U.S. patent application number 13/459558 was filed with the patent office on 2012-11-29 for anti-mn antibodies and methods of using same.
This patent application is currently assigned to BAYER HEALTHCARE LLC. Invention is credited to Lila Adnane, Sha Ha, Timothy McCabe, Gerald Ranges, Paul Tamburini, Pamela Trail.
Application Number | 20120301468 13/459558 |
Document ID | / |
Family ID | 38163479 |
Filed Date | 2012-11-29 |
United States Patent
Application |
20120301468 |
Kind Code |
A1 |
Tamburini; Paul ; et
al. |
November 29, 2012 |
ANTI-MN ANTIBODIES AND METHODS OF USING SAME
Abstract
The invention provides antibodies having an antigenic binding
site specifically directed against an MN protein, and methods of
using such antibodies in treating and diagnosing an MN-related
disorder.
Inventors: |
Tamburini; Paul;
(Kensington, CT) ; Ranges; Gerald; (Hamden,
CT) ; Adnane; Lila; (Madison, CT) ; McCabe;
Timothy; (Branford, CT) ; Trail; Pamela;
(Edmonds, WA) ; Ha; Sha; (Lansdale, PA) |
Assignee: |
BAYER HEALTHCARE LLC
Tarrytown
NY
|
Family ID: |
38163479 |
Appl. No.: |
13/459558 |
Filed: |
April 30, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12086320 |
Nov 23, 2009 |
8168758 |
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PCT/US2006/047445 |
Dec 12, 2006 |
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13459558 |
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60749716 |
Dec 12, 2005 |
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Current U.S.
Class: |
424/135.1 ;
424/133.1; 424/136.1; 424/174.1; 424/178.1; 435/7.23; 530/387.3;
530/389.7; 530/391.7 |
Current CPC
Class: |
A61K 47/6871 20170801;
C07K 2317/92 20130101; A61K 39/395 20130101; A61K 47/6851 20170801;
A61K 39/395 20130101; A61K 2039/505 20130101; A61K 47/6817
20170801; C07K 16/30 20130101; A61P 35/00 20180101; C07K 2317/565
20130101; A61P 37/00 20180101; C07K 2317/55 20130101; C07K 16/40
20130101; C07K 2317/732 20130101; C07K 2317/77 20130101; A61K
47/6803 20170801; A61P 43/00 20180101; A61K 2300/00 20130101; C07K
2317/21 20130101 |
Class at
Publication: |
424/135.1 ;
530/389.7; 530/387.3; 424/174.1; 530/391.7; 424/178.1; 435/7.23;
424/136.1; 424/133.1 |
International
Class: |
C07K 16/30 20060101
C07K016/30; G01N 33/574 20060101 G01N033/574; A61P 35/00 20060101
A61P035/00; A61K 39/395 20060101 A61K039/395 |
Claims
1-3. (canceled)
4. An antibody or antibody fragment having an antigenic binding
site specifically directed against an MN protein, wherein the
antigenic binding site comprises a set of CDR sequences [3ef2] SEQ
ID NOS: 58, 64, 71, 107, 109, and 111; [1e4] SEQ ID NOS: 59, 65,
72, 107, 109, and 111; [3a4] SEQ ID NOS: 60, 66, 73, 108, 110, and
112; [3ab4] SEQ ID NOS: 61, 67, 74, 87, 91, and 95; [3ah10] SEQ ID
NOS: 61, 68, 75, 88, 92, and 96; [3bb2] SEQ ID NOS: 62, 69, 76, 98,
100, and 102; [1aa1] SEQ ID NOS: 77, 78, 79, 86, 90, and 94; [5a6]
SEQ ID NOS: 80, 82, 84, 99, 101, and 103; and [5aa3] SEQ ID NOS:
81, 83, 85, 104, 105, and 106.
5-6. (canceled)
7. The antibody or antibody fragment according to claim 4, wherein
the antibody or antibody fragment binds to the MN protein with a
dissociation constant of about 0.15 nM to about 50 nM.
8. The antibody or antibody fragment according to claim 4, wherein
the antibody is an IgG.
9. The antibody or antibody fragment according to claim 4, wherein
the antibody is an IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA,
IgM Fab fragment, F(ab').sub.2 fragment, scFv fragment, Fv
fragment, a diabody, linear antibody, single-chain antibody,
bispecific antibody, chimeric antibody, or multispecific
antibody.
10. The antibody or antibody fragment according to claim 4, wherein
the antibody or antibody fragment is humanized.
11. The antibody or antibody fragment according to claim 4, wherein
the CDR1, CDR2, and CDR3 are non-human.
12. A composition comprising an antibody or antibody fragment
thereof according to claim 4 and one or more pharmaceutical
auxiliary substance.
13-19. (canceled)
20. A composition reactive against MN protein comprising an
antibody or antibody fragment having an antigenic binding site
specifically directed against an MN protein conjugated to a
cytotoxic agent, wherein the antigenic binding site comprises a set
of CDR sequences [3ef2] SEQ ID NOS: 58, 64, 71, 107, 109, and 111;
[1e4] SEQ ID NOS: 59, 65, 72, 107, 109, and 111; [3a4] SEQ ID NOS:
60, 66, 73, 108, 110, and 112; [3ab4] SEQ ID NOS: 61, 67, 74, 87,
91, and 95; [3ah10] SEQ ID NOS: 61, 68, 75, 88, 92, and 96; [3bb2]
SEQ ID NOS: 62, 69, 76, 98, 100, and 102; [1aa1] SEQ ID NOS: 77,
78, 79, 86, 90, and 94; [5a6] SEQ ID NOS: 80, 82, 84, 99, 101, and
103; and [5aa3] SEQ ID NOS: 81, 83, 85, 104, 105, and 106.
21-22. (canceled)
23. The composition according to claim 20, wherein the cytotoxic
agent is selected from the group consisting of:
monomethylauristatin-E or functional analog thereof,
monomethylauristatin-F or functional analog thereof, aplidin,
azaribine, anastrozole, azacytidine, bleomycin, bortezomib,
bryostatin-1, busulfan, calicheamycin, camptothecin,
10-hydroxycamptothecin, carmustine, celebrex, chlorambucil,
cisplatin, irinotecan (CPT-1 1), SN-38, carboplatin, cladribine,
cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin,
daunomycin glucuronide, daunorubicin, dexamethasone,
diethylstilbestrol, doxorubicin, doxorubicin glucuronide,
epirubicin glucuronide, ethinyl estradiol, estramustine, etoposide,
etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),
3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,
fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone
caproate, hydroxyurea, idarubicin, ifosfamide, L-asparaginase,
leucovorin, lomustine, mechlorethamine, medroprogesterone acetate,
megestrol acetate, melphalan, mercaptopurine, 6-mercaptopurine,
methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane,
phenyl butyrate, prednisone, procarbazine, paclitaxel, pentostatin,
PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol,
testosterone propionate, thalidomide, thioguanine, thiotepa,
teniposide, topotecan, uracil mustard, velcade, vinblastine,
vinorelbine, vincristine, ricin, abrin, ribonuclease, onconase,
rapLR1, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral
protein, gelonin, diphtheria toxin, Pseudomonas exotoxin,
Pseudomonas endotoxin, and combinations thereof.
24. The composition according to claim 20, wherein the cytotoxic
agent is monomethylauristatin-E or functional analog thereof.
25. The composition according to claim 20, wherein the antibody or
antibody fragment binds to the MN protein with a dissociation
constant of about 0.15 nM to about 50 nM.
26. The composition according to claim 20, wherein the antibody is
an IgG.
27. The composition according to claim 20, wherein the antibody or
antibody fragment is an IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE,
IgA, or IgM isotype, an Fab fragment, F(ab')2 fragment, scFv
fragment, Fv fragment, a diabody, linear antibody, single-chain
antibody, bispecific antibody, a chimeric antibody or multispecific
antibody.
28. The composition according to claim 20, wherein the antibody or
antibody fragment humanized.
29. The antibody or antibody fragment according to claim 20,
wherein the CDR1, CDR2, and CDR3 are non-human.
30-32. (canceled)
33. A composition for treating a subject having an MN-related
cancer, comprising an anti-cancer agent and an antibody or antibody
fragment thereof having an antigenic binding site specifically
directed against an MN protein conjugated to a cytotoxic agent,
wherein the antigenic binding site comprises a set of CDR sequences
[3ef2] SEQ ID NOS: 58, 64, 71, 107, 109, and 111; [1e4] SEQ ID NOS:
59, 65, 72, 107, 109, and 111; [3a4] SEQ ID NOS: 60, 66, 73, 108,
110, and 112; [3ab4] SEQ ID NOS: 61, 67, 74, 87, 91, and 95;
[3ah10] SEQ ID NOS: 61, 68, 75, 88, 92, and 96; [3bb2] SEQ ID NOS:
62, 69, 76, 98, 100, and 102; [1aa1] SEQ ID NOS: 77, 78, 79, 86,
90, and 94; [5a6] SEQ ID NOS: 80, 82, 84, 99, 101, and 103; and
[5aa3] SEQ ID NOS: 81, 83, 85, 104, 105, and 106.
34-35. (canceled)
36. The composition according to claim 33, wherein the anti-cancer
agent is selected from the group consisting of: capecitabine,
bleomycin, docetaxel (Taxotere), doxorubicin, edatrexate, erlotinib
(Tarceva), etoposide, finasteride (Proscar), flutamide (Eulexin),
gemcitabine (Gemzar), genitinib (Irresa), goserelin acetate
(Zoladex), granisetron (Kytril), imatinib (Gleevec), irinotecan
(Campto/Camptosar), ondansetron (Zofran), paclitaxel (Taxol),
pegaspargase (Oncaspar), pilocarpine hydrochloride (Salagen),
porfimer sodium (Photofrin), interleukin-2 (Proleukin), rituximab
(Rituxan), topotecan (Hycamtin), trastuzumab (Herceptin), Triapine,
vincristine, vinorelbine tartrate (Navelbine), and therapeutic
antibodies or fragments thereof.
37. The composition according to claim 33, wherein the anti-cancer
agent is an anti-angiogenic agent selected from the group
consisting of angiostatin, bevacizumab (Avastin.RTM.), sorafenib
(Nexavar.RTM.), baculostatin, canstatin, maspin, anti-VEGF
antibodies or peptides, anti-placental growth factor antibodies or
peptides, anti-Flk-1 antibodies, anti-Flt-1 antibodies or peptides,
laminin peptides, fibronectin peptides, plasminogen activator
inhibitors, tissue metalloproteinase inhibitors, interferons,
interleukin 12, IP-10, Gro-.beta., thrombospondin,
2-methoxyoestradiol, proliferin-related protein,
carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate,
angiopoietin 2, interferon-alpha, herbimycin A, PNU145156E, 16K
prolactin fragment, Linomide, thalidomide, pentoxifylline,
genistein, TNP-470, endostatin, paclitaxel, accutin, cidofovir,
vincristine, bleomycin, AGM-1470, platelet factor 4 and
minocycline.
38. The composition according to claim 33, wherein the anti-cancer
agent is an agent that blocks or inhibits a multi-drug resistance
phenotype selected from the group consisting of tamoxifen,
verapamil and cyclosporin A.
39. The composition according to claim 33, wherein the cytotoxic
agent is monomethylauristatin-E or functional analog thereof.
40. (canceled)
41. The composition according to claim 33, wherein the antibody or
antibody fragment binds to the MN protein with a dissociation
constant of about 0.15 nM to about 50 nM.
42. The composition according to claim 33, wherein the antibody is
an IgG.
43. The composition according to claim 33, wherein the antibody or
antibody fragment is an IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE,
IgA, or IgM isotype, a Fab fragment, F(ab').sub.2 fragment, scFv
fragment, Fv fragment, a diabody, linear antibody, single-chain
antibody, bispecific antibody, chimeric antibody or multispecific
antibody.
44. The composition according to claim 33, wherein the antibody or
antibody fragment is humanized.
45. The composition according to claim 33, wherein the CDR1, CDR2,
and CDR3 are non-human.
46-49. (canceled)
50. The composition according to claim 33, wherein the cancer is in
the form of a solid tumor.
51. The composition according to claim 50, wherein the solid tumor
is in or originating from the breast, respiratory tract, lung,
brain, reproductive organ, digestive tract, colon, urinary tract,
kidney, esophagus, cervix, eye, liver, skin, head, neck, thyroid,
and parathyroid.
52. A method of diagnosing an MN-related disorder characterized by
abnormal MN levels comprising comparing the level of MN in a
suspected diseased tissue or cell with the level of MN in a
corresponding healthy tissue or cell, wherein an abnormal MN level
in the suspected diseased tissue or cell is an indication of an
MN-related disorder, said step of comparing further comprising
detecting the level of MN in the diseased tissue and the healthy
tissue with the antibody or antibody fragment according to claim
4.
53. The method according to claim 52, wherein the MN-related
disorder is a cancer.
54. The method according to claim 52, wherein the cancer is in the
form of a solid tumor.
55. The method according to claim 52, wherein the solid tumor is in
or originating from the breast, respiratory tract, lung, brain,
reproductive organ, digestive tract, colon, urinary tract, kidney,
esophagus, cervix, eye, liver, skin, head, neck, thyroid, and
parathyroid.
56. The method according to claim 52, wherein the step of comparing
further comprises the steps of: a) detecting the level of MN
protein in the healthy tissue; b) detecting the level of MN protein
in the suspected diseased tissue; and c) comparing the levels of MN
protein from (a) and (b), wherein an elevated level of MN protein
in the suspected diseased tissue as compared to the level of MN
protein in the healthy tissue is indicative of the presence of an
MN-related disorder.
57. The method according to claim 52, wherein the step of detecting
is achieved by a nuclear imaging modality.
58. The method according to claim 57, wherein the nuclear imaging
modality is SPECT or PET.
59. A kit comprising the antibody or antibody fragment according to
claim 4, and a set of instructions for using the kit in a method of
treating an MN-related disorder.
60. A kit comprising the composition according to claim 20 and a
set of instructions for using the kit in a method of treating an
MN-related disorder.
61. A kit comprising the composition according to claim 33 and a
set of instructions for using the kit in a method of treating an
MN-related disorder.
62-68. (canceled)
69. A method of treating an MN-related disorder characterized by an
abnormal level of MN in a subject comprising administering a
therapeutically effective amount of an antibody or antibody
fragment having an antigenic binding site specifically directed
against an MN protein, wherein the antigenic binding site comprises
a set of CDR sequences [3ef2] SEQ ID NOS: 58, 64, 71, 107, 109, and
111; [1e4] SEQ ID NOS: 59, 65, 72, 107, 109, and 111; [3a4] SEQ ID
NOS: 60, 66, 73, 108, 110, and 112; [3ab4] SEQ ID NOS: 61, 67, 74,
87, 91, and 95; [3ah10] SEQ ID NOS: 61, 68, 75, 88, 92, and 96;
[3bb2] SEQ ID NOS: 62, 69, 76, 98, 100, and 102; [1aa1] SEQ ID NOS:
77, 78, 79, 86, 90, and 94; [5a6] SEQ ID NOS: 80, 82, 84, 99, 101,
and 103; and [5aa3] SEQ ID NOS: 81, 83, 85, 104, 105, and 106.
70-71. (canceled)
72. The method according to claim 69, wherein the antibody or
antibody fragment binds to the MN protein with a dissociation
constant of about 0.15 nM to about 50 nM.
73. The method according to claim 69, wherein the antibody is an
IgG.
74. The method according to claim 69, wherein the antibody or
antibody fragment is an IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE,
IgA, or IgM, a Fab fragment, F(ab').sub.2 fragment, scFv fragment,
Fv fragment, a diabody, linear antibody, single-chain antibody,
bispecific antibody, chimeric antibody or multispecific
antibody.
75. The method according to claim 69, wherein the antibody or
antibody fragment is humanized.
76. The method according to claim 69, wherein the CDR1, CDR2, and
CDR3 are non-human.
77-80. (canceled)
81. The method according to claim 69, wherein the antibody or
antibody fragment is conjugated to a cytotoxic agent.
82. The method according to claim 81, wherein the cytotoxic agent
is selected from the group consisting of: monomethylauristatin-E or
functional analog thereof, monomethylauristatin-F or functional
analog thereof, aplidin, azaribine, anastrozole, azacytidine,
bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin,
camptothecin, 10-hydroxycamptothecin, carmustine, celebrex,
chlorambucil, cisplatin, irinotecan (CPT-1 1), SN-38, carboplatin,
cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel,
dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone,
diethylstilbestrol, doxorubicin, doxorubicin glucuronide,
epirubicin glucuronide, ethinyl estradiol, estramustine, etoposide,
etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),
3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,
fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone
caproate, hydroxyurea, idarubicin, ifosfamide, L-asparaginase,
leucovorin, lomustine, mechlorethamine, medroprogesterone acetate,
megestrol acetate, melphalan, mercaptopurine, 6-mercaptopurine,
methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane,
phenyl butyrate, prednisone, procarbazine, paclitaxel, pentostatin,
PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol,
testosterone propionate, thalidomide, thioguanine, thiotepa,
teniposide, topotecan, uracil mustard, velcade, vinblastine,
vinorelbine, vincristine, ricin, abrin, ribonuclease, onconase,
rapLR1, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral
protein, gelonin, diphtheria toxin, Pseudomonas exotoxin,
Pseudomonas endotoxin, and combinations thereof.
83. The method according to claim 82, wherein the cytotoxic agent
is monomethylauristatin-E or functional analog thereof.
84. The method according to claim 69, further comprising
co-administering a-cancer agent.
85. The method according to claim 84, wherein the anti-cancer agent
is selected from the group consisting of bleomycin, docetaxel
(Taxotere), doxorubicin, edatrexate, erlotinib (Tarceva),
etoposide, finasteride (Proscar), flutamide (Eulexin), gemcitabine
(Gemzar), genitinib (Irresa), goserelin acetate (Zoladex),
granisetron (Kytril), imatinib (Gleevec), irinotecan
(Campto/Camptosar), ondansetron (Zofran), paclitaxel (Taxol),
pegaspargase (Oncaspar), pilocarpine hydrochloride (Salagen),
porfimer sodium (Photofrin), interleukin-2 (Proleukin), rituximab
(Rituxan), topotecan (Hycamtin), trastuzumab (Herceptin), Triapine,
vincristine, vinorelbine tartrate (Navelbine), and therapeutic
antibodies or fragments thereof.
86. The method according to claim 84, wherein the anti-cancer agent
is an agent that blocks or inhibits a multi-drug resistance
phenotype selected from the group consisting of tamoxifen,
verapamil and cyclosporin A.
87. The method according to claim 69, wherein the MN-related
disorder is a cancer.
88. The method according to claim 87, wherein the cancer is a solid
tumor cancer.
89. The method according to claim 88, wherein the solid tumor is in
or originating from the breast, respiratory tract, lung, brain,
reproductive organ, digestive tract, colon, urinary tract, kidney,
esophagus, cervix, eye, liver, skin, head, neck, thyroid, and
parathyroid.
90. An antibody or antibody fragment having an antigenic binding
site specifically directed against an MN protein, wherein the
antigenic binding site comprises a pair of heavy chain variable and
light chain variable regions the heavy chain variable region of SEQ
ID NO:133 and the light chain variable region of SEQ ID NO:134; the
heavy chain variable region of SEQ ID NO:135 and the light chain
variable region of SEQ ID NO:136; the heavy chain variable region
of SEQ ID NO:137 and the light chain variable region of SEQ ID
NO:138; the heavy chain variable region of SEQ ID NO:139 and the
light chain variable region of SEQ ID NO:140; the heavy chain
variable region of SEQ ID NO:141 and the light chain variable
region of SEQ ID NO:142; the heavy chain variable region of SEQ ID
NO:143 and the light chain variable region of SEQ ID NO:144; the
heavy chain variable region of SEQ ID NO:147 and the light chain
variable region of SEQ ID NO:148; the heavy chain variable region
of SEQ ID NO:149 and the light chain variable region of SEQ ID
NO:150; and the heavy chain variable region of SEQ ID NO:151 and
the light chain variable region of SEQ ID NO:152.
91. An anti-MN IgG antibody encoded by the nucleotide sequence of
SEQ ID NO: 153.
Description
INCORPORATION BY REFERENCE
[0001] This application claims the benefit of U.S. patent
application Ser. No. 12/086,320, submitted Jun. 10, 2008, and U.S.
Provisional Patent Application No. 60/749,716, filed Dec. 12, 2005.
Reference is also made to International Application No.
PCT/US02/33470, filed on Oct. 18, 2002 and published as WO
03/033674 on Apr. 24, 2003, which is incorporated herein in its
entirety by reference.
[0002] The file named SEQUENCE-LISTING.txt submitted herewith that
is 56,958 bytes and was created on Apr. 30, 2012 is hereby
incorporated by reference herein in its entirety. The foregoing
application, and all documents cited therein and all documents
cited or referenced therein, and all documents cited or referenced
herein ("herein cited documents"), and all documents cited or
referenced in herein cited documents, together with any
manufacturer's instructions, descriptions, product specifications,
and product sheets for any products mentioned herein or in any
document incorporated by reference herein, are hereby incorporated
herein by reference, and may be employed in the practice of the
invention.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to antibodies and/or fragments
thereof having specificity for MN protein. The invention further
relates to antibody and/or immunoconjugate compositions and their
use in treating, preventing, and/or diagnosing MN-related
disorders, e.g. cancer.
[0005] 2. Background
[0006] The occurrence of cancer is most commonly associated with
aging whereby 65% of all new cases of cancer are recorded for
patients aged 65 and over. Cancer is the second leading cause of
death in the United States, exceeded only by heart disease. Indeed,
the American Cancer Society has estimated that 1 in 4 people will
die from cancer in the U.S., assuming current mortality rates
remain static. In the U.S. alone, 1,399,790 new cases and 564,830
deaths from cancer are expected in 2006. The majority of these new
cases are expected to be cancers of the colon (106,680), lung
(172,570) and breast (214,640). Moreover, both the incidence and
prevalence of cancer is predicted to increase approximately 15%
over the next ten years, reflecting an average growth rate of 1.4%
(American Cancer Society, 2006).
[0007] One recently identified tumor associated antigen, MN, a cell
surface protein, has been found to be expressed in a number of
clinical carcinomas. For example, MN has been found to be
ectopically expressed in 100% of renal cell carcinomas (Liao, S Y,
Cancer Res., 1997, 57:2827-2831), 100% of carcinomas of the
esophagus (Turner J R, Hum. Pathol., 199, 28:740-744), greater than
90% of cervical carcinomas (Liao, S Y, Cancer Res., 1997,
57:2827-2831), 76% of malignant colon carcinomas (Saarnio, J. et
al., Am. J. Path., 1997, 153:279-285), 80% of non-small cell lung
carcinomas (Vermylen P. et al., Eur. Respir. J., 1999, 14:806-811),
and in 48% of breast cancers (Chia S K et al., J. Clin. Oncol.,
2001, 19:3660-3668). Like other tumor associated antigens, the MN
protein is also present on cells of a limited number of normal
tissues, including, for example, stomach, bile duct mucosa and the
highly proliferate normal cells located in the small intestine
(Saarnio J. et al., J. Histochem. Cytochem., 1998, 46:497-504).
[0008] Human MN cDNA has been cloned and sequenced (Pastorek, et
al., Oncogene, 1994, 9:2877-2888). The predicted protein consists
of a signal peptide, a proteoglycan-related sequence, a carbonic
anhydrase domain (carbonic anhydrase IX or CA IX), a transmembrane
segment, and a short intracellular tail. The carbonic anhydrase IX
domain catalyzes the reversible hydration of carbon dioxide to
carbonic acid. This activity may have a role in regulating the
local acidification of the extracellular portion of the tumor
environments, which may consequently lead to the activation of
proteases and finally, metastasis.
[0009] The regulation of MN expression has also been investigated.
In one aspect, for example, MN expression is up-regulated by
hypoxia. The transcriptional complex known as hypoxia-inducible
factor-1 (HIF-1) is a regulator of MN expression. Accordingly, MN
is known as a HIF-1 responsive gene which has implications in the
understanding of tumor response to hypoxia (Wykoff C C et al.,
Cancer Res., 2000, 60:7075-7083). Furthermore, MN expression
correlates with tumor hypoxia levels and is a prognostic indicator
of overall survival and metastasis-free survival in cervical cancer
(Loncaster, J A et al., Cancer Res., 2000, 60:7075-7083). MN
expression also correlates with a high mean vessel density,
advanced cancer stage, degree of necrosis in head and neck
carcinoma (Beasley N J P et al., Cancer Res., 2001, 61:5262-5267),
poor survival in nasopharyngeal carcinoma (Hui E P et al., Clin.
Cancer Res., 2002, 8:2595-2604), tumor necrosis, higher grade,
negative estrogen receptor status, higher relapse rate, and poor
survival for invasive breast carcinoma (Chia S K et al., J. Clin.
Oncol., 2001, 19:3660-3668). Therefore expression of MN antigen is
correlates with poor survival prognosis, and cancers of higher
grade.
[0010] New and improved therapies for these aggressive cancers, in
particular, those that target MN expression, are highly desirable
and would represent an advancement in the art. As such, the present
invention discloses new antibody compositions and immunoconjugates
thereof that are useful in the treatment, prevention and/or
diagnosis of MN-related cancers.
SUMMARY OF THE INVENTION
[0011] The present invention relates to antibodies, e.g.,
monoclonal antibodies, or antibody fragments that bind to the
cell-surface protein MN and which can be used in the treatment,
prevention and/or diagnosis of cancer. The antibodies of the
invention can further be conjugated to cytotoxic agents, e.g.,
monomethylauristatin-E, and/or co-administered or formulated with
one or more additional anti-cancer agents. The anti-MN antibodies
and immunoconjugates of the invention can be used in the methods of
the invention to treat and/or diagnose and/or monitor cancers, e.g.
solid tumors.
[0012] In one aspect, the present invention provides an antibody or
antibody fragment, or a composition that includes the antibody or
antibody fragment, wherein the antibody or fragment has an
antigenic binding site that is specifically directed against an MN
protein. The antigenic binding site may include at least one CDR1,
CDR2, or CDR3, or a CDR1 together with a CDR2 or a CDR3, or a CDR2
together with a CDR 1 or a CDR3, or a CDR3 together with a CDR1 or
CDR 2, or any combination thereof. The CDR1 can be selected from
the group consisting of SEQ ID NOS: 57, 58, 59, 60, 61, 62, 77, 80,
81, 86, 87, 88, 89, 98, 99, 104, 107, and 108. The CDR2 can be
selected from the group consisting of SEQ ID NOS: 63, 64, 65, 66,
67, 68, 69, 78, 82, 83, 90, 91, 92, 93, 100, 101, 105, 109, and
110. The CDR3 can selected from the group consisting of SEQ ID NOS:
70, 71, 72, 73, 74, 75, 76, 79, 84, 85, 94, 95, 96, 97, 102, 103,
106, 111, and 112. The CDR sequences of this aspect of the
invention can also include amino acid sequences that have
preferably greater than about 80% sequence identity, more
preferably greater than about 85% sequence identity, even more
preferably about 90% sequence identity, still more preferably about
95% or even about 99% sequence identity, and even up to about 100%
sequence identity to any of the above sequences indicated for each
of CDR1, CDR2 or CDR3.
[0013] In another aspect, the antigenic binding site can have a
heavy chain variable region CDR that is selected from the group
consisting of: SEQ ID NOS: 57-85 and an amino acid sequence having
greater than about 80% sequence identity to any of SEQ ID NOS:
57-85.
