U.S. patent application number 15/221049 was filed with the patent office on 2017-01-19 for anti-notch3 antibodies and antibody-drug conjugates.
This patent application is currently assigned to Pfizer Inc.. The applicant listed for this patent is Pfizer Inc.. Invention is credited to Yijie Gao, Kenneth G. Geles, Puja Sapra, Lioudmila Gennadievna Tchistiakova, Bin-Bing Stephen Zhou.
Application Number | 20170014526 15/221049 |
Document ID | / |
Family ID | 49585464 |
Filed Date | 2017-01-19 |
United States Patent
Application |
20170014526 |
Kind Code |
A1 |
Geles; Kenneth G. ; et
al. |
January 19, 2017 |
ANTI-NOTCH3 ANTIBODIES AND ANTIBODY-DRUG CONJUGATES
Abstract
The present invention provides for anti-Notch3 antibodies,
anti-Notch3 antibody-drug conjugates and methods for preparing and
using the same.
Inventors: |
Geles; Kenneth G.; (Nyack,
NY) ; Gao; Yijie; (Chestnut Hill, MA) ; Sapra;
Puja; (River Edge, NJ) ; Tchistiakova; Lioudmila
Gennadievna; (Stoneham, MA) ; Zhou; Bin-Bing
Stephen; (Rohnert Park, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pfizer Inc. |
New York |
NY |
US |
|
|
Assignee: |
Pfizer Inc.
New York
NY
|
Family ID: |
49585464 |
Appl. No.: |
15/221049 |
Filed: |
July 27, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14073273 |
Nov 6, 2013 |
9433687 |
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15221049 |
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61889744 |
Oct 11, 2013 |
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61723772 |
Nov 7, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/3061 20130101;
C07K 2317/51 20130101; A61K 47/6817 20170801; C07K 2317/567
20130101; C07K 2317/66 20130101; C07K 16/3015 20130101; A61K
47/6855 20170801; A61K 39/395 20130101; A61K 47/6869 20170801; A61K
47/6851 20170801; A61K 47/6867 20170801; C07K 16/32 20130101; A61K
47/6863 20170801; A61P 35/02 20180101; C07K 16/3069 20130101; C07K
2317/565 20130101; C07K 16/3046 20130101; C07K 2317/515 20130101;
C07K 2317/34 20130101; A61K 47/6857 20170801; A61K 39/3955
20130101; C07K 16/28 20130101; A61P 35/00 20180101; C07K 16/30
20130101; C07K 2317/94 20130101; C07K 2317/77 20130101; C07K
16/3023 20130101; C07K 2317/732 20130101; A61K 47/6849 20170801;
C07K 2317/24 20130101; A61K 47/6889 20170801; A61K 2039/505
20130101; C07K 2317/33 20130101; C07K 16/2866 20130101; C07K
2317/75 20130101; C07K 2317/76 20130101; C07K 2317/56 20130101;
C07K 2317/92 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; C07K 16/30 20060101 C07K016/30; C07K 16/32 20060101
C07K016/32 |
Claims
1.-45. (canceled)
46. A method of treating a condition associated with Notch3
expression in a patient in need thereof, comprising administering
to the patient an effective amount of a pharmaceutical composition
comprising an antibody-drug conjugate of the formula Ab-(L-D)p,
wherein: (a) Ab is an antibody, or antigen-binding fragment
thereof, that binds to Notch3; (b) L-D is a linker-drug moiety,
wherein L is a linker and D is a drug, and wherein D is an
auristatin; and (c) p is an integer from 1 to 12.
47. The method of claim 46, wherein Ab comprises: a VH CDR1, a VH
CDR2, and a VH CDR3 of a heavy chain variable region (VH)
comprising the amino acid sequence set forth in SEQ ID NO: 13; and
a VL CDR1, a VL CDR2, and a VL CDR3 of a light chain variable
region (VL) comprising the amino acid sequence set forth in SEQ ID
NO: 25; or a VH CDR1, a VH CDR2, and a VH CDR3 of a heavy chain
variable region (VH) comprising the amino acid sequence set forth
in SEQ ID NO: 37; and a VL CDR1, a VL CDR2, and a VL CDR3 of a
light chain variable region (VL) comprising the amino acid sequence
set forth in SEQ ID NO: 49.
48. The method of claim 46, wherein Ab comprises: a VH CDR1
comprising the amino acid sequence of SEQ ID NO: 15 or 16; a VH
CDR2 comprising the amino acid sequence of SEQ ID NO: 19 or 20; a
VH CDR3 comprising the amino acid sequence of SEQ ID NO: 23; a VL
CDR1 comprising the amino acid sequence of SEQ ID NO: 27; a VL CDR2
comprising the amino acid sequence of SEQ ID NO: 29; and a VL CDR3
comprising the amino acid sequence of SEQ ID NO: 31; or a VH CDR1
comprising the amino acid sequence of SEQ ID NO: 39 or 40; a VH
CDR2 comprising the amino acid sequence of SEQ ID NO: 43 or 44; a
VH CDR3 comprising the amino acid sequence of SEQ ID NO: 47; a VL
CDR1 comprising the amino acid sequence of SEQ ID NO: 51; a VL CDR2
comprising the amino acid sequence of SEQ ID NO: 53; and a VL CDR3
comprising the amino acid sequence of SEQ ID NO: 55.
49. The method of claim 48, wherein Ab comprises: a heavy chain
variable region having an amino acid sequence that is at least 90%
identical to SEQ ID NO: 13 or a light chain variable region having
an amino acid sequence that is at least 90% identical to SEQ ID NO:
25; or a heavy chain variable region having an amino acid sequence
that is at least 90% identical to SEQ ID NO: 37 or a light chain
variable region having an amino acid sequence that is at least 90%
identical to SEQ ID NO: 49.
50. The method of claim 49, wherein Ab comprises a heavy chain
variable region amino acid sequence of SEQ ID NO: 13; or a heavy
chain variable region amino acid sequence of SEQ ID NO: 37.
51. The method of claim 49, wherein Ab comprises a light chain
variable region amino acid sequence of SEQ ID NO: 25 or a light
chain variable region amino acid sequence of SEQ ID NO: 49.
52. The method of claim 50, wherein Ab comprises a heavy chain
amino acid sequence of SEQ ID NO: 33; or a heavy chain amino acid
sequence of SEQ ID NO: 57.
53. The method of claim 51, wherein Ab comprises a light chain
amino acid sequence of SEQ ID NO: 35; or a light chain amino acid
sequence of SEQ ID NO: 59.
54. The method of claim 46, wherein L is selected from the group
consisting of
maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc),
maleimidocaproyl (mc), maleimido-heptanoyl (me) and
maleimido-Peg6C2 (MalPeg6C2).
55. The method of claim 46, wherein D is a drug selected from the
group consisting of: (i) 0101 having the formula: ##STR00016## (ii)
6780 having the formula: ##STR00017## (iii) 0131 having the
formula: ##STR00018## (iv) 3377 having the formula: ##STR00019##
and (v) 8261 having the formula: ##STR00020##
56. The method of claim 46, wherein L-D is selected from the group
consisting of: vc0101 having the formula: ##STR00021## and vc6780
having the formula: ##STR00022##
57. The method of claim 46, wherein the condition is cancer.
58. The method of claim 57, wherein the cancer is a solid tumor
cancer.
59. The method of claim 58, wherein the solid tumor cancer is
selected from the group consisting of lung cancer, breast cancer,
ovarian cancer, stomach cancer, esophageal cancer, cervical cancer,
head and neck cancer, bladder cancer, liver cancer, skin cancer and
sarcoma.
60. The method of claim 57, wherein the cancer is a blood
cancer.
61. The method of claim 60 wherein the blood cancer is selected
from the group consisting of T-cell malignancies, T-cell leukemia,
T-cell lymphoma, T-cell acute lymphoblastic leukemia, multiple
myeloma, B-cell malignancies, myeloid malignancies, acute myeloid
leukemia and chronic myeloid leukemia.
62. A method of inhibiting tumor growth or progression in a patient
having a Notch3 expressing cancer, comprising administering to the
patient an effective amount of a pharmaceutical composition
comprising an antibody-drug conjugate of the formula Ab-(L-D)p,
wherein: (a) Ab is an antibody, or antigen-binding fragment
thereof, that binds to Notch3; (b) L-D is a linker-drug moiety,
wherein L is a linker and D is a drug, and wherein D is an
auristatin; and (c) p is an integer from 1 to 12.
63. The method of claim 62, wherein Ab comprises: a VH CDR1, a VH
CDR2, and a VH CDR3 of a heavy chain variable region (VH)
comprising the amino acid sequence set forth in SEQ ID NO: 13; and
a VL CDR1, a VL CDR2, and a VL CDR3 of a light chain variable
region (VL) comprising the amino acid sequence set forth in SEQ ID
NO: 25; or a VH CDR1, a VH CDR2, and a VH CDR3 of a heavy chain
variable region (VH) comprising the amino acid sequence set forth
in SEQ ID NO: 37; and a VL CDR1, a VL CDR2, and a VL CDR3 of a
light chain variable region (VL) comprising the amino acid sequence
set forth in SEQ ID NO: 49.
