical carrier when the
pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable solutions.
Suitable pharmaceutical excipients include, for example, starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol, and the like. The composition, if desired, can also
contain minor amounts of wetting or emulsifying agents, or pH
buffering agents. These compositions can take the form of
solutions, suspensions, emulsion, tablets, pills, capsules,
powders, sustained-release formulations and the like. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc. Examples of suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin. Such compositions will
contain a therapeutically effective amount of the nucleic acid or
protein, typically in purified form, together with a suitable
amount of carrier so as to provide the form for proper
administration to the patient. The formulations correspond to the
mode of administration.
[0281] In typical embodiments, the pharmaceutical composition is
formulated in accordance with routine procedures as a
pharmaceutical composition adapted for intravenous administration
to human beings. Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous buffer.
Where necessary, the pharmaceutical can also include a buffering
agent (e.g., a phosphate, citrate or amino acid, such as
histidine), a solubilizing agent (e.g., nonionic detergents such as
a polysorbate, triton, or polyoxamer; or an amino acid) and/or a
local anesthetic such as lignocaine to ease pain at the site of the
injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachette indicating the
quantity of active agent. Where the pharmaceutical is to be
administered by infusion, it can be dispensed with an infusion
bottle containing sterile pharmaceutical grade water or saline.
Where the pharmaceutical is administered by injection, an ampoule
of sterile water for injection or saline can be provided so that
the ingredients can be mixed prior to administration.
[0282] In certain embodiments, the pharmaceutical compositions
comprising the anti-CD70 ADC or ADC derivative can further comprise
a second therapeutic agent (e.g., a second ADC or ADC derivative or
a non-conjugated cytotoxic or immunosuppressive agent such as, for
example, any of those described herein).
[0283] Further, the pharmaceutical composition can be provided as a
pharmaceutical kit comprising (a) a container containing an
anti-CD70 ADC or ADC derivative in lyophilized form and (b) a
second container containing a pharmaceutically acceptable diluent
(e.g., sterile water) for injection. The pharmaceutically
acceptable diluent can be used for reconstitution or dilution of
the lyophilized ADC or ADC derivative. Optionally associated with
such container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration.
[0284] The amount of the ADC or ADC derivative that is effective in
the treatment or prevention of an immunological disorder or
CD70-expressing cancer can be determined by standard clinical
techniques. In addition, in vitro assays may optionally be employed
to help identify optimal dosage ranges. The precise dose to be
employed in the formulation will also depend on the route of
administration, and the stage of immunological disorder or
CD70-expressing cancer, and should be decided according to the
judgment of the practitioner and each patient's circumstances.
Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0285] For example, toxicity and therapeutic efficacy of the ADCs
or ADC derivatives can be determined in cell cultures or
experimental animals by standard pharmaceutical procedures for
determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. ADCs or ADC derivatives
that exhibit large therapeutic indices are preferred. Where an ADC
or ADC derivative exhibits toxic side effects, a delivery system
that targets the ADC or ADC derivative to the site of affected
tissue can be used to minimize potential damage non-CD70-expressing
cells and, thereby, reduce side effects.
[0286] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of the anti-CD70 ADC or ADC derivative typically
lies within a range of circulating concentrations that include the
ED.sub.50 with little or no toxicity. The dosage may vary within
this range depending upon the dosage form employed and the route of
administration utilized. For any ADC or ADC derivative used in the
method, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose can be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound that achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma can
be measured, for example, by high performance liquid
chromatography.
[0287] Generally, the dosage of an anti-CD70 ADC or ADC derivative
administered to a patient with an immunological disorder or
CD70-expressing cancer is typically 0.1 mg/kg to 100 mg/kg of the
subject's body weight. More typically, the dosage administered to a
subject is 0.1 mg/kg to 50 mg/kg of the subject's body weight, even
more typically 1 mg/kg to 30 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to
15 mg/kg, 1 mg/kg to 12 mg/kg, 1 mg/kg to 10 mg/kg, or 1 mg/kg to
7.5 mg/kg of the subject's body weight. Generally, human antibodies
have a longer half-life within the human body than antibodies from
other species due to the immune response to the foreign proteins.
Thus, lower dosages of ADCs comprising humanized, chimeric or human
antibodies and less frequent administration is often possible.
[0288] A dose of an anti-CD70 ADC or ADC derivative can be
administered, for example, daily, once per week (weekly), twice per
week, thrice per week, four times per week, five times per week,
biweekly, monthly or otherwise as needed.
[0289] In some embodiments, the dosage of an anti-CD70 ADC or ADC
derivative corresponds to a sub-optimal dosage (i.e., below the
EC.sub.50 for the anti-CD70 ADC or ADC derivative). For example,
the dosage of an anti-CD70 ADC or ADC derivative can comprise a
dosage selected from the lowest 25%, lowest 15%, lowest 10% or
lowest 5% of the therapeutic window. As used herein, the term
"therapeutic window" refers to the range of dosage of a drug or of
its concentration in a bodily system that provides safe and
effective therapy.
[0290] In some embodiments, the dosage of an anti-CD70 ADC or ADC
derivative is from about 0.05 mg/kg to about 2 mg/kg, about 0.05
mg/kg to about 1 mg/kg, or about 0.1 mg/kg to about 0.9 mg/kg, or
about 0.15 to about 0.75 mg/kg of the subject's body weight. Such a
dosage can be administered from 1 to about 15 times per week. Each
dose can be the same or different. For example, a dosage of about
0.15 mg/kg of an anti-CD70 ADC or ADC derivative can be
administered from 1 to 10 times per four day, five day, six day or
seven day period.
[0291] The anti-CD70 ADC or ADC derivative can be administered in
combination with one or more other therapeutic agents for the
treatment or prevention of immunological disorders or
CD70-expressing cancers. For example, combination therapy can
include a second cytostatic, cytotoxic, or immunosuppressive agent
(for example, an unconjugated cytostatic, cytotoxic, or
immunosuppressive agent such as those conventionally used for the
treatment of cancers or immunological disorders (e.g., standard of
care therapies)). Combination therapy can also include, e.g.,
administration of an agent that targets a receptor or receptor
complex other than CD70 on the surface of activated lymphocytes,
dendritic cells or CD70-expressing cancer cells. An example of such
an agent is a second, non-CD70 antibody that binds to a molecule at
the surface of an activated lymphocyte, dendritic cells or
CD70-expressing cancer cells. Another example is a ligand that
targets such a receptor or receptor complex. Typically, such an
antibody or ligand binds to a cell surface receptor on activated
lymphocytes, dendritic cells or CD70-expressing cancer cells and
enhances the cytotoxic or cytostatic effect of the anti-CD70
antibody by delivering a cytostatic or cytotoxic signal to the
activated lymphocytes, dendritic cells or CD70-expressing cancer
cells.
[0292] In certain embodiments, the therapeutic agent is an
anti-VEGF agent, such as AVASTIN (bevacizumab) or NEXAVAR
(Sorafenib); a PDGF blocker, such as SUTENT (sunitinib malate); an
immunomodulatory agent such as Revlimid (lenalidomide); a cytokine
such as G-CSF, GM-CSF or IL-2; VELCADE (bortezomib); a kinase
inhibitor, such as NEXAVAR (sorafenib tosylateor); a steroid; or an
immunosuppressants, such as Rapamycin (Sirolimus). In some
embodiments, the therapeutic agent can be a combined therapy, such
as CHOP (Cyclophosphamide, Doxorubicin, Prednisolone and
Vincristine), CHOP-R (Cyclophosphamide, Doxorubicin Vincristine,
Prednisolone, and rituximab) or ABVD (Doxorubicin, Bleomycin,
Vinblastine and Dacarbazine). In other embodiments, the therapeutic
agent can be an antibody, such as an anti-CD40 antibody (see, e.g.,
U.S. Pat. No. 6,838,261) or RITUXAN (Rituximab).
[0293] Such combinatorial administration can have an additive or
synergistic effect on disease parameters (e.g., severity of a
symptom, the number of symptoms, or frequency of relapse).
[0294] With respect to therapeutic regimens for combinatorial
administration, in a specific embodiment, an anti-CD70 ADC or ADC
derivative is administered concurrently with a second therapeutic
agent. In another specific embodiment, the second therapeutic agent
is administered prior or subsequent to administration of the
anti-CD70 ADC or ADC derivative, by at least an hour and up to
several months, for example at least an hour, five hours, 12 hours,
a day, a week, a month, or three months, prior or subsequent to
administration of the ADC or ADC derivative.
[0295] The present invention is not to be limited in scope by the
specific embodiments described herein. Various modifications of the
invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying figures. Such modifications are intended to fall
within the scope of the appended claims.
[0296] The invention is further described in the following
examples, which are in not intended to limit the scope of the
invention. Cell lines described in the following examples were
maintained in culture according to the conditions specified by the
American Type Culture Collection (ATCC) or Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany
(DMSZ). Cell culture reagents were obtained from Invitrogen Corp.,
Carlsbad, Calif.
EXAMPLE 1
Sequence Analysis of Anti-CD70 Monoclonal Antibodies 1F6 and
2F2
[0297] To determine the cDNA sequences encoding the light (V.sub.L)
and heavy (V.sub.H) chain variable regions of 1F6 and 2F2 mAb,
total RNA was isolated from the 1F6 and 2F2 hybridomas using the
TRIzol.RTM. Reagent (Invitrogen, Carlsbad, Calif.) according to the
manufacturer's instructions. The gene-specific primers mIgcK1
5'-CTT CCA CTT GAC ATT GAT GTC TTT G-3' (SEQ ID NO:41) and mIgG1
5'-CAG GTC ACT GTC ACT GGC TCA G-3' (SEQ ID NO:42) were applied to
reverse transcribe the light chain variable (V.sub.L) and heavy
chain variable (V.sub.H) first strand cDNAs from both RNA
preparations, respectively. First strand cDNA reactions were run
using the SuperScript.TM. First Strand Synthesis System for RT-PCR
from Invitrogen (Carlsbad, Calif.). The V.sub.L and V.sub.H cDNAs
were then poly-G tailed using terminal deoxynucleotidyl transferase
(TdT) (Invitrogen) and the supplied TdT buffer in conditions
specified by the manufacturer. Poly-G tailed V.sub.L and V.sub.H
first strand cDNAs were then subjected to PCR amplification. The
forward primer for both the V.sub.L and V.sub.H PCRs was ANCTAIL 5'
GTC GAT GAG CTC TAG AAT TCG TGC CCC CCC CCC CCC C-3' (SEQ ID
NO:43). The reverse primer for amplifying the V.sub.L was BBS-mck
5'-CGT CAT GTC GAC GGA TCC AAG CTT CAA GAA GCA CAC GAC TGA GGC
AC-3' (SEQ ID NO:44). The reverse primer for amplifying the V.sub.H
was HBS-mG1 5'-CGT CAT GTC GAC GGA TCC AAG CTT GTC ACC ATG GAG TTA
GTT TGG GC-3' (SEQ ID NO:45). PCRs were run with Ex Taq (Fisher
Scientific, Pittsburgh, Pa.) and the supplied reaction buffer in
conditions specified by the manufacturer. The V.sub.L and V.sub.H
PCR products were then cut by HindIII and EcoRI and cloned into
HindIII/EcoRI-cut pUC19. Recombinant plasmid clones were
identified, and the nucleotide sequences for the 1F6 and 2F2
hybridomas were determined.
[0298] Complementarity determining regions (CDRs) in the heavy and
light chains of 1F6 and 2F2 mAbs were determined according to the
criteria described in Kabat et al., 1991, Sequences of Proteins of
Immunological Interest, Washington D.C., US Department of Health
and Public Services; Chothia and Lesk, 1987, J. Mol. Biol.
196:901-17 (FIGS. 1 and 2). Sequence alignments at both the cDNA
and amino acid levels revealed closely related light chain genes
were probably utilized in both hybridomas. There is a 92% sequence
identity between 1F6 V.sub.L and 2F2 V.sub.L on the amino acid
levels. Sequence comparison of the CDRs shows that 1F6 CDR-L1 is
identical to 2F2 CDR-L1, only one divergent substitution is present
between 1F6 CDR-L2 and 2F2 CDR-L2, and only 2 conservative
substitutions are present between 1F6 CDR-L3 and 2F2 CDR-L3 (FIG.
3). On the other hand, a higher degree of sequence diversity is
present between 1F6 V.sub.H and 2F2 V.sub.H, about 66 of 137 amino
acid residues are different between the 2 V.sub.Hs. Sequence
comparison of the CDRs shows that 5 of 10 residues are different
between 1F6 CDR-H1 and 2F2 CDR-H1 (3 of the 5 substitutions are
divergent), 12 of 17 residues are different between 1F6 CDR-H2 and
2F2 CDR-H2 (9 of the 12 substitutions are divergent), and 5 of 9
residues are different between 1F6 CDR-H3 and 2F2 CDR-H3 (4 of the
5 substitutions are divergent) (FIG. 3).