[0014] The antigenic binding site can also have a light chain
variable region CDR selected from the group consisting of: SEQ ID
NOS: 86-112 and an amino acid sequence having greater than about
80% sequence identity to any of SEQ ID NOS: 86-112.
[0015] The antigenic binding site can also be selected from a set
of specific CDR sequences that include the following sets of six
CDRs:
[0016] (a) [3ee9] SEQ ID NOS: 57, 63, 70, 89, 93, and 97;
[0017] (b) [3ef2] SEQ ID NOS: 58, 64, 71, 107, 109, and 111;
[0018] (c) [1e4] SEQ ID NOS: 59, 65, 72, 107, 109, and 111;
[0019] (d) [3a4] SEQ ID NOS: 60, 66, 73, 108, 110, and 112;
[0020] (e) [3ab4] SEQ ID NOS: 61, 67, 74, 87, 91, and 95;
[0021] (f) [3ah10] SEQ ID NOS: 61, 68, 75, 88, 92, and 96;
[0022] (g) [3bb2] SEQ ID NOS: 62, 69, 76, 98, 100, and 102;
[0023] (h) [1aa1] SEQ ID NOS: 77, 78, 79, 86, 90, and 94;
[0024] (i) [5a6] SEQ ID NOS: 80, 82, 84, 99, 101, and 103; and
[0025] (j) [5aa3] SEQ ID NOS: 81, 83, 85, 104, 105, and 106.
[0026] The antigenic binding site may also include a set of heavy
chain CDR sequences selected from the group consisting of:
[0027] (a) [3ee9] SEQ ID NOS: 57, 63, and 70;
[0028] (b) [3ef2] SEQ ID NOS: 58, 64, and 71;
[0029] (c) [1e4] SEQ ID NOS: 59, 65, and 72;
[0030] (d) [3a4] SEQ ID NOS: 60, 66, and 73;
[0031] (e) [3ab4] SEQ ID NOS: 61, 67, and 74;
[0032] (f) [3ah10] SEQ ID NOS: 61, 68, and 75;
[0033] (g) [3bb2] SEQ ID NOS: 62, 69, and 76;
[0034] (h) [1aa1] SEQ ID NOS: 77, 78, and 79;
[0035] (i) [5a6] SEQ ID NOS: 80, 82, and 84; and
[0036] (j) [5aa3] SEQ ID NOS: 81, 83, and 85.
[0037] The antigenic binding site can also include a set of light
chain CDR sequences selected from the group consisting of:
[0038] (a) [3ee9] SEQ ID NOS: 89, 93, and 97;
[0039] (b) [3ef2] SEQ ID NOS: 107, 109, and 111;
[0040] (c) [1e4] SEQ ID NOS: 107, 109, and 111;
[0041] (d) [3a4] SEQ ID NOS: 108, 110, and 112;
[0042] (e) [3ab4] SEQ ID NOS: 87, 91, and 95;
[0043] (f) [3ah10] SEQ ID NOS: 88, 92, and 96;
[0044] (g) [3bb2] SEQ ID NOS: 98, 100, and 102;
[0045] (h) [1aa1] SEQ ID NOS: 86, 90, and 94;
[0046] (i) [5a6] SEQ ID NOS: 99, 101, and 103; and
[0047] (j) [5aa3] SEQ ID NOS: 104, 105, and 106.
[0048] In yet another aspect, the present invention provides an
antibody or antibody fragment that has an antigenic binding site
that contains a pair of heavy chain variable and light chain
variable regions selected from the group consisting of:
[0049] (a) the heavy chain variable region of SEQ ID NO:133 and the
light chain variable region of SEQ ID NO:134;
[0050] (b) the heavy chain variable region of SEQ ID NO:135 and the
light chain variable region of SEQ ID NO:136;
[0051] (c) the heavy chain variable region of SEQ ID NO:137 and the
light chain variable region of SEQ ID NO:138;
[0052] (d) the heavy chain variable region of SEQ ID NO:139 and the
light chain variable region of SEQ ID NO:140;
[0053] (e) the heavy chain variable region of SEQ ID NO:141 and the
light chain variable region of SEQ ID NO:142;
[0054] (f) the heavy chain variable region of SEQ ID NO:143 and the
light chain variable region of SEQ ID NO:144;
[0055] (g) the heavy chain variable region of SEQ ID NO:145 and the
light chain variable region of SEQ ID NO:146;
[0056] (h) the heavy chain variable region of SEQ ID NO:147 and the
light chain variable region of SEQ ID NO:148;
[0057] (i) the heavy chain variable region of SEQ ID NO:149 and the
light chain variable region of SEQ ID NO:150; and
[0058] (j) the heavy chain variable region of SEQ ID NO:151 and the
light chain variable region of SEQ ID NO:152.
[0059] The antibodies or antibody fragments of the invention can
bind to the MN protein with a dissociation constant of preferably
about 0.15 nM to about 50 nM.
[0060] In another aspect, the antibodies or fragments of the
invention are IgG antibodies or IgG fragments. The antibodies or
fragments can also be IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA,
or IgM antibodies, Fab fragments, F(ab').sub.2 fragments, scFv
fragments, Fv fragments, a diabodies, linear antibodies,
single-chain antibodies, biospecific antibodies, multispecific
antibodies, or chimeric antibodies (e.g. comprising a human
antibody scaffold grafted to a human or non-human antibody binding
region, or a non-human antibody scaffold grafted to a human or
non-human antibody binding region). The chimeric antibodies can
include, for example, antibody scaffold regions from non-human
sources, such as, for example, cow, mouse, llama, camel, or rabbit.
Further information on the engineering of antibodies can be found
in the literature, for example, Holliger and Hudson, Nature
Biotechnology, (September 2005) 23:1126-1136, which is incorporated
herein by reference. The aforementioned fragments can be obtained
from an immunoglobulin or produced by a suitable means, e.g.
recombinant expression, in a fragment form.
[0061] The antibodies or antibody fragments of the invention can
also be humanized,
[0062] wherein the CDR sequences or regions (e.g. CDR1, CDR2, CDR3)
can be non-human, e.g. murine.
[0063] The antibodies or antibody fragments of the invention, or
compositions including the antibodies or fragments, can include a
cytoxic agent that is conjugated to the antibody or fragment. In
one aspect, the cytotoxic agent is monomethylauristatin-E (MMAE),
however, other cytoxic agents are also provided, which can include,
for example, functional analogs of MMAE (e.g.
monomethylauristatin-F), and other cytotoxic agents, e.g., aplidin,
azaribine, anastrozole, azacytidine, bleomycin, bortezomib,
bryostatin-1, busulfan, calicheamycin, camptothecin,
10-hydroxycamptothecin, carmustine, celebrex, chlorambucil,
cisplatin, irinotecan (CPT-1 1), SN-38, carboplatin, cladribine,
cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin,
daunomycin glucuronide, daunorubicin, dexamethasone,
diethylstilbestrol, doxorubicin, doxorubicin glucuronide,
epirubicin glucuronide, ethinyl estradiol, estramustine, etoposide,
etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),
3',5'-.beta.-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,
fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone
caproate, hydroxyurea, idarubicin, ifosfamide, L-asparaginase,
leucovorin, lomustine, mechlorethamine, medroprogesterone acetate,
megestrol acetate, melphalan, mercaptopurine, 6-mercaptopurine,
methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane,
phenyl butyrate, prednisone, procarbazine, paclitaxel, pentostatin,
PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol,
testosterone propionate, thalidomide, thioguanine, thiotepa,
teniposide, topotecan, uracil mustard, velcade, vinblastine,
vinorelbine, vincristine, ricin, abrin, ribonuclease, onconase,
rapLR1, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral
protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and
Pseudomonas endotoxin, or combinations thereof. Any of the cytoxic
agents can also include functional analogs thereof.
[0064] The compositions of the invention can include in addition to
the antibodies and fragments (with or without the aforementioned
conjugated cytoxic agents) various anti-cancer agents, which can
include, for example, bleomycin, docetaxel (Taxotere), doxorubicin,
edatrexate, erlotinib (Tarceva), etoposide, finasteride (Proscar),
flutamide (Eulexin), gemcitabine (Gemzar), genitinib (Irresa),
goserelin acetate (Zoladex), granisetron (Kytril), imatinib
(Gleevec), irinotecan (Campto/Camptosar), ondansetron (Zofran),
paclitaxel (Taxol), pegaspargase (Oncaspar), pilocarpine
hydrochloride (Salagen), porfimer sodium (Photofrin), interleukin-2
(Proleukin), rituximab (Rituxan), topotecan (Hycamtin), trastuzumab
(Herceptin), Triapine, vincristine, and vinorelbine tartrate
(Navelbine), or therapeutic antibodies or fragments thereof, or
anti-angiogenic agent, such as, for example, angiostatin,
bevacizumab (Avastin.RTM.), sorafenib (Nexavar.RTM.), baculostatin,
canstatin, maspin, anti-VEGF antibodies or peptides, anti-placental
growth factor antibodies or peptides, anti-Flk-1 antibodies,
anti-Flt-1 antibodies or peptides, laminin peptides, fibronectin
peptides, plasminogen activator inhibitors, tissue
metalloproteinase inhibitors, interferons, interleukin 12, IP-10,
Gro-.beta., thrombospondin, 2-methoxyoestradiol, proliferin-related
protein, carboxiamidotriazole, CM101, Marimastat, pentosan
polysulphate, angiopoietin 2, interferon-alpha, herbimycin A,
PNU145156E, 16K prolactin fragment, Linomide, thalidomide,
pentoxifylline, genistein, TNP-470, endostatin, paclitaxel,
accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet
factor 4 or minocycline.
[0065] The present invention further provides in another aspect a
method for treating an MN-related disorder by administering a
therapeutically effective amount of the antibodies and/or fragments
of the invention, or the compositions of the invention which
include the antibodies and/or fragments of the invention. The
MN-related disorder can include, for example, cancer, such as, a
solid tumor cancer. The solid tumor can be in or originating from
the breast, respiratory tract, lung, brain, reproductive organ,
digestive tract, colon, urinary tract, kidney, esophagus, cervix,
eye, liver, skin, head, neck, thyroid, and parathyroid.
[0066] In another aspect, the present invention provides a method
of diagnosing an MN-related disorder characterized abnormal MN
levels comprising comparing the level of MN in a suspected diseased
tissue or cell with the level of MN in a corresponding healthy
tissue or cell, wherein an abnormal MN level in the suspected
diseased tissue or cell is an indication of an MN-related disorder,
said step of comparing further comprising detecting by immunoassay
the level of MN in the diseased tissue and the healthy tissue with
the antibodies or antibody fragments of the invention.
[0067] In a particular aspect, the invention provides a method of
diagnosing an MN-related disorder where the immunoassay includes
the steps of: (a) detecting the level of MN protein in the healthy
tissue; (b) detecting the level of MN protein in the suspected
diseased tissue; and (c) comparing the levels of MN protein from
(a) and (b). An elevated level of MN protein in the suspected
diseased tissue as compared to the level of MN protein in the
healthy tissue is indicative of the presence of an MN-related
disorder.
[0068] Also provided by the present invention is a kit comprising
the antibodies or antibody fragments of the invention, or
alternatively, the compositions of the invention, and a set of
instructions for using the kit in a method of treating an
MN-related disorder or for diagnosing a an MN-related disorder.
[0069] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0070] The following detailed description, given by way of example,
but not intended to limit the invention solely to the specific
embodiments described, may best be understood in conjunction with
the accompanying drawings, in which:
[0071] FIGS. 1a, 1b, 1c and 1d depict DNA sequences of the antibody
complementarity determining regions (CDRs);
[0072] FIGS. 2a, 2b and 2c depict the protein sequences of the
antibody complementarity determining regions (CDRs);
[0073] FIGS. 3a, 3b, 3c, 3d and 3e depict the DNA sequences of
specific antibody light and heavy chain variable regions, each of
which contains both CDRs and framework regions;
[0074] FIGS. 4a, 4b and 4c depict protein sequences of specific
antibody light (VL) and heavy (VH) chain variable regions, each of
which contains both the CDRs and the framework regions;
[0075] FIG. 5 depicts the MN-binding properties for the MN
antibodies of the present invention;
[0076] FIG. 6 depicts the prevention of tumor cell adhesion to MN
coated plates produced by incubation with an anti-MN antibody;
[0077] FIG. 7 depicts the in vivo anti-tumor activity in a
xenograft model comprising the MaTu tumor resulting from treatment
with an immunoconjugate comprising the anti-MN monoclonal antibody
1e4;
[0078] FIG. 8 depicts the FACS measurement of binding of an anti-MN
antibody to the PC3 mm2 cell line which expresses the MN protein on
its surface;
[0079] FIG. 9 schematically depicts the antibody conjugate (anti-MN
antibody conjugated to MMAE);
[0080] FIG. 10a shows FACS plots depicting 3ee9/MMAE binding to MN+
(MaTu) cells, but not to MN- (DLD) cells;
[0081] FIG. 10b shows FACS plots depicting 1E4 immunoconjugate
binding to MN+ (PC3 mm2) cells, but not to MN- (DLD) cells;
[0082] FIG. 10c shows FACS plots depicting 1aa1 immunoconjugate
binding to MN+ (PC3 mm2) cells, but not to MN- (DLD) cells;
[0083] FIG. 11a shows immunofluorescence images depicting
internalization of 3ee9/MMAE by MN+ cells and lack of
internalization by MN- cells. Internalized 3ee9/MMAE is shown as
fluorescence;
[0084] FIG. 11b shows immunofluorescence images depicting
internalization of the 1E4 immunoconjugate by MN+ cells and lack of
internalization by MN- cells. Internalized 1E4 immunoconjugate is
shown as fluorescence;
[0085] FIG. 12 shows an immunoblot depicting specific
immunoprecipitation of biotinylated cell surface proteins by MN
antibodies;
[0086] FIG. 13a graphically depicts the cytotoxicity of 3ee9/MMAE
against MN+, but against MN-, cells;
[0087] FIG. 13b graphically depicts the cytotoxicity of the 1E4
immunoconjugate against MN+, but against MN-, cells;
[0088] FIG. 13c graphically depicts the cytotoxicity of the 1aa1
immunoconjugate against MN+, but against MN-, cells;
[0089] FIG. 14 shows immunofluorescence images depicting
3ee9/MMAE's prevention of normal spindle formation by tubulin
inhibition;
[0090] FIG. 15 graphically depicts the anti-tumor efficacy of
3ee9/MMAE against MaTu xenografts;
[0091] FIG. 16 graphically depicts the anti-tumor efficacy of the 1
E4 immunoconjugate against established MaTu breast tumors;
[0092] FIG. 17 graphically depicts the anti-tumor efficacy of the
1aa1 immunoconjugate against MaTu xenografts;
[0093] FIG. 18 graphically depicts the therapeutic index (TI) of
3ee9/MMAE against MaTu xenografts;
[0094] FIG. 19 graphically depicts the anti-tumor efficacy of
3ee9/MMAE and free MMAE against HT-29 xenografts;
[0095] FIG. 20 graphically depicts the anti-tumor efficacy of
3ee9/MMAE against HT-29 xenografts, following the Q7Dx2
schedule;
[0096] FIG. 21 graphically depicts the anti-tumor efficacy of
3ee9/MMAE against HT-29 xenografts, following the Q1Dx1
schedule;
[0097] FIG. 22 graphically depicts the anti-tumor efficacy of
3ee9/MMAE against PC3 mm2 xenografts;
[0098] FIG. 23 graphically depicts the anti-tumor efficacy of
3ee9/MMAE against Colo-205 xenografts;
[0099] FIG. 24 graphically depicts the anti-tumor efficacy of
3ee9/MMAE against HCT-15 xenografts;
[0100] FIG. 25 graphically depicts the anti-tumor efficacy of
3ee9/MMAE against MN- (in left-hand graph) and MN+ (in right-hand
graph) MIAPaca2 xenografts;
[0101] FIGS. 26a and 26b graphically depict the anti-tumor efficacy
of 3ee9/MMAE in combination with Xeloda.RTM. against Colo-205 CRC
xenografts at varying doses;
[0102] FIG. 27 shows immunofluorescence images depicting in vivo
localization of 3ee9 in huMN- MIAPaca2 and MIAPaca2 tumors; and
[0103] FIG. 28 show immunofluorescence images (of histological
samples) depicting 3ee9/MMAE inhibition of tubulin polymerization
in HT-29 CRC xenografts at two doses.
DETAILED DESCRIPTION OF THE INVENTION
[0104] It is to be understood that present invention as described
herein is not to be limited to the particular details set forth
herein regarding any aspect of the present invention, including,
anti-MN antibodies, immunoconjugates, methods of treatment,
protocols, cell lines, animal species or genera, constructs, and
reagents described and, as such, may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to
limit the scope of the present invention.
DEFINITIONS
[0105] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by one of ordinary
skill in the art to which this invention belongs. The following
references, however, can provide one of skill in the art to which
this invention pertains with a general definition of many of the
terms used in this invention, and can be referenced and used so
long as such definitions are consistent the meaning commonly
understood in the art. Such references include, but are not limited
to, Singleton et al., Dictionary of Microbiology and Molecular
Biology (2d ed. 1994); The Cambridge Dictionary of Science and
Technology (Walker ed., 1988); Hale & Marham, The Harper
Collins Dictionary of Biology (1991); and Lackie et al., The
Dictionary of Cell & Molecular Biology (3d ed. 1999); and
Cellular and Molecular Immunology, Eds. Abbas, Lichtman and Pober,
2.sup.nd Edition, W.B. Saunders Company. Any additional technical
resources available to the person of ordinary skill in the art
providing definitions of terms used herein having the meaning
commonly understood in the art can be consulted. For the purposes
of the present invention, the following terms are further defined.
Additional terms are defined elsewhere in the description.
[0106] As used herein and in the appended claims, the singular
forms "a," "and," and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, reference to
"a gene" is a reference to one or more genes and includes
equivalents thereof known to those skilled in the art, and so
forth.
[0107] As used herein, the term "antibody" includes immunoglobulin
molecules (e.g., any type, including IgG, IgE, IgM, IgD, IgA and
IgY, and/or any class, including, IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2) isolated from nature or prepared by recombinant means.
Antibodies also are meant to encompass antigen-binding antibody
fragments, such as Fab, F(ab')2, scFv (single chain Fvs), Fv,
single chain antibodies, diabodies, disulfide-linked Fvs (sdFv),
and fragments comprising a VL or VH domain, which are prepared from
intact immunoglobulins or prepared by recombinant means.
[0108] The antibodies and/or antigen-binding antibody fragments of
the present invention may be monospecific (e.g. monoclonal),
bispecific, trispecific or of greater multi specificity.
Multispecific antibodies may be specific for different epitopes of
an antigen or may be specific for epitopes of more than one
antigen. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO
91/00360; 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;
5,601,819; Kostelny et al., 1992, J. Immunol. 148:1547 1553, each
of which are incorporated herein by reference.
[0109] Antigen-binding antibody fragments may comprise the variable
region(s) alone or in combination with the entirety or a portion of
the following: hinge region, CH1, CH2, CH3 and CL domains. Also
included in the invention are antigen-binding antibody fragments
also comprising any combination of variable region(s) with a hinge
region, CH1, CH2, CH3 and CL domain.
[0110] Preferably, the antibodies or antigen-binding antibody
fragments are human, humanized, murine (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. No. 5,939,598 by Kucherlapati et al. The term antibody also
extends to other protein scaffolds that are able to orient antibody
CDR inserts into the same active binding conformation as that found
in natural antibodies such that binding of the target antigen
observed with these chimeric proteins is maintained relative to the
binding activity of the natural antibody from which the CDRs were
derived.
[0111] As used herein, the term "humanized" forms of non-human
(e.g., murine) antibodies are chimeric antibodies which contain
minimal sequence derived from non-human immunoglobulin. For the
most part, humanized antibodies are human immunoglobulins
(recipient antibody) in which hypervariable region residues (e.g.
the complementarity determining regions "CDR") of the recipient are
replaced by hypervariable region residues (CDRs) from a non-human
species (donor antibody) such as mouse, rat, rabbit, or nonhuman
primate having the desired specificity, affinity, and capacity. In
some instances, framework region (FR) residues of the human
immunoglobulin may be replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues which are
not found in the recipient antibody or in the donor antibody. Such
modifications are made to further refine antibody performance. In
general, the humanized antibody may comprise substantially all of
at least one or typically two variable domains, in which all or
substantially all of the hypervariable regions correspond to those
of a non-human immunoglobulin and all or substantially all of the
FRs are those of a human immunoglobulin sequence. The humanized
antibody optionally also may comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For a review, see Jones, et al., (Nature
321:522-525, 1986); Reichmann, et al., (Nature 332:323-329, 1988);
and Presta, (Curr. Op. Struct. Biol. 2:593-596, 1992). The
preparation of humanized antibodies can be found in U.S. Pat. Nos.
7,049,135, 6,828,422, 6,753,136, 6,706,484, 6,696,248, 6,692,935,
6,667,150, 6,653,068, 6,300,064, 6,294,353, and 5,514,548, each of
which are incorporated herein in their entireties.
[0112] As used herein, the term "single-chain Fv" or "sFv" antibody
fragments comprise the VH and VL domains of an antibody, wherein
these domains are present in a single polypeptide chain. Generally,
the Fv polypeptide further comprises a polypeptide linker between
the V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review, see Pluckthun
(The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and
Moore eds. Springer-Verlag, New York, pp. 269-315, 1994), which is
incorporated herein in its entirety by reference.
[0113] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger, et al., (Proc.
Natl. Acad. Sci. USA 90:6444-6448, 1993), each of which are
incorporated by reference.
[0114] The expression "linear antibodies" refers to the antibodies
described in the art, for example, in Zapata, et al., (Protein Eng.
8(10):1057-1062, 1995), which is incorporated by reference.
Briefly, such antibodies comprise a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0115] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, that is, individual antibodies comprising an identical
population except for possible naturally occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly
specific, that is, directed against a single antigenic site.
Furthermore, in contrast to conventional (polyclonal) antibody
preparations which typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. The
modifier "monoclonal" indicates the character of the antibody as
being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler, et al.,
(Nature 256:495, 1975), or may be made by recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may
also be isolated from phage antibody libraries using the techniques
described in, for example, Clackson, et al., (Nature 352:624-628,
1991) and Marks, et al., (J. Mol. Biol. 222:581-597, 1991).
[0116] The monoclonal antibodies herein also include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(see, e.g., U.S. Pat. No. 4,816,567; and Morrison, et al., Proc.
Natl. Acad. Sci. USA 81:6851-6855, 1984, each of which are
incorporated by reference).
[0117] As used herein, the terms "biological sample" or "patient
sample" as used herein, refers to a sample obtained from an
organism or from components (e.g., cells) of an organism. The
sample may be of any biological tissue or fluid. The sample may be
a "clinical sample" which is a sample derived from a patient. Such
samples include, but are not limited to, sputum, blood, serum,
plasma, blood cells (e.g., white cells), tissue samples, biopsy
samples, urine, peritoneal fluid, and pleural fluid, saliva, semen,
breast exudate, cerebrospinal fluid, tears, mucous, lymph,
cytosols, ascites, amniotic fluid, bladder washes, and
bronchioalveolar lavages or cells therefrom, among other body fluid
samples. The patient samples may be fresh or frozen, and may be
treated with heparin, citrate, or EDTA. Biological samples may also
include sections of tissues such as frozen sections taken for
histological purposes.
[0118] The term "cancer" includes, but is not limited to, solid
tumors, such as cancers of the breast, respiratory tract, brain,
reproductive organs, digestive tract, urinary tract, eye, liver,
skin, head and neck, thyroid, parathyroid, and their distant
metastases. The term also includes sarcomas, lymphomas, leukemias,
and plasma cell myelomas.
[0119] Examples of breast cancer include, but are not limited to,
invasive ductal carcinoma, invasive lobular carcinoma, ductal
carcinoma in situ, and lobular carcinoma in situ. Examples of
cancers of the respiratory tract include, but are not limited to,
small-cell and non-small-cell lung carcinoma, as well as bronchial
adenoma and pleuropulmonary blastoma. Examples of brain cancers
include, but are not limited to, brain stem and hypophtalmic
glioma, cerebellar and cerebral astrocytoma, medulloblastoma,
ependymoma, as well as neuroectodermal and pineal tumor. Tumors of
the male reproductive organs include, but are not limited to,
prostate and testicular cancer. Tumors of the female reproductive
organs include, but are not limited to, endometrial, cervical,
ovarian, vaginal, and vulvar cancer, as well as sarcoma of the
uterus. Tumors of the digestive tract include, but are not limited
to, anal, colon, colorectal, esophageal, gallbladder, gastric,
pancreatic, rectal, small-intestine, and salivary gland cancers.
Tumors of the urinary tract include, but are not limited to,
bladder, penile, kidney, renal pelvis, ureter, and urethral
cancers. Eye cancers include, but are not limited to, intraocular
melanoma and retinoblastoma. Examples of liver cancers include, but
are not limited to, hepatocellular carcinoma (liver cell carcinomas
with or without fibrolamellar variant), cholangiocarcinoma
(intrahepatic bile duct carcinoma), and mixed hepatocellular
cholangiocarcinoma. Skin cancers include, but are not limited to,
squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma,
Merkel cell skin cancer, and non-melanoma skin cancer.
Head-and-neck cancers include, but are not limited to,
laryngeal/hypopharyngeal/nasopharyngeal/oropharyngeal cancer, and
lip and oral cavity cancer. Lymphomas include, but are not limited
to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell
lymphoma, Hodgkin's disease, and lymphoma of the central nervous
system. Sarcomas include, but are not limited to, sarcoma of the
soft tissue, osteosarcoma, malignant fibrous histiocytoma,
lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not
limited to, acute myeloid leukemia, acute lymphoblastic leukemia,
chronic lymphocytic leukemia, chronic myelogenous leukemia, and
hairy cell leukemia.
[0120] As used in this invention, the term "epitope" means any
antigenic determinant on an antigen, e.g. MN protein, to which the
antibody binds through an antigenic binding site. Determinants or
antigenic determinants usually consist of chemically active surface
groupings of molecules such as amino acids or sugar side chains and
usually have specific three dimensional structural characteristics,
as well as specific charge characteristics.
[0121] The term "specifically immunoreactive" refers to a binding
reaction between an antibody and a protein, compound, or antigen,
having an epitope recognized by the antigenic binding site of the
antibody. This binding reaction is determinative of the presence of
a protein, antigen or epitope having the recognized epitope amongst
the presence of a heterogeneous population of proteins and other
biologics. In the context of an immunoassay, specifically
immunoreactive antibodies can bind to a protein having the
recognized epitope and bind, if at all, to a detectably lesser
degree to other proteins lacking the epitope which are present in
the sample. In an in vivo context, "specifically immunoreactive"
can refer to the conditions under which in an animal forms an
immune response against a vaccine or antigen, e.g. a humoral
response to the antigen (the production of antibodies, against a
vaccine, protein, compound, or antigen presented thereto under
immunologically reactive conditions) or a cell-mediated (also
herein as "cellular immune response", i.e. a response mediated by T
lymphocytes against the vaccine, protein, compound or antigen
presented thereto).