64. The method of claim 62, wherein Ab comprises: a VH CDR1
comprising the amino acid sequence of SEQ ID NO: 15 or 16; a VH
CDR2 comprising the amino acid sequence of SEQ ID NO: 19 or 20; a
VH CDR3 comprising the amino acid sequence of SEQ ID NO: 23; a VL
CDR1 comprising the amino acid sequence of SEQ ID NO: 27; a VL CDR2
comprising the amino acid sequence of SEQ ID NO: 29; and a VL CDR3
comprising the amino acid sequence of SEQ ID NO: 31; or a VH CDR1
comprising the amino acid sequence of SEQ ID NO: 39 or 40; a VH
CDR2 comprising the amino acid sequence of SEQ ID NO: 43 or 44; a
VH CDR3 comprising the amino acid sequence of SEQ ID NO: 47; a VL
CDR1 comprising the amino acid sequence of SEQ ID NO: 51; a VL CDR2
comprising the amino acid sequence of SEQ ID NO: 53; and a VL CDR3
comprising the amino acid sequence of SEQ ID NO: 55.
65. The method of claim 64, wherein Ab comprises: a heavy chain
variable region having an amino acid sequence that is at least 90%
identical to SEQ ID NO: 13 or a light chain variable region having
an amino acid sequence that is at least 90% identical to SEQ ID NO:
25; or a heavy chain variable region having an amino acid sequence
that is at least 90% identical to SEQ ID NO: 37 or a light chain
variable region having an amino acid sequence that is at least 90%
identical to SEQ ID NO: 49.
66. The method of claim 65, wherein Ab comprises a heavy chain
variable region amino acid sequence of SEQ ID NO: 13; or a heavy
chain variable region amino acid sequence of SEQ ID NO: 37.
67. The method of claim 65, wherein Ab comprises a light chain
variable region amino acid sequence of SEQ ID NO: 25 or a light
chain variable region amino acid sequence of SEQ ID NO: 49.
68. The method of claim 66, wherein Ab comprises a heavy chain
amino acid sequence of SEQ ID NO: 33; or a heavy chain amino acid
sequence of SEQ ID NO: 57.
69. The method of claim 67, wherein Ab comprises a light chain
amino acid sequence of SEQ ID NO: 35; or a light chain amino acid
sequence of SEQ ID NO: 59.
70. The method of claim 62, wherein L is selected from the group
consisting of
maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc),
maleimidocaproyl (mc), maleimido-heptanoyl (me) and
maleimido-Peg6C2 (MalPeg6C2).
71. The method of claim 62, wherein D is a drug selected from the
group consisting of: (i) 0101 having the formula: ##STR00023## (ii)
6780 having the formula: ##STR00024## (iii) 0131 having the
formula: ##STR00025## (iv) 3377 having the formula: ##STR00026##
and (v) 8261 having the formula: ##STR00027##
72. The method of claim 62, wherein L-D is selected from the group
consisting of: vc0101 having the formula: ##STR00028## and vc6780
having the formula: ##STR00029##
73. The method of claim 62, wherein the cancer is a solid tumor
cancer.
74. The method of claim 73, wherein the solid tumor cancer is
selected from the group consisting of lung cancer, breast cancer,
ovarian cancer, stomach cancer, esophageal cancer, cervical cancer,
head and neck cancer, bladder cancer, liver cancer, skin cancer and
sarcoma.
75. The method of claim 62, wherein the cancer is a blood
cancer.
76. The method of claim 75 wherein the blood cancer is selected
from the group consisting of T-cell malignancies, T-cell leukemia,
T-cell lymphoma, T-cell acute lymphoblastic leukemia, multiple
myeloma, B-cell malignancies, myeloid malignancies, acute myeloid
leukemia and chronic myeloid leukemia.
Description
RELATED APPLICATIONS
[0001] This application claims the benefits of U.S. Provisional
Application No. 61/723,772 filed Nov. 7, 2012 and U.S. Provisional
Application No. 61/889,744 filed Oct. 11, 2013, which are hereby
incorporated by reference in their entireties.
SEQUENCE LISTING
[0002] This application is being filed electronically via EFS-Web
and includes an electronically submitted sequence listing in .txt
format. The .txt file contains a sequence listing entitled
"PC071980A_Sequence_Listing.txt" created on Oct. 11, 2013, and
having a size of 61 KB. The sequence listing contained in this .txt
file is part of the specification and is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to anti-Notch3 antibodies and
anti-Notch3 antibody-drug conjugates. The present invention further
relates to methods of using such antibodies and antibody-drug
conjugates for the treatment of cancer.
BACKGROUND
[0004] Notch signaling is triggered by extracellular receptor and
ligand interactions. Notch receptors control normal cellular
proliferation, differentiation, and death in multicellular
organisms through a signaling cascade that is triggered by
ligand-induced proteolysis. After furin-like protease cleavage at
site S1, the mature Notch heterodimer is translocated into the cell
membrane where it is held in an auto-inhibited state by a
juxtamembrane Negative Regulatory Region (NRR) consisting of three
Lin12/Notch repeats (LNR-A, B, C) and the heterodimerization (HD)
domain. The HD domain is divided into N-terminal (HD1) and
C-terminal (HD2) halves after cleavage at site S1. Through an
uncertain mechanism, binding of ligands of the Delta/Serrate/Lag-2
(DSL) family to the extracellular EGF-repeat region relieves this
inhibition and induces two additional cleavage events. First,
ADAM-type metalloproteinase mediate cleavage at site S2 near the
C-terminal region of the HD-2 domain, thereby releasing the
extracellular domain (ECD) from the cell surface which then
undergoes trans-endocytosis into the ligand-expressing cell. Next,
gamma-secretase mediates cleavage at site S3 within the
transmembrane domain which releases the intracellular domain of
Notch (Notch-ICD) from the membrane, permitting it to translocate
to the nucleus and activate the transcription of target genes
(Bray, S., Nature Reviews Molecular Cell Biology, 2006, volume 7,
678-689).
[0005] The X-ray crystal structure of the human Notch2-NRR domain
in an auto-inhibited conformation revealed extensive interactions
between the LNR repeats and heterdimerization domains within the
NRR burying the metalloprotease S2 site, suggesting that a
substantial conformational movement is necessary to expose the site
during activation by ligand (Gordon, W. R., et. al, Nature
Structural & Molecular Biology, 2007, volume 14, 295-300).
Studies suggest that stabilization of the interactions within the
NRR may prevent ligand-induced Notch activation. The availability
of structural information on the Notch auto-inhibited conformation
provided new opportunities for the development of therapeutics,
particularly antibodies that target Notch signaling. (Li, K., et.
al, Journal of Biological Chemistry, 2008, volume 283, 8046-8054;
Aste-Amezaga, M, et. al, PLOS ONE, 2010, volume 5, e9094; Wu, Y.,
et. al, Nature, 2010, volume 464, 1052-1057;).
[0006] In mammalian cells, there are four known Notch receptors.
Notch-1, -2, -3 and -4 have broad, overlapping patterns of
expression in embryonic and adult tissues, and fulfill
non-redundant roles during hematopoietic stem cell specification, T
cell development, intestinal crypt cell specification and vascular
development. Notch3 is expressed primarily in vascular smooth
muscle cells (vSMC), various thymocyte subpopulations and the
developing nervous system. Consistent with its restricted tissue
distribution, targeted deletion of murine Notch3 does not lead to
embryonic lethality like Notch1 and Notch2 deletion. Instead,
Notch3-null mice are viable, but have defects in the maturation and
differentiation of vSMCs (Domenga, V., et. al, Genes and
Development, 2004, volume 18, 2730-2735).
[0007] Notch activation is oncogenic in many contexts;
constitutively active, intracellular forms of all four Notch
homologues function as oncogenes in vitro and in transgenic mouse
models. Recent studies indicate that Notch3 is often amplified and
overexpressed in various human solid tumors and the over-expression
of developmental signaling pathways, such as Notch3, in human
cancers implicates them as key mediators of tumorigenesis. Several
strategies are in development to block Notch signaling for
therapeutic purposes in cancer; however there is still a need in
the art for more potent and efficacious anti-Notch targeted
therapies for the treatment of cancer.
[0008] Antibody-drug conjugates (ADCs) combine the specificity and
targeting of high affinity antibodies with the cytotoxicity of a
therapeutic agent, such as cytotoxic agents, biological response
modifiers, enzymes, apoptosis-inducing agents, and radioisotopes.
Release of therapeutic agents from the antibody can require
trafficking and localization of the antibody-drug conjugate to
lysosomes and both Notch3-ECD and Notch3-ICD undergo lysosomal
degradation, thus antibodies that bind Notch3 are expected to
traffic to the lysosome (Jia L, et. al, International Journal of
Biochemistry and Cell Biology, 2009, volume 41, 2594-2598). The
present invention provides novel anti-Notch3 antibodies and
antibody-drug conjugates that fulfill an unmet clinical need in the
diagnosis and therapeutic use in the treatment of cancer.
SUMMARY
[0009] The present invention provides for anti-Notch3 antibodies
and antibody-drug conjugates (ADCs). The present invention also
provides for methods of using such anti-Notch3 antibodies and
antibody-drug conjugates for the treatment of cancer.
[0010] In one embodiment, the present invention provides for
isolated antibodies, or antigen-binding fragment thereof, that bind
to Notch3, having a CDR1, a CDR2, and a CDR3 of SEQ ID NO: 13 and,
a light chain variable region having a CDR1, a CDR2, and a CDR3 of
SEQ ID NO: 25.