EXAMPLE 2
Synthesis of Anti-CD70 Drug Conjugates
[0299] The ability of anti-CD70 to deliver a potent cytotoxic drug
in the form of an antibody drug conjugate (ADC) to eliminate
CD70-expressing cells was tested. Monomethyl auristatin E (MMAE),
auristatin phenylalanine phenylenediamine (AFP), and monomethyl
auristatin phenylalanine (MMAF) were used as the targeted cytotoxic
drugs for this study. The drugs were linked to the anti-CD70 mAb
1F6 by the valine-citruline (vc) dipeptide linker to give the
1F6-vcMMAE, 1F6-vcAFP conjugates and 1F6-vcMMAF.
[0300] A. Synthesis of F6-vcMMAE ##STR22##
[0301] The synthesis of the ADC 1F6-vcMMAE, the general structure
of which is depicted above, is described below.
[0302] 1. Drug-Linker Compound Synthesis ##STR23##
[0303] Synthesis of auristatin E has been previously described
(U.S. Pat. No. 5,635,483; Pettit, 1999, Prog. Chem. Org. Nat. Prod.
70:1-79). The monomethyl derivative of Auristatin E (MMAE) was
prepared by replacing a protected form of monomethylvaline for
N,N-dimethylvaline in the synthesis of auristatin E (Senter et al.,
U.S. Provisional Application No. 60/400,403; and PCT/US03/24209
(supra)).
[0304] To prepare the drug-linker compound, MMAE (1.69 g, 2.35
mmol), maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol
p-nitrophenylcarbonate (2.6 g, 3.52 mmol, 1.5 eq., prepared as
described in Dubowchik et al., 2002, Bioconjugate Chem. 13:855-869)
and HOBt (64 mg, 0.45 mmol, 0.2 eq.) were diluted with DMF (25 mL).
After 2 min, pyridine (5 mL) was added and the reaction was
monitored using reverse-phase HPLC. The reaction was shown to be
complete in 24 hr. The reaction mixture was concentrated to provide
a dark oil, which was diluted with 3 mL of DMF. The DMF solution
was purified using flash column chromatography (silica gel, eluant
gradient:100% dichloromethane to 4:1 dichloromethane-MeOH). The
relevant fractions were combined and concentrated to provide an oil
that solidified under high vacuum to provide a mixture of the
desired drug-linker compound and unreacted MMAE as a dirty yellow
solid (R.sub.f 0.40 in 9:1 dichloromethane-MeOH). The dirty yellow
solid was diluted with DMF and purified using reverse-phase
preparative-HPLC (Varian Dynamax C18 column 41.4 mm.times.25 cm,
8.mu., 100 .ANG., using a gradient run of MeCN and 0.1% aqueous TFA
at 45 mL/min from 10% to 100% over 40 min followed by 100% MeCN for
20 min) to provide the desired drug-linker compound as an amorphous
white powder (Rf 0.40 in 9:1 dichloromethane-MeOH) which was
>95% pure by HPLC and which contained less than 1% of MMAE.
Yield: 1.78 g (57%); ES-MS m/z 1316.7 [M+H]+; UV .lamda..sub.max
215, 248 nm.
[0305] 2. Conjugate Preparation
[0306] Antibody Reduction. To 0.7 mL 1F6 (10 mg/mL) was added 90
.mu.L of 500 mM sodium borate/500 mM NaCl, pH 8.0, followed by 90
.mu.L of 100 mM DTT in water, and 20 .mu.L of H.sub.2O. After
incubation at 37.degree. C. for 30 min, the buffer was exchanged by
elution over G25 resin equilibrated and eluted with PBS containing
1 mM DTPA (Aldrich). The thiol/Ab value was found to be 9.8 by
determining the reduced antibody concentration from the solution's
280 nm absorbance, and the thiol concentration by reaction with
DTNB (Aldrich) and determination of the absorbance at 412 nm.
[0307] Conjugation of the Reduced Antibody. The reduced mAb was
chilled on ice. The drug-linker compound was used as a DMSO
solution of known concentration, and the quantity of drug-linker
added to the reaction mixture was calculated as follows: L stock
solution=V.times.[Ab].times.Fold Excess/[Drug-Linker], where V and
[Ab] are the volume and molar concentration of the reduced antibody
solution, respectively. 0.904 .mu.L cold H.sub.2O was added to the
reduced antibody solution, followed by 500 .mu.L cold acetonitrile.
30.5 .mu.L of 10.2 mM drug-linker compound stock solution was
diluted into 1.47 mL acetonitrile. The acetonitrile drug-linker
solution was chilled on ice, then added to the reduced antibody
solution. The reaction was terminated after 0.5 hr by the addition
of a 20 fold molar excess of cysteine over maleimide. The reaction
mixture was concentrated by centrifugal ultrafiltration and
purified by elution through de-salting G25 in PBS at 4.degree. C.
1F6-vcMMAE was then filtered through 0.2 micron filters under
sterile conditions and immediately frozen at -80.degree. C.
1F6-vcMMAE was analyzed for 1) concentration, by UV absorbance; 2)
aggregation, by size exclusion chromatography; 3) drug/Ab, by
measuring unreacted thiols with DTNB, and 4) residual free drug, by
reverse phase HPLC.
[0308] B. Synthesis of 1F6-vcAFP ##STR24##
[0309] The synthesis of the ADC 1F6-vcAFP, the general structure of
which is depicted above, is described below.
[0310] 1. AFP Synthesis ##STR25##
[0311] Boc-phenylalanine (1.0 g, 3.8 mmol) was added to a
suspension of 1,4-diaminobenzene.HCl (3.5 g, 19.0 mmol, 5.0 eq.) in
triethylamine (10.7 mL, 76.0 mmol, 20 eq.) and dichloromethane (50
mL). To the resulting solution was added DEPC (3.2 mL, 19.0 mmol,
5.0 eq.) via syringe. HPLC showed no remaining Boc-phe after 24 hr.
The reaction mixture was filtered, and the filtrate was
concentrated to provide a dark solid. The dark solid residue was
partitioned between 1:1 EtOAc-water, and the EtOAc layer was washed
sequentially with water and brine. The EtOAc layer was dried and
concentrated to provide a dark brown/red residue that was purified
using HPLC (Varian Dynamax column 41.4 mm.times.25 cm, 5.mu., 100
.ANG. using a gradient run of MeCN and water at 45 mL/min form 10%
to 100% over 40 min followed by 100% MeCN for 20 min). The relevant
fractions were combined and concentrated to provide a red-tan solid
intermediate. Yield: 1.4 g (100%); ES-MS m/z 355.9 [M+H]+; UV
.lamda.max 215, 265 nm; .sup.1H NMR (CDCl.sub.3) .delta. 7.48 (1H,
br s), 7.22-7.37 (5H, m), 7.12 (2H, d, J=8.7 Hz), 7.61 (2H, d,
J=8.7 Hz), 5.19 (1H, br s), 4.39-4.48 (1H, m), 3.49 (2H, s), 3.13
(2H, d, J=5.7 Hz), 1.43 (9H, s).
[0312] The red-tan solid intermediate (0.5 g, 1.41 mmol) and
diisopropylethylamine (0.37 mL, 2.11 mmol, 1.5 eq.) were diluted
with dichloromethane (10 mL), and to the resulting solution was
added Fmoc-Cl (0.38 g, 1.41 mmol). The reaction was allowed to
stir, and a white solid precipitate formed after a few minutes. The
reaction was complete according to HPLC after 1 hr. The reaction
mixture was filtered, and the filtrate was concentrated to provide
an oil. The oil was precipitated with EtOAc, resulting in a
reddish-white intermediate product, which was collected by
filtration and dried under vacuum. Yield: 0.75 g (93%); ES-MS m/z
578.1 [M+H].sup.+, 595.6 [M+NH.sub.4].sup.+.
[0313] The reddish-white intermediate (0.49 g, 0.85 mmol), was
diluted with 10 mL of dichloromethane, and then treated with 5 mL
of trifluoroacetic acid. The reaction was complete in 30 min
according to reverse-phase HPLC. The reaction mixture was
concentrated and the resulting residue was precipitated with ether
to provide an off-white solid. The off-white solid was filtered and
dried to provide an amorphous powder, which was added to a solution
of Boc-Dolaproine (prepared as described in Tetrahedron, 1993,
49(9):1913-1924) (0.24 g, 0.85 mmol) in dichloromethane (10 mL). To
this solution was added triethylamine (0.36 mL, 2.5 mmol, 3.0 eq.)
and PyBrop (0.59 g, 1.3 mmol, 1.5 eq.). The reaction mixture was
monitored using reverse-phase HPLC. Upon completion, the reaction
mixture was concentrated, and the resulting residue was diluted
with EtOAc, and sequentially washed with 10% aqueous citric acid,
water, saturated aqueous sodium bicarbonate, water, and brine. The
EtOAc layer was dried (MgSO.sub.4), filtered, and concentrated. The
resulting residue was purified using flash column chromatography
(silica gel) to provide an off-white powdered intermediate. Yield:
0.57 g (88%); ES-MS m/z 764.7 [M+NH.sub.4].sup.+; UV
.lamda..sub.max 215, 265 nm; .sup.1H NMR (DMSO-d.sub.6) .delta.
10.0-10.15 (1H, m), 9.63 (1H, br s), 8.42 (1/2H, d, J=8.4 Hz), 8.22
(1/2H, d, J=8.4 Hz), 7.89 (2H, d, J=7.2 Hz), 7.73 (2H, d, J=7.6
Hz), 7.11-7.55 (13H, m), 4.69-4.75 (1H, m), 4.46 (2H, d, J=6.8 Hz),
4.29 (1H, t, J=6.4 Hz), 3.29 (3H, s), 2.77-3.47 (7H, m), 2.48-2.50
(3H, m), 2.25 (2/3H, dd, J=9.6, 7.2 Hz), 1.41-1.96 (4H, m), 1.36
(9H, s), 1.07 (1H, d, J=6.4 Hz, rotational isomer), 1.00 (1H, d,
J=6.4 Hz, rotational isomer).
[0314] The white solid intermediate (85 mg, 0.11 mmol) and
N-Methylval-val-dil-O-t-butyl (55 mg, 0.11 mmol, prepared as
described in Pettit et al. 1996, J. Chem. Soc. Perk. I p. 859) were
diluted with dichloromethane (5 mL), and then treated with 2.5 mL
of trifluoroacetic acid under a nitrogen atmosphere for two hours
at room temperature. The reaction completion was confirmed by
RP-HPLC. The solvent was removed in vacuo and the resulting residue
was azeotropically dried twice with toluene, and then dried under
high vacuum for 12 hours.
[0315] The residue was diluted with dichloromethane (2 mL),
diisopropylethylamine (3 eq.) was added, followed by DEPC (1.2
eq.). After the reaction was completed, the reaction mixture was
concentrated under reduced pressure, the resulting residue was
diluted with EtOAc, and washed sequentially with 10% aqueous citric
acid, water, saturated aqueous sodium bicarbonate, and brine. The
EtOAc layer was dried, filtered and concentrated to provide a
yellow oil.
[0316] The yellow oil was diluted with dichloromethane (10 mL) and
to the resulting solution diethylamine (5 mL) was added. According
to HPLC, reaction was completed after 2 hr. The reaction mixture
was concentrated to provide an oil. The oil was diluted with DMSO,
and the DMSO solution was purified using reverse phase
preparative-HPLC (Varian Dynamax column 21.4 mm.times.25 cm, 5.mu.,
100 .ANG., using a gradient run of MeCN and 0.1% TFA at 20 mL/min
from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The
relevant fractions were combined and concentrated to provide the
desired drug as an off-white solid. Overall yield: 42 mg (44%
overall); ES-MS m/z 837.8 [M+H].sup.+, 858.5 [M+Na]+; UV
.lamda..sub.max 215, 248 nm.