[0122] As used herein, the term "immunologically reactive
conditions" is used in the context of an immunoassay or an in vitro
reaction wherein the physical conditions of the reaction,
including, for example, the temperature, salt concentration, pH,
reagents and their concentrations, and the concentrations of
antigen and cognate antibody that is specifically immunoreactive to
the antigen, are provided or adjusted to allow binding of the
cognate antibody to the antigen. Immunologically reactive
conditions are dependent upon the format of the antibody binding
reaction and, typically are those utilized in immunoassay
protocols. See Harlow and Lane (1988) Antibodies: A Laboratory
Manual, Cold Spring Harbor Publications, New York, for a
description of immunoassay formats and conditions.
[0123] The term "patient" or "subject" as used herein includes
mammals (e.g., humans and animals).
Antibodies
[0124] The present invention relates to antibodies that bind to MN.
These antibodies may be useful for a variety of therapeutic and
diagnostic purposes.
[0125] It will be generally appreciated by those skilled in the art
that the most critical determinants of antibody selectivity and
binding affinity are the sequences and resulting conformations of
the complementarity regions (CDRs). Most antibodies contain six
CDRs, three within the heavy chain variable region (VH) and three
within the light chain variable region (VL). The intervening
sequences between the CDRs within VH and VL are the framework
regions which orient the CDRs. The CDRs together form the antigenic
binding sites within antibodies. The critical role of these CDRs in
determining the functional properties of antibodies has long been
exploited in the processes of antibody humanization and antibody
optimization. In the former process, the CDRs from a monoclonal
antibody, for example, a mouse antibody are transferred to a human
antibody of similar framework design thereby resulting in an
antibody with the same functional properties and reduced
immunogencity in man.
[0126] The success of this process is evident from the number of
humanized antibodies that have been successfully commercialized as
human therapeutics and include Herceptin.RTM. (trastuzumab,
Genentech, Inc., South San Francisco, Calif.), Synagis.RTM.
(palivizumab, Medimmune, Inc., Gaithersburg, Md.), Campath.RTM.
(alemtuzumab, Genzyme Oncology, Cambridge, Mass.), Zenapax.RTM.
(daclizumab, Roche Pharmaceuticlas, Nutley, N.J.), Xolair.RTM.
(omalizumab, Genentech, Inc., South San Francisco, Calif.),
Raptiva.RTM. (efalizumab, Genentech, Inc., South San Francisco,
Calif.), Avastin.RTM. (bevacizumab, Genentech, Inc., South San
Francisco, Calif.), and Mylotarg.RTM. (gemtuzumab ozogamicin,
Wyeth-Ayerst, Madison, N.J.). Other examples have been described in
Singer, et al., (J. Immunol, 150:2844-2857, 1993); Luo, et al., (J.
Immunol. Meth., 275:31-40, 2002); and Kostelny, et al., (Int. J.
Cancer 93; 556-565, 2001).
[0127] The process of antibody optimization focuses on improvements
in antibody selectivity or binding affinity through specific
alteration in the sequence of the CDRs. It is well accepted within
the field of antibody development that the CDRs encode binding
affinity and selectivity properties required for therapeutic and
diagnostic uses, and these CDR sequences may be used to confer such
desirable functional properties to a wide variety of alternate
antibody frameworks using standard procedures known to those
skilled in the art. It is also possible to transfer antibody
binding activity by grafting antibody CDRs onto other proteins that
possess immunoglobulin-like folds such as other proteins within the
immunoglobulin superfamily and non-immunoglobulins with similar
folds to immunoglobulins (Nicaise, et al., Protein Science
13:1882-1891, 2004).
[0128] The present invention contemplates any known or suitable
technology for the preparation of the antibodies and antigen
binding antibody fragments of the invention.
[0129] For example, phage display technology is useful for
obtaining high affinity anti-MN antibodies or antigen binding
antibody fragments of the invention for the use in diagnosis and/or
treatment of an MN-related disorder, such as, an MN-related cancer.
The technology, referred to as affinity maturation, employs
mutagenesis or CDR walking and re-selection using the MN antigen to
identify antibodies that bind with higher affinity to the antigen
when compared with the initial or parental antibody (see, e.g.,
Glaser et al., 1992, J. Immunology 149:3903). Mutagenizing entire
codons rather than single nucleotides results in a semi-randomized
repertoire of amino acid mutations. Libraries can be constructed
consisting of a pool of variant clones each of which differs by a
single amino acid alteration in a single CDR and which contain
variants representing each possible amino acid substitution for
each CDR residue. Mutants with increased binding affinity for the
antigen can be screened by contact with the immobilized mutants
containing labeled antigen. Any screening method known in the art
can be used to identify mutant antibodies with increased avidity to
the antigen (e.g., ELISA) (See Wu et al., 1998, Proc Natl. Acad.
Sci. USA 95:6037; Yelton et al., 1995, J. Immunology 155:1994,
incorporated by reference). CDR walking may also be used to
randomize the light chain (See Schier et al., 1996, J. Mol. Bio.
263:551, incorporated by reference).
[0130] In a particular example, MorphoSys AG (Germany) provides a
phage-antibody technology that may be used to generate fully human
antibodies. The Morphosys HuCAL GOLD library provides a number of
advancements over earlier versions of the technology (Knappik, et
al., J. Mol. Biol. 296:57-86, 2000, incorporated by reference)
including the HuCAL-Fab 1 library described in Rauchenberger, et
al., (J. Biol. Chem. 278:38194-38205, 2003, incorporated by
reference). Like earlier versions, HuCAL GOLD incorporates a human
antibody design, that features human consensus framework sequences
and patterns of CDR variability that mimic the natural human
sequence diversity. However, diversity is extended in HuCAL GOLD to
include all six antibody CDRs. Moreover, recovery of high affinity
antibodies is augmented through the CysDisplay.TM. feature
(Kretzschmar and von Ruden, Curr. Opin. Biotechnol. 13:598-602,
2002). Antibodies derived with this technology exhibit a greatly
reduced probability of immunogenicity.
[0131] As an example, antibodies with MN binding and cell
adhesion-neutralizing activity may be identified using the
MorphoSys technology. The MN protein may be coated on microtiter
plate or a magnetic bead and incubated with the MorphoSys
HuCAL-GOLD Fab phage library. Phage-linked Fabs that do not bind to
MN may be washed from the plate, leaving only phage that bind to
MN. The bound phage may be eluted by addition of a thiol reducing
agent such as dithiotreitol (DTT) resulting in cleavage of the
disulfide bond linking the antibody to the phage. The recovered
population of phage may be enriched with phage expressing MN
binding antibody fragments and may be amplified by infection of E.
coli hosts. This panning process may be repeated using the enriched
population of phage to further enrich for a population of
phage-linked antibodies that bind to MN. The gene sequence encoding
the Fabs may then be excised using standard cloning techniques and
transferred to an E. coli expression vector which is used to
transform an E. coli expression cell line. Fabs from the enriched
pool may then be expressed and purified.
[0132] Alternatively, panning may be performed using MN expressing
cells as antigen. For example, cells transfected with MN antigen
may be labeled with biotin. These transfected cells may then be
mixed with unlabeled, non-MN transfected cells at a labeled to
unlabeled ratio of 1:10. The phage library is added to the cells,
and the biotinylated, MN-bearing cells are captured with
streptavidin-bound magnetic beads that are bound to a magnet.
Non-specific phage are washed away, and the MN-bearing cells are
specifically eluted by removing the magnetic field. These
specifically bound phage may be amplified for further rounds of
cell panning or may be alternated with peptide and/or protein
panning.
[0133] Antibodies may be produced by a variety of other techniques
as described below. For example, another approach for obtaining
antibodies is to screen a DNA library from B cells as described by
Dower, et al., (WO 91/17271, incorporated by reference) and
McCafferty, et al., (WO 92/01047) (each of which is incorporated by
reference in its entirety). In these methods, libraries of phage
are produced in which members display different antibodies on their
outer surfaces. Antibodies are usually displayed as Fv or Fab
fragments. Phage displaying antibodies are selected by affinity
enrichment for binding to a selected protein.
[0134] In a variation of the phage-display method, human antibodies
having the binding specificity of a selected murine antibody may be
produced (e.g., WO 92/20791, incorporated by reference). In this
method, either the heavy or light chain variable region of the
selected murine antibody (e.g., 5C7.29) may be used as a starting
material. If, for example, a light chain variable region is
selected as the starting material, a phage library may be
constructed in which members displays the same light chain variable
region (i.e., the murine starting material) and a different heavy
chain variable region. The heavy chain variable regions may be
obtained from a library of rearranged human heavy chain variable
regions. A phage showing strong specific binding for a protein
(e.g., at least 10 nM or at least 1 nM) is selected. The human
heavy chain variable region from this phage then serves as a
starting material for constructing a further phage library. In this
library, each phage displays the same heavy chain variable region
(i.e., the region identified from the first display library) and a
different light chain variable region. The light chain variable
regions may be obtained from a library of rearranged human variable
light chain regions. Again, phage showing strong specific binding
are selected.
[0135] As another example, antibodies may also be produced using
trioma methodology. The basic approach and an exemplary cell fusion
partner, SPAZ-4 for use in this approach, have been described by
Oestberg, et al., (Hybridoma 2:361-367, 1983; U.S. Pat. No.
4,634,664, each incorporated by reference); and Engleman, et al.,
(U.S. Pat. No. 4,634,666, incorporated by reference). The
antibody-producing cell lines obtained by this method are called
triomas, because they are descended from three cells--two human and
one mouse. Initially, a mouse myeloma line is fused with a human
B-lymphocyte to obtain a non-antibody-producing xenogeneic hybrid
cell, such as the SPAZ-4 cell line. The xenogeneic cell is then
fused with an immunized human B-lymphocyte to obtain an
antibody-producing trioma cell line. Triomas have been found to
produce antibodies more stably than ordinary hybridomas made from
human cells.
[0136] The B-lymphocytes are obtained from the blood, spleen, lymph
nodes, or bone marrow of a human donor. In vivo immunization of a
living human with protein is usually undesirable because of the
risk of initiating a harmful response. Thus, B-lymphocytes are
usually immunized in vitro with a protein (e.g., MN) or an
antigenic fragment thereof, or a cell bearing said protein (e.g.,
MN). Specific epitopic fragments consisting essentially of the
amino acid segments that bind to one of the exemplified murine
antibodies may be used for in vitro immunization. B-lymphocytes are
typically exposed to antigen for a period of 7-14 days in a media
such as RPMI-1640 (see, e.g., U.S. Pat. No. 4,634,666) supplemented
with 10% human serum.
[0137] The immunized B-lymphocytes may be fused to a xenogeneic
hybrid cell such as SPAZ-4 by methods known in the art. For
example, the cells may be treated with 40-50% polyethylene glycol
of MW 1,000-4,000 for about 5-10 minutes at about 37.degree. C.
Cells may be separated from the fusion mixture and propagated in
media selective for the desired hybrids (e.g., HAT or AH). Clones
secreting antibodies having the required binding specificity may be
identified by assaying the trioma culture medium for the ability to
bind to a protein (e.g., MN) using the same methods as discussed
above for non-human antibodies. Triomas producing human antibodies
having the desired specificity may be sub-cloned, for example, by
the limiting dilution technique and grown in vitro in culture
medium.
[0138] Although triomas are genetically stable, they may not
produce antibodies at very high levels. Expression levels may be
increased by cloning antibody genes from the trioma into one or
more expression vectors, and transforming the vector into a cell
line such as the cell lines for expression of recombinant or
humanized immunoglobulins.
[0139] Human antibodies cross-reactive with a protein (e.g., MN)
may also be produced from non-human transgenic mammals having
transgenes encoding at least a segment of the human immunoglobulin
locus. Usually, the endogenous immunoglobulin locus of such
transgenic mammals is functionally inactivated. The segment of the
human immunoglobulin locus may include unrearranged sequences of
heavy and light chain components. Both inactivation of endogenous
immunoglobulin genes and introduction of exogenous immunoglobulin
genes may be achieved by targeted homologous recombination, or by
introduction of YAC chromosomes. The transgenic mammals resulting
from this process are capable of functionally rearranging the
immunoglobulin component sequences, and expressing a repertoire of
antibodies of various isotypes encoded by human immunoglobulin
genes, without expressing endogenous immunoglobulin genes. The
production and properties of mammals having these properties are
described in detail, for example, by Lonberg, et al., (WO
93/12227); and Kucherlapati, (WO 91/10741) (each of which is
incorporated by reference in its entirety). Cross-reacting MN human
antibodies may be obtained by immunizing a transgenic non-human
mammal as described above. Monoclonal antibodies may be prepared,
for example, by fusing B-cells from such mammals to suitable
myeloma cell lines using conventional Kohler-Milstein technology
(Kohler and Milstein, Nature 256:495-497, 1975, incorporated by
reference).
[0140] Mouse or other non-human antibodies that are cross-reactive
with a protein (e.g., MN) may be obtained using a variety of
immunization strategies. In some strategies, non-human animals
(usually non-human mammals such as mice) may be immunized with MN
antigens. Immunogens may include cells stably transfected with MN
and expressing MN on their cell surface, and MN protein or epitopic
fragments containing the segments of these molecules that bind to
the exemplified cross-reacting antibodies. Antibody-producing cells
obtained from the immunized animals may be immortalized and
selected for the production of an antibody which specifically binds
to MN (e.g., Harlow & Lane, Antibodies, A Laboratory Manual,
C.S.H.P., N.Y., 1988, incorporated by reference). Binding may be
detected, for example, using an appropriate secondary antibody
bearing a second label. Cross-reacting antibodies may then be
further screened for their capacity to direct selective cellular
cytotoxicity to cells expressing MN.
[0141] The present invention also relates to humanized antibodies
having similar binding specificity and affinity to selected mouse
or other non-human antibodies. Humanized antibodies may be formed
by linking CDR regions (e.g., CDR1, CDR2, and CDR3) of non-human
antibodies to a human framework and constant regions by recombinant
DNA techniques (e.g., Queen, et al., Proc. Natl. Acad. Sci. USA
86:10029-10033, 1989; WO 90/07861; each incorporated by reference
in their entirety), i.e. CDR grafting. These humanized
immunoglobulins have variable region framework residues
substantially from a human immunoglobulin (referred to as an
acceptor immunoglobulin) and complementarity determining regions
(CDRs) substantially from a mouse immunoglobulin (referred to as a
donor immunoglobulin). The constant region(s), if present, may also
be substantially from a human immunoglobulin.
[0142] In principal, a framework sequence from any human antibody
may serve as the template for CDR grafting. However, it has been
demonstrated that straight CDR replacement onto such a framework
often leads to significant loss of binding affinity to the antigen
(Glaser, et al., J. Immunol. 149:2606, 1992); Tempest, et al.,
Biotechnology 9:266, 1992; Shalaby, et al., J. Exp. Med. 17: 217,
1992). The more homologous a human antibody is to the original
murine antibody, the less likely combining the murine CDRs with the
human framework will be to introducing distortions into the CDRs
that could reduce affinity. Therefore, homology (i.e., percent
sequence identity) between the humanized antibody variable region
framework and the donor antibody variable region framework of
preferably at least about 65%, more preferably at least about 75%,
still more preferably at least about 85%, and yet more preferably
about 95% or about 99% is suggested.
[0143] The heavy and light chain variable region framework residues
may be derived from the same or different human antibody sequences.
However, heavy chain and light chain framework sequences chosen
from the same human antibody reduce the possibility of
incompatibility in assembly of the two chains. The human antibody
sequences may be the sequences of naturally occurring human
antibodies or may be consensus sequences of several human
antibodies (e.g., WO 92/22653, incorporated by reference). Certain
amino acids from the human variable region framework residues may
be selected for substitution based on their possible influence on
CDR conformation and/or binding to antigen. Analysis of such
possible influences may be accomplished by modeling, examination of
the characteristics of the amino acids at particular locations, or
empirical observation of the effects of substitution or mutagenesis
of particular amino acids.
[0144] For example, when an amino acid differs between a murine
variable region framework residue and a selected human variable
region framework residue, the human framework amino acid may be
substituted by the equivalent framework amino acid from the mouse
antibody when it is reasonably expected that the amino acid: [0145]
(1) contacts antigen directly, [0146] (2) is adjacent to a CDR
region in the sequence, or [0147] (3) otherwise interacts with a
CDR region (e.g., is within about 4-6 .ANG. of a CDR region).
[0148] Other candidates for substitution are, for example, acceptor
human framework amino acids that are unusual for a human
immunoglobulin at that position. These amino acids may be
substituted with amino acids from the equivalent position of the
donor antibody or from the equivalent positions of more typical
human immunoglobulins. The variable region frameworks of humanized
immunoglobulins may show, for example, at least preferably about
85% sequence identity, more preferably at least about 90% sequence
identity, still more preferably at least about 95% sequence
identity, and even more preferably at least about 99% sequence
identity, to a human variable region framework sequence or
consensus of such sequences.
[0149] The present invention also relates to bispecific or
bifunctional antibodies that have one binding site that
specifically binds to a protein (e.g., MN) and a second binding
site that specifically binds to a second moiety. In bispecific
antibodies, one heavy and light chain pair is usually from, for
example, an MN binding antibody and the other pair from an antibody
raised against another epitope. This results in multi-functional
valency, that is, an ability to bind at least two different
epitopes simultaneously.
Binding Assays
[0150] Any useful means to describe the strength of binding (or
affinity) between an antibody or antibody fragment of the invention
and an antigen of the invention (MN protein) can be used. For
example, the dissociation constant, K.sub.d
(K.sub.d=k2/k1=[antibody][antigen]/[antibody-antigen complex]) can
be determined by standard kinetic analyses that are known in the
art. It will be appreciated by those of ordinary skill in the art
that the dissociation constant indicates the strength of binding
between an antibody and an antigen in terms of how easy it is to
separate the complex. If a high concentration of antibody and
antigen are required to form the complex, the strength or affinity
of binding is low, resulting in a higher K.sub.d. It follows that
the smaller the K.sub.d (as expressed in concentration units, e.g.
molar or nanomolar), the stronger the binding.
[0151] Affinity can be assessed and/or measured by a variety of
known techniques and immunoassays, including, for example,
enzyme-linked immunospecific assay (ELISA), Bimolecular Interaction
Analysis (BIA) (e.g., Sjolander and Urbaniczky, Anal. Chem.
63:2338-2345, 1991; Szabo, et al., Curr. Opin. Struct. Biol.
5:699-705, 1995, each incorporated herein by reference), and
fluorescence-activated cell sorting (FACS) for quantification of
antibody binding to cells that express MN. BIA is a technology for
analyzing biospecific interactions in real time, without labeling
any of the interactants (e.g., BIAcore.TM.). BIAcore is based on
determining changes in the optical phenomenon surface plasmon
resonance (SPR) in real-time reactions between biological
molecules, such as, an antibody of the invention and an MN protein
antigen. References relating to BIAcore technology can be further
found in U.S. Published Application Nos: 2006/0223113,
2006/0134800, 2006/0094060, 2006/0072115, 2006/0019313,
2006/0014232, and 2005/0199076, each of which are incorporated
herein in their entireties by reference.
[0152] Antibodies or antigen binding antibody fragments of the
invention that specifically bind to a protein (e.g., MN) provide a
detection signal, for example, preferably at least about 5-fold
higher, more preferably at least about 10-fold higher, and still
more preferably at least about 20-fold higher than a detection
signal provided for other proteins when used in an immunochemical
assay. As such, these antibodies may be used to immunoprecipitate a
protein (e.g., MN) from solution.
[0153] The antibodies and fragments of the invention may be assayed
for immunospecific binding (or binding that is "specifically
immunoreactive," which is herein defined) by any suitable method
known in the art. Assays involving an antibody and an antigen are
known as "immunoassays," which can be employed in the present
invention to characterize both the antibodies and the antigens of
the invention. The immunoassays which can be used include but are
not limited to 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, 1994, Current Protocols in Molecular Biology, Vol. 1,
John Wiley & Sons, Inc., New York, which is incorporated by
reference herein in its entirety) and can be performed without
undue experimentation. Exemplary immunoassays are described briefly
below (but are not intended by way of limitation).
[0154] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding the antibody of interest to the
cell lysate, incubating for a period of time (e.g., 1 4 hours) at
4.degree. C., adding protein A and/or protein G sepharose beads to
the cell lysate, incubating for about an hour or more at 4.degree.
C., washing the beads in lysis buffer and resuspending the beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a particular antigen can be assessed by, e.g.,
western blot analysis. One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase
the binding of the antibody to an antigen and decrease the
background (e.g., pre-clearing the cell lysate with sepharose
beads). For further discussion regarding immunoprecipitation
protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols
in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York
at 10.16.1, which is incorporated herein by reference.
[0155] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8% 20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with primary antibody (the antibody of
interest) diluted in blocking buffer, washing the membrane in
washing buffer; blocking the membrane with a secondary antibody
(which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
.sub.32P or .sub.125I) diluted in blocking buffer, washing the
membrane in wash buffer, and detecting the presence of the antigen.
One of skill in the art would be knowledgeable as to the parameters
that can be modified to increase the signal detected and to reduce
the background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols
in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York
at 10.8.1, which is incorporated herein by reference.
[0156] ELISAs typically comprise preparing antigen, coating the
well of a 96 well microtiter plate with the antigen, adding the
antibody of interest conjugated to a detectable compound such as an
enzymatic substrate (e.g., horseradish peroxidase or alkaline
phosphatase) to the well and incubating for a period of time, and
detecting the presence of the antigen. In ELISAs the antibody of
interest does not have to be conjugated to a detectable compound;
instead, a second antibody (which recognizes the antibody of
interest) conjugated to a detectable compound may be added to the
well. Further, instead of coating the well with the antigen, the
antibody may be coated to the well. In this case, a second antibody
conjugated to a detectable compound may be added following the
addition of the antigen of interest to the coated well. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art. For further discussion
regarding ELISAs see, e.g., Ausubel et al., eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York at 11.2.1, which is incorporated herein by
reference.
[0157] In one aspect, the present invention embodies a number of
different antibodies having MN binding characteristics identified
by screening the MorphoSys HuCAL GOLD Fab library. In one aspect of
the invention, the amino acid sequences of three CDRs of the VH
region of a human antibody were identified (SEQ ID NOS: 57-85; FIG.
2). In another embodiment of the invention, the amino acid
sequences of three CDRs of the VL region of a human MN antibody
were identified (SEQ ID NOS: 86-112; FIG. 2). The present invention
also relates to combinations of CDRs, frameworks, and VH/VL pairs.
Examples of such combinations are shown in FIG. 3 (SEQ ID NOS:
113-132 for the encoding nucleotide sequences) and in FIG. 4) SEQ
ID NOS 133-152 for the protein sequences). Antibodies that have MN
binding are also shown in FIG. 4. Details of the screening process
are described in the examples described herein. Other selection
methods for highly active specific antibodies or antibody fragments
may be envisioned by those skilled in the art and used to identify
human MN antibodies.
[0158] Antibodies and/or antigen binding antibody fragments of the
invention may also be purified from any cell that expresses the
antibodies, including host cells that have been transfected with
antibody-encoding expression constructs. The host cells may be
cultured under conditions whereby the antibodies are expressed.
Purified antibodies and/or antigen binding antibody fragments may
be separated from other cellular components that may associate with
the antibodies in the cell, such as certain proteins,
carbohydrates, or lipids using methods well known in the art. Such
methods include, but are not limited to, size exclusion
chromatography, ammonium sulfate fractionation, ion exchange
chromatography, affinity chromatography, and preparative gel
electrophoresis. Purity of the preparations may be assessed by any
means known in the art, such as SDS-polyacrylamide gel
electrophoresis. A preparation of purified antibodies may contain
more than one type of antibody (e.g., antibodies with the MN
binding and neutralizing characteristics).
[0159] Alternatively, antibodies may be produced using chemical
methods to synthesize its amino acid sequence or portions of the
antibody sequence (e.g. CDR sequences), such as by direct peptide
synthesis using solid-phase techniques (e.g., Merrifield, J. Am.
Chem. Soc. 85:2149-2154, 1963; Roberge, et al., Science
269:202-204, 1995, each of which are incorporated herein by
reference). Protein synthesis may be performed using manual
techniques or by automation. Automated synthesis may be achieved,
for example, using Applied Biosystems 431A Peptide
[0160] Synthesizer (Perkin Elmer). Optionally, fragments of
antibodies may be separately synthesized and combined using
chemical methods to produce a full-length molecule.
[0161] Once an antibody or fragment in accordance with the
invention is identified or obtained, for example, by any of the
methods herein described, for example, including by traditional
methods of antibody production (e.g. animal immunization methods),
monoclonal production, or by recombinant DNA means, it may be
preferable to carry out further steps to characterize and/or purify
and/or modify the antibody. For example, it may be desirable to
prepare an immunoreactive antibody fragment or to prepare a
purified, high-titer composition of the identified, desirable
antibody or to test the immunoreactivity of the identified antibody
or fragment thereof. The present invention contemplates any known
and suitable methods for characterizing, purifying, or assaying the
antibodies of the present invention and it is expected the any
person of ordinary skill in the art to which the invention pertains
will have the requisite level of technical know-how and resources,
e.g. technical manuals or treatises, to accomplish any further
characterization, purification and/or assaying of the antibodies of
the invention without undue experimentation.
[0162] The term "isolating" in the context of antibodies refers to
any process known in the art for purifying antibodies. Methods for
purifying antibodies are well known in the art and the present
invention contemplates any suitable method that would be known to
the skilled person and which would not require undue
experimentation. For example, chromatographic methods, such as, for
example, immuno-affinity chromatography (immobilized ligand to bind
and trap antibody of interest), affinity chromatography, protein
precipitation, ion exchange chromatography, hydrophobic interaction
chromatography, size-exclusion chromatography, as well as
electrophoresis, can be found described in the technical
literature, for example, in Methods in Enzymology, Volume 182,
Guide to Protein Purification, Eds. J. Abelson, M. Simon, Academic
Press, 1.sup.st Edition, 1990, which is incorporated herein by
reference. Thus, suitable materials for performing such
purification steps, such as chromatographic steps, are known to
those skilled in the art. Such methods are suitable for
purification of any of the antibodies, antigens (e.g. alpha-,
gamma-, or alpha/gamma-PGA antigens) or any fragments thereof that
are in accordance with the invention as described herein.
[0163] Certain embodiments may require the purification or
isolation of expressed proteins or antibodies or fragments thereof
from a host cell or a portion thereof. Conventional procedures for
isolating recombinant proteins from transformed host cells are
contemplated by the present invention. Such methods include, for
example, isolation of the protein or fragments of interest by
initial extraction from cell pellets or from cell culture medium,
followed by salting-out, and one or more chromatography steps,
including aqueous ion exchange chromatography, size exclusion
chromatography steps, high performance liquid chromatography
(HPLC), and affinity chromatography may be used to isolate the
recombinant protein or fragment. Guidance in the procedures for
protein purification can be found in the technical literature,
including, for example, Methods in Enzymology, Volume 182, Guide to
Protein Purification, Eds. J. Abelson, M. Simon, Academic Press,
1st Edition, 1990, which is already incorporated by reference.
[0164] Chemically-synthesized molecules may be substantially
purified by preparative high performance liquid chromatography
(see, e.g., Creighton, Proteins: Structures and Molecular
Principles, WH Freeman and Co., New York, N.Y., 1983, incorporated
herein by reference). The composition of a synthetic polypeptide
may be confirmed by amino acid analysis or sequencing (e.g., using
Edman degradation).
Polynucleotides
[0165] In another aspect, the present invention relates to
polynucleotides encoding the antibodies (e.g., antibodies against
MN) or the antigen binding antibody fragments of the invention.
These polynucleotides may be used, for example, to produce
quantities of the antibodies for therapeutic or diagnostic use or
the produce samples of antibodies or fragments for use in
immunoassays of the invention.