[0011] In another embodiment, the present invention provides for
isolated antibodies, or antigen-binding fragment thereof, that bind
to Notch3, wherein the antibody or antigen-binding fragment: (a)
internalizes into a cell, (b) does not inhibit Notch3 signaling, or
(c) does not activate Notch3 signaling. In a further embodiment,
the present invention provides for isolated antibodies, or
antigen-binding fragment thereof, that bind to Notch3, wherein the
antibody or antigen-binding fragment: (a) binds to the LNR-C and
HD-1 domains of the Notch3 NRR, (b) does not maintain the Notch3
NRR in an auto-inhibitory conformation, or (c) does not inhibit
S2-cleavage.
[0012] In a further embodiment, the present invention provides for
isolated antibodies, or antigen-binding fragment thereof, that bind
to Notch3, having: (a) a heavy chain CDR1 comprising SEQ ID NO: 15
or 16; (b) a heavy chain CDR2 comprising SEQ ID NO: 19 or 20; (c) a
heavy chain CDR3 comprising SEQ ID NO: 23; (d) a light chain CDR1
comprising SEQ ID NO: 27; (e) a light chain CDR2 comprising SEQ ID
NO: 29; and, (f) a light chain CDR3 comprising SEQ ID NO: 31.
[0013] The present invention also provides for isolated antibodies,
or antigen-binding fragment thereof, that bind to Notch3, having a
heavy chain variable region amino acid sequence that is at least
90% identical to SEQ ID NO: 13 or a light chain variable region
amino acid sequence that is at least 90% identical to SEQ ID NO:
25.
[0014] The present invention also provides for isolated antibodies,
or antigen-binding fragment thereof, that bind to Notch3 having a
heavy chain variable region amino acid sequence of SEQ ID NO: 13
and isolated antibodies, or antigen-binding fragment thereof, that
bind to Notch3 having a light chain variable region amino acid
sequence of SEQ ID NO: 25. The present invention also provides for
isolated antibodies, or antigen-binding fragment thereof, that bind
to Notch3 having a heavy chain amino acid sequence of SEQ ID NO: 33
and isolated antibodies, or antigen-binding fragment thereof, that
bind Notch3 having a light chain amino acid sequence of SEQ ID NO:
35.
[0015] In another embodiment, the present invention provides for
isolated antibodies, or antigen-binding fragment thereof, that bind
to Notch3, having a heavy chain variable region comprising a CDR1,
a CDR2, and a CDR3 of SEQ ID NO: 37 and, a light chain variable
region having a CDR1, a CDR2, and a CDR3 of SEQ ID NO: 49.
[0016] In further embodiment, the present invention provides for
isolated antibodies, or antigen-binding fragment thereof, that bind
to Notch3, having: (a) a heavy chain CDR1 comprising SEQ ID NO: 39
or 40; (b) a heavy chain CDR2 comprising SEQ ID NO: 43 or 44; (c) a
heavy chain CDR3 comprising SEQ ID NO: 47; (d) a light chain CDR1
comprising SEQ ID NO: 51; (e) a light chain CDR2 comprising SEQ ID
NO: 53; and, (f) a light chain CDR3 comprising SEQ ID NO: 55.
[0017] The present invention also provides for isolated antibodies,
or antigen-binding fragment thereof, that bind to Notch3, having a
heavy chain variable region amino acid sequence that is at least
90% identical to SEQ ID NO: 37 or a light chain variable region
amino acid sequence that is at least 90% identical to SEQ ID NO:
49.
[0018] The present invention also provides for isolated antibodies,
or antigen-binding fragment thereof, that bind to Notch3 having a
heavy chain variable region amino acid sequence of SEQ ID NO: 37
and isolated antibodies, or antigen-binding fragment thereof, that
bind to Notch3 having a light chain variable region amino acid
sequence of SEQ ID NO: 49. The present invention also provides for
isolated antibodies, or antigen-binding fragment thereof, that bind
to Notch3 having a heavy chain amino acid sequence of SEQ ID NO: 57
and isolated antibodies, or antigen-binding fragment thereof, that
bind to Notch3 having a light chain amino acid sequence of SEQ ID
NO: 59.
[0019] The invention further provides for isolated antibodies that
compete with an antibody, or antigen-binding fragment thereof, of
the present invention for specific binding to Notch3.
[0020] The present invention further provides for antibody-drug
conjugates having a cytotoxic agent conjugated to any antibody, or
antigen-binding fragment thereof, of the present invention.
[0021] In another embodiment, the present invention provides for
antibody-drug conjugates of the formula: Ab-(L-D)p, or a
pharmaceutically acceptable salt thereof wherein; Ab is an
antibody, or antigen-binding fragment thereof, that binds to
Notch3; L-D is a linker-drug moiety, wherein L is a linker, and D
is a drug; and p is an integer from 1 to about 12.
[0022] In a further embodiment, the present invention provides
antibody-drug conjugates of the formula: Ab-(L-D)p, or a
pharmaceutically acceptable salt thereof wherein; Ab is any
antibody, or antigen-binding fragment thereof, of the present
invention; L-D is a linker-drug moiety, wherein L is a linker, and
D is a drug; and p is an integer from 1 to about 12.
[0023] In another embodiment, the present invention provides
antibody-drug conjugates wherein L is selected from the group
consisting of vc, mc, me and MalPeg6C2.
[0024] In another embodiment, the present invention provides
antibody-drug conjugates wherein D is selected from the group
consisting of: (a) 0101
(2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-me-
thyl-3-oxo-3-{[(1
S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-met-
hyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), (b) 6780
(2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1
S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl-
]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinami-
de), (c) 0131
(2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1
S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolid-
in-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,
trifluoroacetic acid salt), (d) 3377 (N,2-dimethylalanyl-N-{(1
S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1
S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolid-
in-1-yl}-2-methoxy-1-[(1
S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,
trifluoroacetic acid salt), and (e) 8261
(2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1
S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolid-
in-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide).
[0025] The present invention further provides for antibody-drug
conjugates wherein L-D is selected from the group consisting
of:
[0026] vc0101 having the formula:
##STR00001##
[0027] and vc6780 having the formula:
##STR00002##
[0028] In another embodiment, the present invention provides for
antibody-drug conjugates, wherein Ab comprises (a) a heavy chain
variable region having a CDR1, a CDR2, and a CDR3 of SEQ ID NO: 13;
and, (b) a light chain variable region having a CDR1, a CDR2, and a
CDR3 of SEQ ID NO: 25.
[0029] The present invention further provides for antibody-drug
conjugates, wherein Ab comprises (a) a heavy chain CDR1 comprising
SEQ ID NO: 15; (b) a heavy chain CDR2 comprising SEQ ID NO: 19; (c)
a heavy chain CDR3 comprising SEQ ID NO: 23; (d) a light chain CDR1
comprising SEQ ID NO: 27; (e) a light chain CDR2 comprising SEQ ID
NO: 29; and, (f) a light chain CDR3 comprising SEQ ID NO: 31.
[0030] In another embodiment, the present invention provides for
antibody-drug conjugates, wherein Ab comprises (a) a heavy chain
variable region comprising a CDR1, a CDR2, and a CDR3 of SEQ ID NO:
37; and, (b) a light chain variable region comprising a CDR1, a
CDR2, and a CDR3 of SEQ ID NO: 49.
[0031] The present invention further provides for antibody-drug
conjugates, wherein Ab comprises (a) a heavy chain CDR1 comprising
SEQ ID NO: 39; (b) a heavy chain CDR2 comprising SEQ ID NO: 43; (c)
a heavy chain CDR3 comprising SEQ ID NO: 47; (d) a light chain CDR1
comprising SEQ ID NO: 51; (e) a light chain CDR2 comprising SEQ ID
NO: 53; and, (f) a light chain CDR3 comprising SEQ ID NO: 55.
[0032] The present invention further provides for antibody-drug
conjugates, wherein Ab comprises an engineered human IgG1 heavy
chain constant domain (Cy) polypeptide selected from the group
consisting of: (a) one amino acid substitution at position L443 as
set forth in SEQ ID NO: 61 and (b) two amino acid substitutions at
positions L443 and K392 as set forth in SEQ ID NO: 65, according to
the EU index of Kabat.
[0033] The present invention further provides for antibody-drug
conjugates, wherein Ab comprises an engineered human kappa light
chain constant domain (CK) polypeptide having one amino acid
substitution at position .kappa.K183 as set forth in SEQ ID NO: 63,
according to the EU index of Kabat.
[0034] The present invention further provides a pharmaceutical
composition having any antibody, or antigen-binding fragment
thereof, of the present invention or any antibody-drug conjugate of
the present invention and a pharmaceutically acceptable
carrier.
[0035] Further, the present invention provides for a method of
treating a condition associated with Notch3 expression in a patient
in need thereof, comprising administering to the patient any
antibody-drug conjugate or a pharmaceutical composition of the
present invention. The present invention also provides for a method
of treating cancer, wherein the cancer is a solid tumor cancer.
Further, the present invention provides for methods treating solid
tumor cancers including, but not limited to, lung cancer, breast
cancer, ovarian cancer, stomach cancer, esophageal cancer, cervical
cancer, head and neck cancer, bladder cancer, liver cancer, skin
cancer and sarcoma. The present invention also provides for methods
of treating cancer, wherein the cancer is a blood cancer including,
but limited to, T-cell malignancies, T-cell leukemia, T-cell
lymphoma, T-cell acute lymphoblastic leukemia, multiple myeloma,
B-cell malignancies, myeloid malignancies, acute myeloid leukemia
and chronic myeloid leukemia.