[0317] 2. Preparation of Drug-Linker Compound ##STR26##
[0318] The trifluoroacetate salt of AFP (0.37 g, 0.39 mmol, 1.0
eq.) and Fmoc-val-cit (0.30 g, 0.58 mmol, 1.5 eq., prepared
according to Dubowchik et al., 2002, Bioconjugate Chem. 13:855-896)
were diluted with DMF (5 mL, 0.1 M), and to the resulting solution
was added pyridine (95 .mu.L, 1.2 mmol, 3.0 eq.). HATU (0.23 g,
0.58 mmol, 1.5 eq.) was then added as a solid, and the reaction
mixture was allowed to stir under argon atmosphere while being
monitored using HPLC. The reaction progressed slowly, and 4 hr
later, 1.0 eq. of diisopropylethylamine was added. The reaction was
complete in 1 hr. The reaction mixture was concentrated in vacuo
and the resulting residue was purified using prep-HPLC (Varian
Dynamax C18 column 41.4 mm.times.25 cm, 5.mu., 100 .ANG., using a
gradient run of MeCN and 0.1% aqueous TFA at 45 mL/min from 10% to
100% over 40 min followed by 100% MeCN for 20 min) to provide a
faint pink solid intermediate.
[0319] The pink solid intermediate was diluted with DMF (30 mL) and
to the resulting solution was added diethylamine (15 mL). Reaction
was complete by HPLC in 2 hr. The reaction mixture was concentrated
and the resulting residue was washed twice with ether. The solid
intermediate was dried under high vacuum and then used directly in
the next step.
[0320] The solid intermediate was diluted with DMF (20 mL) and to
the resulting solution was added
6-(2,5-dioxy-2,5-dihydro-pyrrol-1-yl)-hexanoic acid
2,5-dioxy-pyrrolidin-1-yl ester (0.12 g, 0.39 mmol, 1.0 eq.) (EMCS,
Molecular Biosciences Inc., Boulder, Colo.). After 4 days, the
reaction mixture was concentrated to provide an oil which was
purified using prep-HPLC (Varian Dynamax C18 column 41.4
mm.times.25 cm, 5.mu., 100 .ANG., using a gradient run of MeCN and
0.1% aqueous TFA at 45 mL/min from 10% to 100% over 40 min followed
by 100% MeCN for 20 min) to provide the desired drug-linker
compound as a white flaky solid. Yield: 0.21 g (38% overall); ES-MS
m/z 1285.9 [M+H].sup.+; 13.07.8 [M+Na].sup.+; UV .lamda..sub.max
215, 266 nm.
[0321] 3. Conjugate Preparation
[0322] Antibody Reduction. To 0.48 mL 1F6 (10.4 mg/mL) was added 75
mL of 500 mM sodium borate/500 mM NaCl, pH 8.0, followed by 75
.mu.L of 100 mM DTT in water, and 20 .mu.L PBS. After incubation at
37.degree. C. for 30 min, the buffer was exchanged by elution over
G25 resin equilibrated and eluted with PBS containing 1 mM DTPA
(Aldrich). The thiol/Ab value was found to be 10.3 by determining
the reduced antibody concentration from the solution's 280 nm
absorbance, and the thiol concentration by reaction with DTNB
(Aldrich) and determination of the absorbance at 412 nm.
[0323] Conjugation of the Reduced Antibody. The reduced mAb was
chilled on ice. The drug-linker compound was used as a DMSO
solution of known concentration, and the quantity of drug-linker
added to the reaction mixture was calculated as follows: L stock
solution=V.times.[Ab].times.Fold Excess/[Drug-Linker], where V and
[Ab] are the volume and molar concentration of the reduced antibody
solution, respectively. 984 .mu.L cold PBS/DTPA was added to the
reduced antibody solution, followed by 400 .mu.L acetonitrile, and
the mixture chilled on ice. 26.4 .mu.L of 8.3 mM drug-linker
compound stock solution was then added to the reduced antibody/DMSO
solution. The reaction was terminated after 1 hr by the addition of
a 40 .mu.L of 100 mM cysteine. The reaction mixture was
concentrated by centrifugal ultrafiltration and purified by elution
through de-salting G25 in PBS at 4.degree. C. 1F6-vcAFP was then
filtered through 0.2 micron filters under sterile conditions and
immediately frozen at -80.degree. C. 1F6-vcAFP was analyzed for 1)
concentration, by UV absorbance; 2) aggregation, by size exclusion
chromatography; 3) drug/Ab, by measuring unreacted thiols by
treatment with DTT, followed by DTNB, and 4) residual free drug, by
reverse phase HPLC.
[0324] C. Synthesis of 1F6-vcMMAF ##STR27##
[0325] The synthesis of the ADC 1F6-vcMMAF, the general structure
of which is depicted above, is described below.
[0326] 1. MMAF Synthesis ##STR28##
[0327] MMAF was prepared from its t-butyl ester (Compound 1) as is
described below. Phenylalanine t-butyl ester HCl salt (868 mg, 3
mmol), N-Boc-Dolaproine (668 mg, 1 eq.), DEPC (820 .mu.L, 1.5 eq.),
and DIEA (1.2 mL) were diluted with dichloromethane (3 mL). After 2
hours (h) at room temperature (about 28 degrees Celsius), the
reaction mixture was diluted with dichloromethane (20 mL), washed
successively with saturated aqueous (aq.) NaHCO.sub.3 (2.times.10
mL), saturated aq. NaCl (2.times.10 mL). The organic layer was
separated and concentrated. The resulting residue was re-suspended
in ethyl acetate and was purified via flash chromatography on
silica gel in ethyl acetate. The relevant fractions were combined
and concentrated to provide the dipeptide as a white solid: 684 mg
(46%). ES-MS m/z 491.3 [M+H].sup.+.
[0328] For selective Boc cleavage in the presence of t-butyl ester,
the above dipeptide (500 mg, 1.28 mmol) was diluted with dioxane (2
mL). 4M HCl/dioxane (960 .mu.L, 3 eq.) was added, and the reaction
mixture was stirred overnight at room temperature. Almost complete
Boc deprotection was observed by RP-HPLC with minimal amount of
t-butyl ester cleavage. The mixture was cooled down on an ice bath,
and triethylamime (500 .mu.L) was added. After 10 min., the mixture
was removed from the cooling bath, diluted with dichloromethane (20
mL), washed successively with saturated aq. NaHCO.sub.3 (2.times.10
mL), saturated aq. NaCl (2.times.10 mL). The organic layer was
concentrated to give a yellow foam: 287 mg (57%). The intermediate
was used without further purification.
[0329] The tripeptide N-Fmoc-N-Methylval-val-dil-O-t-butyl (0.73
mmol) was treated with TFA (3 mL), dichloromethane (3 mL) for 2 h
at room temperature. The mixture was concentrated to dryness, the
residue was co-evaporated with toluene (3.times.20 mL), and dried
in vacuum overnight. The residue was diluted with dichloromethane
(5 mL) and added to the deprotected dipeptide Dap-phe-O-t-butyl
(287 mg, 0.73 mmol), followed by DIEA (550 .mu.L, 4 eq.), DEPC (201
.mu.L, 1.1 eq.). After 2 h at room temperature the reaction mixture
was diluted with ethyl acetate (50 mL), washed successively with
10% aq. citric acid (2.times.20 mL), saturated aq. NaHCO.sub.3
(2.times.10 mL), saturated aq. NaCl (10 mL). The organic layer was
separated and concentrated. The resulting residue was re-suspended
in ethyl acetate and was purified via flash chromatography in ethyl
acetate. The relevant fractions were combined and concentrated to
provide N-Fmoc-N-Methylval-val-dil-dap-phe-O-t-butyl as a white
solid: 533 mg (71%). R.sub.f 0.4 (EtOAc). ES-MS m/z 1010.6
[M+H].sup.+.
[0330] The product (200 mg, 0.2 mmol) was diluted with
dichloromethane (3 mL), diethylamine (1 mL). The reaction mixture
was stirred overnight at room temperature. Solvents were removed to
provide an oil that was purified by flash silica gel chromatography
in a step gradient 0-10% MeOH in dichloromethane to provide
Compound 1 (below) as a white solid: 137 mg (87%). R.sub.f 0.3 (10%
MeOH/CH.sub.2Cl.sub.2). ES-MS m/z 788.6 [M+H].sup.+. ##STR29##
[0331] MMAF was prepared from Compound 1 (30 mg, 0.038 mmol) by
treatment with 4M HCl/dioxane (4 ml) for 7 h at room temperature.
The solvent was removed, and the residue was dried in a vacuum
overnight to give provide MMAF as a hydroscopic white solid: 35 mg
(120% calculated for HCl salt). ES-MS m/z 732.56 [M+H].sup.+.
[0332] 2. Preparation of Drug-Linker Compound
[0333] Compound 1 (83 mg, 0.11 mmol),
maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol
p-nitrophenylcarbonate (85 mg, 0.12 mmol, 1.1 eq.), and HOBt (2.8
mg, 21 .mu.mol, 0.2 eq.) were taken up in dry DMF (1.5 mL) and
pyridine (0.3 mL) while under argon. After 30 h, the reaction was
found to be essentially complete by HPLC. The mixture was
evaporated, taken up in a minimal amount of DMSO and purified by
prep-HPLC (C.sub.12-RP column, 5.mu., 100 .ANG., linear gradient of
MeCN in water (containing 0.1% TFA) 10 to 100% in 40 min followed
by 20 min at 100%, at a flow rate of 25 mL/min) to provide
MC-Val-Cit-PAB-MMAF-O-t-butyl ester as a white solid. Yield: 103 mg
(71%). ES-MS m/z 1387.06 [M+H].sup.+, 1409.04 [M+Na].sup.+; UV
.lamda..sub.max 205, 248 nm.
[0334] MC-Val-Cit-PAB-MMAF-O-t-butyl ester (45 mg, 32 .mu.mol) was
suspended in methylene chloride (6 mL) followed by the addition of
TFA (3 mL). The resulting solution stood for 2 h. The reaction
mixture was concentrated in vacuo and purified by prep-HPLC
(C.sub.12--RP column, 5.mu., 100 .ANG., linear gradient of MeCN in
water (containing 0.1% TFA) 10 to 100% in 40 min followed by 20 min
at 100%, at a flow rate of 25 mL/min). The desired fractions were
concentrated to provide MC-Val-Cit-PAB-MMAF as an off-white solid.
Yield: 11 mg (25%). ES-MS m/z 1330.29 [M+H].sup.+, 1352.24
[M+Na].sup.+; UV .lamda..sub.max 205, 248 nm.
[0335] 3. Conjugate Preparation
[0336] Antibody Reduction. To 0.75 mL 1F6 (4.2 mg/mL) was added 50
L of 500 mM sodium borate/500 mM NaCl, pH 8.0, followed by 160
.mu.L of 100 mM DTT in water, and 100 .mu.L PBS. After incubation
at 37.degree. C. for 30 min, the buffer was exchanged by elution
over G25 resin equilibrated and eluted with PBS containing 1 mM
DTPA (Aldrich). The thiol/Ab value was found to be 8.9 by
determining the reduced antibody concentration from the solution's
280 nm absorbance, and the thiol concentration by reaction with
DTNB (Aldrich) and determination of the absorbance at 412 nm.
[0337] Conjugation of the Reduced Antibody. The reduced mAb was
chilled on ice. The drug-linker compound was used as a DMSO
solution of known concentration, and the quantity of drug-linker
added to the reaction mixture was calculated as follows: L stock
solution=V.times.[Ab].times.Fold Excess/[Drug-Linker], where V and
[Ab] are the volume and molar concentration of the reduced antibody
solution, respectively. The reduced antibody (785 .mu.l of 1.77
mg/ml) was added to the mixture of 7.2 .mu.l of 12.9 mM MCvcMMAF in
92 .mu.l of acetonitrile and mixed rapidly. The reaction mixture
was allowed to incubate on ice for 1 h and followed by addition of
a 40 .mu.L of 100 mM cysteine. The reaction mixture was purified by
G25 column equilibrated in PBS at 4.degree. C. 1F6-vcAFP was then
filtered through 0.2 micron filters under sterile conditions and
immediately frozen at -80.degree. C. 1F6-vcMMAF was analyzed for 1)
concentration, by UV absorbance; 2) aggregation, by size exclusion
chromatography; 3) drug/Ab, by measuring unreacted thiols by
treatment with DTT, followed by DTNB, and 4) residual free drug, by
reverse phase HPLC.
EXAMPLE 3
Expression of CD70 on Hematologic Cell Lines
[0338] Surface expression of CD70 was examined using flow
cytometry. In general, 0.2.times.10.sup.6 cells were incubated with
50 .mu.l of staining medium (RPMI-1640 supplemented with 5-10% FBS)
containing a fluorochrome-conjugated mAb (10 .mu.g/ml) for
single-color flow cytometry or a cocktail of
fluorochrome-conjugated mAbs for multiple-color flow cytometry.