[0166] Polynucleotides that may be used to encode, for example,
each of three CDRs within antibody VH regions are described by SEQ
ID NOS: 1-29. Polynucleotides that may be used to encode, for
example, each of three CDRs within antibody VL regions are
described by SEQ ID NOS: 30-56. Polynucleotides that encode, for
example, complete heavy chain and light chain variable regions of
antibodies are described by SEQ ID NOS: 113-132 (FIG. 3).
[0167] Polynucleotides of the present invention may also be
isolated from host cells, free of other cellular components such as
membrane components, proteins, and lipids according to any known or
suitable method in the art. Polynucleotides may be isolated using
standard nucleic acid purification techniques, or synthesized using
an amplification technique such as the polymerase chain reaction
(PCR), or by using an automatic synthesizer. Methods for isolating
polynucleotides are routine and are known in the art. Any such
technique for obtaining a polynucleotide may be used to obtain
isolated polynucleotides encoding antibodies of the invention. For
example, restriction enzymes and probes may be used to isolate
polynucleotides which encode antibodies.
[0168] Antibody-encoding cDNA molecules may be made with standard
molecular biology techniques, using mRNA as a template. Thereafter,
cDNA molecules may be replicated using molecular biology techniques
known in the art and disclosed in manuals such as Sambrook, et al.,
(Molecular Cloning: A Laboratory Manual, (Second Edition, Cold
Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989,
Vol. 1-3, incorporated herein by reference). An amplification
technique, such as PCR, may be used to obtain additional copies of
the polynucleotides.
[0169] Alternatively, synthetic chemistry techniques may be used to
synthesize polynucleotides encoding antibodies of the invention.
The degeneracy of the genetic code allows alternate nucleotide
sequences to be synthesized that will encode an antibody having,
for example, one of the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1,
VL-CDR2, VL-CDR3, complete VH or complete VL amino acid sequences
(e.g., SEQ ID NOS: 57-112 and 133-152).
[0170] To express a polynucleotide encoding an antibody, the
polynucleotide may be inserted into an expression vector that
contains the necessary elements for the transcription and
translation of the inserted coding sequence. Methods that are well
known to those skilled in the art may be used to construct
expression vectors containing sequences encoding antibodies and
appropriate transcriptional and translational control elements.
These methods include in vitro recombinant DNA techniques,
synthetic techniques, and in vivo genetic recombination. Such
techniques are described, for example, in Sambrook, et al. (1989)
and in Ausubel, et al., (Current Protocols in Molecular Biology,
John Wiley & Sons, New York, N.Y., 1995, incorporated herein by
reference).
[0171] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding antibodies. These
include, but are not limited to, microorganisms, such as bacteria
transformed with recombinant bacteriophage, plasmid, or cosmid DNA
expression vectors; yeast transformed with yeast expression
vectors; insect cell systems infected with virus expression vectors
(e.g., baculovirus); plant cell systems transformed with virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV); or bacterial expression vectors (e.g., Ti or
pBR322 plasmids), or animal cell systems.
[0172] The control elements or regulatory sequences are those
non-translated regions of the vector--enhancers, promoters, 5' and
3' untranslated regions--which interact with host cellular proteins
to carry out transcription and translation. Such elements may vary
in strength and specificity. Depending on the vector system and
host utilized, any number of suitable transcription and translation
elements, including constitutive and inducible promoters, may be
used. For example, when cloning in bacterial systems, inducible
promoters such as the hybrid lacZ promoter of the BLUESCRIPT
phagemid (Stratagene, LaJolla, Calif.), pSPORT1 plasmid (Life
Technologies), or the like can be used. The baculovirus polyhedrin
promoter may be used in insect cells. Promoters or enhancers
derived from the genomes of plant cells (e.g., heat shock, RUBISCO,
and storage protein genes) or from plant viruses (e.g., viral
promoters or leader sequences) may be cloned into the vector. In
mammalian cell systems, promoters from mammalian genes or from
mammalian viruses may be used. If it is necessary to generate a
cell line that contains multiple copies of a nucleotide sequence
encoding an antibody, vectors based on SV40 or EBV may be used with
an appropriate selectable marker.
[0173] General texts describing additional molecular biological
techniques useful herein, including the preparation of antibodies
include Berger and Kimmel (Guide to Molecular Cloning Techniques,
Methods in Enzymology, Vol. 152, Academic Press, Inc.); Sambrook,
et al., (Molecular Cloning: A Laboratory Manual, (Second Edition,
Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.;
1989) Vol. 1-3); Current Protocols in Molecular Biology, (F. M.
Ausabel et al. [Eds.], Current Protocols, a joint venture between
Green Publishing Associates, Inc. and John Wiley & Sons, Inc.
(supplemented through 2000)); Harlow et al., (Monoclonal
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press (1988), Paul [Ed.]); Fundamental Immunology, (Lippincott
Williams & Wilkins (1998)); and Harlow, et al., (Using
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press (1998)), all of which are incorporated herein by
reference.
[0174] In another embodiment, the present invention also relates to
the use of quantitative immunoassays to measure levels of MN
protein in patient samples. Many formats may be adapted for use
with the methods of the present invention. For example, the
detection and quantitation of MN protein in patient samples may be
performed, by enzyme-linked immunosorbent assays,
radioimmunoassays, dual antibody sandwich assays, agglutination
assays, fluorescent immunoassays, immunoelectron and scanning
microscopy, among other assays commonly known in the art. The
quantitation of MN protein in such assays may be adapted by
conventional methods known in the art. In one embodiment, serial
changes in circulating MN protein levels may be detected and
quantified by a sandwich assay in which the capture antibody has
been immobilized using conventional techniques on the surface of
the support.
[0175] Suitable supports include, for example, synthetic polymer
supports, such as polypropylene, polystyrene, substituted
polystyrene, polyacrylamides (such as polyamides and
polyvinylchloride), glass beads, agarose, and nitrocellulose.
[0176] An example of an ELISA sandwich immunoassay that may be used
in the methods of the present invention, uses mouse anti-human MNN
monoclonal antibody as the capture antibody and biotinylated goat
anti-human MN polyclonal antibody as the detector antibody. The
capture monoclonal antibody is immobilized on microtiter plate
wells. Diluted human serum/plasma samples or MN standards
(recombinant wild-type MN protein) are incubated in the wells to
allow binding of MN antigen by the capture monoclonal antibody.
After washing of wells, the immobilized MN antigen is exposed to a
biotinylated detector antibody after which the wells are again
washed. A streptavidin-horseradish peroxidase conjugate is then
added. After a final wash, TMB Blue Substrate is added to the wells
to detect bound peroxidase activity. The reaction is stopped by the
addition of 2.5 N sulfuric acid, and the absorbance is measured at
450 nm. Correlating the absorbance values of samples with the MN
standards allows the determination of a quantitative value of MN in
pg/ml of serum or plasma.
[0177] The antibodies useful to identify MN proteins may be labeled
in any conventional manner. An example of a label is horseradish
peroxidase, and an example of a method of labeling antibodies is by
using biotin-strepavidin complexes.
[0178] As appropriate, antibodies used in the immunoassays of this
invention that are used as tracers may be labeled in any manner,
directly or indirectly, that results in a signal that is visible or
can be rendered visible. Detectable marker substances include
radionuclides, such as .sup.3H, .sup.125I, and .sup.131I;
fluorescers, such as, fluorescein isothiocyanate and other
fluorochromes, phycobiliproteins, phycoerythin, rare earth
chelates, Texas red, dansyl and rhodamine; colorimetric reagents
(chromogens); electron-opaque materials, such as colloidal gold;
bioluminescers; chemiluminescers; dyes; enzymes, such as,
horseradish peroxidase, alkaline phosphatases, glucose oxidase,
glucose-6-phosphate dehydrogenase, acetylcholinesterase, alpha-,
beta-galactosidase, among others; coenzymes; enzyme substrates;
enzyme cofactors; enzyme inhibitors; enzyme subunits; metal ions;
free radicals; or any other immunologically active or inert
substance which provides a means of detecting or measuring the
presence or amount of immunocomplex formed. Exemplary of enzyme
substrate combinations are horseradish peroxidase and tetramethyl
benzidine (TMB), and alkaline phosphatases and paranitrophenyl
phosphate (pNPP).
[0179] Another detection and quantitation systems according to this
invention produce luminescent signals, bioluminescent (BL) or
chemiluminescent (CL). In chemiluminescent (CL) or bioluminescent
(BL) assays, the intensity or the total light emission is measured
and related to the concentration of the unknown analyte. Light can
be measured quantitatively using a luminometer (photomultiplier
tube as the detector) or charge-coupled device, or qualitatively by
means of photographic or X-ray film. The main advantages of using
such assays is their simplicity and analytical sensitivity,
enabling the detection and/or quantitation of very small amounts of
analyte.
[0180] Exemplary luminescent labels are acridinium esters,
acridinium sulfonyl carboxamides, luminol, umbelliferone,
isoluminol derivatives, photoproteins, such as aequorin, and
luciferases from fireflies, marine bacteria, Vargulla and Renilla.
Luminol can be used optionally with an enhancer molecule such as
4-iodophenol or 4-hydroxy-cinnamic acid. Typically, a CL signal is
generated by treatment with an oxidant under basic conditions.
[0181] Additional luminescent detection systems are those wherein
the signal (detectable marker) is produced by an enzymatic reaction
upon a substrate. CL and BL detection schemes have been developed
for assaying alkaline phosphatases (AP), glucose oxidase, glucose
6-phosphate dehydrogenase, horseradish peroxidase (HRP), and
xanthine-oxidase labels, among others. AP and HRP are two enzyme
labels which can be quantitated by a range of CL and BL reactions.
For example, AP can be used with a substrate, such as an adamantyl
1,2-dioxetane aryl phosphate substrate (e.g. AMPPD or CSPD; Kricka,
L.J., "Chemiluminescence and Bioluminescence, Analysis by,"
Molecular Biology and Biotechnology: A Comprehensive Desk Reference
(ed. R. A. Meyers) (VCH Publishers; N.Y., N.Y.; 1995)); for
example, a disodium salt of
4-methoxy-4-(3-phosphatephenyl)spiro[1,2-dioxetane-3,2'-adamantane],
with or without an enhancer molecule such as 1-(trioctylphosphonium
methyl)-4-(tributylphosphonium methyl)benzene diochloride. HRP is
may be used with substrates, such as,
2',3',6'-trifluorophenyl-methoxy-10-methylacridan-9-carboxylate.
[0182] CL and BL reactions may be adapted for analysis not only of
enzymes, but also of other substrates, cofactors, inhibitors, metal
ions, and the like. For example, luminol, firefly luciferase, and
marine bacterial luciferase reactions are indicator reactions for
the production or consumption of peroxide, ATP, and NADPH,
respectively. They may be coupled to other reactions involving
oxidases, kinases, and dehydrogenases, and may be used to measure
any component of the coupled reaction (enzyme, substrate,
cofactor).
[0183] The detectable marker may be directly or indirectly linked
to an antibody used in an assay of this invention. Exemplary of an
indirect linkage of the detectable label is the use of a binding
pair between an antibody and a marker or the use of a signal
amplification system.
[0184] Examples of binding pairs that may be used to link
antibodies to detectable markers are biotin/avidin, streptavidin,
or anti-biotin; avidin/anti-avidin; thyroxine/thyroxine-binding
globulin; antigen/antibody; antibody/anti-antibody;
carbohydrate/lectins; hapten/anti-hapten antibody; dyes and
hydrophobic molecules/hydrophobic protein binding sites; enzyme
inhibitor, coenzyme or cofactor/enzyme; polynucleic acid/homologous
polynucleic acid sequence; fluorescein/anti-fluorescein;
dinitrophenol/anti-dinitrophenol; vitamin B12/intrinsic factor;
cortisone, cortisol/cortisol binding protein; and ligands for
specific receptor protein/membrane associated specific receptor
proteins.
[0185] Various means for linking labels directly or indirectly to
antibodies are known in the art. For example, labels may be bound
either covalently or non-covalently. Exemplary antibody conjugation
methods are described in Avarmeas, et al., Scan. J. Immunol.
8(Suppl. 7): 7, 1978); Bayer, et al., Meth. Enzymol. 62:308, 1979;
Chandler, et al., J. Immunol. Meth. 53:187, 1982; Ekeke and
Abuknesha, J. Steroid Biochem. 11:1579, 1979; Engvall and Perlmann,
J. Immunol. 109:129, 1972; Geoghegan, et al., Immunol. Comm. 7:1,
1978; and Wilson and Nakane, Immunofluorescence and Related
Techniques, Elsevier/North Holland Biomedical Press; Amsterdam
(1978), each of which are incorporate herein by reference.
[0186] Depending upon the nature of the label, various techniques
may be employed for detecting and quantitating the label. For
fluorescers, a large number of fluorometers are available. For
chemiluminescers, luminometers or films are available. With
enzymes, a fluorescent, chemiluminescent, or colored product may be
determined or measured fluorometrically, luminometrically,
spectrophotometrically, or visually.
[0187] Various types of chemiluminescent compounds having an
acridinium, benzacridinium, or acridan type of heterocyclic ring
systems are other examples of labels. Examples of acridinium esters
include those compounds having heterocyclic rings or ring systems
that contain the heteroatom in a positive oxidation state including
such ring systems as acridinium, benz[a]acridinium,
benz[b]acridinium, benz[c]acridinium, a benzimidazole cation,
quinolinium, isoquinolinium, quinolizinium, a cyclic substituted
quinolinium, phenanthridinium, and quinoxalinium.
[0188] The tracer may be prepared by attaching to the selected
antibody either directly or indirectly a reactive functional group
present on the acridinium or benzacridinium ester, as is well known
to those skilled in the art (see, e.g., Weeks, et al., Clin. Chem.
29(8):1474-1479, 1983). Examples of compounds are acridinium and
benzacridinium esters with an aryl ring leaving group and the
reactive functional group present in either the para or the meta
position of the aryl ring. (e.g., U.S. Pat. No. 4,745,181 and WO
94/21823, incorporated herein by reference).
Methods of Use
[0189] The term "treatment" includes any process, action,
application, therapy, or the like, wherein a subject (or patient),
including a human being, is provided medical aid with the object of
improving the subject's condition, directly or indirectly, or
slowing the progression of a condition or disorder in the subject,
or ameliorating at least one symptom of the disease or disorder
under treatment.
[0190] The term "combination therapy" or "co-therapy" means the
administration of two or more therapeutic agents to treat a
disease, condition, and/or disorder. Such administration
encompasses co-administration of two or more therapeutic agents in
a substantially simultaneous manner, such as in a single capsule
having a fixed ratio of active ingredients or in multiple, separate
capsules for each inhibitor agent. In addition, such administration
encompasses use of each type of therapeutic agent in a sequential
manner. The order of administration of two or more sequentially
co-administered therapeutic agents is not limited.
[0191] The phrase "therapeutically effective amount" means the
amount of each agent administered that will achieve the goal of
improvement in a disease, condition, and/or disorder severity,
and/or symptom thereof, while avoiding or minimizing adverse side
effects associated with the given therapeutic treatment.
[0192] The term "pharmaceutically acceptable" means that the
subject item is appropriate for use in a pharmaceutical
product.
[0193] The antibodies of this invention are expected to be valuable
as therapeutic agents. Accordingly, an embodiment of this invention
includes a method of treating the various conditions in a patient
(including mammals) which comprises administering to said patient a
composition containing an amount of an antibody of the invention
that is effective in treating the target condition.
[0194] The antibodies of the present invention may be used in the
treatment or prevention of diseases and/or behaviors that are
associated with the MN protein. These diseases and/or behaviors
include, for example, cancer, such as, carcinomas of the kidney,
esophagus, breast, cervix, colon, and lung. The present invention
also relates to methods of ameliorating symptoms of a disorder in
which MN is elevated or otherwise abnormally expressed. These
disorders include, without limitation, carcinomas of the kidney,
esophagus, breast, cervix, colon, and lung (see, e.g., (Liao,
Cancer Res. 57:2827-2831, 1997; Turner, Hum. Pathol. 28:740-744,
1997; Liao, et al., Am. J. Pathol. 145:598-609, 1994; Saarnio, et
al., Am. J. Pathol. 153:279-285, 1998; Vermylen, et al., Eur.
Respir. J. 14:806-811, 1999). In one embodiment of the invention, a
therapeutically effective dose of an antibody of the invention is
administered to a patient having a disorder in which MN is
elevated.
[0195] Antibodies of the present invention may be administered
alone or in combination with one or more additional therapeutic
agents. Combination therapy includes administration of a single
pharmaceutical dosage formulation which contains an antibody of the
present invention and one or more additional therapeutic agents, as
well as administration of the antibody of the present invention and
each additional therapeutic agents in its own separate
pharmaceutical dosage formulation. For example, an antibody of the
present invention and a therapeutic agent may be administered to
the patient together in a single oral dosage composition or each
agent may be administered in separate oral dosage formulations.
[0196] Where separate dosage formulations are used, the antibody of
the present invention and one or more additional therapeutic agents
may be administered at essentially the same time (e.g.,
concurrently) or at separately staggered times (e.g.,
sequentially). The order of administration of the agents is not
limited.
[0197] For example, in one aspect, co-administration of an anti-MN
antibody or antibody fragment of the invention together with one or
more anti-cancer agents to potentiate the effect of either the
antibody/fragment or the anti-cancer agent(s) or both is
contemplated for use in treating MN-related disorders, such as,
cancer.
[0198] Such combination therapies may also be used to prevent
cancer, prevent the recurrence of cancer, prevent the spread or
metastasis of a cancer, or reduce or ameliorate the symptoms
associated with cancer.
[0199] The one or more anti-cancer agents can include any known and
suitable compound in the art, such as, for example, chemoagents,
other immunotherapeutics, cancer vaccines, anti-angiogenic agents,
cytokines, hormone therapies, gene therapies, and radiotherapies. A
chemoagent (or "anti-cancer agent" or "anti-tumor agent" or "cancer
therapeutic") refers to any molecule or compound that assists in
the treatment of a cancer. Examples of chemoagents contemplated by
the present invention include, but are not limited to, cytosine
arabinoside, taxoids (e.g., paclitaxel, docetaxel), anti-tubulin
agents (e.g., paclitaxel, docetaxel, epothilone B, or its
analogues), macrolides (e.g., rhizoxin) cisplatin, carboplatin,
adriamycin, tenoposide, mitozantron, discodermolide, eleutherobine,
2-chlorodeoxyadenosine, alkylating agents (e.g., cyclophosphamide,
mechlorethamine, thioepa, chlorambucil, melphalan, carmustine
(BSNU), lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin, thio-tepa),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, anthramycin), antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, flavopiridol,
5-fluorouracil, fludarabine, gemcitabine, dacarbazine,
temozolamide), asparaginase, Bacillus Calmette and Guerin,
diphtheria toxin, hexamethylmelamine, hydroxyurea, LYSODREN.RTM.,
nucleoside analogues, plant alkaloids (e.g., Taxol, paclitaxel,
camptothecin, topotecan, irinotecan (CAMPTOSAR, CPT-11),
vincristine, vinca alkyloids such as vinblastine), podophyllotoxin
(including derivatives such as epipodophyllotoxin, VP-16
(etoposide), VM-26 (teniposide)), cytochalasin B, colchine,
gramicidin D, ethidium bromide, emetine, mitomycin, procarbazine,
mechlorethamine, anthracyclines (e.g., daunorubicin (formerly
daunomycin), doxorubicin, doxorubicin liposomal),
dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D,
procaine, tetracaine, lidocaine, propranolol, puromycin,
anti-mitotic agents, abrin, ricin A, pseudomonas exotoxin, nerve
growth factor, platelet derived growth factor, tissue plasminogen
activator, aldesleukin, allutamine, anastrozle, bicalutamide,
biaomycin, busulfan, capecitabine, carboplain, chlorabusil,
cladribine, cylarabine, daclinomycin, estramusine, floxuridhe,
gamcitabine, gosereine, idarubicin, itosfamide, lauprolide acetate,
levamisole, lomusline, mechlorethamine, magestrol, acetate,
mercaptopurino, mesna, mitolanc, pegaspergase, pentoslatin,
picamycin, riuxlmab, campath-1, straplozocin, thioguanine,
tretinoin, vinorelbine, or any fragments, family members, or
derivatives thereof, including pharmaceutically acceptable salts
thereof. Compositions comprising one or more chemoagents (e.g.,
FLAG, CHOP) are also contemplated by the present invention. FLAG
comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP
comprises cyclophosphamide, vincristine, doxorubicin, and
prednisone.
[0200] The chemoagent can be an anti-angiogenic agent, such as, for
example, angiostatin, bevacizumab (Avastin.RTM.), sorafenib
(Nexavar.RTM.), baculostatin, canstatin, maspin, anti-VEGF
antibodies or peptides, anti-placental growth factor antibodies or
peptides, anti-Flk-1 antibodies, anti-Flt-1 antibodies or peptides,
laminin peptides, fibronectin peptides, plasminogen activator
inhibitors, tissue metalloproteinase inhibitors, interferons,
interleukin 12, IP-10, Gro-.beta., thrombospondin,
2-methoxyoestradiol, proliferin-related protein,
carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate,
angiopoietin 2, interferon-alpha, herbimycin A, PNU145156E, 16K
prolactin fragment, Linomide, thalidomide, pentoxifylline,
genistein, TNP-470, endostatin, paclitaxel, accutin, cidofovir,
vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline.
Without being bound by theory, the coadministration of an
anti-angiogenic agent advantageously may lead to the increase in MN
expression in a tumor, thereby making the tumor more susceptible to
the antibodies and antibody conjugates of the invention.
[0201] In one aspect, said chemoagent is gemcitabine at a dose
ranging from 100 to 1000 mg/m.sup.2/cycle. In one embodiment, said
chemoagent is dacarbazine at a dose ranging from 200 to 4000
mg/m.sup.2 cycle. In another aspect, said dose ranges from 700 to
1000 mg/m.sup.2/cycle. In yet another aspect, said chemoagent is
fludarabine at a dose ranging from 25 to 50 mg/m.sup.2/cycle. In
another aspect, said chemoagent is cytosine arabinoside (Ara-C) at
a dose ranging from 200 to 2000 mg/m.sup.2/cycle. In still another
aspect, said chemoagent is docetaxel at a dose ranging from 1.5 to
7.5 mg/kg/cycle. In yet another aspect, said chemoagent is
paclitaxel at a dose ranging from 5 to 15 mg/kg/cycle. In a further
aspect, said chemoagent is cisplatin at a dose ranging from 5 to 20
mg/kg/cycle. In a still further aspect, said chemoagent is
5-fluorouracil at a dose ranging from 5 to 20 mg/kg/cycle. In
another aspect, said chemoagent is doxorubicin at a dose ranging
from 2 to 8 mg/kg/cycle. In yet a further aspect, said chemoagent
is epipodophyllotoxin at a dose ranging from 40 to 160 mg/kg/cycle.
In yet another aspect, said chemoagent is cyclophosphamide at a
dose ranging from 50 to 200 mg/kg/cycle. In a further aspect, said
chemoagent is irinotecan at a dose ranging from 50 to 150
mg/m.sup.2/cycle. In a still further aspect, said chemoagent is
vinblastine at a dose ranging from 3.7 to 18.5 mg/m.sup.2/cycle. In
another aspect, said chemoagent is vincristine at a dose ranging
from 0.7 to 2 mg/m.sup.2/cycle. In one aspect, said chemoagent is
methotrexate at a dose ranging from 3.3 to 1000
mg/m.sup.2/cycle.
[0202] In another aspect, the anti-MN antibodies and/or antibody
fragments of the present invention are administered in combination
with one or more immunotherapeutic agents, such as antibodies or
immunomodulators, which include, but are not limited to,
Herceptin.RTM., Retuxan.RTM.., OvaRex, Panorex, BEC2, IMC-C225,
Vitaxin, Campath I/H, Smart MI95, LymphoCide, Smart I D10, and
Oncolym, rituxan, rituximab, gemtuzumab, or trastuzumab.
[0203] The invention also contemplates administering the anti-MN
antibodies and/or antibody fragments of the present invention with
one or more anti-angiogenic agents, which includes, but is not
limited to, angiostatin, thalidomide, kringle 5, endostatin, Serpin
(Serine Protease Inhibitor) anti-thrombin, 29 kDa N-terminal and a
40 kDa C-terminal proteolytic fragments of fibronectin, 16 kDa
proteolytic fragment of prolactin, 7.8 kDa proteolytic fragment of
platelet factor-4, a n-amino acid peptide corresponding to a
fragment of platelet factor-4 (Maione et al., 1990, Cancer Res.
51:2077), a 14-amino acid peptide corresponding to a fragment of
collagen I (Tolma et al., 1993, J. Cell Biol. 122:497), a 19 amino
acid peptide corresponding to a fragment of Thrombospondin I
(Tolsma et al., 1993, J. Cell Biol. 122:497), a 20-amino acid
peptide corresponding to a fragment of SPARC (Sage et al., 1995, J.
Cell. Biochem. 57:1329-), or any fragments, family members, or
derivatives thereof, including pharmaceutically acceptable salts
thereof.
[0204] Other peptides that inhibit angiogenesis and correspond to
fragments of laminin, fibronectin, procollagen, and EGF have also
been described (See the review by Cao, 1998, Prog. Mol. Subcell.
Biol. 20:161). Monoclonal antibodies and cyclic pentapeptides,
which block certain integrins that bind RGD proteins (i.e., possess
the peptide motif Arg-Gly-Asp), have been demonstrated to have
anti-vascularization activities (Brooks et al., 1994, Science
264:569; Hammes et al., 1996, Nature Medicine 2:529). Moreover,
inhibition of the urokinase plasminogen activator receptor by
antagonists inhibits angiogenesis, tumor growth and metastasis (Min
et al., 1996, Cancer Res. 56:2428-33; Crowley et al., 1993, Proc
Natl Acad. Sci. USA 90:5021). Use of such anti-angiogenic agents is
also contemplated by the present invention.
[0205] In another aspect, the anti-MN antibodies and/or antibody
fragments of the present invention are administered in combination
with a regimen of radiation.
[0206] The anti-MN antibodies and/or antibody fragments of the
present invention can also be administered in combination with one
or more cytokines, which includes, but is not limited to,
lymphokines, tumor necrosis factors, tumor necrosis factor-like
cytokines, lymphotoxin-.alpha., lymphotoxin-.beta.,
interferon-.beta., macrophage inflammatory proteins, granulocyte
monocyte colony stimulating factor, interleukins (including, but
not limited to, interleukin-1, interleukin-2, interleukin-6,
interleukin-12, interleukin-15, interleukin-18), OX40, CD27, CD30,
CD40 or CD137 ligands, Fas-Pas ligand, 4-1BBL, endothelial monocyte
activating protein or any fragments, family members, or derivatives
thereof, including pharmaceutically acceptable salts thereof.
[0207] The anti-MN antibodies and/or antibody fragments of the
present invention can also be administered in combination with a
cancer vaccine, examples of which include, but are not limited to,
autologous cells or tissues, non-autologous cells or tissues,
carcinoembryonic antigen, alpha-fetoprotein, human chorionic
gonadotropin, BCG live vaccine, melanocyte lineage proteins (e.g.,
gp100, MART-1/MelanA, TRP-1 (gp75), tyrosinase, widely shared
tumor-associated, including tumor-specific, antigens (e.g., BAGE,
GAGE-1, GAGE-2, MAGE-1, MAGE-3, N-acetylglucosaminyltransferase-V,
p15), mutated antigens that are tumor-associated (.beta.-catenin,
MUM-1, CDK4), nonmelanoma antigens (e.g., HER-2/neu (breast and
ovarian carcinoma), human papillomavirus-E6, E7 (cervical
carcinoma), MUC-1 (breast, ovarian and pancreatic carcinoma). For
human tumor antigens recognized by T-cells, see generally Robbins
and Kawakami, 1996, Curr. Opin. Immunol. 8:628. Cancer vaccines may
or may not be purified preparations.