[0036] The present invention provides any antibody-drug conjugate
or pharmaceutical composition of the present invention for use in
therapy. The present invention further provides for use in a
therapy, wherein the cancer is a solid tumor cancer. The present
invention also provides for use in therapy wherein the solid tumor
cancers includes but is not limited lung cancer, breast cancer,
ovarian cancer, stomach cancer, esophageal cancer, cervical cancer,
head and neck cancer, bladder cancer, liver cancer, skin cancer and
sarcoma. The present invention also provides for use in a therapy,
wherein the cancer is a blood cancer including, but limited to,
T-cell malignancies, T-cell leukemia, T-cell lymphoma, T-cell acute
lymphoblastic leukemia, multiple myeloma, B-cell malignancies,
myeloid malignancies, acute myeloid leukemia and chronic myeloid
leukemia. The invention further provides for use of any
antibody-drug conjugate of the present invention in the manufacture
of a medicament for therapy. The invention further provides the use
of any antibody-drug conjugate of the present invention, wherein
said use is for the treatment of a Notch3 expressing cancer.
[0037] The invention further provides a nucleic acid that encodes
Notch3 antibodies, or antibody-binding fragments thereof, of the
present invention, a vector comprising said nucleic acid, and a
host cell comprising said vector. The invention also provides a
process for producing Notch3 antibodies of the present invention
wherein said process comprises cultivating the host cell comprising
the above mentioned vector and recovering the antibody from the
cell culture.
[0038] In another embodiment, the invention provides a process for
producing antibody-drug conjugates of the present invention
comprising: linking L to D; conjugating the L-D to an antibody
recovered from the culture of the present invention; and purifying
the antibody-drug conjugate.
[0039] The invention further provides for antibody-drug conjugates
having antibody, or antigen-binding fragments thereof, of the
present invention that specifically binding to Notch3.
[0040] In a further embodiment, the present invention provides a
method for predicting whether a subject with cancer will respond to
any antibody-drug conjugates of the present invention by
determining whether a biological sample from the subject expresses
Notch3.
[0041] The invention further provides a process of determining the
level of Notch3 in a biological sample comprising the steps of:
contacting a sample from a subject suspected to have cancer with
any antibody, or antigen-binding fragment thereof, of the present
invention; determining the cell surface levels of Notch3 on the
sample; and comparing the cell surface levels of Notch3 with that
of a reference subject or standard.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 shows a schematic diagram of recombinant, S1-cleaved,
heterodimeric Notch3 NRR protein immunogen with Avi and His
tags.
[0043] FIG. 2 shows the amino acid and nucleotide sequences of
purified human and mouse Notch3 NRR recombinant proteins.
[0044] FIG. 3 shows the amino acid and nucleotide sequences of r28
and r75 antibody variable regions (CDRs underlined).
[0045] FIGS. 4A through 4C show the amino acid and nucleotide
sequences of [A] hu28 VH 1.0 and CDRs (Kabat and Chothia), [B] hu28
VL 1.0 and CDRs (Kabat and Chothia), and [C] hu28 HC 1.0 and LC
1.0.
[0046] FIGS. 5A through 5C show the amino acid and nucleotide
sequences of [A] hu75 VH 1.9 and CDRs (Kabat and Chothia), [B] hu75
VL 1.3 and CDRs (Kabat and Chothia), and [C] hu75 HC 1.9 and LC
1.3.
[0047] FIG. 6 shows recombinant human Notch1 NRR and Notch3 NRR
domain swap chimeric constructs for epitope mapping of the
anti-Notch3 antibodies ch28 and ch75.
[0048] FIG. 7 shows Western blot analysis of Notch3 positive and
negative cell lines using the D11B8 antibody.
[0049] FIGS. 8A through 8E show [A] cell membrane localization of
anti-Notch3 antibody hu75-Alexa 488 and labeling of acidic vesicles
with pHrodo.TM. red dextran in MDA-MB-468 breast cancer cells at
hour 0, [B] Intracellular trafficking of anti-Notch3 antibody
hu75-Alexa 488 and co-localization with pHrodo.TM. red dextran in
MDA-MB-468 breast cancer cells at hour 5 (arrows), [C] cell
membrane localization of anti-Notch3 antibody hu28-DyLight650 and
labeling of acidic vesicles with pHrodo.TM. red dextran
(intracellular puncta) in MDA-MB-468 breast cancer cells at hour 0,
[D] intracellular trafficking of anti-Notch3 antibody
hu28-DyLight650 and co-localization with pHrodo.TM. red dextran in
MDA-MB-468 breast cancer cells at hour 8 (arrows) and [E] Pearson's
correlation coefficient demonstrating the degree of overlap or
co-localization of hu28-DyLight650 and pHrodo.TM. red dextran
fluorescent labels in MDA-MB-468 cells over time.
[0050] FIG. 9 shows Western blot analysis of S2-cleavage assay
using HCC2429 and MDA-MB-468 cells treated with anti-Notch3 hu28
and hu75. M. W.=molecular weight.
[0051] FIG. 10 shows the treatment of OVCAR3 ovarian cancer cells
with anti-Notch3 hu28-vc0101 disrupts microtubules (stained with
anti-alpha-tubulin antibody) in mitotic cells that were identified
by phospho-Histone H3 staining.
[0052] FIG. 11 shows Western blot analysis of Notch3-ECD from
xenografts harvested from 2-3 mice (M).
[0053] FIG. 12 shows the efficacy of anti-Notch3 hu28-vc0101 and
hu75-vc0101 at a dose of 3 mg/kg compared to Cisplatin at a dose of
5 mg/kg in the 37622A1 NSCLC patient derived xenograft model.
[0054] FIGS. 13A and 13B show [A] the efficacy of anti-Notch3
hu28-vc0101 in the MDA-MB-468 breast model and [B] the efficacy of
anti-Notch3 hu75-vc0101 in the MDA-MB-468 breast model.
[0055] FIGS. 14A and 14B show [A] the efficacy of anti-Notch3
hu28-vc6780 in the MDA-MB-468 breast model and [B] the efficacy of
anti-Notch3 hu75-vc6780 in the MDA-MB-468 breast model.
[0056] FIG. 15 shows the efficacy of anti-Notch3 hu28-vc0101 in the
OVCAR3 ovarian model.
[0057] FIGS. 16A through 16E show [A] the efficacy of rat-human
chimeric anti-Notch3 antibody-drug conjugates dosed at 5 mg/kg in
HCC2429 lung xenografts; [B and C] the efficacy of rat-human
chimeric anti-Notch3 antibody-drug conjugates dosed at 5 mg/kg in
MDA-MB-468 breast xenografts; [D and E] the efficacy of rat-human
chimeric anti-Notch3 antibody-drug conjugates dosed at 5 mg/kg in
N87 gastric xenografts.
[0058] FIGS. 17A and 17B show the amino acid and nucleotide
sequences of [A] single cysteine mutants hu28 HC 1.0 L443C and hu28
LC 1.0 .kappa.K183C and [B] double cysteine mutants hu28 HC 1.0
L443C/K392C.
DETAILED DESCRIPTION OF THE INVENTION
[0059] The present invention provides anti-Notch3 antibodies, or
antigen-binding fragment thereof, and antibody-drug conjugates
(ADCs) for the treatment of cancer. In order that the present
invention is more readily understood, certain terms and general
techniques are first defined.
[0060] All amino acid abbreviations used in this disclosure are
those accepted by the United States Patent and Trademark Office as
set forth in 37 C.F.R. .sctn.1.822 (d)(1).
[0061] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. Generally, nomenclature used in connection with, and
techniques of, cell and tissue culture, molecular biology,
immunology, microbiology, genetics and protein and nucleic acid
chemistry and hybridization described herein are those well known
and commonly used in the art.
[0062] The methods and techniques of the present invention are
generally performed according to conventional methods well known in
the art and as described in various general and more specific
references that are cited and discussed throughout the present
specification unless otherwise indicated. See, e.g., Sambrook J.
& Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(2000); Ausubel et al., Short Protocols in Molecular Biology: A
Compendium of Methods from Current Protocols in Molecular Biology,
Wiley, John & Sons, Inc. (2002); Harlow and Lane Using
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short
Protocols in Protein Science, Wiley, John & Sons, Inc.
(2003).
[0063] "Notch3" or "Notch-3" refers to native, variants, isoforms
and species homologs of human Notch3 protein. Native human Notch3
protein, for example, is made up of a leader peptide, a large
epidermal growth factor (EGF)-like repeat region, three Lin12
repeats, a N terminal heterodimerization domain (HD-1), a C
terminal heterodimerization domain (HD-2), a transmembrane (TM)
sequence and an intracellular domain (Notch3.sup.ICD). The
NCBI/GenBank accession number of the full length human Notch3 is
NM_000435.2.
[0064] "Notch3 negative regulatory region", or "Notch3 NRR" as used
herein, unless otherwise indicated, refers to any native or
synthetic polypeptide region of Notch3 consisting of the three
Lin12 domains and the amino acid sequence or sequences located
between the three Lin12 domains, plus the HD1 and HD2 domains of
Notch3. In one embodiment, the "Notch3 NRR" includes the three
Lin12 domains and two heterodimerization domains HD-1, and HD-2,
wherein the HD-1 and HD-2 domains of Notch3 are covalently bonded
and not yet cleaved by the furin-like protease (before S1
cleavage). In another embodiment, the "Notch3 NRR" includes the
three Lin12 domains and the two heterodimerization domains HD-1,
and HD-2, wherein the HD-1 and HD-2 domains are non-covalently
bonded (after S1 cleavage). In one aspect of this embodiment, the
S2 site within the HD-2 domain has not been cleaved by the
ADAM-type metalloproteases. In another particular aspect of this
embodiment, the S2 site within the HD-2 domain is being cleaved or
has already been cleaved by the ADAM-type metalloproteases.