Incubations were carried out on ice for 20-30 minutes. Cells were
then washed 3 times with the staining buffer and fixed in PBS
containing 1% of paraformaldehyde. Flow cytometric analysis was
performed with a FACScan (BD Immunocytometry, San Jose, Calif.) and
data were analyzed by either the CellQuest (BD Immunocytometry) or
the WinMDI software. The anti-human CD70 monoclonal antibodies
(mAb) 1F6 and 2F2 were obtained from the Central Laboratory of the
Netherlands Red Cross Blood Transfusion Service (Amsterdam, The
Netherlands). 1F6 was conjugated to AlexaFluor488 (AF) (Molecular
Probes, Eugene, Oreg.) according to the manufacturer's instruction,
and AF-conjugated 1F6 was used to detect CD70 by flow cytometry
whenever necessary. The anti-CD70 mAb Ki-24 and anti-CD3 mAb were
purchased from BD PharMingen (San Diego, Calif.).
[0339] The expression of cell surface CD70 was surveyed on a panel
of hematologic cell lines. This panel included both Epstein-Barr
Virus (EBV) negative American Burkitt's lymphoma (BL) and EBV.sup.+
African Burkitt's lymphoma lines, a variety of non-Hodgkin's
lymphoma (NHL) lines which are EBV.sup.-, 2 Hodgkin's disease (HD)
lines, 2 EBV-transformed B lymphoblastoid cell lines (EBV-LCL), and
an acute T cell leukemia line Jurkat (FIG. 4). The cell lines were
maintained in culture conditions specified by the ATCC or DSMZ. For
flow cytometric analysis, AF-conjugated or a commercially available
anti-CD70 (clone Ki-24) purchased from BD PharMingen was used. Cell
lines on which anti-CD70 showed greater than 2-fold binding
compared to the control IgG binding, based on mean fluorescence
intensities, were arbitrarily defined as CD70-expressing. Based on
this criterion, all of the BL lines, both EBV.sup.+ and EBV.sup.-,
examined expressed detectable, albeit low levels of CD70. Five out
of ten NHL cell lines expressed CD70. Both HD and EBV-LCL also
expressed CD70 at levels that were generally higher than those of
BL and NHL lines. The acute T leukemia cell line Jurkat was found
to be CD70.sup.-.
EXAMPLE 4
Proliferation Inhibitory and Cytotoxic Effects of Anti-CD70 ADCs on
CD70.sup.+ Hematologic Lines
[0340] A subset of the CD70.sup.+ hematologic lines listed in FIG.
4 was tested for sensitivity to the 1F6 ADCs. These lines were
seeded at 5,000 cells per well in 96-well plates in quadruplicates
in a total of 200 .mu.l of culture medium containing graded doses
of the ADCs as indicated in the FIG. 5 and the accompanying figure
legend. Proliferation assays were carried out for 96 hours.
Tritiated thymidine (3H-TdR) incorporation during the last 16 hours
of incubation was used to assess DNA synthesis. The responses of a
CD70.sup.+ B lymphoma cell line WSU-NHL and the CD70.sup.- Jurkat
cells are shown in the upper panels of FIG. 5. Both 1F6-vcMMAE and
1F6-vcAFP exerted dose-dependent inhibitory effects on the
proliferation of WSU-NHL cells. Proliferation inhibitory effects
were apparent when the ADCs were used at concentrations higher than
0.005 .mu.g/ml. The non-binding IgG control ADCs did not exert any
significant effects at concentrations lower than 0.5 .mu.g/ml. On
the other hand, proliferation of the CD70.sup.- Jurkat acute T
leukemia cell line was not affected by either 1F6-vcMMAE or
1F6-vcAFP at concentrations as high as 2 .mu.g/ml. Hence, the
proliferation inhibitory activity of the 1F6 ADCs on target
positive cells resulted from specific binding of ADCs to CD70
expressed on the target cells. The responses of other CD70.sup.+
cell lines tested are summarized in the lower panel of FIG. 5.
Significant proliferation inhibition by the 1F6 ADCs was observed
in 2 of the 3 B lineage non-Hodgkin's lymphoma cell lines (MC116
and WSU-NHL), 2 of 2 Hodgkin's disease lines, and the
EBV-transformed B lymphoblastoid cell line CESS. In all the
responsive cell lines tested, 1F6-vcAFP was demonstrated to exert
more potent inhibitory activity than 1F6-vcMMAE. These results
suggest that anti-CD70 ADCs can potentially be applied in
immunotherapy of CD70.sup.+ lymphomas. Moreover, EBV-LCLs are
phenotypically very similar to activated normal B cells. Both
EBV-LCLs and activated normal B cells are also efficient antigen
presenting cells possessing strong T cell stimulatory activities.
The observation that anti-CD70 ADC can efficiently suppress the
proliferation of EBV-LCL CESS cells indicates that anti-CD70 ADCs
can also be applied to suppress immune responses by eliminating
activated B cells.
EXAMPLE 5
Expression of CD70 Transcripts in Solid Tumors
[0341] Hodgkin's disease (HD) and nasopharyngeal carcinoma (NPC)
are two examples of conditions that frequently associate with EBV
and a prominent lymphoid stroma at the tumor sites. The role of
this lymphoid stroma is uncertain and may represent infiltration of
lymphocytes to tumors as part of the immune response to
tumor-associated antigens. Alternatively, the recruitment of
lymphocytes by tumor cells may be a mechanism through which tumor
cells derive cytokine and growth factors from the infiltrating
lymphocytes in a paracrine fashion. Some of these growth factors
may contribute to tumor development by supporting tumor cell
proliferation as hypothesized for HD (Gruss et al., 1997, Immunol.
Today 18:156-63). CD70 has been found to be present on the
Reed-Sternberg cells in HD, and FIG. 4 demonstrates the expression
of CD70 on HD cell lines. In a study on frozen tumor biopsies from
EBV-associated undifferentiated nasopharyngeal carcinomas, 80% (16
of 20 cases) showed in situ expression of the CD70 protein on tumor
cells (Agathanggelou et al., 1995, Am. J. Pathol. 147:1152-60).
Others also reported the expression of CD70 on EBV-tumors including
thymic carcinomas (Hishima et al., 2000, Am. J. Surg. Path.
24:742-746), gliomas, and meningiomas (Held-Feindt and Mentlein,
2002, Int. J. Cancer 98:352-56).
[0342] The potential expression of CD70 in additional carcinoma
types was further surveyed using the Cancer Profiling Array (CPA)
(BD Biosciences Clontech, Palo Alto, Calif.). The CPA includes
normalized cDNA from 241 tumor and corresponding normal tissues
from individual patients. In order to examine the expression of
CD70 message in these patients, a piece of cDNA corresponding to 3'
untranslated region of nucleotides 734-874 of the CD70 message was
amplified using the reverse transcriptase polymerase chain reaction
(RT-PCR) approach. First strand cDNA was synthesized from total RNA
isolated from the Burkitt's lymphoma cell line Ramos (ATCC) using
the SuperScript.TM. First Strand Synthesis System for RT-PCR from
Invitrogen (Carlsbad, Calif.). The forward primer 5'-CCA CTG CTG
CTG ATT AG-3' (SEQ ID NO:46), the reverse primer 5'-CAA TGC CTT CTC
TTG TCC-3' (SEQ ID NO:47), and the Advantage 2 PCR Kit (Clontech,
Palo Alto, Calif.) were used for the PCR. The PCR product was
cloned into the pCR4-TOPO vector and sequence verified. To generate
a probe for hybridization, PCR was run using the cloned cDNA as the
template, the above primer pairs, and the Advantage 2 PCR Kit
(Clontech). To label the probe with .sup.32P-dGTP, 200 ng of the
purified PCR product were combined with 75 ng of random hexamer, 33
.mu.M each of dATP, dTTP and dCTP, 5 .mu.l of .alpha.-.sup.32P dGTP
(approximately 3000 Ci/mmol, 10 mCi/ml, Amersham Pharmacia Biotech,
Piscataway, N.J.), 1 .mu.l of Klenow fragment (New England Biolabs,
Beverly, Mass.), and 1.times.EcoPol buffer (New England Biolabs) in
a total volume of 50 .mu.l. The reaction mixture was incubated at
room temperature for 15 minutes, and EDTA was added to a final
concentration of 10 mM to stop the reaction. Labeled probe was
purified using ProbeQuant G-50 Micro Columns (Amersham Pharmacia
Biotech, Piscataway, N.J.). This probe was hybridized to patient
cDNA spotted on a CPA using the BD ExpressHyb hybridization
solution (BD Biosciences) according to the manufacturer's
instructions. Hybridization signals were quantified by
phospho-imaging on a PhosphoImager SI (Amersham). Reprobing the
same CPA for the housekeeping EF-1 gene gave tumor:normal ratio of
approximately one for each sample pair, confirming comparable
loading of cDNA. Therefore ratios between the CD70 hybridization
signals obtained from tumor and normal cDNAs were used as a
semi-quantitative measurement to CD70 transcript expression.
[0343] Differential hybridization of the CD70 probe to tumor cDNA
was observed in several cancer types. Most notably, cDNAs from 9 of
20 cases of kidney carcinoma and one case each of colon, stomach,
and rectum carcinoma showed more intense hybridization signals than
cDNAs from the corresponding normal tissues. The 9 cases (45%) of
kidney cancer in FIG. 6, all classified as RCC, showed more than
2-fold (range of 2.3 to 8.9) over-expression of CD70 transcripts.
In the single case of stomach, colon, and rectum cancer showing
differential CD70 cDNA hybridization (FIG. 6), the tumor:normal
ratios were 2.9, 8.9, and 3.2, respectively.
[0344] Quantitative PCR (QPCR) was used to quantify CD70 transcript
expression in 8 RCC and 4 normal kidney cDNA samples, all obtained
from BioChain (Hayward, Calif.). The RCC cDNA samples were from
donors independent of those on the CPA in FIG. 6. QPCR analysis was
performed using TaqMan.TM. MGB chemistry (Applied Biosystems,
Foster City, Calif.) containing a reporter dye at the 5' end (FAM
or VIC) and a non-fluorescent quencher (NFQ) at the 3' end on an
ABI PRISM 7000 Sequence Detection System (Applied Biosystems).
Assays specific for either CD70 (Applied Biosystems Assay#
Hs00174297_m1) or control gene GAPDH (Applied Biosystems PDAR#
4326317E) were used to evaluate samples. Comparison of CD70
transcript expression between normal kidney and RCC tissues was
conducted based on the comparative threshold cycle (Ct) method as
previously reported (Winer et al., 1999, Anal. Biochem. 270: 41-49;
Aarskog and Vedeler, 2000, Hum. Genet. 107: 494-498). Ct values for
the CD70 and a housekeeping gene GAPDH from two independent
reactions were generated. The Ct values for the GAPDH transcripts
from normal kidney and RCC samples did not differ significantly
from each other (<1 cycle, student's t-test: p=0.405),
suggesting that CD70 expression can be normalized against GAPDH for
tumor:normal comparison. .DELTA.Ct values for CD70 expression in
RCC and normal kidney samples were calculated by subtracting
Ct.sub.GAPDH from Ct.sub.CD70 to give .DELTA.Ct.sub.Tumor and
.DELTA.Ct.sub.Normal, respectively. .DELTA..DELTA.Ct values were
calculated by subtracting .DELTA.Ct.sub.Normal from
.DELTA.Ct.sub.Tumor. CD70 transcript expression in RCC expressed as
fold increase over normal kidneys was then expressed as 2
(-.DELTA..DELTA.Ct). CD70 transcript over-expression (>2-fold)
was observed in 6 of the 8 RCC cDNA samples, consistent with the
results obtained form the CPA experiment (FIG. 6). Over-expression
of CD70 transcripts ranged from 2.5- to 133.7-fold (FIG. 7).
EXAMPLE 6
Expression of CD70 Protein on Renal Cell Carcinoma
[0345] Immunohistochemistry was used to determine if
over-expression of CD70 transcript in RCC was paralleled by CD70
protein expression. Frozen RCC tissue sections and normal adjacent
tissue sections from two patients independent of those used in CD70
transcript analysis (FIGS. 6 and 7) were fixed in acetone at
-20.degree. C. for 10 minutes. After rehydration in PBS, sections
were blocked with PBS containing 5% normal goat serum and Avidin D
for 30 minutes at room temperature, and washed in PBS. Sections
were incubated with the anti-CD70 mAb 2F2 or non-binding IgG at 2
.mu.g/ml in PBS containing 5% normal goat serum with biotin for 60
minutes at room temperature. After washes with PBS, biotinylated
anti-mouse IgG (VECTASTAIN ABC Kit, Vector Laboratories,
Burlingame, Calif.) at a 1:250 dilution in PBS containing 5% normal
goat serum was used to detect bound primary antibody by incubating
for 30 minutes at room temperature. Excess biotinylated anti-mouse
IgG was removed by PBS washes. Sections were quenched with 1%
H.sub.2O.sub.2 in PBS for 30 minutes at room temperature, washed,
and then incubated with the VECTASTAIN ABC complex made up
according to the manufacturer's instructions. After PBS washes,
sections were incubated for 30 minutes at room temperature with the
DAB solution (VECTASTAIN ABC Kit) made up according to the
manufacturer's instructions for color development. Sections were
then washed in water, counterstained with hematoxylin for 2
minutes, mounted in VectaMount (Vector Laboratories), and then
observed using 40.times. light microscopy.