[0208] In yet another embodiment, the anti-MN antibodies and/or
antibody fragments of the present invention are used in association
with a hormonal treatment. Hormonal therapeutic treatments comprise
hormonal agonists, hormonal antagonists (e.g., flutamide,
tamoxifen, leuprolide acetate (LUPRON), LH-RH antagonists),
inhibitors of hormone biosynthesis and processing, and steroids
(e.g., dexamethasone, retinoids, betamethasone, cortisol,
cortisone, prednisone, dehydrotestosterone, glucocorticoids,
mineralocorticoids, estrogen, testosterone, progestins),
antigestagens (e.g., mifepristone, onapristone), and antiandrogens
(e.g., cyproterone acetate).
[0209] The anti-MN antibodies and/or fragments of the invention can
be used in combination with, e.g. co-administered with, an anti-MDR
(multidrug resistance) phenotype agent.
[0210] Many human cancers intrinsically express or spontaneously
develop resistance to several classes of anticancer drugs at the
same time, notwithstanding that each of the drug classes have
different structures and mechanisms of action. This phenomenon,
which can be mimicked in cultured mammalian cells, is generally
referred to as multidrug resistance ("MDR") or the multidrug
resistance phenotype. The MDR phenotype presents significant
obstacles to the successful chemotherapeutic treatments for cancers
in human patients. Resistance of malignant tumors to multiple
chemotherapeutic agents is a major cause of treatment failure
(Wittes et al., Cancer Treat. Rep. 70:105 (1986); Bradley, G. et
al., Biochim. Biophys. Acta 948:87 (1988); Griswald, D. P. et al.,
Cancer Treat. Rep. 65(52):51 (1981); Osteen, R. T. (ed.), Cancer
Manual, (1990)). Tumors initially sensitive to cytotoxic agents
often recur or become refractory to multiple chemotherapeutic drugs
(Riordan et al., Pharmacol. Ther. 28:51 (1985); Gottesman et al.,
Trends Pharmacol. Sci. 9:54 (1988); Moscow et al., J. Natl. Cancer
Inst. 80:14 (1988); Croop, J. M. et al., J. Clin. Invest. 81:1303
(1988)). Cells or tissues obtained from tumors and grown in the
presence of a selecting cytotoxic drug can result in
cross-resistance to other drugs in that class as well as other
classes of drugs including, but not limited to, anthracyclines,
Vinca alkaloids, and epipodophyllotoxins (Riordan et al.,
Pharmacol. Ther. 28:51 (1985); Gottesman et al., J. Biol. Chem.
263:12163 (1988)). Thus, acquired resistance to a single drug
results in simultaneous resistance to a diverse group of drugs that
are structurally and functionally unrelated. Such resistance can be
a problem for both solid-form and liquid-form tumors (e.g. blood or
lymph-based cancers).
[0211] One major mechanism of multidrug resistance in mammalian
cells involves the increased expression of the 170 kDa plasma
membrane glycoprotein pump system (Juranka et al., FASEB J 3:2583
(1989); Bradley, G. et al., Biochem. Biophys. Acta 948:87 (1988)).
The gene encoding this pump system, sometimes referred to as a
multidrug transporter, has been cloned from cultured human cells
and is generally referred to as mdr1. This gene is expressed in
several classes of normal tissues, but physiological substrates
transported for the mdr1 gene product in these tissues have not
been identified. The MDR1 product is a member of the ABC
Transporter Protein superfamily, a group of proteins having
energy-dependent export function.
[0212] The protein product of the mdr1 gene, generally known as
P-glycoprotein ("P-170", "P-gp"), is a 170 kDa trans-plasma
membrane protein that constitutes the aforementioned
energy-dependent efflux pump. Expression of P-gp on the cell
surface is sufficient to render cells resistant to multiple
cytotoxic drugs, including many anti-cancer agents. P-gp-mediated
MDR appears to be an important clinical component of tumor
resistance in tumors of different types, and mdr1 gene expression
correlates with resistance to chemotherapy in different types of
cancer.
[0213] The nucleotide sequence of the mdr1 gene (Gros, P. et al.,
Cell 47:371 (1986); Chen, C. et al., Cell 47:381 (1986)) indicates
that it encodes a polypeptide similar or identical to
P-glycoprotein and that these are members of the highly conserved
class of membrane proteins similar to bacterial transporters and
involved in normal physiological transport processes. Sequence
analysis of the mdr1 gene indicates that Pgp consists of 1280 amino
acids distributed between two homologous (43% identity) halves.
Each half of the molecule has six hydrophobic transmembrane domains
and each has an ATP binding site within the large cytoplasmic
loops. Only about 8% of the molecule is extracellular, and the
carbohydrate moiety (approximately 30 kDa) is bound to sites in
this region.
[0214] Thus, it will be appreciated that mammalian cells having a
"multidrug-resistance" or "multidrug-resistant" phenotype are
characterized by the ability to sequester, export or expel a
plurality of cytotoxic substances (e.g., chemotherapeutic drugs)
from the intracellular milieu. Cells may acquire this phenotype as
a result of selection pressure imposed by exposure to a single
chemotherapeutic drug (the selection toxin). Alternatively, cells
may exhibit the phenotype prior to toxin exposure, since the export
of cytotoxic substances may involve a mechanism in common with
normal export of cellular secretion products, metabolites, and the
like. Multidrug resistance differs from simple acquired resistance
to the selection toxin in that the cell acquires competence to
export additional cytotoxins (other chemotherapeutic drugs) to
which the cell was not previously exposed. For example, Mirski et
al. (1987), 47 Cancer Res. 2594-2598, describe the isolation of a
multidrug-resistant cell population by culturing the H69 cell line,
derived from a human small cell lung carcinoma, in the presence of
adriamycin (doxorubicin) as a selection toxin. Surviving cells were
found to resist the cytotoxic effects of anthracycline analogs
(e.g., daunomycin, epirubicin, menogaril and mitoxantrone),
acivicin, etoposide, gramicidin D, colchicine and Vinca-derived
alkaloids (vincristine and vinblastine) as well as of adriamycin.
Similar selection culturing techniques can be applied to generate
additional multidrug-resistant cell populations.
Accordingly, the pharmaceutical compositions of the invention can
additionally include compounds which act to inhibit the MDR
phenotype and/or conditions associated with MDR phenotype. Such
compounds can include any known MDR inhibitor compounds in the art,
such as, antibodies specific for MDR components (e.g. anti-MDR
transporter antibodies) or small molecule inhibitors of MDR
transporters, including specifically, tamoxifen, verapamil and
cyclosporin A, which are agents known to reverse or inhibit
multidrug resistance. (Lavie et al. J. Biol. Chem. 271:
19530-10536, 1996, incorporated herein by reference). Such
compounds can be found in U.S. Pat. Nos. 5,773,280, 6,225,325, and
5,403,574, each of which are incorporated herein by reference. Such
MDR inhibitor compounds can be co-administered with the anti-MN
antibodies and/or fragments of the invention for various purposes,
including, reversing the MDR phenotype following the detection of
the MDR phenotype to assist or enhance a chemotherapeutic
treatment. The MDR inhibitor, such as, for example, tamoxifen,
verapamil or cyclosporin A, may be used in conjuction with the
compounds of the invention to assist in the detection of the MDR
phenotype. In accordance with this aspect, an MDR inhibitor can
enhance the uptake and accumulation of a compound of the invention
in an MDR cancer cell since the capacity of the MDR transport
system in transporting or "pumping out" the imaging compound
vis-a-vis the substrate domain would be diminished in the presence
of an MDR inhibitor.
[0215] In yet another embodiment, the anti-MN antibodies and/or
antibody fragments of the present invention are used in association
with a gene therapy program in the treatment of cancer. Gene
therapy with recombinant cells secreting interleukin-2 can be
administered in combination with the inventive antibodies to
prevent or treat cancer, particularly breast cancer (See, e.g.,
Deshmukh et al., 2001, J. Neurosurg. 94:287).
[0216] To assess the ability of a particular antibody to be
therapeutically useful to treat cancer, as an example, the antibody
may be tested in vivo in a mouse xenograft tumor model. If desired,
MN antibodies may be converted into IgG.sub.1 antibodies before
therapeutic assessment. This conversion is described in Example 5,
and an example of a therapeutic model is detailed in Example 9.
Antibody activity may also be tested using an antibody dependent
cell-mediated cytotoxicity assay as described in Example 12.
[0217] The present invention also provides diagnostic methods with
which MN may be detected in a patient sample or biological sample.
Such diagnostic methods may be used, for example, to diagnose
disorders in which MN is elevated. Such disorders include, but are
not limited to, carcinomas of the kidney, esophagus, breast,
cervix, colon, and lung. When used for diagnosis, detection of an
amount of the antibody-MN complex in a sample from a patient which
is greater than an amount of the complex in a normal sample
identifies the patient as likely to have the disorder. An
immunohistochemical method for the detection of MN in cancer
tissues is described in Example 11.
[0218] In another aspect of the invention, a method is provided for
detecting and/or visualizing an MN-related cancer having an
abnormally amount of expressed MN protein. Such methods can
comprise contacting the MN-related cancer cell with an anti-MN
antibody or fragment of the invention, and making an image using a
medical imaging modality, wherein the anti-MN antibody or fragment
comprises a label domain capable of being detected by the medical
imaging modality.
[0219] The detection methods of the invention can be performed in
vitro. The cancer cell or tissue can be from any suitable source,
such as for example, a biopsy or a cell or tissue culture. Methods
for obtaining biopsies and maintaining and/or propagating the
removed tissues and/or cells will be well known to the skilled
artisan. In vitro detection of multidrug resistance can have
various applications, such as, for example, determining whether a
particular subject's cancer, either before, during or after
treatment, has developed a multidrug phenotype.
[0220] The type of imaging modality used to detect the compounds of
the invention will depend on the particular label domain used in
the inventive compounds. For example, if the label domain comprises
a gadolinium chelate, then typically MRI could be used to detect
the imaging agent of the invention. If a radionuclide chelate is
used as the label domain, a nuclear imaging method could be used
(e.g. PET). If a fluorescence-based label domain is used, an
optical imaging system could be used, such as, for example a FACS
system or fluorescence microscopy or a fluorescence automated plate
reader. Choosing an appropriate imaging modality for use in the in
vitro detection methods of the invention are completely within the
knowledge of the skilled artisan.
[0221] In addition, the amount of imaging agent used in the in
vitro detection methods of the invention will be determined by one
of ordinary skill in the art and can depend on the degree to which
the MDR phenotype is present, e.g. the level of expression of the
MDR transport system (e.g. the P-glycoprotein). The skilled artisan
can determine what amount of the novel imaging compounds that is
sufficient for detecting a MDR phenotype without undue
experimentation, i.e. a detectably sufficient amount.
[0222] The type of imaging modality used is not limited to any
particular type, and can include, for example MRI, nuclear imaging
(e.g. PET or SPECT), optical imaging, sonoluminence imaging or
photoacoustic imaging (ultrasound). The skilled artisan will
appreciate that the particular label domain of the imaging
compounds of the invention should be compatible with the particular
imaging modality being used.
[0223] In a preferred embodiment, the methods of detection utilize
anti-MN antibodies or fragments thereof and appropriate labels that
are capable of being detected by MRI. For example, the antibodies
or fragments of the invention can comprise a label domain that is a
MR contrast agent, such as, for example a paramagnetic metal
chelate or chelates or any of those described herein. The imaging
agent can also comprise a radionuclide label domain for imaging or
detecting by a nuclear imaging modality, such as, positron emission
tomography (PET) or single photon emission computer tomography
(SPECT), e.g. a radionuclide such as, for example, .sup.199Au,
.sup.72As, .sup.141Ce, .sup.67Cu, .sup.60Cu, .sup.52Fe, .sup.67Ga,
68Ga, .sup.51Gr, .sup.111In, .sup.177Lu, .sup.51Mn, .sup.203Pb,
.sup.188Re, .sup.97Ru, .sup.47Sc, .sup.177mSn, .sup.94mTc,
.sup.167Tm, and .sup.90Y. The radionuclide can be chelated by a
suitable chelator, or by multiple chelators, such as, for example
HYNIC, DRPA, EDTA, DOTA, TETA, DTPA and BAT. Conditions under which
a chelator will coordinate a metal are described, for example, by
Gansow et al., U.S. Pat. Nos. 4,831,175, 4,454,106 and 4,472,509,
each of which are incorporated herein by reference. .sup.99mTc
(Technetium-99m) is a particularly attractive radioisotope for
therapeutic and diagnostic applications, as it is generally
available to nuclear medicine departments, is inexpensive, gives
minimal patient radiation doses, and has ideal nuclear imaging
properties.
[0224] The patient sample may be contacted with an antibody of the
invention, and the patient sample may then be assayed for the
presence of an antibody-MN complex. As described above, the
antibody may comprise a detectable label, such as a fluorescent,
radioisotopic, chemiluminescent, or enzymatic label, such as
horseradish peroxidase, alkaline phosphatase, or luciferase.
[0225] Optionally, the antibody may be bound to a solid support,
which may accommodate automation of the assay. Suitable solid
supports include, but are not limited to, glass or plastic slides,
tissue culture plates, microtiter wells, tubes, silicon chips, or
particles such as beads (including, but not limited to, latex,
polystyrene, or glass beads). Any method known in the art may be
used to attach the antibody to the solid support, including use of
covalent and non-covalent linkages, passive absorption, or pairs of
binding moieties attached to the antibody and the solid support.
Binding of MN and the antibody may be accomplished in any vessel
suitable for containing the reactants. Examples of such vessels
include microtiter plates, test tubes, and microcentrifuge
tubes.
Pharmaceutical Compositions and Dosages
[0226] The antibodies described herein may be provided in a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier. The pharmaceutically acceptable carrier may be
non-pyrogenic. The compositions may be administered alone or in
combination with at least one other agent, such as stabilizing
compound, which may be administered in any sterile, biocompatible
pharmaceutical carrier including, but not limited to, saline,
buffered saline, dextrose, and water. A variety of aqueous carriers
may be employed including, but not limited to saline, glycine, or
the like. These solutions are sterile and generally free of
particulate matter. These solutions may be sterilized by
conventional, well-known sterilization techniques (e.g.,
filtration).
[0227] Generally, the phrase "pharmaceutically acceptable carrier"
is art recognized and includes a pharmaceutically acceptable
material, composition or vehicle, suitable for administering
compounds of the present invention to mammals. The carriers include
liquid or solid filler, diluent, excipient, solvent or
encapsulating material, involved in carrying or transporting the
subject agent from one organ, or portion of the body, to another
organ, or portion of the body. Each carrier must be "acceptable" in
the sense of being compatible with the other ingredients of the
formulation and not injurious to the patient. Some examples of
materials which can serve as pharmaceutically acceptable carriers
include: sugars, such as lactose, glucose and sucrose; starches,
such as corn starch and potato starch; cellulose, and its
derivatives, such as sodium carboxymethyl cellulose, ethyl
cellulose and cellulose acetate; powdered tragacanth; malt;
gelatin; talc; excipients, such as cocoa butter and suppository
waxes; oils, such as peanut oil, cottonseed oil, safflower oil,
sesame oil, olive oil, corn oil and soybean oil; glycols, such as
propylene glycol; polyols, such as glycerin, sorbitol, mannitol and
polyethylene glycol; esters, such as ethyl oleate and ethyl
laurate; agar; buffering agents, such as magnesium hydroxide and
aluminum hydroxide; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and other non-toxic compatible substances employed in
pharmaceutical formulations.
[0228] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
antibody compositions of the invention.
[0229] Examples of pharmaceutically acceptable antioxidants
include: water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0230] The compositions may contain pharmaceutically acceptable
auxiliary substances as required to approximate physiological
conditions such as pH adjusting and buffering agents, and the like.
The concentration of the antibody of the invention in such
pharmaceutical formulation may vary widely, and may be selected
primarily based on fluid volumes, viscosities, etc., according to
the particular mode of administration selected. If desired, more
than one type of antibody may be included in a pharmaceutical
composition (e.g., an antibody with different K.sub.d for MN
binding).
[0231] The compositions may be administered to a patient alone, or
in combination with other agents, drugs or hormones. In addition to
the active ingredients, these pharmaceutical compositions may
contain suitable pharmaceutically acceptable carriers comprising
excipients and auxiliaries that facilitate processing of the active
compounds into preparations which may be used pharmaceutically.
Pharmaceutical compositions of the invention may be administered by
any number of routes including, but not limited to, oral,
intravenous, intramuscular, intra-arterial, intramedullary,
intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal, parenteral, topical, sublingual, or
rectal means.
[0232] The compositions of the invention additionally contemplate
suitable immunocarriers, such as, proteins, polypeptides or
peptides such as albumin, hemocyanin, thyroglobulin and derivatives
thereof, particularly bovine serum albumin (BSA) and keyhole limpet
hemocyanin (KLH), polysaccharides, carbohydrates, polymers, and
solid phases. Other protein-derived or non-protein derived
substances are known to those skilled in the art.
[0233] In aspects involving vaccines, e.g. cancer vaccines together
with the antibodies of the invention, the compositions of the
invention can be administered with or without an adjuvant.
Administration can be carried out in the absence of an adjuvant in
order to avoid any adjuvant-induced toxicity. The person of
ordinary skill in the art to which this invention pertains, e.g. a
medical doctor specializing in cancer, will appreciate and
understand how to ascertain whether an adjuvant should or should
not be used and can dependent upon the medical history of a
subject, family data, toxicity data, allergy-related test results,
etc. In embodiments where an adjuvant is used, it is advantageous
that the adjuvant promotes the formation of protective antibodies,
such as protective IgG antibodies. Any suitable adjuvant known to
one of ordinary skill in the art is contemplated by the present
invention and are readily adapted to this invention. Suitable
adjuvants for use in vaccinating animals can include, but are not
limited to, aluminum hydroxide, aluminum hydroxide, saponin and its
purified component Quit A, complete Freund's adjuvant (CFA) and
incomplete Freund's adjuvant (IFA). Dextran sulfate has been shown
to be a potent stimulator of IgG2 antibody against staphylococcal
cell surface antigens, and also is suitable as an adjuvant. It will
be appreciated by the skilled person that some adjuvants can be
more preferable for veterinary application, whereas other adjuvants
will be preferable for use in humans, and that adjuvant toxicities
are a consideration that should be made by the skilled person prior
to administration of the compound to a human.
[0234] Formulations suitable for parenteral, subcutaneous,
intravenous, intramuscular, and the like; suitable pharmaceutical
carriers; and techniques for formulation and administration may be
prepared by any of the methods well known in the art (see, e.g.,
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., 20.sup.th edition, 2000). Liquid dosage forms for oral
administration of the compounds of the invention include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
ingredient, the liquid dosage forms may contain inert diluent
commonly used in the art, such as, for example, water or other
solvents, solubilizing agents and emulsifiers, such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
oils (in particular, cottonseed, groundnut, corn, germ, olive,
castor and sesame oils), glycerol, tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and
mixtures thereof.
[0235] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0236] The determination of a therapeutically effective dose is
well within the capability of those skilled in the art. A
therapeutically effective dose refers to the amount of an antibody
that may be used to effectively treat a disease (e.g., cancer)
compared with the efficacy that is evident in the absence of the
therapeutically effective dose.
[0237] The therapeutically effective dose may be estimated
initially in animal models (e.g., rats, mice, rabbits, dogs, or
pigs). The animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information may then be used to determine useful doses and routes
for administration in humans.
[0238] Therapeutic efficacy and toxicity (e.g., ED.sub.50--the dose
therapeutically effective in 50% of the population and
LD.sub.50--the dose lethal to 50% of the population) of an antibody
may be determined by standard pharmaceutical procedures in cell
cultures or experimental animals. The dose ratio of toxic to
therapeutic effects is the therapeutic index, and it may be
expressed as the ratio, LD.sub.50/ED.sub.50. The data obtained from
animal studies may used in formulating a range of dosage for human
use. The dosage contained in such compositions may be within a
range of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage varies within this range
depending upon the dosage form employed, sensitivity of the
patient, and the route of administration.
[0239] The exact dosage may be determined by the practitioner, in
light of factors related to the patient who requires treatment.
Dosage and administration may be adjusted to provide sufficient
levels of the antibody or to maintain the desired effect. Factors
that may be taken into account include the severity of the disease
state, general health of the subject, age, weight, and gender of
the subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy.
[0240] Polynucleotides encoding antibodies of the invention may be
constructed and introduced into a cell either ex vivo or in vivo
using well-established techniques including, but not limited to,
transferrin-polycation-mediated DNA transfer, transfection with
naked or encapsulated nucleic acids, liposome-mediated cellular
fusion, intracellular transportation of DNA-coated latex beads,
protoplast fusion, viral infection, electroporation, "gene gun,"
and DEAE- or calcium phosphate-mediated transfection.
[0241] Effective in vivo dosages of an antibody are in the range of
about 5 .mu.g to about 500 .mu.g/kg of patient body weight. For
administration of polynucleotides encoding the antibodies,
effective in vivo dosages are in the range of about 100 ng to about
500 .mu.g of DNA.
[0242] The mode of administration of antibody-containing
pharmaceutical compositions of the present invention may be any
suitable route which delivers the antibody to the host. As an
example, pharmaceutical compositions of the invention may be useful
for parenteral administration (e.g., subcutaneous, intramuscular,
intravenous, or intranasal administration).
[0243] All patents and patent applications cited in this disclosure
are expressly incorporated herein by reference. The above
disclosure generally describes the present invention. A more
complete understanding can be obtained by reference to the
following specific examples, which are provided for purposes of
illustration only and are not intended to limit the scope of the
invention.
EXAMPLES
[0244] The structures, materials, compositions, and methods
described herein are intended to be representative examples of the
invention, and it will be understood that the scope of the
invention is not limited by the scope of the examples. Those
skilled in the art will recognize that the invention may be
practiced with variations on the disclosed structures, materials,
compositions and methods, and such variations are regarded as
within the ambit of the invention
Example 1
Construct of Human Combinatorial Fab Library (HuCAL Gold)
[0245] HuCAL.RTM. GOLD is an antibody library based on HuCAL.RTM.
technology (Human Combinatorial Antibody Library; MorphoSys AG,
Martinsried/Planegg, Germany). The library combines a synthetic,
fully human antibody library in the Fab format featuring six
diversified CDR regions with an display technology for the
selection of high affinity antibodies, CysDisplay.TM. (MorphoSys
AG, Martinsried/Planegg, Germany). CysDisplay.TM. is a monovalent
phage display technology based on a phenotype-genotype linkage via
disulfide bonds, which allows the recovery of specific antibodies
with high affinities.
[0246] The phage display vector pMORPH.RTM.23 is a phagemid vector
allowing monovalent CysDisplay.TM. of Fab fragments. It encodes the
full length gIIIp, the Fd chain (V.sub.H-C.sub.H1), and the light
chain (V.sub.L-C.sub.L), all of which are equipped with different
secretory signal sequences which directs the corresponding protein
chain to the periplasm of E. coli. Both ompA and phoA signal
sequences are utilized to transport the heavy and light chains to
the periplasm, where the chains assemble via non-covalent
interactions occurs. This display vector carries an inducible lac
promotor/operator region. The laqI.sup.q gene product for
repression of expression is supplied by the E. coli host strain
TG1. Induction of Cys-gIIIp and Fab expression is achieved by the
addition of IPTG (isopropyl-.beta.-D-thio-galacto-pyranoside). The
enriched Fab pools were sub-cloned from pMORPH.RTM.23 to the Fab
expression vector pMORPH.RTM.x9 using the restriction enzymes XbaI
and EcoRI. By this step, the cysteine and the linker-His6 part at
the C-terminus of the Fd chain were removed and the gIIIp is
excised. The expression vector pMORPH.RTM.x9_Fab_FH provides two
C-terminal tags a FLAG and 6.times.His thus facilitating detection
and purification of the Fab proteins.
Phagemid Rescue and Phage Amplification
[0247] The HuCAL GOLD Fabs phagemid in TG1 cells were amplified in
2xTY media supplemented with 34 .mu.g/ml chloramphenicol and 1%
glucose. After helper phage infection (VCSM13
.about.2-6.times.10.sup.11 pfu/ml) at 37.degree. C. for 30 minutes,
TG1 cells were concentrated by centrifugation. The phage were
amplified by incubation of the infected TG1 cells at 22.degree. C.
overnight in 2xTY media containing 34 .mu.g/ml chloramphenicol, 50
.mu.g/ml kanamycin, and 0.25 mM IPTG. The phage containing
supernatant was used for the phase panning.
Example 2
Solid Phase Panning
[0248] Solid phase panning was carried out by coating MaxiSorp.TM.
plates (Nalgene Nunc International, Rochester, N.Y.) or
Dynabeads.TM. (Invitrogen, Carlsbad, Calif.) with human MN protein
in PBS (1 .mu.g/well or 1 .mu.g/1 mg of beads). The MN protein
represents the entire extra-cellular domain of the protein with a
C-terminal histag for purification. The MN protein was expressed in
a mammalian cell line HKB-11 and purified by Ni-NTA chromatography
using standard methods that are well known to those skilled in the
art. Wells containing bound MN protein were blocked with 5% milk in
PBS, washed in PBS followed by a 2 hour incubation at room
temperature with an aliquot of pre-blocked HuCAL GOLD phage library
containing 1.times.10.sup.12 HuCAL GOLD Fab phage. Bound phage were
washed and then eluted with 20 mM DTT in 10 mM Tris buffer at pH
8.0. Three rounds of panning were performed with phage
amplification conducted between each round as described above. Wash
stringency was increased between each round of panning to decrease
nonspecific binding.
Example 3
Subcloning of Selected Fab Fragments for Expression in E. Coli
[0249] The selected Fabs were cloned from the pMORPH.RTM.23 display
vector to the pMORPH.RTM.x9_Fab_FH expression vector to facilitate
rapid expression of soluble Fab for ELISA screening. The DNA
preparation of the pMORPH.RTM.23 vector was digested with EcoRI and
XbaI, thus cutting out the entire Fab-encoding fragment (ompA-VL-VL
and phoA-Fd). After subsequent purification, the fragment was
ligated to the prepared pMORPHx9_Fab_FH vector (also digested with
EcoRI/XbaI and purified). The vector containing the Fab insert was
transfected into competent E. coli TG1 F-cells by electroporation.
The transformed E. coli cell were grown on LB plates containing 34
.mu.g/ml chloramphenicol and 1% glucose o/n at 37.degree. C.
Colonies were picked and placed in culture media containing 34
.mu.g/ml chloramphenicol and 1% glucose. Glycerol was added to 20%
and these stock starter cultures were stored at -80.degree. C.
until needed for ELISA. To obtain purified Fabs, E. coli
transformants carrying this vector were grown typically in
multiples of 1 L shake flask cultures, harvested, and purified
using Ni-NTA affinity chromatography using methods well known to
those skilled in the art.