(Gordon, W. R., et. al, Nature Structural & Molecular Biology,
2007, volume 14, 295-300).
[0065] An "antibody" or "Ab" is an immunoglobulin molecule capable
of specific binding to a target, such as a carbohydrate,
polynucleotide, lipid, polypeptide, etc., through at least one
antigen recognition site, located in the variable region of the
immunoglobulin molecule. As used herein, the term "antibody"
encompasses not only intact polyclonal or monoclonal antibodies,
but also any antigen binding portion (e.g., "antigen-binding
fragment") thereof of an intact antibody that retains the ability
to specifically bind to a given antigen (e.g., target Notch3) or
single chain thereof, fusion proteins comprising an antibody, and
any other modified configuration of the immunoglobulin molecule
that comprises an antigen recognition site, for example without
limitation, Fab; Fab'; F(ab').sub.2; an Fd fragment; an Fv
fragment; a single domain antibody (dAb) fragment; an isolated
complementarity determining region (CDR); single chain (scFv) and
single domain antibodies (e.g., shark and camelid antibodies),
maxibodies, minibodies, intrabodies, diabodies, triabodies,
tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson,
2005, Nature Biotechnology 23(9): 1126-1136). An antibody includes
an antibody of any class, such as IgG, IgA, or IgM (or sub-class
thereof), and the antibody need not be of any particular class.
Depending on the antibody amino acid sequence of the constant
region of its heavy chains, immunoglobulins can be assigned to
different classes. There are five major classes of immunoglobulins:
IgA, IgD, IgE, IgG, and IgM, and several of these may be further
divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4,
IgA1 and IgA2. The heavy chain (HC) constant regions that
correspond to the different classes of immunoglobulins are called
alpha, delta, epsilon, gamma, and mu, respectively. The subunit
structures and three-dimensional configurations of different
classes of immunoglobulins are well known.
[0066] An "isolated antibody" refers to an antibody that is
substantially free of other antibodies having different antigenic
specificities (e.g., an isolated antibody that specifically binds
Notch3 is substantially free of antibodies that specifically bind
antigens other than Notch3). An isolated antibody that specifically
binds Notch3 may, however, have cross-reactivity to other antigens,
such as Notch3 molecules from other species. Moreover, an isolated
antibody may be substantially free of other cellular material
and/or chemicals.
[0067] A "variable region" of an antibody refers to the variable
region of the antibody light chain (VL) or the variable region of
the antibody heavy chain (VH), either alone or in combination. As
known in the art, the variable regions of the heavy and light chain
each consist of four framework regions (FRs) connected by three
complementarity determining regions (CDR1, CDR2, CDR3) also known
as hypervariable regions, contribute to the formation of the
antigen binding site of antibodies. If variants of a subject
variable region are desired, particularly with substitution in
amino acid residues outside of a CDR region (i.e., in the framework
region), appropriate amino acid substitution, preferably,
conservative amino acid substitution, can be identified by
comparing the subject variable region to the variable regions of
other antibodies which contain CDR1 and CDR2 sequences in the same
canonincal class as the subject variable region (Chothia and Lesk,
J Mol Biol 196(4): 901-917, 1987). When choosing FR to flank
subject CDRs, e.g., when humanizing or optimizing an antibody, FRs
from antibodies which contain CDR1 and CDR2 sequences in the same
canonical class are preferred.
[0068] A "CDR" of a variable domain are amino acid residues within
the variable region that are identified in accordance with the
definitions of the Kabat, Chothia, the accumulation of both Kabat
and Chothia, AbM, contact, and/or conformational definitions or any
method of CDR determination well known in the art. Antibody CDRs
may be identified as the hypervariable regions originally defined
by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of
Proteins of Immunological Interest, 5th ed., Public Health Service,
NIH, Washington D.C. The positions of the CDRs may also be
identified as the structural loop structures originally described
by Chothia and others. See, e.g., Chothia et al., 1989, Nature
342:877-883. Other approaches to CDR identification include the
"AbM definition," which is a compromise between Kabat and Chothia
and is derived using Oxford Molecular's AbM antibody modeling
software (now Accelrys.RTM.), or the "contact definition" of CDRs
based on observed antigen contacts, set forth in MacCallum et al.,
1996, J. Mol. Biol., 262:732-745. In another approach, referred to
herein as the "conformational definition" of CDRs, the positions of
the CDRs may be identified as the residues that make enthalpic
contributions to antigen binding. See, e.g., Makabe et al., 2008,
Journal of Biological Chemistry, 283:1156-1166. Still other CDR
boundary definitions may not strictly follow one of the above
approaches, but will nonetheless overlap with at least a portion of
the Kabat CDRs, although they may be shortened or lengthened in
light of prediction or experimental findings that particular
residues or groups of residues or even entire CDRs do not
significantly impact antigen binding. As used herein, a CDR may
refer to CDRs defined by any approach known in the art, including
combinations of approaches. The methods used herein may utilize
CDRs defined according to any of these approaches. For any given
embodiment containing more than one CDR, the CDRs may be defined in
accordance with any of Kabat, Chothia, extended, AbM, contact,
and/or conformational definitions.
[0069] The terms "IgG Fc region", "Fc region", "Fc domain" and
"Fc", as interchangeably used herein refer to the portion of an IgG
molecule that correlates to a crystallizable fragment obtained by
papain digestion of an IgG molecule. The Fc region consists of the
C-terminal half of the two heavy chains of an IgG molecule that are
linked by disulfide bonds. It has no antigen binding activity but
contains the carbohydrate moiety and the binding sites for
complement and Fc receptors, including the FcRn receptor (see
below). The Fc fragment contains the entire second constant domain
CH2 (residues 231-340 of human IgG1, according to the Kabat
numbering system) and the third constant domain CH3 (residues
341-447).
[0070] By "engineered Fc polypeptide", "engineered Fc region" and
"engineered Fc" as the terms are interchangeably used herein, is
meant an Fc polypeptide, or portion thereof, comprising at least
one mutation, e.g., an amino acid substitution, introducing a site
for conjugation. Preferably, the mutation introduces a cysteine in
place of the naturally-occurring amino acid residue at that
position, where the mutation creates a reactive site (e.g., a
reactive sulfhydryl group) for conjugation of a moiety to the
Fc.
[0071] The term "monoclonal antibody" or "mAb" refers to an
antibody that is derived from a single copy or clone, including
e.g., any eukaryotic, prokaryotic, or phage clone, and not the
method by which it is produced. Preferably, a monoclonal antibody
of the invention exists in a homogeneous or substantially
homogeneous population.
[0072] "Humanized" antibody refers to forms of non-human (e.g. rat)
antibodies that are chimeric immunoglobulins, immunoglobulin
chains, or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2
or other antigen-binding subsequences of antibodies) that contain
minimal sequence derived from non-human immunoglobulin. Preferably,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a complementary determining region (CDR) of
the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat, or rabbit having the
desired specificity, affinity, and capacity.
[0073] "Human antibody or fully human antibody" refers to those
antibodies derived from transgenic mice carrying human antibody
genes or from human cells.
[0074] The term "chimeric antibody" is intended to refer to
antibodies in which the variable region sequences are derived from
one species and the constant region sequences are derived from
another species, such as an antibody in which the variable region
sequences are derived from a rat antibody and the constant region
sequences are derived from a human antibody.
[0075] A "therapeutic agent" is an agent that exerts a cytotoxic,
cytostatic, and/or immunomodulatory effect on cancer cells or
activated immune cells. Examples of therapeutic agents include
cytotoxic agents, chemotherapeutic agents, cytostatic agents, and
immunomodulatory agents.
[0076] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer.
[0077] A "cytotoxic effect" refers to the depletion, elimination
and/or the killing of a target cell(s). A "cytotoxic agent" refers
to an agent that has a cytotoxic and/or cytostatic effect on a
cell.
[0078] A "cytostatic effect" refers to the inhibition of cell
proliferation. A "cytostatic agent" refers to an agent that has a
cytostatic effect on a cell, thereby inhibiting the growth and/or
expansion of a specific subset of cells.
[0079] "Antibody-drug conjugate" or "ADC" refers to antibodies or
antibody fragments thereof, including antibody derivatives that
bind to Notch3 and are conjugated to cytotoxic, cytostatic, and/or
therapeutic agents.
[0080] "Anti-Notch3 antibody-drug conjugate" or "anti-Notch3 ADC"
refers to an anti-Notch3 antibody or antigen binding fragment
thereof, as described herein linked to a drug (D) via a linker
(L).
[0081] "Linker (L)" describes the direct or indirect linkage of the
antibody to the drug. Attachment of a linker to an antibody can be
accomplished in a variety of ways, such as through surface lysines,
reductive-coupling to oxidized carbohydrates, and through cysteine
residues liberated by reducing interchain disulfide linkages. A
variety of antibody-drug conjugate linkage systems are known in the
art, including hydrazone-, disulfide- and peptide-based
linkages.
[0082] "Drug (D)" is any substance having biological or detectable
activity, for example, therapeutic agents, detectable labels,
binding agents, etc., and prodrugs, which are metabolized to an
active agent in vivo. The terms drug, payload and compound are used
interchangeably.
[0083] "L-D" is a linker-drug moiety resulting from a drug (D)
linked to a linker (L).