[0346] Intense binding of 2F2 to RCC tumor sections from 2
different donors was observed (FIG. 8A, right panels). In contrast,
a control IgG did not bind to serial tumor sections, confirming the
specificity of 2F2 for CD70 (FIG. 8A, left panels). 2F2 also did
not demonstrate binding beyond that of the control IgG to normal
adjacent tissues from the same RCC samples (FIG. 8B), suggesting
minimal or no CD70 protein expression in normal kidneys. These
results confirm the over-expression of CD70 transcripts in RCC and
provide direct evidence for in situ CD70 protein expression in
RCC.
[0347] Expression of the CD70 protein on the RCC cell surface was
next examined. A panel of 7 RCC lines was evaluated using flow
cytometry. The RCC lines Hs 835.T, Caki-1, Caki-2, 786-O, 769-P,
and ACHN were obtained from the ATCC (Manassas, Va.) and were
maintained in conditions specified by the ATCC. The RCC lines CAL54
and A498 were obtained from Deutsche Sammlung von Mikroorganismen
und Zellkulturen GmbH (Braunschweig, Germany). Cell lines were
maintained in the conditions specified the vendors. The RCC lines
SK-RC-6 and SK-RC-7 have been reported previously (Murakami et al.,
1984, Hepatology 42:192-8). SK-RC-6 and SK-RC-7 was maintained in
DMEM supplemented with 10% fetal bovine serum (FBS). Two normal
human renal tubule epithelial lines, RPTEC and HRCE (Cambrex, East
Rutherford, N.J.) were also included in the analysis. FIG. 9A shows
that the RCC lines 786-O, Caki-1, and Caki-2 expressed CD70 while
Hs 835.T did not. Low, but detectable, levels of CD70 expression
were also observed on the normal kidney tubule epithelial lines
PRTEC and HRCE. FIG. 9B summarizes the relative levels of CD70
expressed on RCC lines and normal kidney epithelial cells lines
based on the mean fluorescence intensity obtain from flow
cytometry. Of the 10 RCC lines tested 9 were found to be CD70
expressing. These results demonstrate that the presence of CD70
transcripts and protein in kidney carcinomas was accompanied by
surface expression of CD70 on the cancer cells.
EXAMPLE 7
Proliferation Inhibitory and Cytotoxic Effects of Anti-CD70 ADCs on
CD70.sup.+ Renal Cell Carcinoma Cell Lines
[0348] To examine the responses of CD70.sup.+ RCC lines to 1F6
ADCs, cells were seeded between 1,000 to 3,000 cells per well in
100 .mu.l of medium. An additional 100 .mu.l of culture medium
containing graded doses of ADCs were added to the wells after cells
were allowed to attached overnight. In addition to DNA synthesis,
cell viability, as a reflection of the cytotoxic activities of
ADCs, was also assayed by the reduction of alamarBlue.TM.
(Biosource International, Camarillo, Calif.). The alamarBlue.TM.
dye was added at a one to four dilution during the last 4 to 24
hours of the incubation. Dye reduction was assessed by fluorescence
spectrometry using the excitation and emission wavelengths of 535
nm and 590 nm, respectively. For analysis, the amount of
.sup.3H-TdR incorporated or the extent of alamarBlue.TM. reduction
by the treated cells was compared to that of the untreated control
cells. FIG. 10 shows the effects of 1F6-vcAFP on the proliferation
(.sup.3H-TDR incorporation) and viability (alamarBlue.TM.
reduction) on two representative CD70.sup.+ RCC lines. 1F6-vcAFP
demonstrated potent proliferation inhibitory (FIG. 10, upper panel)
and cytotoxic (FIG. 10, lower panel) activities on Caki-1 and 786-O
cells, maximal effects were achieved at concentrations lower than
0.1 .mu.g/ml. In contrast, non-binding control IgG-vcAFP at
concentrations lower than 1 .mu.g/ml exerted minimal effects on
these cells.
[0349] FIG. 11 summarizes the IC.sub.50s for 1F6-vcMMAE, 1F6-vcAFP,
and 1F6-vcMMAF on inhibiting the proliferation and inducing
cytotoxicity in RCC and normal kidney tubule epithelial cells.
1F6-vcMMAE was very effective in suppressing the proliferation of
Caki-1 and Caki-2 cells with IC.sub.50 values lower than 20 ng/ml.
However, it was not active on other CD70.sup.+ RCC lines, the
CD70.sup.- Hs 835.T cells, or the normal kidney epithelial lines
RPTEC and HRCE. IC.sub.50 values for proliferation inhibition by
1F6-vcAFP ranged from 2-247 ng/ml for CD70.sup.+ RCC lines.
Substantially higher IC.sub.50 values were obtained for 1F6-vcAFP
on the CD70.sup.- Hs 835.T, suggesting specific targeting of
CD70.1F6-vcMMAF were also active in inhibiting proliferation in all
CD70.sup.+ RCC lines tested, with IC.sub.50 values lower than 30
ng/ml. Proliferation inhibition was paralleled with cytotoxicity.
IC.sub.50 values for 1F6-vcAFP to induce cytotoxicity were below 20
ng/ml in 4 of the 7 CD70.sup.+ RCC lines tested, while those for
1F6-vcMMAF were below 30 ng/ml in 5 of 7 CD70.sup.+ RCC lines
tested. Specific targeting by the 1F6 ADC to CD70 was confirmed by
the much higher IC.sub.50 values obtained for the control
non-binding IgG ADCs, >1000 ng/ml for all the lines tested.
Moreover, RCC appeared to be differentially more sensitive to
anti-CD70 ADC-mediated proliferation inhibition and cytotoxicity,
as despite detectable expression of CD70, normal kidney epithelial
cells were very insensitive to 1F6 ADCs when compared to the
CD70.sup.+ RCC lines.
EXAMPLE 8
In Vivo Efficacy of 1F6-vcAFP in a Xenograft Model of RCC
[0350] Subcutaneous xenografts of Caki-1 cells have been
successfully used as models for RCC to test the efficacy of
antibody-based therapeutics including an anti-VEGF antibody
(Dagnaes-Hansen et al., 2003, Anticancer Res. 23: 1625-30) as well
as an ADC consisting of calicheamicin thetal1 conjugated to an
anti-gamma-glutamyltransferase antibody (Knoll et al., 2000, Cancer
Res. 60: 6089-94). Using xenografts of the Caki-1 line in nude
mice, the in vitro antitumor activity of 1F6-vcAFP was tested. To
establish Caki-1 tumors, nude mice were injected subcutaneously
with 5.times.10.sup.6 Caki-1 cells in 0.2 ml PBS. Average tumor
size increased to more than 500 mm.sup.3 within 55 days post
injection. Caki-1-containing tumors were excised from tumor bearing
mice and tumor tissue blocks of approximately 30 mm.sup.3 were
prepared. Naive nude mice to be used for evaluating in vivo
antitumor activity of anti-CD70 ADC were each implanted
subcutaneously with one tumor block of 30 mm.sup.3. Following
implantation, tumor sizes increased to more than 800 mm.sup.3
within 40 days in the absence of any treatment (FIG. 12). Treatment
was initiated when the average tumor size within a group was
approximately 100 mm.sup.3 (FIG. 12, arrows). Very little
therapeutic activity was detected in 1F6, as tumor growth rate in
mice treated with 1F6 was virtually identical to either the
untreated control or the IgG-treated group. However, treatment of
mice with 1F6-vcAFP significantly inhibited tumor growth. The
average tumor size in mice treated with 1 mg/ml of 1F6-vcAFP was
<600 mm.sup.3 56 days after treatment initiation, whereas in the
group treated with IgG-vcAFP at the same dose the average tumor
size was already >600 mm=20 days after treatment initiation
(FIG. 12, upper panel). Increasing the dose of 1F6-vcAFP to 3 mg/ml
further suppressed tumor growth; average tumor size was kept under
200 mm.sup.3 70 days after treatment initiation while IgG-vc-AFP at
the same dose resulted in tumors >400 mm.sup.3 42 days after
treatment initiation (FIG. 12, upper panel). The potent, specific
in vivo antitumor activity of 1F6-vcAFP at 3 mg/kg was from a
separate experiment is shown in FIG. 12, lower panel.
EXAMPLE 9
Expression of CD70 on Activated T Cells
[0351] The generation and maintenance of the T cell clones have
been described in U.S. Provisional Patent Application No.
60/331,750 and in International Patent Application PCT/US02/37223,
both of which are incorporated herein by reference in their
entireties. To activate resting T cell clones, 5.times.10.sup.6 T
cells were incubated in RPMI-1640 supplemented with 10% FBS and 2
mM L-glutamine with PHA-L (1-2 .mu.g/ml) (Sigma), 10.times.10.sup.6
irradiated feeder cells (CESS), rhIL-2 (200 IU/ml) (Proleukin,
Chiron, Emeryville, Calif.), and IL-4 (10 ng/ml) (R&D Systems,
Minneapolis, Minn.). T cell clones were usually allowed to expand
for 10-14 days before re-stimulation. A panel of antigen
non-specific T cell clones was examined for the expression of CD70.
Table 2 summarizes the characteristics of the T cell clones
examined. This panel contains both CD4.sup.+ and CD8.sup.+ T cells
clones that appear to belong to different T cell differentiation
pathways as suggested by their cytokine profiles. Significant
levels of CD70 were detectable on all of the clones examined. T
cell clones were activated by PHA, irradiated feeder cells (CESS),
and rIL-2 and the expression of CD25 and CD70 was monitored by flow
cytometric analysis. Clone C1A, representative of other T cell
clones, showed extensive upregulation of CD25, which peaked on day
2 (FIG. 13). This is indicative of T lymphocyte activation.
Expression of CD25 gradually declined in the following days. The
induction of CD70 expression paralleled that of CD25. The peak CD70
induction was also observed after 2 days of stimulation.
Considerable CD70 expression was still detectable on day 8. All T
cell clones examined, including those listed in Table 2, showed
similar kinetics and magnitudes of activation-induced expression of
CD70. TABLE-US-00005 TABLE 2 T cell clone phenotypes and
activation-induced expression of CD70 Phenotype T cell T-lineage
CD70 clone marker expression* Cytokine profile Designation 3.27.2
CD3, CD4 15 IL-4, IL5, IL-13, IFN.gamma. Th0 4.01.1 CD3, CD4 7
IL-4, IL5, IL-13, IFN.gamma. Th0 20G5 CD3, CD4 3 IL-4, IL-13 Th2
40D8 CD3, CD4 8 IL-4, IL-13 Th2 C1A CD3, CD8 3 IL-4, IL-13,
IFN.gamma. Tc0 C2A CD3, CD8 12 IL-4, IL-13, IFN.gamma. Tc0 *Ratio
between the mean log fluorescence intensities for anti-CD70 binding
and control IgG binding
EXAMPLE 10
Proliferation Inhibitory Effects of Anti-CD70 ADCs on Activated T
Cells
[0352] The effects of 1F6 ADCs on the proliferation of activated T
cells were examined using activated T cell clones, mixed lymphocyte
reactions (MLR), and antigen-primed T cells.
[0353] Standard proliferation assays in 96-well format in
quadruplicates were used to evaluate the effects of anti-CD70 ADCs
on activated T cells. For MLR, PBMCs were seeded at 50,000
cells/well with 50,000 irradiated allogeneic CESS cells in a total
volume of 2001 of medium. For tetanus toxoid-induced T cell
proliferation, tetanus toxoid-reactive T cells were seeded at
20,000 cells/well with 2,000 autologous DCs in a total volume of
200 .mu.l. Dialyzed and clarified tetanus toxoid (Colorado Serum
Company, Denver, Colo.) was used at a final concentration of 1:50
dilution. Two-day activated T cell clones were seeded at 10,000
cells per well. Proliferation assays was usually carried out for 72
to 120 hours. Tritiated thymidine (.sup.3H-TdR) incorporation
during the last 16 hours of incubation and scintillation counting
were used to assess DNA synthesis. For analysis, the amount of
thymidine incorporated by the treated cells was compared to that of
the untreated control cells.