Example 4
Identification of MN-Binding Fab Fragments by ELISA
[0250] Maxisorp 96-well ELISA plates were coated with 100
.mu.l/well of a solution of purified human MN protein at a
concentration of 5 .mu.g/ml in PBS. Fabs were expressed using E.
coli transformants grown in 2.times.TY media containing 34 .mu.g/ml
chloramphenicol from started stock cultures. Twelve (12) hours
prior to harvest Fab expression was induced by addition of IPTG
(0.5 mM final concentration). Cells were lysed and 100 .mu.l lysate
was incubated within the well of an MN-precoated and milk-blocked
Maxisorp plate for 2 hours at room temperature. Plates were then
washed in TBS and Tween containing-TBS to remove nonspecific
binding. Bound Fabs were detected with goat anti-human (Fab').sub.2
antibody conjugated to alkaline phosphatase (Pierce Chemical,
Rockford, Ill.). The substrate AttoPhos (Roche Diagnostics,
Alameda, Calif.) was used as directed in the manufacturers
instructions, excitation was at 430 nm and emission was read at 535
nm.
[0251] A large number of E. coli transformants expressed Fabs that
exhibited a signal to noise ration in the ELISA of >10. DNA
sequencing of the VH and VL regions identified over 50 unique
MN-binding Fabs. The DNA and protein sequences of the entire VH and
VL of ten antibodies based upon their functional properties is
shown in FIGS. 3 and 4, respectively. Each of these ten disclosed
antibodies bound by ELISA to the purified MN protein isolated from
a mammalian expression cell line, HKB-11 (FIG. 5). The antibodies
examined also reacted with purified MN isolated from sf9 insect
cells that had been infected with a baculovirus encoding the
extra-cellular domain of MN, (FIG. 5) demonstrating that the ELISA
interactions were specific for the MN protein and not any trace
contaminants in the protein preparations. In a BIAcore.TM. assay,
antibodies of the present invention specifically bind to human MN
with a K.sub.d in the range from 1 nM (1.times.10.sup.-9 M) to
about 50 nM (5.0.times.10.sup.-8 nM) (FIG. 5).
Example 5
Identification of MN-Binding Fabs and IgGs
[0252] The binding interactions between MN protein and the
antibodies of this invention were analyzed using surface plasmon
resonance technology on a BIAcore 3000 instrument (BIAcore,
Uppsala, Sweden). For the binding of full-length IgGs (1e4, 1aa1,
and 3ee9), goat anti-human IgG Fc was covalently coupled to the CM5
biosensor chip at high density using a standard amine coupling kit
(BIAcore, Uppsala, Sweden). A capture assay was then used to bind
these IgG to the anti-human IgG Fc-immobilized surface, aiming for
a 300 response unit BIAcore signal. The BIAcore was operated at
25.degree. C. and a flow rate of 10 .mu.l/min running buffer
containing PBS/0.05% Tween 20, or containing 10 mM Na-HEPES, pH
7.5/150 mM NaCl/3 mM EDTA/0.005% Tween 20. After ligand binding,
the flow rate was then increased to 25 .mu.l/min and various
concentrations of MN analyte (e.g., range of 50 nM to 1 nM) were
flowed over the surface for 10 minutes such that the association
phase could be monitored. Dissociation then proceeded for 35
minutes, followed by regeneration of the anti-human IgG
Fc-immobilized surface at 100 .mu.l/min with 100 .mu.l of 10 mM
phosphoric acid. Sensorgrams were fitted using a 1:1 Langmuir
binding model to calculate rate constants. The binding of Fabs to
MN was similarly determined except that an anti-human-Fab IgG was
immobilized onto the chip and used to capture the Fabs prior to the
assessment of MN binding. FIG. 5 shows the resulting binding
constants for the binding of the disclosed antibodies to purified
MN protein. Each of the antibodies bound MN exhibiting Kd values
that ranged between 0.15 and 50 nM.
Example 6
Construction of HuCAL Immunoglobulin Expression Vectors
[0253] Heavy chain expression vector. The multiple cloning site of
pcDNA3.1+ (Invitrogen, Carlsbad, Calif.) was removed (NheI/ApaI),
and a site compatible with the restriction sites used for HuCAL was
inserted for the ligation of the leader sequences (NheI/EcoRI),
VH-domains (EcoRI/BlpI), and the immunoglobulin constant regions
(BlpI/ApaI). The leader sequence (EMBL M83133) was equipped with a
Kozak sequence (Kozak, Nucleic Acid Res. 15:8125-8148, 1987). The
constant regions of human IgG1 (PIR J00228), IgG4 (EMBL K01316),
and serum IgA1 (EMBL J00220) were dissected into overlapping
oligonucleotides with lengths of about 70 bases. Silent mutations
were introduced to remove restriction sites non-compatible with the
HuCAL design. The oligonucleotides were spliced by overlap
extension-PCR.
[0254] Light chain expression vectors. The multiple cloning site of
pcDNA3.1/Zeo+ (Invitrogen, Carlsbad, Calif.) was replaced by two
different sites. The .kappa.-site provided restriction sites for
insertion of a .kappa.-leader (NheI/EcoRV), HuCAL-scFv Vk-domains
(EcoRV/BsiWI) and the .kappa.-chain constant region (BsiWI/ApaI).
The corresponding restriction sites in the .lamda.-site were
NheI/EcoRV (1-leader), EcoRV/Hpal (VI-domains), and HpaI/ApaI
(.lamda.-chain constant region). The .kappa.-leader (EMBL Z00022)
as well as the .lamda.-leader (EMBL L27692) were both equipped with
Kozak sequences. The constant regions of the human .kappa.-chain
(EMBL J00241) and .lamda.-chain (EMBL M18645) were assembled by
overlap extension-PCR as described above.
[0255] Generation of full-length IgG from Fabs. Fab heavy chain
sequence contained within the E. coli expression vector
pMORPHx9_Fab_FH was excised by cutting with MfeI/BlpI and ligated
into the heavy chain expression vector described above that had
been cut with EcoRI/BlpI. Fab kappa light chain sequence contained
within pMORPHx9_Fab_FH was excised with EcoRV/BsiWI and ligated
into the Kappa light chain expression vector described above that
had also been cut with EcoRV/BsiWI. Fab lambda light chain sequence
contained within pMORPHx9_Fab_FH was excised with EcoRV/Hpal and
ligated into the lambda expression vector described above.
[0256] Large scale transient expression of full-length IgGs.
Cellbag 20L/0 (Wave Biotech LLC, Somerset, N.J.) were seeded with
0.25.times.10.sup.6 293F cells/ml (Invitrogen) in 9.3 L Freestyle
293 expression medium (Invitrogen). Cells were grown to a density
of 1.times.10.sup.6/ml and transfected by the addition of 5 mg each
of the light chain expression vector and heavy chain expression
vector encoding the full length antibody in 350 ml Optimem
(Invitrogen) containing 293fectin reagent (Invitrogen). Following
fermentation for 96 hours at 37.degree. C., cell culture
supernatant was harvested by centrifugation, sterile filtered,
concentrated by tangential flow filtration adjusted to pH 7.6 and
then subjected to standard protein A column chromatography
(Amersham Pharmacia Biotech, Piscataway, N.J.).
Example 7
Cell Adhesion Assay
[0257] Fifty (50) .mu.L of a 1 .mu.g/mL solution of purified MN in
PBS was adsorbed onto a non-treated 96-well plate overnight at
4.degree. C. The solution was removed, and the wells rinsed
3.times. with PBS. The wells were blocked for 1 hour with 200 .mu.L
50% FBS in RPMI1640 media. The wells were then treated with 100
.mu.g 1e4 anti-MN antibody in 1% BSA in PBS, control IgG in 1% BSA
in PBS or 1% BSA in PBS. After washing with PBS, 5000 MaTu cells
(MN+ cells) were added to the wells and the plate incubated
overnight at 37.degree. C. at 5% CO.sub.2. The ability of anti-MN
antibodies to block adhesion of MaTu cells to MN-coated wells was
assessed after washing with PBS. An example of this experiment is
shown in FIG. 6 where 100 .mu.g of anti-MN antibody 1e4 inhibits
cell adhesion, whereas control IgG or the buffer vehicle does
not.
Example 8
Subcutaneous Xenograft Cancer Model with Immunoconjugate
[0258] Anti-MN antibodies were conjugated to cytotoxic small
molecules using protocols that are known in the art (e.g., Liu, et
al., Proc. Natl. Acad. Sci. 93:8618-8623, 1996). Human mammary
xenograft, MaTu cells were maintained as adherent cultures in RPMI
supplemented with 10% FBS. Ncr nude mice (8-12 weeks of age) were
inoculated subcutaneously in the right flank with 5.times.10.sup.6
cells in 0.1 mL of 80% matrigel/20% HBSS. When tumors reached an
average size of .about.180 mg (6 days), treatment was initiated.
Monoclonal antibodies conjugated to cytotoxic small molecules were
administered i.v. once every four days (Q4Dx3) at a dose of 10
mg/kg. Control mice were treated with PBS or an unconjugated
monoclonal antibody. Daily examinations into the health status of
each animal were conducted. Each experimental group consisted of 10
mice and the dosing volume was 0.1 mL/10 g body weight. The length
and width of each tumor was measured by using an electronic caliper
2-3 times per week and tumor weights (mg) were calculated based on
the formula of [length (mm).times.width (mm).sup.2]/2. All data,
including daily observations, obtained throughout the course of the
study were documented. Tumor growth inhibition (TGI) was calculated
as 1-T/C.times.100, where T=final tumor weights from a treated
group, and C=final tumor weights from the control group. FIG. 7
shows that the monoclonal IgG1 1e4 when conjugated to a cytotoxic
drug produced a significant anti-tumor effect at 30 mg/kg
immunoconjugate, whereas the unconjugated antibody had no effect.
These data demonstrate that the therapeutic utility of antibodies
directed against MN protein as vehicles for cytotoxic drug delivery
to tumors.
Example 9
Fluorescence-Activated Cell Sorting Assay (FACS Assay)
[0259] Cells may be assayed for MN expression as a diagnostic tool.
Adherent MN-expressing PC-3 mm2 cells and non-MN expressing DLD1
cells were detached from their flasks with 1:10 trypsin/Versene in
PBS solution for 5 to 10 minutes. Cells were spun down (1000 rpm, 5
minutes), washed once with ice cold RPMI 10% FBS, and resuspended
in ice-cold staining buffer (Ca.sup.+ Mg.sup.+-free PBS, 2% BSA,
and 0.05% sodium azide) at 6.times.10.sup.6 cells/ml. Primary
antibody, human anti-MN IgG1, or control human IgG1 antibody at 25
.mu.g/mL were incubated with 6.times.10.sup.5 cells on ice for 1
hour. The unbound antibody was washed from the cells with the
ice-cold staining buffer. The cells were fixed with 2% formaldehyde
in PBS for 10 minutes, then washed twice with staining buffer. The
cell pellet was resuspended in 100 .mu.l ice cold staining buffer
containing anti-human Alexa fluor 488 secondary antibody (final
conc. 1:200, Molecular Probes/Invitrogen, Carlsbad, Calif.), and
incubated on ice for 1 hour. The unbound antibody was washed from
the cells two times with flow buffer (PBS containing 2% BSA), and
the cells were resuspended in 1 mL flow buffer. FACS analysis of
the resuspended cells was performed on a Beckman FACS Caliber
instrument. FIG. 8 shows that PC-3 mm2 human prostate cancer cells
expressed MN as assayed by FACS, whereas DLD-1 cells did not.
Example 10
Antibody-Dependent Cell Mediated Cytotoxicity Assays (ADCC
Assays)
[0260] Anti-tumor activity of anti-MN IgGs may be mediated by ADCC
activity. MN-expressing PC-3 mm2 cells and non-MN expressing
HCT-116 cells are incubated with 250 ng/mL, 1000 ng/mL, or 2000
ng/mL human anti-MN IgG1, or control human IgG1 anti-digoxin
antibody. Human PBMCs are added to these cells at effector: target
ratios of 50:1, 25:1, and 5:1 ratios. A chromium-51 release assay
is performed to determine the level of target cell lysis. A small
amount of lysis is observed upon incubation of control antibody or
no antibody in the presence of DLD and PC-3 mm2 cells. This
spontaneous level of lysis is 10-15%, 5-10%, or 2-3% for 50:1,
25:1, and 5:1 target effector ratios, respectively. Similarly,
lysis of non-MN expressing DLD cells was in the 0-10% range when
incubated with the anti-MN antibodies. However, lysis of PC-3 mm2
cells when incubated with the human anti-MN IgGs was significantly
higher than the controls. Lysis of 40, 50, and 60% was observed
when using 250 ng/mL, 1000 ng/mL, and 2000 ng/mL at 50:1
target:effector ratios. Similarly, 30, 33, and 38% lysis was
observed at 25:1 ratios, and finally, 8, 10, and 15% lysis was
observed at 5:1 target:effector ratios. These experiments show that
human anti-MN antibodies mediate anti-tumor ADCC activity and may
be used for the therapeutic treatment of cancer.
Example 11
Immunoconjugate
Preparation
[0261] A human antibody directed at the MN cell surface antigen was
generated using a phage display library that encodes a diversity of
human Fabs (Morphosys). This antibody was conjugated to
Monomethylauristatin E (MMAE) (Fransisco et al., Blood, 2003,
102:1458-1465) (FIG. 9).
In Vitro Activity and Selectivity
[0262] The antibody portion of 3ee9/MMAE (3ee9) was identified by
in vitro "panning" of a purified extracellular domain of human MN
against the MorphoSys phage library composed of 10.sup.10 human Fab
fragments (Fabs are the antigen binding portions of antibodies).
The active Fabs were further examined for their capacity to
selectively bind and undergo internalization upon addition to MN
positive cells. The resulting active Fabs were then converted to
full length human IgG1 antibodies, expressed in CHO cells, purified
and then conjugated to the toxophore, MMAE (Liu et al, Proc Natl
Acad Sci, 1996, 93:8618-8623). The conjugated antibodies were then
tested for their ability to kill MN expressing cells. From a panel
of seven full-length antibodies tested, 3ee9/MMAE was selected
based on its binding properties, selectivity and potency in both in
vitro and in vivo assays.
Surface Plasmon Resonance (Biacore) Materials and Methods
[0263] Binding interactions between HKB11-expressed human MN
protein and human full-length anti-MN MAbs were analyzed using
surface plasmon resonance technology on a BIAcore 3000 instrument
(BIAcore, Uppsala, Sweden). For chip preparation, goat anti-human
IgG Fc was covalently coupled to the CM5 biosensor chip using the
standard amine coupling kit (BIAcore, Uppsala, Sweden). A capture
assay was then used to bind the antibodies of interest to the
anti-human IgG Fc-immobilized surface, aiming for a 300 response
unit BIAcore signal. The BIAcore was operated at 25.degree. C. with
a flow rate of 10 ul/min and running buffer containing 10 mM
Na-HEPES, pH 7.5/150 mM NaCl/3 mM EDTA/0.005% Tween 20. After
ligand binding, flow rate was increased to 25 ul/min and MN analyte
(50 nM to 1 nM) was flowed over the surface for 10 min such that
the association phase could be monitored. Dissociation then
proceeded for 35 min, followed by regeneration of the anti-human
IgG Fc-immobilized surface at 100u1/min with 10 mM phosphoric
acid.
[0264] Sensorgrams were fitted using a 1:1 Langmuir binding model
to calculate rate constants (Table 1, below).
TABLE-US-00001 TABLE 1 Affinity of panning-derived antibodies to
the soluble MN determined by surface plasmon resonance (Biacore) 1
E4 3EE9 1AA1 3A4 5AA3 3EF2 Affinity 5 4 27 1 1 6 (Kd, nM)
Antibody Binding
[0265] 3ee9/MMAE was shown to have a kD of 3.6 nM for purified MN
protein using Biacore technology, which was the same as the
affinity of the unconjugated antibody 3ee9. The binding to MN(CA
IX) appeared specific, as there was no detectable binding to 13
other carbonic anhydrases. Binding to mitochondria-associated CA5
was observed, but this isozyme would be inaccessible to the
antibody in vivo.
[0266] By FACS analysis, 3ee9/MMAE was shown to bind to
MN-expressing MaTu cells, but not MN-negative DLD cells (FIG. 10a).
The MN antibody G250, a humanized IgG1 antibody, (Wilex) was used
as a reference throughout the in vitro studies. It bound MN with a
Kd of 5.3 nM and exhibited a similar binding profile to MN+ and MN-
cell lines as 3ee9/MMAE.
[0267] Similar results were obtained for the antibody 1 E4 and 1aa1
immunoconjugates (FIGS. 10b and 10c, respectively), which exhibited
binding to MN+ cells, but not to MN- cells.
Antibody Internalization
[0268] 3ee9/MMAE was selectively internalized by MN-expressing
cells (PC3 mm2), but not by MN-negative cells (DLD1), as measured
by Cellomics (FIG. 11a). Similarly, the 1E4 immunoconjugate was
found to be internalized by MN+ cells (PC3 mm2), but not by MN-
(DLD1) cells (FIG. 11b).
Immunoprecipitation
[0269] To further explore specificity, various candidate antibodies
including the antibody portion of 3ee9/MMAE (3ee9) and the
reference antibody G250 were incubated with cell lysates of MN+
(Pc3 mm2) and MN- (DLD-1) cell lines that had been selectively
labeled with biotin. Complexes between the antibodies and cellular
proteins were immuno-precipitated and the co-precipitated antigens
visualized using immunoblots developed with enzyme-linked
streptavidin. Both G250 and the 3ee9 antibody of 3ee9/MMAE
selectively bound and co-immunoprecipitated with a single band of
the same size as MN from MN+ cells (FIG. 12). Less specific
antibodies such as 3ef2, 5aa3 and 5A6 co-immunoprecipitated several
other proteins in addition to MN.
Cytotoxicity
[0270] In vitro cytotoxicity assays were carried out. 3ee9/MMAE was
found to be highly cytotoxic to MN-expressing PC3 mm2 cells
(EC50=50 nM), but not to MN-negative MIAPaca2 cells (FIG. 13a).
Less than 10% killing of MN-negative cells was seen even at doses
as high as 1 uM. The 1A4 immunoconjugate was also found to be
selectively cytotoxic for MN+ cells (PC3 mm2 and MaTu), but not for
MN- cells (MIAPaca2 and DLD1) in the in vitro cytotoxicity assays
(FIG. 13b). Additionally, the 1aa1 immunoconjugate was found to be
selectively cytotoxic for MN+ cells (PC3 mm2), but not for MN-
cells (MIAPaca2) in the in vitro cytotoxicity assays (FIG.
13c).
[0271] The cytotoxic drug MMAE delivered by 3ee9/MMAE is a tubulin
inhibitor that prevents spindle formation during cell mitosis
resulting in G2/M arrest. The effect of treatment of MN-expressing
cells (Pc3 mm2) and MN non-expressing cells (H460) with 3ee9/MMAE
is shown in FIG. 14. The tubulin is stained with florescence
labeled antibody. Untreated cells showed normal spindle formation,
while the treated MN expressing cells showed fragmented fibers
resulting from tubulin binding with MMAE and prevention of normal
spindle formation. No activity was seen in MN non-expressing cells.
These studies confirmed that the antibody drug conjugate 3ee9/MMAE
kills cell through targeted tubulin disruption.
Example 12
In Vivo Activity of Immunoconiuqate
[0272] In Vivo Pharmacology of 3ee9/MMAE Against Subcutaneously
Implanted Human Xenograft Cancer Models
[0273] 3ee9/MMAE exhibits significant and consistent anti-tumor
effects on the growth of multiple human xenograft tumor models in
athymic mice when administered via the intravenous route in an
intermittent schedule. The in vivo anti-tumor effect of 3ee9/MMAE
was examined in 6 different human xenograft tumor models. These
models were established through subcutaneous implantation of human
tumor cells into female athymic NCr (nu/nu) mice (Taconic, N.Y.).
The human tumor xenograft models evaluated included the MaTu human
mammary carcinoma model, the HT-29 and Colo-205 human colo-rectal
carcinoma (CRC) models, PC3MM2 prostate carcinoma model, the HCT-15
multi-drug resistant (P-gp) CRC model, and the MiaPaCa2 human
pancreatic model. PBS was used as a vehicle, and dosing solutions
were prepared fresh daily. The dosing volume was 0.1 mL/10 g (10
mL/kg).
[0274] The length and width of each tumor were measured using an
electronic caliper 2-3 times per week, and tumor weights (mg) were
calculated based on the formula of [length (mm).times.width
(mm).sup.2]/2. All data, including daily observations, obtained
throughout the course of the study were documented in Anti-tumor
Data Acquisition System (ADAS). The maximum tolerated dose (MTD) is
defined as the highest dose that does not produce greater than 20%
lethality and/or 20% net body weight loss. Tumor growth inhibition
(TGI) was calculated as (1-T/C).times.100, where T=final tumor
weights from a treated group after the last dose, and C=final tumor
weights from the control group after the last dose. In addition,
anti-tumor efficacy was measured as the incidence of responses (or
responders or regressions) defined as tumors with .ltoreq.50% of
their initial size. A minimum duration of 7 days is required for a
response to be considered durable.
Efficacy of 3ee9/MMAE in MaTu Xenograft Model
[0275] Human mammary xenograft, MaTu cells were maintained as
adherent cultures in RPMI supplemented with 10% FBS. NCr nude mice
(8-12 weeks of age) were inoculated subcutaneously in the right
flank with 5.times.10.sup.6 cells in 0.1 mL of 80% matrigel/20%
HBSS. When tumors reached an average size of .about.180 mg (7
days), treatment was initiated. BAY 79-4620 (3ee9-IC) was
administered i.v. once every four days (Q4Dx3) at a dose of 1, 3
and 10 mg/kg. Control mice were treated with PBS or an
un-conjugated monoclonal antibody at a dose of 10 mg/kg.
[0276] Daily examinations into the health status of each animal
were conducted. Each experimental group consisted of 10 mice, and
the dosing volume was 0.1 mL/10 g body weight. The length and width
of each tumor was measured by using an electronic caliper 2-3 times
per week, and tumor weights (mg) were calculated based on the
formula of [length (mm).times.width (mm).sup.2]/2. All data,
including daily observations, obtained throughout the course of the
study were documented. Tumor growth inhibition (TGI) was calculated
as 1-T/C.times.100, where T=final tumor weights from a treated
group, and C=final tumor weights from the control group.
[0277] 3ee9/MMAE was well-tolerated at all doses examined with all
the treated animals exhibiting no significant weight loss.
Representative efficacy of 3ee9/MMAE in the MaTu tumor model is
illustrated in FIG. 15. Tumors from both the untreated and
vehicle-treated control groups grew progressively in all animals.
The mean doubling time for animals in control and vehicle groups
were 11.2 days. At the end of dosing, 3ee9/MMAE showed robust
anti-tumor efficacy at all doses examined. More specifically, BAY
79-4620 (3ee9-IC) yielded 67, 72 and 78% TGI at 1, 3 and 10 mg/kg,
respectively. In comparison, the unconjugated 3ee9 mAb had no
significant effect in inhibiting the growth of this mammary
xenograft tumor.
[0278] Following the completion of the pre-determined dosing
regimen (Q4Dx3), the effect of 3ee9/MMAE on tumor growth delay and
regression was determined. As shown in FIG. 15, treatment of
3ee9/MMAE resulted in significant tumor growth delay and
regression. At the lowest dose examined (1 mg/kg), following
cessation of treatment, tumors remained stable for .about.2 weeks
and began to grow back thereafter. Overall, 30% of the tumors were
responsive to this treatment of 1 mg/kg. In comparison, 100% of the
animals responded when challenged with 3 and 10 mg/kg. Of note,
even after .about.3 months following the cessation of treatment,
only 3 and 1 tumors showed signs of re-growth in the 3 and 10 mg/kg
groups, respectively, showing that 70 and 90% of the animals
remained tumor-free even after .about.90 days after the cessation
of treatment.
[0279] The monoclonal IgG1 1E4, when conjugated to a cytotoxic
drug, produced a significant anti-tumor effect against the human
mammary xenograft, MaTu, when dosed at 30 mg/kg immunoconjugate,
whereas the unconjugated antibody had no effect (FIG. 16). Further
experiments using the same protocol, but substituting conjugated
forms of antibody 1aa1 (FIG. 17), 3ee9 and 5aa3, gave similar
results. Additionally, when using the conjugated form of 3ee9,
anti-tumor effects were seen in other human xenograft models
derived from various histological type including, HT-29 and
Colo-205 human colo-rectal cancer xenograft models, and PC3-mm2, a
human prostate xenograft model. These data indicate that the
therapeutic utility of antibodies directed against MN protein as
vehicles for cytotoxic drug delivery to tumors.
Example 13
Therapeutic Index Determination of 3ee9/MMAE in MaTu Xenograft
Model
[0280] The maximum tolerated dose (MTD), minimum efficacious dose
(MED), and therapeutic index (TI) of 3ee9/MMAE was determined using
MaTu xenograft tumor-bearing mice. The TI is defined as the ratio
of the MTD divided by MED. 3ee9/MMAE was administered intravenously
once every 4.sup.th day for a total of 3 injections (Q4Dx3).
3ee9/MMAE was administered at dose levels of 0.625, 1.25, 2.5, 5.0,
10, 30 and 60 mg/kg. Control mice were treated with PBS or an
unconjugated monoclonal antibody at a dose of 60 mg/kg. 3ee9/MMAE
dosed at 60 mg/kg appeared to be the MTD, as there was 10%
lethality and .about.20% body weight loss in response to this
treatment. All other treatment groups were well tolerated.
[0281] The anti-tumor activity of 3ee9/MMAE is presented in FIG.
18. At the end of dosing, 3ee9/MMAE at a dose of 0.625 and 1.25
mg/kg resulted in 62 and 81% inhibition, respectively. Doses of
2.5, 5, 10, 30 and 60 mg/kg resulted in a greater inhibition of
tumor growth (.about.90%) with majority of tumors starting to show
regressions. Based on data observed, the MED of 3ee9/MMAE was 0.625
mg/kg. Following the completion of the pre-determined dosing
regimen (Q4Dx3), the effect of 3ee9/MMAE on tumor growth delay and
regression was determined. No tumor regression was seen for the
0.625 mg/kg dose. In contrast, 80% of the animals exhibited tumor
regressions in response to 1.25 mg/kg. Moreover, at doses of 2.5
mg/kg and higher, 100% of the animals responded to treatment. The
therapeutic index of 3ee9/MMAE was determined to be .about.96.
Example 14
Efficacy of 3ee9/MMAE in HT-29 Xenograft Model
[0282] Human CRC xenograft, HT-29 cells were inoculated
subcutaneously in the right flank with 5.times.10.sup.6 cells in
0.1 mL of HBSS. When tumors reached an average size of .about.120
mg (5 days), treatment was initiated. 3ee9/MMAE was administered
i.v. once every four days (Q4Dx3) at a dose of 0.625, 1.25, 2.5,
5.0, 10 mg/kg. Control mice were treated with PBS or an
un-conjugated monoclonal antibody at a dose of 10 mg/kg. In
addition, MMAE was assessed as a free drug at a dose of 0.1, 0.2
and 1 mg/kg. The 0.1 and 0.2 mg/Kg doses of MMAE represent
equivalent amounts of this drug to those present on 5 and 10 mg/Kg
3ee9/MMAE respectively.
[0283] 3ee9/MMAE was well tolerated at all doses examined with all
treated animals exhibiting no significant weight loss. Similarly,
lower doses of MMAE, i.e., 0.1 and 0.2 mg/kg, were also well
tolerated with non-significant, minimal weight loss. However, at
the top dose of 1 mg/kg, 50% lethality was observed with the
remaining animals exhibiting severe weight loss and thus was
considered toxic.