[0084] The term "epitope" refers to that portion of a molecule
capable of being recognized by and bound by an antibody at one or
more of the antibody's antigen-binding regions. Epitopes often
consist of a chemically active surface grouping of molecules such
as amino acids or sugar side chains and have specific
three-dimensional structural characteristics as well as specific
charge characteristics. The term "antigenic epitope" as used
herein, is defined as a portion of a polypeptide to which an
antibody can specifically bind as determined by any method well
known in the art, for example, by conventional immunoassays. A
"nonlinear epitope" or "conformational epitope" comprises
noncontiguous polypeptides (or amino acids) within the antigenic
protein to which an antibody specific to the epitope binds. Once a
desired epitope on an antigen is determined, it is possible to
generate antibodies to that epitope, e.g., using the techniques
described in the present specification. During the discovery
process, the generation and characterization of antibodies may
elucidate information about desirable epitopes. From this
information, it is then possible to competitively screen antibodies
for binding to the same epitope. An approach to achieve this is to
conduct competition and cross-competition studies to find
antibodies that compete or cross-compete with one another e.g., the
antibodies compete for binding to the antigen.
[0085] The term "binding affinity (K.sub.D)" as used herein, is
intended to refer to the dissociation rate of a particular
antigen-antibody interaction. The K.sub.D is the ratio of the rate
of dissociation, also called the "off-rate (k.sub.d)", to the
association rate, or "on-rate (k.sub.a)". Thus, K.sub.D equals
k.sub.d/k.sub.a and is expressed as a molar concentration (M). It
follows that the smaller the K.sub.D, the stronger the affinity of
binding. Therefore, a K.sub.D of 1 .mu.M indicates weak binding
affinity compared to a K.sub.D of 1 nM. K.sub.D values for
antibodies can be determined using methods well established in the
art. One method for determining the K.sub.D of an antibody is by
using surface plasmon resonance, typically using a biosensor system
such as a Biacore.RTM. system.
[0086] An antibody, an antibody conjugate, or a polypeptide that
"preferentially binds" or "specifically binds" (used
interchangeably herein) to a target (e.g., Notch3 protein) is a
term well understood in the art, and methods to determine such
specific or preferential binding are also well known in the art. A
molecule is said to exhibit "specific binding" or "preferential
binding" if it reacts or associates more frequently, more rapidly,
with greater duration and/or with greater affinity with a
particular cell or substance than it does with alternative cells or
substances. An antibody "specifically binds" or "preferentially
binds" to a target if it binds with greater affinity, avidity, more
readily, and/or with greater duration than it binds to other
substances. For example, an antibody that specifically or
preferentially binds to a Notch3 epitope is an antibody that binds
this epitope with greater affinity, avidity, more readily, and/or
with greater duration than it binds to other Notch3 epitopes or
non-Notch3 epitopes. It is also understood that by reading this
definition, for example, an antibody (or moiety or epitope) that
specifically or preferentially binds to a first target may or may
not specifically or preferentially bind to a second target. As
such, "specific binding" or "preferential binding" does not
necessarily require (although it can include) exclusive binding.
Generally, but not necessarily, reference to binding means
preferential binding. "EC.sub.50" is a measurement of binding
capacity and is defined as the half maximal effective concentration
of an antibody or antibody-drug conjugate that is needed to produce
a response halfway between the baseline and maximum.
[0087] "Pharmaceutically acceptable salt" as used herein refers to
pharmaceutically acceptable organic or inorganic salts of a
molecule or macromolecule.
[0088] The term "potency" is a measurement of biological activity
and may be designated as IC.sub.50, or inhibitory concentration of
an antibody or antibody drug conjugate to the antigen Notch3,
needed to inhibit 50% of Notch3-dependent reporter gene activity or
growth of a Notch3 positive cell line as described in Examples 9
and 12 respectively.
[0089] The phrase "effective amount" or "therapeutically effective
amount" as used herein refers to an amount necessary (at dosages
and for periods of time and for the means of administration) to
achieve the desired therapeutic result. An effective amount is at
least the minimal amount, but less than a toxic amount, of an
active agent which is necessary to impart therapeutic benefit to a
subject.
[0090] The terms "inhibit" or "neutralize" as used herein with
respect to bioactivity of an antibody of the invention mean the
ability of the antibody to substantially antagonize, prohibit,
prevent, restrain, slow, disrupt, eliminate, stop, reduce or
reverse e.g. progression or severity of that which is being
inhibited including, but not limited to, a biological activity.
[0091] The term "compete" or "competes", as used herein with regard
to an antibody, means that a first antibody, or an antigen-binding
fragment thereof, binds to an epitope in a manner sufficiently
similar to the binding of a second antibody, or an antigen-binding
fragment thereof, such that the result of binding of the first
antibody with its cognate epitope is detectably decreased in the
presence of the second antibody compared to the binding of the
first antibody in the absence of the second antibody. The
alternative, where the binding of the second antibody to its
epitope is also detectably decreased in the presence of the first
antibody, can, but need not be the case. That is, a first antibody
can inhibit the binding of a second antibody to its epitope without
that second antibody inhibiting the binding of the first antibody
to its respective epitope. However, where each antibody detectably
inhibits the binding of the other antibody with its cognate epitope
or ligand, whether to the same, greater, or lesser extent, the
antibodies are said to "cross-compete" with each other for binding
of their respective epitope(s). Both competing and cross-competing
antibodies are encompassed by the present invention. Regardless of
the mechanism by which such competition or cross-competition occurs
(e.g., steric hindrance, conformational change, or binding to a
common epitope, or fragment thereof), the skilled artisan would
appreciate, based upon the teachings provided herein, that such
competing and/or cross-competing antibodies are encompassed and can
be useful for the methods disclosed herein.
[0092] The terms "polynucleotide" or "nucleic acid molecule", as
used herein, are intended to include DNA molecules and RNA
molecules. A nucleic acid molecule may be single-stranded or
double-stranded, but preferably is double-stranded DNA.
[0093] The polynucleotides that encode the antibodies of the
present invention may include the following: only the coding
sequence for the variant, the coding sequence for the variant and
additional coding sequences such as a functional polypeptide, or a
signal or secretory sequence or a pro-protein sequence; the coding
sequence for the antibody and non-coding sequence, such as introns
or non-coding sequence 5' and/or 3' of the coding sequence for the
antibody. The term `polynucleotide encoding an antibody"
encompasses a polynucleotide which includes additional coding
sequence for the variant but also a polynucleotide which includes
additional coding and/or non-coding sequence. It is known in the
art that a polynucleotide sequence that is optimized for a specific
host cell/expression system can readily be obtained from the amino
acid sequence of the desired protein (see GENEART AG, Regensburg,
Germany).
[0094] A "host cell" includes an individual cell or cell culture
that can be or has been a recipient for vector(s) for incorporation
of polynucleotide inserts. Host cells include progeny of a single
host cell, and the progeny may not necessarily be completely
identical (in morphology or in genomic DNA complement) to the
original parent cell due to natural, accidental, or deliberate
mutation. A host cell includes cells transfected in vivo with a
polynucleotide(s) of this invention.
[0095] The term "vector" means a construct, which is capable of
delivering, and, preferably, expressing, one or more gene(s) or
sequence(s) of interest in a host cell. Examples of vectors
include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, plasmid, cosmid or phage vectors, DNA or RNA
expression vectors associated with cationic condensing agents, DNA
or RNA expression vectors encapsulated in liposomes, and certain
eukaryotic cells, such as producer cells.
[0096] The term "expression control sequence" means a nucleic acid
sequence that directs transcription of a nucleic acid. An
expression control sequence can be a promoter, such as a
constitutive or an inducible promoter, or an enhancer. The
expression control sequence is operably linked to the nucleic acid
sequence to be transcribed.
[0097] The polynucleotides encoding the antibodies of the present
invention will typically include an expression control
polynucleotide sequence operably linked to the antibody coding
sequences, including naturally-associated or heterologous promoter
regions known in the art. Preferably, the expression control
sequences will be eukaryotic promoter systems in vectors capable of
transforming or transfecting eukaryotic host cells, but control
sequences for prokaryotic hosts may also be used. Once the vector
has been incorporated into the appropriate host cell line, the host
cell is propagated under conditions suitable for expressing the
nucleotide sequences, and, as desired, for the collection and
purification of the antibodies. Preferred eukaryotic cell lines
include CHO cell lines, various COS cell lines, HeLa cells, myeloma
cell lines, transformed B-cells, or human embryonic kidney cell
lines. The most preferred host cell is a CHO cell line.
Antibodies
[0098] Antibodies of the invention can be produced using techniques
well known in the art, e.g., recombinant technologies, phage
display technologies, synthetic technologies or combinations of
such technologies or other technologies readily known in the art
(see, for example, Jayasena, S. D., Clin. Chem., 45: 1628-50 (1999)
and Fellouse, F. A., et al, J. Mol. Biol., 373(4):924-40
(2007)).
[0099] An embodiment of the invention is an antibody that
specifically binds to the same Notch3 epitope as an antibody
comprising a first amino acid sequence that is at least 90%, 92%,
94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13 and a
second amino acid sequence that is at least 90%, 92%, 94%, 95%,
96%, 97%, 98% or 99% identical to SEQ ID NO: 25.
[0100] Another embodiment of the invention is an antibody that
specifically binds to the same Notch3 epitope as an antibody
comprising a first amino acid sequence that is at least 90%, 92%,
94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37 and a
second amino acid sequence that is at least 90%, 92%, 94%, 95%,
96%, 97%, 98% or 99% identical to SEQ ID NO: 49.