[0354] In the first system, the responses of T cell clones to 1F6
ADCs were examined. Resting T cell clones were activated to induce
CD70 expression as described in FIG. 13 and the accompanying figure
legend. After 2 days of activation, a portion of the cells was
analyzed for CD25 and CD70 expression by flow cytometry to confirm
cellular activation and CD70 induction. The remaining cells were
pelleted and resuspended in new medium containing 200 IU/ml of
rIL-2 or 200 IU/ml of IL-2 and 10 ng/ml IL-4. Cells were then
plated out at 10,000 cells/well in a final volume of 200 .mu.l of
medium containing graded concentrations of 1F6 ADCs or the
non-binding IgG ADCs. Cells were incubated for an additional 72
hours with the last 16 hours pulsed with .sup.3H-TdR to assess
cellular DNA synthesis. The results for the responses of three T
cell clones toward ADC treatment was shown in FIG. 14. 1F6 ADCs at
concentrations higher than 0.01 .mu.g/ml significantly inhibited
the proliferation of the T cell clones. 1F6-vcAFP appeared to be
more active than 1F6-vcMMAE; the IC.sub.50s for 1F6-vcAFP on all 3
clones were found to be below 0.05 .mu.g/ml. The control ADC
cIgG-vcMMAE and cIgG-vcAFP did not significantly inhibit
proliferation at concentrations below 2 .mu.g/ml, confirming the
antigen specificity of the 1F6 ADCs.
[0355] In the second system, an MLR was set up between PBMC and an
irradiated allogeneic stimulator CESS. Allogeneic activation of
peripheral blood T cells was achieved by conventional mixed
lymphoycte reactions (MLR). MLR were initiated by mixing PBMCs and
irradiated allogeneic Epstein-Barr virus (EBV)-transformed B
lymphoblastoid CESS cells (ATCC, Manassas, Va.) at a ratio of one T
cell to ten irradiated CESS cells. T cell density was adjusted to
0.25.times.10.sup.6 T cells/ml of RPMI-1640 supplemented with 10%
FBS and 2 mM L-glutamine. Rapid proliferation and expansion of
alloreactive T cells was observed after approximately 96 hours of
culture. After 5 days of stimulation, cells were assessed for CD70
expression using the anti-CD70 mAb 1F6 by flow cytometry. Flow
cytometric analysis showed that most of the cells within the viable
gate were CD3.sup.+ T cells and that CD70 was expressed on the
viable CD3.sup.+ T cells. FIG. 15, upper panel, shows the
expression of CD70 at the end of the MLR (120 hours after
initiation). In parallel cultures in 96-well plates, graded doses
of either 1F6-vcMMAE or 1F6-vcAFP were included in the cultures at
initiation. DNA synthesis was assayed during the last 16 hours of
the 5-day culture by a pulse of .sup.3H-TdR. Both 1F6-vcMMAE and
1F6-vcAFP substantially inhibited T cell proliferation (FIG. 15,
lower panel). 1F6-vcAFP was more active than 1F6-vcMMAE, with an
IC.sub.50 of approximately 0.1 .mu.g/ml.
[0356] In the third system, the effect of 1F6 and 1F6 ADCs on
antigen-induced T cell proliferation was examined. To evaluate
antigen-specific T cell activation, T cells specific against
tetanus toxoid were enriched from PBMC by multiple rounds of
antigenic stimulation with tetanus toxoid in the presence of
autologous DCs. Before antigenic stimulation of T cells, mature
autologous DCs pulsed with tetanus toxoid were prepared as
described above. Autologous PBMC were then co-cultured with
antigen-pulsed mature DCs at a ratio of 10 PBMC to 1 DC. At the
density of 0.25-0.5.times.10.sup.6 PBMCs/ml of RPMI-1640
supplemented with 10% FBS and 2 mM L-glutamine. T cell activation
and expansion were allowed to continue for 7 days. Viable T cells
were harvested from the culture and re-stimulated with tetanus
toxoid-pulsed autologous mature DCs again under conditions
identical to those in first round of activation. Two additional,
identical rounds of activation were conducted to further enrich for
tetanus toxoid-specific T cells.
[0357] Tetanus toxoid-specific T cells were enriched and expanded
by four consecutive rounds of stimulation by tetanus toxoid and
autologous DCs as described above. After the 4th round of
stimulation, resting tetanus toxoid-specific T cells were plated in
96-well plates at 20,000 cells/well in the presence of autologous
DCs (at 2,000 cells/well) and tetanus toxoid. Graded doses of 1F6,
1F6-vcMMAE, and 1F6-vcAFP were also included in the cultures at the
initiation. DNA synthesis was assay during the last 16 hours by
.sup.3H-TdR incorporation. Unconjugated 1F6 did not show a
significant effect on tetanus toxoid-induced T cell proliferation,
whereas both 1F6-vcMMAE and 1F6-vcAFP exerted substantial
inhibition at doses higher than 0.03 .mu.g/ml (FIG. 16).
EXAMPLE 11
CD70 Expression on Activated T cells During an Antigen-Specific In
Vitro Immune Response
[0358] A 9-amino acid peptide (GILGFVFTL, M1 peptide; SEQ ID NO:48)
derived from the influenza virus matrix protein binds to the
peptide-binding groove of the HLA-A0201 molecule. Presentation of
the M1 peptide by HLA-A0201 expressing antigen presenting cells to
autologous T cells specifically stimulates the activation and
expansion of CD8.sup.+ cytotoxic T cells expressing the T cell
receptor V.beta.17 chain (Lehner et al., 1995, J. Exp. Med.
181:79-91), constituting a convenient in vitro experimental system
to track the activation and expansion of antigen-specific T cells
to their cognate antigen.
[0359] To examine CD70 expression on activated antigen-specific T
cells, PBMCs from a normal donor expressing HLA-A0201 were
stimulated with the M1 peptide. PBMCs were seeded at
2.times.10.sup.6 cells/ml with 5 .mu.g/ml of M1 peptide in AIMV
medium supplemented with 5% human AB serum. IL-2 (Proleukin,
Chiron) and IL-15 (R&D Systems, MN) were added to final
concentrations of 20 IU/ml and 5 ng/ml, respectively, once every
two days beginning on day 2 after culture initiation. The expansion
of CD8.sup.+/V.beta.17.sup.+ T cells and induction of CD70 on the
CD8.sup.+/V.beta.17.sup.+ was followed by three-color flow
cytometry. V.beta.17.sup.+ T cells were identified by the
anti-TCRV.beta.17 mAb clone E17.5F3 (Beckman Coulter, Miami, Fla.).
Results from a representative experiment are shown in FIGS. 17A and
B. FIG. 17A, upper panels, shows that only 0.9% of the cells within
the lymphocyte population were CD8.sup.+/V.beta.17.sup.+ two days
after culture initiation. T cell expansion was only evident within
the CD8.sup.+/V.beta.17.sup.+ population. The percentage of
CD8.sup.+/V.beta.17.sup.+ progressively increased to 23% on day 11.
CD70 expression became detectable 3 days after antigen stimulation
(FIG. 17A, lower panel). On day 7, approximately 60% of the
expanding CD8.sup.+/V.beta.17.sup.+ cells expressed CD70 (FIG. 17A,
lower left panel). The highest level of CD70 expression as
indicated by the mean fluorescence intensity (MFI) was also
detected on day 7 (FIG. 17A, lower right panel). The percentage of
CD70.sup.+/CD8.sup.+/V.beta.17.sup.+ cells and the MFI of CD70
expression on the CD8.sup.+/V.beta.17.sup.+ started to decline
thereafter. Whereas CD70 was clearly expressed on the
CD8.sup.+/V.beta.17.sup.+ cells, no CD70 could be detected on the
CD8.sup.+/V.beta.17.sup.- cells (FIG. 17B). These results confirmed
that CD70 induction was restricted to the activated T cells
responding to the antigenic stimulation but not the bystander,
antigen non-specific T cells.
EXAMPLE 12
In Vitro Deletion of CD70.sup.+ Antigen-Specific T Cells by
Anti-CD70 ADCs
[0360] PBMCs from a normal donor expressing HLA-A0201 were
stimulated with the M1 peptide as described in Example 11. On day
5, cells were harvested, washed, and re-seeded at
0.5-1.times.10.sup.6 cells/ml (approximately 100,000 of
CD8.sup.+/V.beta.17.sup.+ T cells) in fresh AIMV medium
supplemented with 5% human AB, 20 IU/ml of IL-2, and 5 ng/ml of
IL-15. 1F6 or control non-binding IgG (cIgG) ADCs were added to
some cultures to a final concentration of 1 .mu.g/ml. Total viable
cell counts were conducted 24, 48, and 76 hours after ADC addition.
Two-color flow cytometry was conducted to determine the percentages
of CD8.sup.+/V.beta.17.sup.+ cells among the viable cells. The
absolute number of CD8.sup.+/VP17.sup.+ T cells in each culture was
then calculated. The number of CD8.sup.+/V.beta.17.sup.+ T cells in
the two control cultures increased to more than 800,000 after 76
hours (FIG. 18, upper panel). Substantial inhibition of
CD8.sup.+/V.beta.17.sup.+ T cell expansion was seen in the 1F6
ADC-treated cultures. The number of CD8.sup.+/V.beta.17.sup.+ T
cells was approximately 400,000 in the 1F6-vcMMAE-treated culture,
compared to >800,000 in the cIgG-vcMMAE-treated culture.
Virtually no expansion of CD8.sup.+/V.beta.17.sup.+ T cells
occurred in the 1F6-vcAFP-treated culture, whereas >500,000 of
CD8.sup.+/V.beta.17.sup.+ T cells were present in the
cIgG-vcAFP-treated culture. The effects of 1F6 ADCs on the
CD8.sup.+/V.beta.17.sup.- cells were also evaluated. FIG. 18, lower
panel, shows that the CD8.sup.+/V.beta.17.sup.+ cells in the
1F6-vcAFP- and 1F6-vcMMAE-treated cultures were <40% and
<20%, respectively, of the control untreated culture at the end
of the experiment. In contrast, the antigen non-specific bystander,
antigen non-specific CD8.sup.+/V.beta.17.sup.- cells in the
ADC-treated cultures were >60% of the untreated culture. These
results demonstrate that 1F6 ADCs specifically targeted the
CD70.sup.+/CD8.sup.+/V.beta.17.sup.+ T cells and exerted limited
effects on the CD70.sup.-/CD8.sup.+/V.beta.17.sup.- T cells that
co-inhabited the same culture, suggesting limited bystander
toxicity of the 1F6 ADCs.
[0361] The potency of three 1F6 ADCs was then compared. HLA-A0201
PBMCs were stimulated with the M1 peptide and re-seeded as
described for FIG. 18 with the various ADCs at the indicated
concentrations (FIG. 19). The percent of CD8.sup.+/V.beta.17.sup.+
T cells after a 96-hour incubation was determined. A dose-dependent
inhibition of CD8.sup.+/VP17.sup.+ T cell expansion was observed
with 1F6-vcMMAE, -vcAFP, and -vcMMAF, while the corresponding IgG
control ADCs at the top concentration of 1 .mu.g/ml showed no
detectable inhibitory activity compared to the no drug control
culture. The IC.sub.50 values for 1F6-vcAFP and 1F6-vcMMAF were
between 0.1 and 0.01 .mu.g/ml. The inhibitory activity of
1F6-vcMMAE appeared to be weaker than both 1F6-vcAFP and
1F6-vcMMAF.
[0362] A re-stimulation assay was used to evaluate the functional
capacity of the bystander, antigen non-specific
CD8.sup.+/V.beta.17.sup.- T cells. HLA-A0201 expressing PBMCs were
stimulated with the M1 peptide and then treated with 1 .mu.g/ml of
1F6-vcMMAF on day 5 to delete the
CD70.sup.+/CD8.sup.+/V.beta.17.sup.+ T cells as described in FIGS.
18 and 19. As expected the number of CD8.sup.+/V.beta.17.sup.+ T
cells in the ADC-treated culture was lower than the untreated
culture, 14% versus 47% (FIG. 20, left column). Responses of the
remaining cells to a mitogenic re-stimulation were assessed. In
order to normalize for the number of V.beta.17.sup.- T cells used
in the re-stimulation assay and minimize the contribution of
CD8.sup.+/V.beta.17.sup.+ T cells to the proliferation response,
V.beta.17.sup.- T cells were enriched by negative immunoselection.