[0284] Representative efficacy of 3ee9/MMAE and free MMAE in the
HT-29 tumor model is illustrated in FIG. 19. Tumors from both the
untreated and vehicle-treated control groups grew progressively in
all animals. The mean doubling time for animals in control and
vehicle groups were 6.4 days. At the end of dosing, 3ee9/MMAE
showed robust anti-tumor efficacy at all doses examined. More
specifically, 3ee9/MMAE at doses of 0.625, 1.25, 2.5, 5 and 10
mg/kg yielded 54, 72, 97, 100 and 100% TGI, respectively. In
comparison, free MMAE of 0.2 mg/kg resulted in significant TGI of
60%, whereas 0.1 mg/kg had no significant effect in inhibiting the
growth of this xenograft model. In terms of tumor responses,
3ee9/MMAE dosed at 1.25 mg/kg resulted in 20% of the animals
showing regressions. At higher doses, the tumor responses were much
greater, with 2.5 mg/kg showing 90% regressions and 5 and 10 mg/kg
inducing 100% responses. In contrast, free MMAE did not induce any
tumor responses as defined above.
[0285] Anti-tumor effect of 3ee9/MMAE was also assessed in varying
schedules, i.e., once a week dosing for total of two doses (Q7Dx2,
FIG. 20) and a single dose at the time of staging (Q1Dx1, FIG. 21).
In both schedules, 3ee9/MMAE was dosed at 0.625, 1.25, 2.5, 5 and
10 mg/kg. Regardless of schedule, 3ee9/MMAE was highly effective in
inhibiting the growth in this CRC xenograft model.
[0286] Table 2, below, summarizes anti-tumor efficacy of 3ee9/MMAE.
The anti-tumor efficacy was very similar to that which was observed
for 3ee9/MMAE in the Q4Dx3 schedule, indicating that in the
schedules examined, the anti-tumor efficacy of 3ee9/MMAE appears to
be schedule-independent.
TABLE-US-00002 TABLE 2 Anti-tumor efficacy of 3ee9/MMAE %
Inhibition Dose Treatment (1-T/D)*100 Percent Compound (mg/kg)
Schedule (Day 18) Regressions Control N/A N/A N/A 0 PBS 0 Q4D
.times. 3 -17 0 3ee9 Ab 10 Q4D .times. 3 -11 0 3EE9IC 0.625 Q4D
.times. 3 54 0 3EE9IC 1.25 Q4D .times. 3 72 2 3EE9IC 2.5 Q4D
.times. 3 97 9 3EE9IC 5 Q4D .times. 3 100 10 3EE9IC 10 Q4D .times.
3 100 10 3EE9IC 0.625 Q7D .times. 2 48 0 3EE9IC 1.25 Q7D .times. 2
71 0 3EE9IC 2.5 Q7D .times. 2 92 4 3EE9IC 5 Q7D .times. 2 96 7
3EE9IC 10 Q7D .times. 2 100 10 3EE9IC 0.625 Q1D .times. 1 42 0
3EE9IC 1.25 Q1D .times. 1 48 0 3EE9IC 2.5 Q1D .times. 1 93 5 3EE9IC
5 Q1D .times. 1 98 8 3EE9IC 10 Q1D .times. 1 100 10 MMAE 1 Q4D
.times. 3 Toxic N/A MMAE 0.1 Q4D .times. 3 27 0 MMAE 0.2 Q4D
.times. 3 60 0
Example 15
Efficacy of 3ee9/MMAE in PC3 mm2 and Colo-205 Xenograft Models
[0287] The anti-tumor activity of 3ee9/MMAE was next evaluated
against human prostate (PC-3 mm2) and CRC (Colo-205) tumor
xenografts. Female NCr nu/nu mice were implanted subcutaneously
(s.c.) either with 5.times.10.sup.6 PC3 mm2 or Colo-205 cells.
Treatment was initiated when tumors were of an average size of
approximately 125-150 mg. The general health of mice was monitored
and recorded daily. Tumor dimensions and body weights were recorded
twice a week starting with the first day of treatment.
[0288] In the PC3 mm2 and Colo-205 studies, 3ee9/MMAE was
administered intravenously (i.v.) once every 4th day for a total of
3 injections (Q4Dx3) at doses of 1, 3, 10 and 30 mg/kg and 1.25,
2.5, 5 and 10 mg/kg, respectively. As in other studies, 3ee9/MMAE
was well tolerated at all doses evaluated. 3ee9/MMAE inhibited the
growth of established PC3MM2 prostate tumors when administered at
dose levels of 10 and 30 mg/kg, with 100% of the animals showing
responses (FIG. 22). At the lower doses of 1 and 3 mg/kg, only
moderate effects were seen, as 45 to 50% TGI was observed at the
end of dosing. 20% of the animals did, however, show responses in
the 3 mg/kg group.
[0289] Similarly in the Colo-205 CRC xenograft model, 3ee9/MMAE was
highly effective in inhibiting tumor growth (FIG. 23). At the end
of dosing, >90% TGI was observed for 5 and 10 mg/kg doses and
>70% TGI was seen in response to 2.5 mg/kg dose. The lowest
dose, 1 mg/kg, of 3ee9/MMAE was relatively ineffective against this
CRC model. In terms of regressions, 3ee9/MMAE dosed at 5 and 10
mg/kg resulted in 40 and 80% responses, respectively.
Example 16
Efficacy of 3ee9/MMAE in HCT-15 Xenograft Model
[0290] To examine the effect of 3ee9/MMAE in a multi-drug
resistance model, female NCr nu/nu mice were implanted
subcutaneously (s.c.) with 5.times.10.sup.6 HCT-15 cells. HCT-15 is
a multi-drug resistant (MDR) line that over-expresses
P-glycoprotein (P-gp). As such, this cell line exhibits a
multi-drug resistance phenotype and is resistant to Taxol.RTM.,
doxorubicin and etoposide. 3ee9/MMAE, Taxol, and Gemcitabine were
administered when mice had established tumors with mean weight of
.about.150 mg. Gemcitabine, a pyrimidine analog that is not a P-gp
substrate was used as a positive control. As expected, Gemcitabine
(120 mg/kg, i.p., QDx10) was highly active in this MDR model (FIG.
24). In contrast, 3ee9/MMAE (MMAE being a known substrate of P-gp)
and Taxol.RTM. failed to produce any significant anti-tumor
effect.
Example 17
Efficacy of 3ee9/MMAE in huMN-MIAPaca2 and MIAPaca2 Xenograft
Models
[0291] To better understand the relationship between MN expression
and antitumor efficacy, xenograft efficacy studies were conducted
using a low MN-expressing pancreatic model, MIAPaCa2 and high
MN-expressing huMN-MIAPaCa2 model. This latter line was derived by
engineering the MIAPaCa2 line to stably express MN. 3ee9/MMAE had
minimal effect in inhibiting the growth of this low MN-expressing
MIAPaCa2 line (FIG. 25). 3ee9/MMAE dosed at 10 mg/kg only produced
50% TGI. In contrast, significant anti-tumor activity was seen for
3ee9/MMAE against huMN-MIAPaCa2 model, where, at the end of dosing,
doses of 2.5, 5 and 10 mg/kg yielded TGI of 63, 82 and 94%,
respectively. Moreover, the 10 mg/kg dose induced 100% responses,
indicating that there is a great shift in sensitivity by
over-expressing MN levels in tumors.
Example 18
Combination with Xeloda.RTM. in Colo-205 Xenograft Model
[0292] The feasibility of using 3ee9/MMAE in conjunction with other
cancer chemotherapeutic agents was investigated. Xeloda.RTM. is
used as first line treatment of patients with metastatic colorectal
carcinoma. The activity and tolerability of combined therapy using
3ee9/MMAE and Xeloda.RTM. was evaluated. 3ee9/MMAE was administered
i.v. on a Q4Dx3 schedule and at dose levels of 1.25, 2.5 and 10
mg/kg. Xeloda.RTM. was administered orally once daily for 9 days at
dose levels of 250 and 500 mg/kg. 3ee9/MMAE administered alone at
2.5 and 10 mg/kg resulted in robust tumor growth inhibition (TGI of
73 and 96%, respectively) (FIG. 26a). The 1.25 mg/kg group of
3ee9/MMAE, however, was relatively ineffective, resulting in only
31% TGI. Xeloda.RTM. administered alone at dose levels of 250 and
500 mg/kg also produced robust TGI of 78 and 86%, respectively.
[0293] In terms of responses, 10 mg/kg group of 3ee9/MMAE induced
80% response rate, whereas zero percent of the animals in both dose
groups of Xeloda showed any regressions. All combination treatment
groups were well tolerated and resulted in significant inhibition
of tumor growth as well response rates. Quantitative data detailing
the TGI and tumor responses by combining these two agents are shown
in FIGS. 26a and 26b, as well as Table 3, below. In the doses
examined, the anti-tumor activity of 3ee9/MMAE in combination with
Xeloda.RTM. was far superior to that of either 3ee9/MMAE or
Xeloda.RTM. administered as single agents. Finally, in regards to
tolerability, administration of these therapeutics was well
tolerated with no adverse reactions.
TABLE-US-00003 TABLE 3 Anti-tumor activity of BAY 79-4620 (3ee9-IC)
in combination with Xeloda .RTM. % Inhibition % Weight Dose (T/C)
Loss Treatment Schedule mg/kg/inj (d 17) (d 17) % Regressions
Untreated -- -- -- 0.3 0 79-4620 (3ee9 cj) Q4D .times. 3; IV 1.25
30.6 0.5 0 79-4620 (3ee9 cj) Q4D .times. 3; IV 2.5 73 0.1 0 79-4620
(3ee9 cj) Q4D .times. 3; IV 10 96.2 0.9 80 Xeloda Q1D .times. 9; PO
250 78 -0.5 0 Xeloda Q1D .times. 9; PO 500 86.1 -5.6 0 Xeloda (250
mg/kg) 79-4620 Q4D .times. 3; IV/Q1 1.25 92.1 -2 50 (3ee9cj) +
Xeloda D .times. 9; PO 79-4620 (3ee9 Q4D .times. 3; IV/Q1 2.5 91.4
-0.9 60 cj) + Xeloda D .times. 9; PO 79-4620 (3ee9 Q4D .times. 3;
IV/Q1 10 97.5 -0.4 100 cj) + Xeloda D .times. 9; PO Xeloda (500
mg/kg) 79-4620 (3ee9 Q4D .times. 3; IV/Q1 1.25 94.2 -0.3 70 cj) +
Xeloda D .times. 9; PO 79-4620 (3ee9 Q4D .times. 3; IV/Q1 2.5 95.7
-3.2 90 cj) + Xeloda D .times. 9; PO 79-4620 (3ee9 Q4D .times. 3;
IV/Q1 10 97.3 -1.4 100 cj) + Xeloda D .times. 9; PO
Example 19
In Vivo Distribution/Tumor Localization of 3ee9 mAb Against
Bilaterally Implanted Human Xenograft Cancer Model
[0294] To determine the in vivo distribution and tumor localization
of the mAb component of 3ee9/MMAE, non-invasive in vivo imaging
studies were conducted using the CRI Maestro.TM. in mice which
received bilateral implantation of tumors exhibiting both high and
low MN expression. The Maestro.TM. in-vivo imaging system (CRI,
Woburn, Mass.) with multi-spectral acquisition and analysis is
designed to eliminate auto-fluorescence background. Mabs 3ee9 of
BAY 79-4620, M75 (mouse monoclonal recognizing MN) and control
human IgG, were conjugated with Alexa Fluor 750 (Invitrogen Cat#
A20011) according to manufacturer's instructions. The ratio of
Protein/AF750 (mol/mol) was 1/5.9 for 3ee9, 8.9 for human IgG, and
6.0 for M75.
[0295] Twelve days before the injection of each
fluorescence-labeled antibody, Harlan Balb/C nudes were implanted
with MIAPaCa-2 (low MN-expressing; 7.5.times.10.sup.6 cells in 50%
Matrigel) subcutaneously in the right flank, and huMN-MIAPaCa2 (MN
transfected stable cell line; 5.times.10.sup.6 cells in 50%
Matrigel) subcutaneously in the left flank. 4 .mu.g of conjugated
antibody was injected on Day 0 (12 days post implant) to each
animal. Imaging was done on Day 4, Day 5 and Day 10. Data were
collected using animals that were anaesthetized (100 mg/kg
ketamine/10 mg/kg xylazine, i.p.), and placed inside the imaging
system. Multi-spectral image cubes (series of images) were acquired
with images spaced every 10 nm throughout the spectral range of 680
to 950 nm, which covered the Near Infrared (NIR) range.
[0296] Each image was exposed for 5 seconds. False-colored images
were synthesized from the spectral cube using Maestro software and
scaled to visible brightness using ImagePro Plus 6.0. Scaling was
identical to all images, with the brightest image slightly below
saturation level. Most fluorescently labeled antibodies tend to
localize to the liver and the bladder. After 4 to 5 days, most of
signal then decreases from the liver and the bladder and stabilizes
in the tumor. Images taken on Day 5 yielded the highest signal to
background ratio (FIG. 27.) After five days, there was very little
signal generated from the hIgG-injected animals, indicating that
hIgG failed to localized into tumor tissue. In contrast, both M75
and 3ee9 localized specifically to high MN-expressing
(huMN-MIAPaCa2) tumors. No localization was seen in low
MN-expressing MIAPaCa2 tumors in the same animals.
Example 20
In Vivo Mechanism of Action
[0297] To explore the in vivo mechanism of action of 3ee9/MMAE,
tumors were implanted subcutaneously at right flank of the mice
with 5.times.10.sup.6 HT-29 cells. Eight days later, athymic mice
bearing HT-29 tumors were treated with vehicle or with 3ee9/MMAE at
1.25 and 5 mg/kg (Q1Dx1). Tumors were collected and fixed in
formalin 4 hours and 1, 3 and 5 hours following administration of
3ee9/MMAE. Samples were then embedded in paraffin, sectioned at 5
.mu.m, de-paraffinized, and stained using standard protocol for
fluorescent immunohistochemistry of human tissues using mouse
antibodies: Anti-.alpha./.beta.-tubulin, Anti-phospho-Histone H3,
and DNA. The slides were then observed under a fluorescent
microscope, and representative images were taken through three
separate color channels.
[0298] 3ee9/MMAE had little effect in affecting cell mechanism in
the 4 hr samples. However, by day 1, increased numbers of cells
were in G2/M arrest, and multi-polar spindle was clearly seen. In
addition, decreased level of tubulin staining could be observed.
These effects were then highly amplified in the day 3 and day 5
samples. In fact, almost all cells in the 5 mg/kg dose group of
3ee9/MMAE on day 5 were severely affected by treatment (FIG. 28).
These data clearly indicate that the 3ee9/MMAE affected the growth
of cancer cells by tubulin inhibition, leading to G2/M arrest and
apoptosis.
[0299] Table 4, below, summarizes the behavior profile of
3ee9/MMAE.
TABLE-US-00004 TABLE 4 3ee9/MMAE profile Assay Result for 3ee9/MMAE
Affinity (Kd) 3.6 nM Cell binding (FACS): MN+/MN- +++/-
Internalization: MN+/MN- +++/- Cytotoxicity: MN+/MN- (ec50) 50
nM/>1 .mu.M Immunoprecipitation specific IHC specific In vivo
distribution normal In vivo activity: MED 1 mg/kg In vivo activity:
MTD 60 mg/kg
[0300] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the above paragraphs is not to be limited to particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention.
Sequence CWU 1
1
153130DNAHomo sapiens 1ggatttacct tttcttctta tggtatgtct
30230DNAHomo sapiens 2ggatttacct tttcttctta tggtatgcat 30330DNAHomo
sapiens 3ggatttacct tttctaataa tgctatgaat 30430DNAHomo sapiens
4ggatttacct tttctgatta ttctattaat 30530DNAHomo sapiens 5ggatttacct
tttcttctta tggtatttct 30630DNAHomo sapiens 6ggatttacct tttctaatta
tggtatttct 30751DNAHomo sapiens 7ggtatctctt ctcttggtag cactacctat
tatgcggata gcgtgaaagg c 51851DNAHomo sapiens 8gctatctctt attcttctag
ctctacctct tatgcggata gcgtgaaagg c 51951DNAHomo sapiens 9ggtatctctt
atgattctag caagacctat tatgcggata gcgtgaaagg c 511051DNAHomo sapiens
10aatatctctt attctggtag ctctacctat tatgcggata gcgtgaaagg c
511151DNAHomo sapiens 11ggtatctctt attctggtag ctctacctat tatgcggata
gcgtgaaagg c 511251DNAHomo sapiens 12tctatctctt attctggtag
caatacctat tatgcggata gcgtgaaagg c 511351DNAHomo sapiens
13gctatctctt attatggtag caatacctat tatgcggata gcgtgaaagg c
511436DNAHomo sapiens 14actggttctc ctggtacttt tatgcatggt gatcat
361524DNAHomo sapiens 15ctttcttata ctggttttgc tgtt 241624DNAHomo
sapiens 16cttacttata ctggtgctta tcgt 241727DNAHomo sapiens
17tttaagtatt ctggtggttc tgattct 271839DNAHomo sapiens 18cttaagcctt
atcgtcataa gaatggttgg tttgattat 391933DNAHomo sapiens 19atgaagccta
tgcgtggtta ttctggtgct gtt 332024DNAHomo sapiens 20cttaagggtg
gttctggttt tgtt 242130DNAHomo sapiens 21ggatatacct ttactactaa
ttatatgcat 302251DNAHomo sapiens 22attatcaatc cgcataatgg ctctacgtct
tacgcgcaga agtttcaggg c 512324DNAHomo sapiens 23ggtcgttatt
ttcttatgga tgtt 242430DNAHomo sapiens 24ggatattcct tttctaagta
ttggattggt 302527DNAHomo sapiens 25ggatattcct ttactggtta tatttct
272651DNAHomo sapiens 26attatctatc cgactgatag ctatacccgt tattctccga
gctttcaggg c 512751DNAHomo sapiens 27attatctatc cgggtgatag
ctataccaat tattctccga gctttcaggg c 512836DNAHomo sapiens
28actcatggtt attataagaa tggtcgtatg gatgtt 362933DNAHomo sapiens
29tattctggtc ctaattggga tgttatggat tct 333033DNAHomo sapiens
30agagcgagcc agaatattct ttcttatctg aat 333133DNAHomo sapiens
31agagcgagcc agaatatttc taattatctg aat 333233DNAHomo sapiens
32agagcgagcc aggatatttc taatcgtctg gct 333333DNAHomo sapiens
33agagcgagcc aggatattaa taattatctg tct 333424DNAHomo sapiens
34tatgctgctt cttctttgca aagc 243524DNAHomo sapiens 35cataaggttt
ctaatttgca aagc 243624DNAHomo sapiens 36tatgatgcta attctttgca aagc
243724DNAHomo sapiens 37tatggtgctt ctaatttgca aagc 243821DNAHomo
sapiens 38cagcagtatg gttctgttcc t 213924DNAHomo sapiens
39cttcagtatg atgattttcc tcgt 244018DNAHomo sapiens 40tttcagtatt
ctggtcct 184124DNAHomo sapiens 41cagcagtatt atggtcgtcc tact
244248DNAHomo sapiens 42agaagcagcc aaagcctggt ttattctaat ggcaatacta
ctctgtct 484348DNAHomo sapiens 43agaagcagcc aaagcctggt tcattctaat
ggctataatt atctgtct 484424DNAHomo sapiens 44tatggtgttt ctaatcgtgc
cagt 244524DNAHomo sapiens 45tatcttggtt ctaatcgtgc cagt
244624DNAHomo sapiens 46cagcagtata attcttttcc tcgt 244724DNAHomo
sapiens 47catcagtatg gtgatttttc tgat 244839DNAHomo sapiens
48agcggcagca gcagcaacat tggttcttat tatgtgaat 394933DNAHomo sapiens
49cttctgattt atgctgatga taagcgtccc tca 335030DNAHomo sapiens
50cagtcttatg attctactaa ggatgattct 305133DNAHomo sapiens
51agcggcgata atcttggttc ttattatgtt cat 335233DNAHomo sapiens
52agcggcgata atcttcctga tttttatgtt cat 335333DNAHomo sapiens
53cttgtgattt atgatgataa taatcgtccc tca 335433DNAHomo sapiens
54cttgtgattt ctgaggataa taagcgtccc tca 335524DNAHomo sapiens
55cagtcttatg attttggtaa ggtt 245630DNAHomo sapiens 56tctacttatg
gttatactta ttcttattct 305710PRTHomo sapiens 57Gly Phe Thr Phe Ser
Ser Tyr Gly Met Ser1 5 105810PRTHomo sapiens 58Gly Phe Thr Phe Ser
Ser Tyr Gly Met His1 5 105910PRTHomo sapiens 59Gly Phe Thr Phe Ser
Asn Asn Ala Met Asn1 5 106010PRTHomo sapiens 60Gly Phe Thr Phe Ser
Asp Tyr Ser Ile Asn1 5 106110PRTHomo sapiens 61Gly Phe Thr Phe Ser
Ser Tyr Gly Ile Ser1 5 106210PRTHomo sapiens 62Gly Phe Thr Phe Ser
Asn Tyr Gly Ile Ser1 5 106317PRTHomo sapiens 63Gly Ile Ser Ser Leu
Gly Ser Thr Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly6417PRTHomo
sapiens 64Ala Ile Ser Tyr Ser Ser Ser Ser Thr Ser Tyr Ala Asp Ser
Val Lys1 5 10 15Gly6517PRTHomo sapiens 65Gly Ile Ser Tyr Asp Ser
Ser Lys Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly6617PRTHomo
sapiens 66Asn Ile Ser Tyr Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser
Val Lys1 5 10 15Gly6717PRTHomo sapiens 67Gly Ile Ser Tyr Ser Gly
Ser Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly6817PRTHomo
sapiens 68Ser Ile Ser Tyr Ser Gly Ser Asn Thr Tyr Tyr Ala Asp Ser
Val Lys1 5 10 15Gly6917PRTHomo sapiens 69Ala Ile Ser Tyr Tyr Gly
Ser Asn Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly7012PRTHomo
sapiens 70Thr Gly Ser Pro Gly Thr Phe Met His Gly Asp His1 5
10718PRTHomo sapiens 71Leu Ser Tyr Thr Gly Phe Ala Val1 5728PRTHomo
sapiens 72Leu Thr Tyr Thr Gly Ala Tyr Arg1 5739PRTHomo sapiens
73Phe Lys Tyr Ser Gly Gly Ser Asp Ser1 57413PRTHomo sapiens 74Leu
Lys Pro Tyr Arg His Lys Asn Gly Trp Phe Asp Tyr1 5 107511PRTHomo
sapiens 75Met Lys Pro Met Arg Gly Tyr Ser Gly Ala Val1 5
10768PRTHomo sapiens 76Leu Lys Gly Gly Ser Gly Phe Val1
57710PRTHomo sapiens 77Gly Tyr Thr Phe Thr Thr Asn Tyr Met His1 5
107817PRTHomo sapiens 78Ile Ile Asn Pro His Asn Gly Ser Thr Ser Tyr
Ala Gln Lys Phe Gln1 5 10 15Gly798PRTHomo sapiens 79Gly Arg Tyr Phe
Leu Met Asp Val1 58010PRTHomo sapiens 80Gly Tyr Ser Phe Ser Lys Tyr
Trp Ile Gly1 5 10819PRTHomo sapiens 81Gly Tyr Ser Phe Thr Gly Tyr
Ile Ser1 58217PRTHomo sapiens 82Ile Ile Tyr Pro Thr Asp Ser Tyr Thr
Arg Tyr Ser Pro Ser Phe Gln1 5 10 15Gly8317PRTHomo sapiens 83Ile
Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe Gln1 5 10
15Gly8412PRTHomo sapiens 84Thr His Gly Tyr Tyr Lys Asn Gly Arg Met
Asp Val1 5 108511PRTHomo sapiens 85Tyr Ser Gly Pro Asn Trp Asp Val
Met Asp Ser1 5 108611PRTHomo sapiens 86Arg Ala Ser Gln Asn Ile Leu
Ser Tyr Leu Asn1 5 108711PRTHomo sapiens 87Arg Ala Ser Gln Asn Ile
Ser Asn Tyr Leu Asn1 5 108811PRTHomo sapiens 88Arg Ala Ser Gln Asp
Ile Ser Asn Arg Leu Ala1 5 108911PRTHomo sapiens 89Arg Ala Ser Gln
Asp Ile Asn Asn Tyr Leu Ser1 5 10908PRTHomo sapiens 90Tyr Ala Ala
Ser Ser Leu Gln Ser1 5918PRTHomo sapiens 91His Lys Val Ser Asn Leu
Gln Ser1 5928PRTHomo sapiens 92Tyr Asp Ala Asn Ser Leu Gln Ser1
5938PRTHomo sapiens 93Tyr Gly Ala Ser Asn Leu Gln Ser1 5947PRTHomo
sapiens 94Gln Gln Tyr Gly Ser Val Pro1 5958PRTHomo sapiens 95Leu
Gln Tyr Asp Asp Phe Pro Arg1 5966PRTHomo sapiens 96Phe Gln Tyr Ser
Gly Pro1 5978PRTHomo sapiens 97Gln Gln Tyr Tyr Gly Arg Pro Thr1
59816PRTHomo sapiens 98Arg Ser Ser Gln Ser Leu Val Tyr Ser Asn Gly
Asn Thr Thr Leu Ser1 5 10 159916PRTHomo sapiens 99Arg Ser Ser Gln
Ser Leu Val His Ser Asn Gly Tyr Asn Tyr Leu Ser1 5 10 151008PRTHomo
sapiens 100Tyr Gly Val Ser Asn Arg Ala Ser1 51018PRTHomo sapiens
101Tyr Leu Gly Ser Asn Arg Ala Ser1 51028PRTHomo sapiens 102Gln Gln
Tyr Asn Ser Phe Pro Arg1 51038PRTHomo sapiens 103His Gln Tyr Gly
Asp Phe Ser Asp1 510413PRTHomo sapiens 104Ser Gly Ser Ser Ser Asn
Ile Gly Ser Tyr Tyr Val Asn1 5 1010511PRTHomo sapiens 105Leu Leu
Ile Tyr Ala Asp Asp Lys Arg Pro Ser1 5 1010610PRTHomo sapiens
106Gln Ser Tyr Asp Ser Thr Lys Asp Asp Ser1 5 1010711PRTHomo
sapiens 107Ser Gly Asp Asn Leu Gly Ser Tyr Tyr Val His1 5
1010811PRTHomo sapiens 108Ser Gly Asp Asn Leu Pro Asp Phe Tyr Val
His1 5 1010911PRTHomo sapiens 109Leu Val Ile Tyr Asp Asp Asn Asn
Arg Pro Ser1 5 1011011PRTHomo sapiens 110Leu Val Ile Ser Glu Asp
Asn Lys Arg Pro Ser1 5 101118PRTHomo sapiens 111Gln Ser Tyr Asp Phe
Gly Lys Val1 511210PRTHomo sapiens 112Ser Thr Tyr Gly Tyr Thr Tyr
Ser Tyr Ser1 5 10113347DNAHomo sapiens 113gaattggttc agagcggcgc
ggaagtgaaa aaaccgggcg cgagcgtgaa agtgagctgc 60aaagcctccg gatatacctt
tactactaat tatatgcatt gggtccgcca agcccctggg 120cagggtctcg
agtggatggg cattatcaat ccgcataatg gctctacgtc ttacgcgcag
180aagtttcagg gccgggtgac catgacccgt gataccagca ttagcaccgc
gtatatggaa 240ctgagcagcc tgcgtagcga agatacggcc gtgtattatt
gcgcgcgtgg tcgttatttt 300cttatggatg tttggggcca aggcaccctg
gtgacggtta gctcagc 347114324DNAHomo sapiens 114gatatccaga
tgacccagag cccgtctagc ctgagcgcga gcgtgggtga tcgtgtgacc 60attacctgca
gagcgagcca gaatattctt tcttatctga attggtacca gcagaaacca
120ggtaaagcac cgaaactatt aatttatgct gcttcttctt tgcaaagcgg
ggtcccgtcc 180cgttttagcg gctctggatc cggcactgat tttaccctga
ccattagcag cctgcaacct 240gaagactttg cggtttatta ttgccagcag
tatggttctg ttcctacctt tggccagggt 300acgaaagttg aaattaaacg tacg
324115347DNAHomo sapiens 115gaattggtgg aaagcggcgg cggcctggtg
caaccgggcg gcagcctgcg tctgagctgc 60gcggcctccg gatttacctt ttctaataat
gctatgaatt gggtgcgcca agcccctggg 120aagggtctcg agtgggtgag
cggtatctct tatgattcta gcaagaccta ttatgcggat 180agcgtgaaag
gccgttttac catttcacgt gataattcga aaaacaccct gtatctgcaa
240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtct
tacttatact 300ggtgcttatc gttggggcca aggcaccctg gtgacggtta gctcagc
347116311DNAHomo sapiens 116gatatcgaac tgacccagcc gccttcagtg
agcgttgcac caggtcagac cgcgcgtatc 60tcgtgtagcg gcgataatct tggttcttat
tatgttcatt ggtaccagca gaaacccggg 120caggcgccag ttcttgtgat
ttatgatgat aataatcgtc cctcaggcat cccggaacgc 180tttagcggat
ccaacagcgg caacaccgcg accctgacca ttagcggcac tcaggcggaa
240gacgaagcgg attattattg ccagtcttat gattttggta aggttgtgtt
tggcggcggc 300acgaagttaa c 311117350DNAHomo sapiens 117gaattggtgg
aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg tctgagctgc 60gcggcctccg
gatttacctt ttctgattat tctattaatt gggtgcgcca agcccctggg
120aagggtctcg agtatgtgag caatatctct tattctggta gctctaccta
ttatgcggat 180agcgtgaaag gccgttttac catttcacgt gataattcga
aaaacaccct gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc
gtgtattatt gcgcgcgttt taagtattct 300ggtggttctg attcttgggg
ccaaggcacc ctggtgacgg ttagctcagc 350118317DNAHomo sapiens
118gatatcgaac tgacccagcc gccttcagtg agcgttgcac caggtcagac