[0101] In some embodiments, the antibody, or antigen-binding
fragment thereof, specifically binds to Notch3, and the antibody,
or antigen-binding fragment thereof, competes with the binding of
an antibody comprising a first amino acid sequence that is at least
90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13
and a second amino acid sequence that is at least 90%, 92%, 94%,
95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 25.
[0102] In some embodiments, the antibody, or antigen-binding
fragment thereof, specifically binds to Notch3, and the antibody,
or antigen-binding fragment thereof, competes with the binding of
an antibody comprising a first amino acid sequence that is at least
90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37
and a second amino acid sequence that is at least 90%, 92%, 94%,
95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 49.
Conjugation of a Drug to an Antibody
[0103] The drug has, or is modified to include, a group reactive
with a conjugation point on the antibody. For example, a drug can
be attached by alkylation (e.g., at the epsilon-amino group lysines
or the N-terminus of antibodies), reductive amination of oxidized
carbohydrate, transesterification between hydroxyl and carboxyl
groups, amidation at amino groups or carboxyl groups, and
conjugation to thiols. In some embodiments, the number of drug, p,
conjugated per antibody molecule ranges from an average of 1 to 12,
1 to 11, 1 to 10, 1 to 9, 1 to 8; 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1
to 3, or to 2. In some embodiments, p ranges from an average of 2
to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2
to 4 or 2 to 3. In other embodiments, p is an average of 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments, p ranges from
an average of about 1 to about 12, about 1 to about 11, about 1 to
about 10, about 1 to about 9, about 1 to about 8; about 1 to about
7, about 1 to about 6, about 1 to about 5, about 1 to about 4,
about 1 to about 3, or about 1 to about 2. In some embodiments, p
ranges from about 2 to about 12, about 2 to about 11, about 2 to
about 10, about 2 to about 9, about 2 to about 8, about 2 to about
7, about 2 to about 6, about 2 to about 5, about 2 to about 4 or
about 2 to about 3. For examples of chemistries that can be used
for conjugation, see, e.g., Current Protocols in Protein Science
(John Wiley & Sons, Inc.), Chapter 15 (Chemical Modifications
of Proteins).
Linkers
[0104] A linker is a bifunctional compound which can be used to
link a drug and an antibody to form an antibody drug conjugate
(ADC). Such conjugates are useful, for example, in the formation of
immunoconjugates directed against tumor associated antigens. Such
conjugates allow the selective delivery of cytotoxic drugs to tumor
cells. Suitable linkers include, for example, cleavable and
non-cleavable linkers. A cleavable linker is typically susceptible
to cleavage under intracellular conditions. Suitable cleavable
linkers include, for example, a peptide linker cleavable by an
intracellular protease, such as lysosomal protease or an endosomal
protease. In exemplary embodiments, the linker can be a dipeptide
linker, such as a valine-citrulline (val-cit), a
phenylalanine-lysine (phe-lys) linker, or
maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl (vc)
linker. Another linker is
Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate
(smcc). Sulfo-smcc conjugation occurs via a maleimide group which
reacts with sulfhydryls (thiols, --SH), while its Sulfo-NHS ester
is reactive toward primary amines (as found in Lysine and the
protein or peptide N-terminus). Yet another linker is
maleimidocaproyl (mc). Other suitable linkers include linkers
hydrolyzable at a specific pH or a pH range, such as a hydrazone
linker. Additional suitable cleavable linkers include disulfide
linkers. The linker may be covalently bound to the antibody to such
an extent that the antibody must be degraded intracellularly in
order for the drug to be released e.g. the mc linker and the
like.
[0105] Linkers of the present invention inlcue
maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl (vc)
and maleimidocaproyl (mc), me and MalPeg6C2.
Engineered Fc Polypeptide
[0106] It has been previously reported that certain residues
presumably present on the surface of the CH2 or CH3 domain of the
heavy chain of antibodies, or on the constant domain of the light
chain, or otherwise accessible, are suitable for the substitution
of the naturally-occurring wild type amino acid with, for example,
cysteine, and are therefore useful to engineer a site capable of
conjugation to various agents, as described in International
Publication No. WO/2013/093809, which is incorporated herein by
reference.
[0107] Amino acid modifications can be made by any method known in
the art and many such methods are well known and routine for the
skilled artisan. For example, but not by way of limitation, amino
acid substitutions, deletions and insertions may be accomplished
using any well-known PCR-based technique. Amino acid substitutions
may be made by site-directed mutagenesis (see, for example, Zoller
and Smith, 1982, Nucl. Acids Res. 10:6487-6500; and Kunkel, 1985,
Proc. Natl. Acad. Sci USA 82:488).
[0108] In some embodiments, the engineered Fc polypeptide of the
disclosure may be used to prepare an antibody, or antigen binding
fragment thereof, such that the antibody or fragment thereof
thereby comprises the engineered Fc region which can be used to
conjugate, at the engineered residue (i.e., the amino acid
substituted compared to wild type unmodified Fc), a wide variety of
moieties.
[0109] In some embodiments, the engineered kappa light chain
constant polypeptide of the disclosure may be used to prepare an
antibody, or antigen binding fragment thereof, such that the
antibody or fragment thereof thereby comprises an engineered CL
region comprising an amino acid mutation, or fragment thereof,
which can be used to conjugate, at the engineered amino acid
residue, a wide variety of moieties.
[0110] It should be noted that a single substitution in an Fc
polypeptide, for example of a cysteine residue, normally results in
the display of two corresponding residues in the resultant IgG
antibody due to the homodimeric nature of IgG antibody molecules.
Thus, the resultant engineered IgG antibodies of the invention may
display at least 1, 2, 3, 4, or more reactive groups for the
purpose of conjugation to a drug or compound. In an embodiment, one
or more of the substitutions is with a cysteine residue, and the
resulting engineered antibodies may display at least 1, 2, 3, 4, or
more thiol groups for the purpose of conjugation to a drug or
compound.
[0111] In some embodiments, the engineered Fc polypeptide of the
present invention comprises one or more substitutions selected from
the positions 443 and 392, of the heavy chain of an antibody, and
wherein the numbering system of the constant region is that of the
EU index as set forth in Kabat et al. (supra).
[0112] In some embodiments, the engineered Fc polypeptide comprises
one amino acid substitution (L443C) as provided in SEQ ID NO: 61.
In another embodiment, the engineered Fc polypeptide comprises two
amino acid substitutions (L443C/K392C) as provided in SEQ ID NO:
65.
Engineered CK Polypeptide
[0113] The anti-Notch3 antibodies of the present invention may
encompass an engineered antibody light chain constant region (LC),
or a fragment thereof, where 1, 2, or 3 amino acids of the antibody
light chain, wherein the numbering system of the light chain
constant region is that of the Kabat numbering system as set forth
in Kabat et al. (1991, NIH Publication 91-3242, National Technical
Information Service, Springfield, Va., hereinafter "Kabat"), of a
parent, native, or wild type antibody, substituted with another
amino acid (including natural and non-natural/synthetic amino
acids).
[0114] In other embodiments, due to the dimeric nature of many
antibodies (e.g., IgGs comprise two light chains and two heavy
chains each heavy chain comprising an Fc polypeptide), an antibody
of the invention may comprise at least one engineered Fc
polypeptide and may further comprise at least one engineered light
chain constant polypeptide thereby providing at least two
site-specific conjugation sites--one in the Fc polypeptide and
another in the CL polypeptide.
[0115] In some embodiments, the engineered CK polypeptide of the
present invention comprises at least one substitution at position
183 of the light chain of the antibody. In some embodiments, the
engineered CK polypeptide comprises one amino acid substitution
(.kappa.K183C) as provided in SEQ ID NO. 63.
Therapy for Cancer
[0116] Cancers, including, but not limited to, a tumor, metastasis,
or other disease or disorder characterized by uncontrolled cell
growth, can be treated or prevented by administration of an
antibody, or antigen-binding fragment thereof, or antibody-drug
conjugate (ADC) of the present invention.
[0117] Exemplary anti-Notch3 antibodies, or antigen-binding
fragments thereof, and antibody-drug conjugates are useful for
treating cancer in which Notch3 is expressed or overexpressed,
relative to a reference subject or standard (e.g., non-cancerous or
normal tissue). Treatment or prevention of a Notch3-expressing
cancer, according to the methods described herein, can be achieved
by administering to a subject in need of such treatment an
effective amount of an anti-Notch3 antibody, or antigen-binding
fragment thereof, and/or antibody-drug conjugate. In some
embodiments, an anti-Notch3 full length antibody, or
antigen-binding fragment thereof, that is conjugated to a cytotoxic
agent will be administered. In some exemplary embodiments, an
anit-Notch3 antibody-drug conjugate of the present invention will
(i) bind to Notch3 expressing cancer cells, and (ii) exert a
cytotoxic or cytostatic effect to, for example, inhibit the
proliferation of the Notch3 expressing cancer cells, or kill Notch3
expressing cancer cells.
[0118] In other embodiments, the anti-Notch3 antibodies, or
antigen-binding fragment thereof, and/or anti-Notch3 antibody-drug
conjugates are co-administered with another therapeutic agent, or
administered sequentially with another therapeutic agent. In some
embodiments, the anti-Notch3 antibodies, or antigen-binding
fragment thereof, and/or anti-Notch3 antibody-drug conjugates are
co-administered with chemotherapeutics, including standard of care
chemotherapeutics, or administered sequentially.