Briefly, day 9 cells were harvested, washed, and resuspended to
5.times.10.sup.7 cells/ml in medium containing an anti-TCRV.beta.17
mAb (clone E17.5F3) at 1-2 .mu.g/10.sup.6 target T cells. After
incubation at 4.degree. C. for 30 minutes, unbound
anti-TCRV.beta.17 mAb was removed by washing the cells twice with
PBS containing 0.1% human serum. The cells were resuspended at a
concentration of 2-5.times.10.sup.6/ml two washes in medium and
Dynabeads M-450 goat anti-mouse IgG paramagnetic beads (Dynal
Biotech Inc., Lake Success, N.Y.) were added at a ratio of 4
paramagnetic beads to 1 target T cell. The mixture was rotated at
4.degree. C. for 30 minutes. Paramagnetic beads with bound
V.beta.17.sup.+ T cells were then separated from the rest of the
cells by a magnetic device. Removal of V.beta.17.sup.+ T cells was
confirmed by two-color flow cytometry. FIG. 20, middle column,
shows that only 3% and 5% of the cells were left in the
1F6-vcMMAF-treated and untreated cultures, respectively. Anti-CD3
and anti-CD28 immobilized onto tissue culture wells at graded
concentrations were used to re-stimulate the V.beta.17.sup.- cells
in the absence of any exogenous IL-2. Twenty thousand
V.beta.17.sup.- cells were seeded per well in 96-well plates in a
total of 200 .mu.l of AIMV medium supplemented with 5% human serum.
Cells were cultured for 96 hours, and DNA synthesis was assayed by
.sup.3H-TdR incorporation during the last 18 hours of culture.
V.beta.17.sup.- T cells enriched from the 1F6-vcMMAF-treated
culture demonstrated a dose-dependent response to anti-CD3 plus
anti-CD28 re-stimulation comparable to that of the untreated cells.
This suggests that, although the V.beta.17.sup.- T cells were
incubated with the CD8.sup.+/V.beta.17.sup.+ cells in the presence
of 1F6-vcMMAF for 4 days, they had retained their proliferation
capacity. More importantly, their ability to mount a proliferation
response in the absence of any exogenous IL-2 illustrates that they
were also functionally intact with regard to their ability to
express and secrete the necessary cytokine(s) needed for their
proliferation. Taken together, these results suggest that anti-CD70
ADCs can selectively delete CD70.sup.+ activated T cells without
causing substantial collateral damage to bystander T cells. This
characteristic suggests that they can be applied as
immunosuppressive agents that may have minimum impact on the immune
repertoire of the host.
EXAMPLE 13
Treatment of Experimental Allergic Encephalomyelitis by
Administration of Anti-CD70 ADCs
[0363] Studies indicate a role for CD70/CD27-mediated T cell-T cell
interactions in enhancing the Th.sub.1-mediated immune responses in
cell-mediated autoimmune diseases, including, for example,
autoimmune demyelinating diseases. In this example, experimental
allergic encephalomyelitis (EAE), an animal model of the
demylelinating disease multiple sclerosis (MS), is treated with an
ADC comprising an antibody that (a) is conjugated to AFP or MMAE
with a val-cit (vc) linker and (b) recognizes an epitope of murine
CD70 corresponding the 1F6 epitope of human CD70.
[0364] Induction and clinical assessment of Experimental allergic
encephalomyelitis (EAE): R-EAE (relapsing EAE) is induced in six-
to seven-week-old female SJL mice by subcutaneous immunization with
100 .mu.l of complete Freund's adjuvant (CFA) emulsion containing
200 .mu.g of Mycobacterium tuberculosis H37Ra and 40 .mu.g of the
immunodominant epitope of proteolipid protein, PLP.sub.139-151. The
signs of EAE are scored on a 0 to 5 scale as follows: (0) normal;
(1) limp tail or hind limb weakness; (2) limp tail and hind-limb
weakness (waddling gait); (3) partial hind-limb paralysis; (4)
complete hind-limb paralysis; and (5) moribund. A relapse is
defined as a sustained increase (more than 2 days) in at least one
full grade in clinical score after the animal had improved
previously at least a full clinical score and had stabilized for at
least 2 days. The data are plotted as the mean clinical score for
all animals in a particular treatment group or as the relapse rate
(total number of relapses in a group divided by the total number of
mice in that group).
[0365] Anti-CD70 ADC Administration Regimens: Anti-CD70 ADC (0.1-3
mg/g body weight) is administered intraperitoneally in a total
volume of 100 .mu.l. Mice are treated 3 times per week for 3
consecutive weeks (9 total treatments). Treatment is initiated
before disease onset (day 7) or at the peak of acute disease (day
14). As a control, one group of EAE-induced mice are left
untreated.
[0366] Inhibition of TNF-.alpha. and IFN-.gamma. induction: The
demonstration of induction of TNF-.alpha. and IFN-.gamma. in brains
of EAE shows an inflammatory disease process indicative of EAE
disease progression and inhibition of these cytokines in brains of
SJL mice treated with anti-CD70 ADC indicates the value of
anti-CD70 ADC therapy in preventing or treating EAE. Brains are
obtained from at least three animals treated preclinically (at day
13, after three treatments, and day 26, after nine treatments) and
at peak of acute disease (at day 20, after three treatments, and
day 33, after nine treatments). Brains are fixed (10% buffered
formalin), and tissues are embedded in paraffin and sectioned.
Sections are then independently stained for TNF-.alpha. or
IFN-.gamma. by incubation with a primary antibody specific for the
respective cytokine, followed by incubation with a secondary
antibody conjugated to FITC. Tissue sections are then mounted in
mounting media and analyzed by immunofluorescence microscopy.
Decreased levels of TNF-.alpha. or INF-.gamma. staining in
ADC-treated mice versus the untreated EAE-induced mice shows
inhibition of inflammatory cytokine induction using anti-CD70 ADC
therapy.
[0367] Inhibition of Disease Symptoms or Relapse Rates: EAE-induced
SJL mice in the treatment group are compared with untreated
EAE-induced mice to assess the efficacy of anti-CD70 ADC therapy in
either preventing disease onset or treating established disease.
For mice treated preclinically, a decrease in the mean score for
EAE disease, as compared to the untreated control group,
demonstrates the efficacy of anti-CD70 ADC therapy in preventing
disease. For mice treated at the peak of acute disease, either (a)
a decrease in the relapse rate or (b) a decrease in the
post-treatment mean score for EAE, as compared to the untreated
control group, demonstrates the efficacy of anti-CD70 ADC therapy
in treating established disease.
EXAMPLE 14
Mouse Xenograft Model of Renal Cell Carcinoma
[0368] A 786-O subcutaneous xenograft model was used to evaluate
the antitumor activity of anti-CD70 ADCs administered at different
dosages and schedules. Subcutaneous 786-O tumors were initiated in
nude mice by implanting tumor fragments (N=5 or 6/group) of
approximated 30 mm.sup.3. Tumor growth was allowed to establish and
treatment began when average tumor size was approximately 100
mm.sup.3. Tumor dimensions were determined by caliper measurements
to monitor growth. Tumor size was calculated using the formula of
(length.times.width.sup.2)/2. In the absence of any treatment, mean
tumor volume increased to approximately 600 mm.sup.3 within 40 to
50 days after tumor implantation (see FIG. 21A). A dose-dependent
effect in tumor growth suppression was observed in mice received
either humanized 1F6 (h1F6)-mcMMAF4 (see U.S. Patent Application
Publication No. 2005-0238649; loaded with an average of four MMAF
molecules per antibody) or h1F6-vcMMAF4 (loaded with an average of
four MMAF molecules per antibody). Detectable delay in tumor growth
was observed even at 0.5 and 0.17 mg/kg of h1F6-mcMMAF4 and
h1F6-vcMMAF4, respectively.
[0369] Tumor growth was also assessed by time needed for tumors to
quadruple in size (see FIG. 21B). Treatment with either
h1F6-mcMMAF4 or h1F6-vcMMAF4 at 0.17 mg/kg significantly delayed
the growth of tumors. This delay was observed when the ADCs were
given on a q4d.times.4 or q4d.times.10 schedule. However,
additional administrations as exemplified by the q4d.times.10
schedule appeared to have a stronger growth inhibitory activity
compared to the q4d.times.4 schedule.
EXAMPLE 15
Expression of CD70 on Multiple Myeloma Cell Lines
[0370] Cell surface CD70 expression was evaluated in a panel of
multiple myeloma cell lines (Table 3). Copy number of CD70
molecules expressed by each cell line was determined by
quantitative flow cytometry using the QIFIKit.RTM. (Dako,
Carpinteria, Calif.). Response of these cells to anti-CD70
ADC-mediated cytotoxicity was determined. Both chimeric
1F6(c1F6)-vcMMAF4 and c1F6-mcMMAF4 were cytotoxic against
CD70-expressing multiple myeloma cells. The IC.sub.50 values
obtained with c1F6-vcMMAF4 ranged from 1.2-160 ng/mL while that
obtained with c1F6-mcMMAF4 ranged from 1.7-500 ng/mL.
TABLE-US-00006 TABLE 3 Cytotoxic Activity of Anti-CD70 ADCs against
Multiple Myeloma Cell Lines IC.sub.50 (ng/mL) Cell Line CD70
copies/cell c1F6-vcMMAF4 c1F6-mcMMAF4 MM.1S 14,000 20 22 MM.1R
25,000 13 20 AMO-1 92,000 16 38 JJN-3 19,000 46 61 L363 13,000 78
210 LB 45,000 80 500 U266 155,000 1.2 1.7 LP-1 34,000 160 155
MOLP-8 9,000 73 33
EXAMPLE 16
Mouse Xenograft Models of Multiple Myeloma
[0371] The in vivo activity of anti-CD70 ADCs in xenograft models
of multiple myeloma was further examined. Human multiple myeloma
cell lines MM-1S or L363 were resuspended in RPMI-1640 medium at
the concentration of 10.times.10.sup.6 cells/300 .mu.L. To
establish tumors 300 .mu.L of cell suspension were injected
intravenously through the tail veins of SCID mice. In the MM-1S
model, untreated mice succumbed to the injected tumor cells and
manifested symptoms around 40 days post tumor implant including
hind limb paralysis, hunched posture, cranial swelling, and/or
scruffy coat. Mice were euthanized when they demonstrated one or
more of these symptoms. Both h1F6-vcMMAF4 and h1F6-mcMMAF4 provided
significant survival benefits to tumor bearing mice compared to
control non-binding IgG-vcMMAF4 and IgG-mcMMAF4 (see FIG. 22A).
Tumor burden in the MM-1S model was also assessed by enumerating
the number of bone marrow cells expressing human CD138, a plasma
cell marker expressed by the MM-1S cells. Bone marrow cells were
recovered from mice that were euthanized due to manifestation of
symptom or at the end of the experiment on day 122, and the number
of CD138-expressing MM-1S cells was determined by flow cytometry.
Compared to untreated mice, both control IgG-vcMMAF4 and
IgG-mcMMAF4 did not significantly reduce the number of
CD138-expressing cells in the bone marrow. On the other hand,
h1F6-vcMMAF4 and h1F6-mcMMAF4 significantly reduce tumor burden as
demonstrated by much lower number of bone marrow CD138-expressing
cells compared to the control ADCs (see FIG. 22B).
[0372] In the L363 model, disseminated tumor masses develop at
multiple locations in mice receiving no treatment, and tumor masses
became palpable around 40 days after tumor injection, at which
tumor bearing mice would be euthanized. Similar to the MM-1S model,
control IgG-vcMMAF4 provided no survival advantage, whereas
h1F6-vcMMAF4 significantly prolonged survival (see FIG. 23A). Since
L363 cells secrete immunoglobulin lambda light chain (.lamda. LC),
tumor burden can be determined by monitoring the level of human
.lamda. LC in the plasma of tumor bearing mice. An ELISA was used
to detect secreted 1 LC. Ninety six-well flat-bottom Immuno plates
(Nunc Maxisorp, #442404, Nalge Nunc international, Rochester, N.Y.)
was coated with 100 .mu.L/well of goat anti-human Ig (Southern
Biotech #2010-01, Birmingham, Ala.) at 2 .mu.g/mL in 0.1M sodium
carbonate/bicarbonate overnight at 4.degree. C. Wells were washed
5.times. with 1.times.PBST (PBS, 0.05% Tween-20), and blocked with
200 .mu.L/well of 1% BSA/PBST (0.05% Tween-20) for 1 hour at room
temperature. After 5 washes with 1.times.PBST, serially diluted
human .lamda. LC-containing mouse serum samples were added.
Purified human .lamda. LC (Bethyl labs, #P80-127, Montgomery, Tex.)
was used as the standard. After one hour of incubation at room
temperature, wells were washed 5 times with 1.times.PBST. HRP-goat
anti-human lambda chain specific F(ab').sub.2 (Southern Biotech
#2072-05) at 1:4000 dilution in 1% BSA/PBST was added. After an
additional one hour incubation at room temperature, wells were
washed 5 times with 1.times.PBST. TMB substrate 100 .mu.L/well
(Sigma, #T8665, St. Louis, Mo.) was used to detect captured .lamda.