cgcgcgtatc 60tcgtgtagcg gcgataatct tcctgatttt tatgttcatt ggtaccagca
gaaacccggg 120caggcgccag ttcttgtgat ttctgaggat aataagcgtc
cctcaggcat cccggaacgc 180tttagcggat ccaacagcgg caacaccgcg
accctgacca ttagcggcac tcaggcggaa 240gacgaagcgg attattattg
ctctacttat ggttatactt attcttattc tgtgtttggc 300ggcggcacga agttaac
317119362DNAHomo sapiens 119gaattggtgg aaagcggcgg cggcctggtg
caaccgggcg gcagcctgcg tctgagctgc 60gcggcctccg gatttacctt ttcttcttat
ggtatttctt gggtgcgcca agcccctggg 120aagggtctcg agtgggtgag
cggtatctct tattctggta gctctaccta ttatgcggat 180agcgtgaaag
gccgttttac catttcacgt gataattcga aaaacaccct gtatctgcaa
240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtct
taagccttat 300cgtcataaga atggttggtt tgattattgg ggccaaggca
ccctggtgac ggttagctca 360gc 362120327DNAHomo sapiens 120gatatccaga
tgacccagag cccgtctagc ctgagcgcga gcgtgggtga tcgtgtgacc 60attacctgca
gagcgagcca gaatatttct aattatctga attggtacca gcagaaacca
120ggtaaagcac cgaaactatt aattcataag gtttctaatt tgcaaagcgg
ggtcccgtcc 180cgttttagcg gctctggatc cggcactgat tttaccctga
ccattagcag cctgcaacct 240gaagactttg cggtttatta ttgccttcag
tatgatgatt ttcctcgtac ctttggccag 300ggtacgaaag ttgaaattaa acgtacg
327121356DNAHomo sapiens 121gaattggtgg aaagcggcgg cggcctggtg
caaccgggcg gcagcctgcg tctgagctgc 60gcggcctccg gatttacctt ttcttcttat
ggtatttctt gggtgcgcca agcccctggg 120aagggtctcg agtgggtgag
ctctatctct tattctggta gcaataccta ttatgcggat 180agcgtgaaag
gccgttttac catttcacgt gataattcga aaaacaccct gtatctgcaa
240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtat
gaagcctatg 300cgtggttatt ctggtgctgt ttggggccaa ggcaccctgg
tgacggttag ctcagc 356122321DNAHomo sapiens 122gatatccaga tgacccagag
cccgtctagc ctgagcgcga gcgtgggtga tcgtgtgacc 60attacctgca gagcgagcca
ggatatttct aatcgtctgg cttggtacca gcagaaacca 120ggtaaagcac
cgaaactatt aatttatgat gctaattctt tgcaaagcgg ggtcccgtcc
180cgttttagcg gctctggatc cggcactgat tttaccctga ccattagcag
cctgcaacct 240gaagactttg cgacttatta ttgctttcag tattctggtc
ctacctttgg ccagggtacg 300aaagttgaaa ttaaacgtac g 321123347DNAHomo
sapiens 123gaattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt ttctaattat ggtatttctt gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cgctatctct tattatggta
gcaataccta ttatgcggat 180agcgtgaaag gccgttttac catttcacgt
gataattcga aaaacaccct gtatctgcaa 240atgaacagcc tgcgtgcgga
agatacggcc gtgtattatt gcgcgcgtct taagggtggt 300tctggttttg
tttggggcca aggcaccctg gtgacggtta gctcagc 347124342DNAHomo sapiens
124gatatcgtga tgacccagag cccactgagc ctgccagtga ctccgggcga
gcctgcgagc 60attagctgca gaagcagcca aagcctggtt tattctaatg gcaatactac
tctgtcttgg 120taccttcaaa aaccaggtca aagcccgcag ctattaattt
atggtgtttc taatcgtgcc 180agtggggtcc cggatcgttt tagcggctct
ggatccggca ccgattttac cctgaaaatt 240agccgtgtgg aagctgaaga
cgtgggcgtg tattattgcc agcagtataa ttcttttcct 300cgtacctttg
gccagggtac gaaagttgaa attaaacgta cg 342125359DNAHomo sapiens
125gaattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt ttcttcttat ggtatgtctt gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cggtatctct tctcttggta
gcactaccta ttatgcggat 180agcgtgaaag gccgttttac catttcacgt
gataattcga aaaacaccct gtatctgcaa 240atgaacagcc tgcgtgcgga
agatacggcc gtgtattatt gcgcgcgtac tggttctcct 300ggtactttta
tgcatggtga tcattggggc caaggcaccc tggtgacggt tagctcagc
359126327DNAHomo sapiens 126gatatccaga tgacccagag cccgtctagc
ctgagcgcga gcgtgggtga tcgtgtgacc 60attacctgca gagcgagcca ggatattaat
aattatctgt cttggtacca gcagaaacca 120ggtaaagcac cgaaactatt
aatttatggt gcttctaatt tgcaaagcgg ggtcccgtcc 180cgttttagcg
gctctggatc cggcactgat tttaccctga ccattagcag cctgcaacct
240gaagactttg cggtttatta ttgccagcag tattatggtc gtcctactac
ctttggccag 300ggtacgaaag ttgaaattaa acgtacg 327127359DNAHomo
sapiens 127gaattggttc agagcggcgc ggaagtgaaa aaaccgggcg aaagcctgaa
aattagctgc 60aaaggttccg gatattcctt ttctaagtat tggattggtt gggtgcgcca
gatgcctggg 120aagggtctcg agtggatggg cattatctat ccgactgata
gctatacccg ttattctccg 180agctttcagg gccaggtgac cattagcgcg
gataaaagca ttagcaccgc gtatcttcaa 240tggagcagcc tgaaagcgag
cgatacggcc atgtattatt gcgcgcgtac tcatggttat 300tataagaatg
gtcgtatgga tgtttggggc caaggcaccc tggtgacggt tagctcagc
359128342DNAHomo sapiens 128gatatcgtga tgacccagag cccactgagc
ctgccagtga ctccgggcga gcctgcgagc 60attagctgca
gaagcagcca aagcctggtt cattctaatg gctataatta tctgtcttgg
120taccttcaaa aaccaggtca aagcccgcag ctattaattt atcttggttc
taatcgtgcc 180agtggggtcc cggatcgttt tagcggctct ggatccggca
ccgattttac cctgaaaatt 240agccgtgtgg aagctgaaga cgtgggcgtg
tattattgcc atcagtatgg tgatttttct 300gatacctttg gccagggtac
gaaagttgaa attaaacgta cg 342129353DNAHomo sapiens 129gaattggttc
agagcggcgc ggaagtgaaa aaaccgggcg aaagcctgaa aattagctgc 60aaaggttccg
gatattcctt tactggttat atttcttggg tgcgccaagc ccctgggaag
120ggtctcgagt ggatgggcat tatctatccg ggtgatagct ataccaatta
ttctccgagc 180tttcagggcc aggtgaccat tagcgcggat aaaagcatta
gcaccgcgta tcttcaatgg 240agcagcctga aagcgagcga tacggccatg
tattattgcg cgcgttattc tggtcctaat 300tgggatgtta tggattcttg
gggccaaggc accctggtga cggttagctc agc 353130323DNAHomo sapiens
130gatatcgtgc tgacccagcc gccttcagtg agtggcgcac caggtcagcg
tgtgaccatc 60tcgtgtagcg gcagcagcag caacattggt tcttattatg tgaattggta
ccagcagttg 120cccgggacgg cgccgaaact tctgatttat gctgatgata
agcgtccctc aggcgtgccg 180gatcgtttta gcggatccaa aagcggcacc
agcgcgagcc ttgcgattac gggcctgcaa 240agcgaagacg aagcggatta
ttattgccag tcttatgatt ctactaagga tgattctgtg 300tttggcggcg
gcacgaagtt aac 323131347DNAHomo sapiens 131gaattggtgg aaagcggcgg
cggcctggtg caaccgggcg gcagcctgcg tctgagctgc 60gcggcctccg gatttacctt
ttcttcttat ggtatgcatt gggtgcgcca agcccctggg 120aagggtctcg
agtatgtgag cgctatctct tattcttcta gctctacctc ttatgcggat
180agcgtgaaag gccgttttac catttcacgt gataattcga aaaacaccct
gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt
gcgcgcgtct ttcttatact 300ggttttgctg tttggggcca aggcaccctg
gtgacggtta gctcagc 347132311DNAHomo sapiens 132gatatcgaac
tgacccagcc gccttcagtg agcgttgcac caggtcagac cgcgcgtatc 60tcgtgtagcg
gcgataatct tggttcttat tatgttcatt ggtaccagca gaaacccggg
120caggcgccag ttcttgtgat ttatgatgat aataatcgtc cctcaggcat
cccggaacgc 180tttagcggat ccaacagcgg caacaccgcg accctgacca
ttagcggcac tcaggcggaa 240gacgaagcgg attattattg ccagtcttat
gattttggta aggttgtgtt tggcggcggc 300acgaagttaa c 311133115PRTHomo
sapiens 133Glu Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
Ser Val1 5 10 15Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr
Asn Tyr Met 20 25 30His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met Gly Ile 35 40 45Ile Asn Pro His Asn Gly Ser Thr Ser Tyr Ala
Gln Lys Phe Gln Gly 50 55 60Arg Val Thr Met Thr Arg Asp Thr Ser Ile
Ser Thr Ala Tyr Met Glu65 70 75 80Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95Gly Arg Tyr Phe Leu Met Asp
Val Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser
115134108PRTHomo sapiens 134Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asn Ile Leu Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Val Pro Thr 85 90 95Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105135115PRTHomo sapiens
135Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu1
5 10 15Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Asn Ala
Met 20 25 30Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
Ser Gly 35 40 45Ile Ser Tyr Asp Ser Ser Lys Thr Tyr Tyr Ala Asp Ser
Val Lys Gly 50 55 60Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr Leu Gln65 70 75 80Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Arg 85 90 95Leu Thr Tyr Thr Gly Ala Tyr Arg Trp
Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser 115136106PRTHomo
sapiens 136Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro
Gly Gln1 5 10 15Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Gly Ser
Tyr Tyr Val 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val
Leu Val Ile Tyr 35 40 45Asp Asp Asn Asn Arg Pro Ser Gly Ile Pro Glu
Arg Phe Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile
Ser Gly Thr Gln Ala Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln
Ser Tyr Asp Phe Gly Lys Val Val 85 90 95Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 105137116PRTHomo sapiens 137Glu Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu1 5 10 15Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asp Tyr Ser Ile 20 25 30Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser Asn 35 40 45Ile Ser Tyr
Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln65 70 75
80Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
85 90 95Phe Lys Tyr Ser Gly Gly Ser Asp Ser Trp Gly Gln Gly Thr Leu
Val 100 105 110Thr Val Ser Ser 115138105PRTHomo sapiens 138Asp Ile
Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln1 5 10 15Thr
Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Pro Asp Phe Tyr Val 20 25
30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Ser
35 40 45Glu Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly
Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln
Ala Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Ser Thr Tyr Gly Tyr
Thr Tyr Ser Tyr 85 90 95Ser Val Phe Gly Gly Gly Thr Lys Leu 100
105139120PRTHomo sapiens 139Glu Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu1 5 10 15Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr Gly Ile 20 25 30Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val Ser Gly 35 40 45Ile Ser Tyr Ser Gly Ser Ser
Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln65 70 75 80Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95Leu Lys Pro
Tyr Arg His Lys Asn Gly Trp Phe Asp Tyr Trp Gly Gln 100 105 110Gly
Thr Leu Val Thr Val Ser Ser 115 120140109PRTHomo sapiens 140Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Ser Asn Tyr 20 25
30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45His Lys Val Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Tyr Asp
Asp Phe Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg Thr 100 105141118PRTHomo sapiens 141Glu Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu1 5 10 15Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr Gly Ile 20 25 30Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser 35 40 45Ile Ser Tyr Ser
Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln65 70 75 80Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90
95Met Lys Pro Met Arg Gly Tyr Ser Gly Ala Val Trp Gly Gln Gly Thr
100 105 110Leu Val Thr Val Ser Ser 115142107PRTHomo sapiens 142Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Arg
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Asp Ala Asn Ser Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Tyr
Ser Gly Pro Thr Phe 85 90 95Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr 100 105143115PRTHomo sapiens 143Glu Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu1 5 10 15Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asn Tyr Gly Ile 20 25 30Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val Ser Ala 35 40 45Ile Ser Tyr Tyr Gly
Ser Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln65 70 75 80Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95Leu
Lys Gly Gly Ser Gly Phe Val Trp Gly Gln Gly Thr Leu Val Thr 100 105
110Val Ser Ser 115144114PRTHomo sapiens 144Asp Ile Val Met Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile
Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser 20 25 30Asn Gly Asn Thr
Thr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu
Leu Ile Tyr Gly Val Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Tyr
85 90 95Asn Ser Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 110Arg Thr145119PRTHomo sapiens 145Glu Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu1 5 10 15Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met 20 25 30Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly 35 40 45Ile Ser
Ser Leu Gly Ser Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln65 70 75
80Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
85 90 95Thr Gly Ser Pro Gly Thr Phe Met His Gly Asp His Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115146109PRTHomo sapiens
146Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Asn
Tyr 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Tyr Gly Arg Pro Thr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr 100 105147119PRTHomo sapiens 147Glu Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu1 5 10 15Lys Ile Ser Cys
Lys Gly Ser Gly Tyr Ser Phe Ser Lys Tyr Trp Ile 20 25 30Gly Trp Val
Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile 35 40 45Ile Tyr
Pro Thr Asp Ser Tyr Thr Arg Tyr Ser Pro Ser Phe Gln Gly 50 55 60Gln
Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln65 70 75
80Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg
85 90 95Thr His Gly Tyr Tyr Lys Asn Gly Arg Met Asp Val Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115148114PRTHomo sapiens
148Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His
Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys His Gln Tyr 85 90 95Gly Asp Phe Ser Asp Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr149117PRTHomo sapiens
149Glu Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu1
5 10 15Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Gly Tyr Ile
Ser 20 25 30Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly
Ile Ile 35 40 45Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe
Gln Gly Gln 50 55 60Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala
Tyr Leu Gln Trp65 70 75 80Ser Ser Leu Lys Ala Ser Asp Thr Ala Met
Tyr Tyr Cys Ala Arg Tyr 85 90 95Ser Gly Pro Asn Trp Asp Val Met Asp
Ser Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
115150107PRTHomo sapiens 150Asp Ile Val Leu Thr Gln Pro Pro Ser Val
Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Ser Tyr 20 25 30Tyr Val Asn Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ala Asp Asp Lys Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65 70 75 80Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Thr Lys 85 90 95Asp Asp Ser
Val Phe Gly Gly Gly Thr Lys Leu 100 105151115PRTHomo sapiens 151Glu
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu1 5 10
15Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met
20 25 30His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser
Ala 35 40 45Ile Ser Tyr Ser Ser Ser Ser Thr Ser Tyr Ala Asp Ser Val
Lys Gly 50 55 60Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu Gln65 70 75 80Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg 85
90 95Leu Ser Tyr Thr Gly Phe Ala Val Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110Val Ser Ser 115152103PRTHomo sapiens 152Asp Ile Glu
Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln1 5 10 15Thr Ala
Arg Ile Ser Cys Ser Gly Asp Asn Leu Gly Ser Tyr Tyr Val 20 25 30His
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40
45Asp Asp Asn Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala
Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Phe Gly
Lys Val Val 85 90 95Phe Gly Gly Gly Thr Lys Leu
1001536675DNAArtificial SequenceDescription of Artificial Sequence
Synthetic nucleotide construct 153aattggaggc tacagtcagt ggagaggact
ttcactgact gactgactgc gtctcaacct 60ggggacaagt ttgtacaaaa aagcaggctc
tgtctagagg gcaccatggt gttgcagacc 120caggtcttca tttctctgtt
gctctggatc tctggtgcct acggggatat ccagatgacc 180cagagcccgt
ctagcctgag cgcgagcgtg ggtgatcgtg tgaccattac ctgcagagcg
240agccaggata ttaataatta tctgtcttgg taccagcaga aaccaggtaa
agcaccgaaa 300ctattaattt atggtgcttc taatttgcaa agcggggtcc
cgtcccgttt tagcggctct 360ggatccggca ctgattttac cctgaccatt
agcagcctgc aacctgaaga ctttgcggtt 420tattattgcc agcagtatta
tggtcgtcct actacctttg gccagggtac gaaagttgaa 480attaaacgta
cggtggctgc accatctgtc ttcatcttcc cgccatctga tgagcagttg
540aaatctggaa ctgcctctgt tgtgtgcctg ctgaataact tctatcccag
agaggccaaa 600gtacagtgga aggtggataa cgccctccaa tcgggtaact
cccaggagag tgtcacagag 660caggacagca aggacagcac ctacagcctc
agcagcaccc tgacgctgtc taaagcagac 720tacgagaaac acaaagtcta
cgcctgcgaa gtcacccatc agggcctgag ctcgcccgtc 780acaaagagct
tcaacagggg agagtgttag gcggccgcgc ctcgactgtg ccttctagtt
840gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
ggtgccactc 900ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
ttgtctgagt aggtgtcatt 960ctattctggg gggtggggtg gggcaggaca
gcaaggggga ggattgggaa gacaatagca 1020ggcatgctgg ggatgcggtg
ggctctatgg gatgcttatc gccacgttcg gcgcgccgtc 1080gacgatgtac
gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
1140tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
ataacttacg 1200gtaaatggcc cgcctggctg accgcccaac gacccccgcc
cattgacgtc aataatgacg 1260tatgttccca tagtaacgcc aatagggact
ttccattgac gtcaatgggt ggactattta 1320cggtaaactg cccacttggc
agtacatcaa gtgtatcata tgccaagtac gccccctatt 1380gacgtcaatg
acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
1440tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
gatgcggttt 1500tggcagtaca tcaatgggcg tggatagcgg tttgactcac
ggggatttcc aagtctccac 1560cccattgacg tcaatgggag tttgttttgg
caccaaaatc aacgggactt tccaaaatgt 1620cgtaacaact ccgccccatt
gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat 1680ataagcagag
ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat
1740acgactcact atagggagac ccaagctggc tagcgccacc atgaaacacc
tgtggttctt 1800cctcctgctg gtggcagctc ccagatgggt cctgtcccag
gtggaattgg tggaaagcgg 1860cggcggcctg gtgcaaccgg gcggcagcct
gcgtctgagc tgcgcggcct ccggatttac 1920cttttcttct tatggtatgt
cttgggtgcg ccaagcccct gggaagggtc tcgagtgggt 1980gagcggtatc
tcttctcttg gtagcactac ctattatgcg gatagcgtga aaggccgttt
2040taccatttca cgtgataatt cgaaaaacac cctgtatctg caaatgaaca
gcctgcgtgc 2100ggaagatacg gccgtgtatt attgcgcgcg tactggttct
cctggtactt ttatgcatgg 2160tgatcattgg ggccaaggca ccctggtgac
ggttagctca gcctccacca agggtccatc 2220ggtcttcccc ctggcaccct
cctccaagag cacctctggg ggcacagcgg ccctgggctg 2280cctggtcaag
gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac
2340cagcggcgtg cacaccttcc cggctgtcct acagtcctca ggactctact
ccctcagcag 2400cgtggtgacc gtgccctcca gcagcttggg cacccagacc
tacatctgca acgtgaatca 2460caagcccagc aacaccaagg tggacaagaa
agttgagccc aaatcttgtg acaaaactca 2520cacatgccca ccgtgcccag
cacctgaact cctgggggga ccgtcagtct tcctcttccc 2580cccaaaaccc
aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt
2640ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg
gcgtggaggt 2700gcataatgcc aagacaaagc cgcgggagga gcagtacaac
agcacgtacc gggtggtcag 2760cgtcctcacc gtcctgcacc aggactggct
gaatggcaag gagtacaagt gcaaggtctc 2820caacaaagcc ctcccagccc
ccatcgagaa aaccatctcc aaagccaaag ggcagccccg 2880agaaccacag
gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag
2940cctgacctgc ctggtcaaag gcttctatcc cagcgacatc gccgtggagt
gggagagcaa 3000tgggcagccg gagaacaact acaagaccac gcctcccgtg
ctggactccg acggctcctt 3060cttcctctac agcaagctca ccgtggacaa
gagcaggtgg cagcagggga acgtcttctc 3120atgctccgtg atgcatgagg
ctctgcacaa ccactacacg cagaagagcc tctccctgtc 3180tccgggtaaa
tgagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
3240ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
gtgccactcc 3300cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
tgtctgagta ggtgtcattc 3360tattctgggg ggtggggtgg ggcaggacag
caagggggag gattgggaag acaatagcag 3420gcatgctggg gatgcggtgg
gctctatggc ttctgaggcg gaaagaacca gcctcacgtg 3480gacccagctt
tcttgtacaa agtggtcccc ctacagagac gactgactga ctgactggaa
3540agaggaaggg ctggaagagg aaggagcttg gcgtaatcat ggtcatagct
gtttcctgtg 3600tgaaattgtt atccgctcac aattccacac aacatacgag
ccggaagcat aaagtgtaaa 3660gcctggggtg cctaatgagt gagctaactc
acattaattg cgttgcgctc actgcccgct 3720ttccagtcgg gaaacctgtc
gtgccagctg cattaatgaa tcggccaacg cgcggggaga 3780ggcggtttgc
gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
3840gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
atccacagaa 3900tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
agcaaaaggc caggaaccgt 3960aaaaaggccg cgttgctggc gtttttccat
aggctccgcc cccctgacga gcatcacaaa 4020aatcgacgct caagtcagag
gtggcgaaac ccgacaggac tataaagata ccaggcgttt 4080ccccctggaa
gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
4140tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
taggtatctc 4200agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
acgaaccccc cgttcagccc 4260gaccgctgcg ccttatccgg taactatcgt
cttgagtcca acccggtaag acacgactta 4320tcgccactgg cagcagccac
tggtaacagg attagcagag cgaggtatgt aggcggtgct 4380acagagttct
tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
4440tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
atccggcaaa 4500caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
agcagattac gcgcagaaaa 4560aaaggatctc aagaagatcc tttgatcttt
tctacggggt ctgacgctca gtggaacgaa 4620aactcacgtt aagggatttt
ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 4680ttaaattaaa
aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
4740agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
tcgttcatcc 4800atagttgcct gactccccgt cgtgtagata actacgatac
gggagggctt accatctggc 4860cccagtgctg caatgatacc gcgagaccca
cgctcaccgg ctccagattt atcagcaata 4920aaccagccag ccggaagggc
cgagcgcaga agtggtcctg caactttatc cgcctccatc 4980cagtctatta
attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
5040aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
tatggcttca 5100ttcagctccg gttcccaacg atcaaggcga gttacatgat
cccccatgtt gtgcaaaaaa 5160gcggttagct ccttcggtcc tccgatcgtt
gtcagaagta agttggccgc agtgttatca 5220ctcatggtta tggcagcact
gcataattct cttactgtca tgccatccgt aagatgcttt 5280tctgtgactg
gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
5340tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
tttaaaagtg 5400ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
ggatcttacc gctgttgaga 5460tccagttcga tgtaacccac tcgtgcaccc
aactgatctt cagcatcttt tactttcacc 5520agcgtttctg ggtgagcaaa
aacaggaagg caaaatgccg caaaaaaggg aataagggcg 5580acacggaaat
gttgaatact catactcttc ctttttcaat attattgaag catttatcag
5640ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
acaaataggg 5700gttccgcgca catttccccg aaaagtgcca cctgacgtct
aagaaaccat tattatcatg 5760acattaacct ataaaaatag gcgtatcacg
aggccctttc gtctcgcgcg tttcggtgat 5820gacggtgaaa acctctgaca
catgcagctc ccggagacgg tcacagcttg tctgtaagcg 5880gatgccggga
gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
5940tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccataa
aattgtaaac 6000gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
tcagctcatt ttttaaccaa 6060taggccgaaa tcggcaaaat cccttataaa
tcaaaagaat agcccgagat agggttgagt 6120gttgttccag tttggaacaa
gagtccacta ttaaagaacg tggactccaa cgtcaaaggg 6180cgaaaaaccg
tctatcaggg cgatggccca ctacgtgaac catcacccaa atcaagtttt
6240ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
ccgatttaga 6300gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
ggaagaaagc gaaaggagcg 6360ggcgctaggg cgctggcaag tgtagcggtc
acgctgcgcg taaccaccac acccgccgcg 6420cttaatgcgc cgctacaggg
cgcgtactat ggttgctttg acgtatgcgg tgtgaaatac 6480cgcacagatg
cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca
6540actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
gcgaaagggg 6600gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
ttcccagtca cgacgttgta 6660aaacgacggc cagtg 6675
* * * * *