LC. Forty days after L363 cell implant serum .lamda. LC levels were
comparable between the untreated mice and the IgG-vcMMAF4-treated
mice (FIG. 23B). In contrast, serum .lamda. LC levels in the
h1F6-vcMMAF4-treated mice were significantly lower (FIG. 23B),
confirming the ability of anti-CD70 ADC to reduce tumor burden in
mice bearing multiple myeloma xenografts.
EXAMPLE 17
Expression of CD70 on Hodgkin's and Glioblastoma Cell Lines
[0373] Cell surface CD70 expression was also evaluated in panels of
Hodgkin's disease (Table 4) and glioblastoma cell lines (Table 5).
The copy number of CD70 molecules expressed by each cell line was
determined by quantitative flow cytometry using the QIFIKit.RTM.
(Dako, Carpinteria, Calif.). The response of these cells to
anti-CD70 ADC-mediated cytotoxicity was determined. Both chimeric
1F6(c1F6)-vcMMAF4 and c1F6-mcMMA 4 were cytotoxic against these
CD70-expressing cell lines. In the Hodgkin's disease panel, the
IC.sub.50 values obtained with c1F6-vcMMAF4 ranged from 0.41-42
ng/mL while that obtained with c1F6-mcMMAF4 ranged from 5.2-310
ng/mL (Table 5). In the glioblastoma panel, the IC.sub.50 values
obtained with h1F6-vcMMAF4 ranged from 2.3-27 ng/mL while that
obtained with h1F6-mcMMAF4 ranged from 15-110 ng/mL (Table 4).
TABLE-US-00007 TABLE 4 Cytotoxic Activity of Anti-CD70 ADCs against
Hodgkin's Disease Cell Lines IC.sub.50 (ng/mL) Cell Line CD70
copies/cell c1F6-vcMMAF4 C1F6-mcMMAF4 RPMI-6666 21,000 42 230 Hs445
64,000 7.3 310 L428 105,000 1.4 35 KMH2 160,000 0.41 5.2 SUP-HD-1
221,000 6.3 53
[0374] TABLE-US-00008 TABLE 5 Cytotoxic Activity of Anti-CD70 ADCs
against Glioblastoma Cell Lines IC.sub.50 (ng/mL) Cell Line CD70
copies/cell c1F6-vcMMAF4 c1F6-mcMMAF4 U251 117,000 5.3 15 SNB-19
90,000 12 27 U373MG 70,000 16 35 GMS-10 64,000 27 110 DBTRG-05MG
59,000 2.3 20
[0375] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims.
[0376] Various references, including patent applications, patents,
and scientific publications, are cited herein, the disclosures of
each of which is incorporated herein by reference in its entirety.
Sequence CWU 1
1
48 1 411 DNA mouse 1 atggcttggg tgtggacctt gctattcctg atggcagctg
cccaaagtgc ccaagcacag 60 atccagttgg tgcagtctgg acctgaggtg
aagaagcctg gagagacagt caagatctcc 120 tgcaaggctt ctgggtatac
cttcacaaac tatggaatga actgggtgaa gcaggctcca 180 ggaaagggtt
taaagtggat gggctggata aacacctaca ctggagagcc aacatatgct 240
gatgccttca agggacggtt tgccttctct ttggaaacct ctgccagcac tgcctatttg
300 cagatcaaca acctcaaaaa tgaggacacg gctacatatt tctgtgcaag
agactacggc 360 gactatggta tggactactg gggtcaagga acctcagtca
ccgtctcctc a 411 2 137 PRT mouse 2 Met Ala Trp Val Trp Thr Leu Leu
Phe Leu Met Ala Ala Ala Gln Ser 1 5 10 15 Ala Gly Ala Gln Ile Gln
Leu Val Gln Ser Gly Pro Glu Val Lys Lys 20 25 30 Pro Gly Glu Thr
Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn
Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu 50 55 60
Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala 65
70 75 80 Asp Ala Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser
Ala Ser 85 90 95 Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu
Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Ala Arg Asp Tyr Gly Asp Tyr
Gly Met Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Ser Val Thr Val Ser
Ser 130 135 3 57 DNA mouse 3 atggcttggg tgtggacctt gctattcctg
atggcagctg cccaaagtgc ccaagca 57 4 19 PRT mouse 4 Met Ala Trp Val
Trp Thr Leu Leu Phe Leu Met Ala Ala Ala Gln Ser 1 5 10 15 Ala Gly
Ala 5 30 DNA mouse 5 gggtatacct tcacaaacta tggaatgaac 30 6 10 PRT
mouse 6 Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn 1 5 10 7 51 DNA
mouse 7 tggataaaca cctacactgg agagccaaca tatgctgatg ccttcaaggg a 51
8 17 PRT mouse 8 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala
Asp Ala Phe Lys 1 5 10 15 Gly 9 27 DNA mouse 9 gactacggcg
actatggtat ggactac 27 10 9 PRT mouse 10 Asp Tyr Gly Asp Tyr Gly Met
Asp Tyr 1 5 11 396 DNA mouse 11 atggagacag acacactcct gttatgggta
ctgctgctct gggttccagg ttccactggt 60 gacattgtgc tgacacagtc
tcctgcttcc ttagctgtat ctctggggca gagggccacc 120 atctcatgca
gggccagcaa aagtgtcagt acatctggct atagttttat gcactggtat 180
caacagaaac caggacagcc acccaaactc ctcatctatc ttgcatccaa cctagaatct
240 ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct
caacatccat 300 cctgtggagg aggaggatgc tgcaacctat tactgtcagc
acagtaggga ggttccgtgg 360 acgttcggtg gaggcaccaa gctggaaatc aaacgg
396 12 132 PRT mouse 12 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Val Leu Thr
Gln Ser Pro Ala Ser Leu Ala 20 25 30 Val Ser Leu Gly Gln Arg Ala
Thr Ile Ser Cys Arg Ala Ser Lys Ser 35 40 45 Val Ser Thr Ser Gly
Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro
Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser 65 70 75 80 Gly
Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90
95 Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys
100 105 110 Gln His Ser Arg Glu Val Pro Trp Thr Phe Gly Gly Gly Thr
Lys Leu 115 120 125 Glu Ile Lys Arg 130 13 60 DNA mouse 13
atggagacag acacactcct gttatgggta ctgctgctct gggttccagg ttccactggt
60 14 20 PRT mouse 14 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly 20 15 45 DNA mouse 15
agggccagca aaagtgtcag tacatctggc tatagtttta tgcac 45 16 15 PRT
mouse 16 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Phe Met
His 1 5 10 15 17 21 DNA mouse 17 cttgcatcca acctagaatc t 21 18 7
PRT mouse 18 Leu Ala Ser Asn Leu Glu Ser 1 5 19 27 DNA mouse 19
cagcacagta gggaggttcc gtggacg 27 20 9 PRT mouse 20 Gln His Ser Arg
Glu Val Pro Trp Thr 1 5 21 411 DNA mouse 21 atggaatgga cctgggtctt
tctcttcctc ctgccagtaa ctgcagatgt ccaatcccag 60 gttcagctgc
aacagtctgg aactgagctg atgacgcctg gggcctcagt gacgatgtcc 120
tgcaagactt ctggctacac attcagtacc tactggatag agtgggtaaa acagaggcct
180 ggacatggcc ttgagtggat tggagaaatt ttacctggaa gtggttatac
tgactacaat 240 gagaagttca aggccaaggc cacattcact gcagatacat
cctccaacac agcctacatg 300 caactcagca gcctggcatc tgaggactct
gccgtctatt actgtgcaag atgggatagg 360 ctctatgcta tggactactg
gggtcaagga acctcagtca ccgtctcctc a 411 22 137 PRT mouse 22 Met Glu
Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Asp 1 5 10 15
Val Gln Ser Gln Val Gln Leu Gln Gln Ser Gly Thr Glu Leu Met Thr 20
25 30 Pro Gly Ala Ser Val Thr Met Ser Cys Lys Thr Ser Gly Tyr Thr
Phe 35 40 45 Ser Thr Tyr Trp Ile Glu Trp Val Lys Gln Arg Pro Gly
His Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Leu Gly Pro Ser Gly
Tyr Thr Asp Tyr Asn 65 70 75 80 Glu Lys Phe Lys Ala Lys Ala Thr Phe
Thr Ala Asp Thr Ser Ser Asn 85 90 95 Thr Ala Tyr Met Gln Leu Ser
Ser Leu Ala Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg
Trp Asp Arg Leu Tyr Ala Met Asp Tyr Trp Gly 115 120 125 Gly Gly Thr
Ser Val Thr Val Ser Ser 130 135 23 57 DNA mouse 23 atggaatgga
cctgggtctt tctcttcctc ctgtcagtaa ctgcagatgt ccaatcc 57 24 19 PRT
mouse 24 Met Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val Thr
Ala Asp 1 5 10 15 Val Gln Ser 25 30 DNA mouse 25 ggctacacat
tcagtaccta ctggatagag 30 26 10 PRT mouse 26 Gly Tyr Thr Phe Ser Thr
Tyr Trp Ile Glu 1 5 10 27 51 DNA mouse 27 gaaattttac ctggaagtgg
ttatactgac tacaatgaga agttcaaggc c 51 28 17 PRT mouse 28 Glu Ile
Leu Gly Pro Ser Gly Tyr Thr Asp Tyr Asn Glu Lys Phe Lys 1 5 10 15
Ala 29 27 DNA mouse 29 tgggataggc tctatgctat ggactac 27 30 9 PRT
mouse 30 Trp Asp Arg Leu Tyr Ala Met Asp Tyr 1 5 31 396 DNA mouse
31 atggagacag acacactcct gttatgggta ctgctgctct gggttccagg
ttccactggt 60 gacattgtgc tgacacagtc tcctgcttcc ttaactgtat
ctctggggca gaagaccacc 120 atctcatgca gggccagcaa gagtgtcagt
acatctggct atagttttat gcactggtac 180 caactgaaac caggacagtc
acccaaactc ctcatctatc ttgcgtccaa cctaccatct 240 ggggtccctg
ccaggttcag tggcagtggg tctgggacag acttcaccct caaaatccat 300
cctgtggagg aggaggatgc tgcaacctat tactgtcagc acagtaggga gattccgtac
360 acgttcggag gggggaccaa gctggaaata acacgg 396 32 132 PRT mouse 32
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5
10 15 Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Thr 20 25 30 Val Ser Leu Gly Gln Lys Thr Thr Ile Ser Cys Arg Ala
Ser Lys Ser 35 40 45 Val Ser Thr Ser Gly Tyr Ser Phe Met His Trp
Tyr Gln Leu Lys Pro 50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile Tyr
Leu Ala Ser Asp Leu Pro Ser 65 70 75 80 Gly Val Pro Ala Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile His Pro
Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Gln His Ser
Arg Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu
Ile Thr Arg 130 33 60 DNA mouse 33 atggagacag acacactcct gttatgggta
ctgctgctct gggttccagg ttccactggt 60 34 20 PRT mouse 34 Met Glu Thr
Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly
Ser Thr Gly 20 35 45 DNA mouse 35 agggccagca agagtgtcag tacatctggc
tatagtttta tgcac 45 36 15 PRT mouse 36 Arg Ala Ser Lys Ser Val Ser
Thr Ser Gly Tyr Ser Phe Met His 1 5 10 15 37 21 DNA mouse 37
cttgcgtcca acctaccatc t 21 38 7 PRT mouse 38 Leu Ala Ser Asn Leu
Pro Ser 1 5 39 28 DNA mouse 39 cagcacagta gggagattcc gtacacgt 28 40
9 PRT mouse 40 Gln His Ser Arg Glu Ile Pro Tyr Thr 1 5 41 25 DNA
Artificial Sequence Primer 41 cttccacttg acattgatgt ctttg 25 42 22
DNA Artificial Sequence Primer 42 caggtcactg tcactggctc ag 22 43 37
DNA Artificial Sequence Primer 43 gtcgatgagc tctagaattc gtgccccccc
ccccccc 37 44 47 DNA Artificial Sequence Primer 44 cgtcatgtcg
acggatccaa gcttcaagaa gcacacgact gaggcac 47 45 47 DNA Artificial
Sequence Primer 45 cgtcatgtcg acggatccaa gcttgtcacc atggagttag
tttgggc 47 46 17 DNA Artificial Sequence Primer 46 ccactgctgc
tgattag 17 47 18 DNA Artificial Sequence Primer 47 caatgccttc
tcttgtcc 18 48 8 PRT virus 48 Gly Ile Leu Gly Val Phe Thr Leu 1
5
* * * * *