[0119] In some embodiments, the other therapeutic agent will be an
agent that is standard of care for the specific disease to be
treated or is part of a salvage regimen for the specific disease to
be treated. Anti-cancer agents and chemotherapeutic regimens
include, for example, anti-cancer antibodies, including, for
example, anti-CD52 antibodies (e.g., Alemtuzumab), anti-CD20
antibodies (e.g., Rituximab), and anti-CD40 antibodies (e.g.,
SGN40); chemotherapeutic regimens including, for example, CHOP
(cyclophosphamide, doxorubicin, vincristine, and prednisone); CVP
(cyclophosphamide, vincristine, and prednisone); RCVP
(Rituximab+CVP); RCHOP (Rituximab+CHOP); RICE (Rituximab+ifosamide,
carboplatin, etoposide); RDHAP, (Rituximab+dexamethasone,
cytarabine, cisplatin); RESHAP (Rituximab+etoposide,
methylprednisolone, cytarabine, cisplatin); gemcitabine;
combination treatment with vincristine, prednisone, and
anthracycline, with or without asparaginase; combination treatment
with daunorubicin, vincristine, prednisone, and asparaginase;
combination treatment with teniposide and Ara-C (cytarabine);
combination treatment with methotrexate and leucovorin; combination
treatment with bleomycin, doxorubicin, etoposide, mechlorethamine,
prednisone, vinblastine, and vincristine; small molecule
inhibitors; and proteosome inhibitors including, for example,
bortezomib.
[0120] In some embodiments, methods for treating cancer including
administering to a patient in need thereof an effective amount of
an anti-Notch3 antibody, or antigen-binding fragment thereof,
and/or anti-Notch3 antibody-drug conjugate in combination with
radiation treatment, and optionally another therapeutic agent. In
some embodiments, the anti-Notch3 antibody, or antigen-binding
fragment thereof, and/or anti-Notch3 antibody-drug conjugate is
administered concurrently or sequentially with an anti-cancer agent
(e.g., a chemotherapeutic agent) and/or with radiation therapy. In
some embodiments, the chemotherapeutic agent or radiation therapy
is administered at least an hour, five hours, 12 hours, a day, a
week, a month, several months (e.g., up to three months), prior or
subsequent to administration of a compound of the present
invention.
[0121] Generally, for administration of an anti-Notch3 antibody
and/or an anti-Notch3 antibody-drug conjugate, an initial candidate
dosage can be about 2 mg/kg. For the purpose of the present
invention, a typical daily dosage might range from about any of 3
.mu.g/kg to 30 .mu.g/kg to 300 .mu.g/kg to 3 mg/kg, to 30 mg/kg, to
100 mg/kg or more, depending on the factors mentioned above. For
example, dosage of about 1 mg/kg, about 2.5 mg/kg, about 5 mg/kg,
about 10 mg/kg, and about 25 mg/kg may be used. For repeated
administrations over several days or longer, depending on the
condition, the treatment is sustained until a desired suppression
of symptoms occurs or until sufficient therapeutic levels are
achieved, for example, to inhibit or delay tumor growth/progression
or metatstasis of cancer cells. An exemplary dosing regimen
comprises administering an initial dose of about 2 mg/kg, followed
by a weekly maintenance dose of about 1 mg/kg of the anti-Notch3
antibody or anti-Notch3 antibody-drug conjugate, or followed by a
maintenance dose of about 1 mg/kg every other week. Other exemplary
dosing regimens comprise administering increasing doses (e.g.,
initial dose of 1 mg/kg and gradual increase to one or more higher
doses every week or longer time period). Other dosing regimens may
also be useful, depending on the pattern of pharmacokinetic decay
that the practitioner wishes to achieve. For example, in some
embodiments, dosing from one to four times a week is contemplated.
In other embodiments dosing once a month or once every other month
or every three months is contemplated as well as weekly, bi-weekly
and every three weeks. The progress of this therapy may be
monitored by conventional techniques and assays. The dosing regimen
(including the anti-Notch3 antibody or the anti-Notch3
antibody-drug conjugate used) can vary over time.
[0122] For the purpose of the present invention, the appropriate
dosage of an anti-Notch3 antibody or an anti-Notch3 antibody-drug
conjugate will depend on the anti-Notch3 antibody or the
anti-Notch3 antibody-drug conjugate (or compositions thereof)
employed, the type and severity of symptoms to be treated, whether
the agent is administered for therapeutic purposes, previous
therapy, the patient's clinical history and response to the agent,
the patient's clearance rate for the administered agent, and the
discretion of the attending physician. The clinician may administer
an anti-Notch3 antibody or an anti-Notch3 antibody-drug conjugate
until a dosage is reached that achieves the desired result and
beyond. Dose and/or frequency can vary over course of treatment,
but may stay constant as well. Empirical considerations, such as
the half-life, generally will contribute to the determination of
the dosage. For example, antibodies that are compatible with the
human immune system, such as humanized antibodies or fully human
antibodies, may be used to prolong half-life of the antibody and to
prevent the antibody being attacked by the host's immune system.
Frequency of administration may be determined and adjusted over the
course of therapy, and is generally, but not necessarily, based on
treatment and/or suppression and/or amelioration and/or delay of
symptoms, e.g., tumor growth inhibition or delay, etc.
Alternatively, sustained continuous release formulations of
anti-Notch3 antibodies or anti-Notch3 antibody-drug conjugates may
be appropriate. Various formulations and devices for achieving
sustained release are known in the art.
[0123] In one embodiment, dosages for anti-Notch3 antibody or
anti-Notch3 antibody-drug conjugate may be determined empirically
in individuals who have been given one or more administration(s) of
the anti-Notch3 antibody or its anti-Notch3 antibody-drug
conjugate. Individuals are given incremental dosages of an
anti-Notch3 antibody or a Notch3 antagonist. To assess efficacy, an
indicator of the disease can be followed.
[0124] Administration of an anti-Notch3 antibody or an anti-Notch3
antibody-drug conjugate in accordance with the method in the
present invention can be continuous or intermittent, depending, for
example, upon the recipient's physiological condition, whether the
purpose of the administration is therapeutic or prophylactic, and
other factors known to skilled practitioners. The administration of
an anti-Notch3 antibody or an anti-Notch3 antibody-drug conjugate
may be essentially continuous over a preselected period of time or
may be in a series of spaced doses.
[0125] In some embodiments, more than one anti-Notch3 antibody or
anti-Notch3 antibody-drug conjugate may be present. At least one,
at least two, at least three, at least four, at least five
different or more anti-Notch3 antibody or anti-Notch3 antibody-drug
conjugate can be present. Generally, those anti-Notch3 antibodies
or anti-Notch3 antibody-drug conjugates may have complementary
activities that do not adversely affect each other. For example,
one or more of the following anti-Notch3 antibody may be used: a
first anti-Notch3 antibody directed to one epitope on Notch3 and a
second anti-Notch3 antibody directed to a different epitope on
Notch3.
[0126] The anti-Notch3 antibodies, or antigen-binding fragment
thereof, and/or anti-Notch3 antibody-drug conjugates of the present
invention can be in the form of a pharmaceutical composition for
administration that are formulated to be appropriate for the
selected mode of administration, and pharmaceutically acceptable
diluent or excipients, such as buffers, surfactants, preservatives,
solubilizing agents, isotonicity agents, stabilizing agents,
carriers, and the like. Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton Pa., 18.sup.th ed., 1995, provides a
compendium of formulation techniques as are generally known to
practitioners.
[0127] These pharmaceutical compositions may be administered by any
means known in the art that achieve the generally intended purpose
to treat cancer. The in one embodiment, the route of administration
is parenteral, defined herein as referring to modes of
administration that include but not limited to intravenous,
intramuscular, intraperitoneal, subcutaneous, and intraarticular
injection and infusion. The dosage administered will be dependent
upon the age, health, and weight of the recipient, kind of
concurrent treatment, if any, frequency of treatment, and the
nature of the effect desired.
[0128] Compositions within the scope of the invention include all
compositions wherein an anti-Notch3 antibody, or antigen-binding
fragment thereof, and/or anti-Notch3 antibody-drug conjugate is
present in an amount that is effective to achieve the desired
medical effect for treating cancer. While individual needs may vary
from one patient to another, the determination of the optimal
ranges of effective amounts of all of the components is within the
ability of the clinician of ordinary skill.
Diagnostic
[0129] The antibodies, or antigen-binding fragment thereof, of the
invention can also be used to detect Notch3 in a biological sample
in vitro or in vivo. In one embodiment, the anti-Notch3 antibodies,
or antigen-binding fragment thereof, of the invention are used to
determine the level of Notch3 in a tissue or in cells derived from
the tissue. In a one embodiment, the tissue is a diseased tissue.
In some embodiments, the tissue is a tumor or a biopsy thereof. In
a one embodiment, the levels of Notch3 in a tissue or biopsy from a
patient can be determined in an immunoassay with the antibodies, or
antigen-binding fragment thereof, of the invention. The tissue or
biopsy thereof can be excised from the patient and can be frozen or
fixed. The same method can be used to determine other properties of
the Notch3 protein, such as its level of cell surface levels, or
cellular localization.
[0130] The above-described method can be used to diagnose a cancer
in a subject known to or suspected to have a cancer, wherein the
level of Notch3 measured in the patient is compared with that of a
reference subject or standard. The method can then be used to
determine whether a tumor expresses Notch3, which may suggest that
the tumor will respond well to treatment with the antibody-drug
conjugates of the present invention. In one embodiment of the
invention, the tumor is a solid tumor cancer, including but not
limited to, lung cancer, breast cancer, ovarian cancer, stomach
cancer, esophageal cancer, cervical cancer, head and neck cancer,
bladder cancer, liver cancer, skin cancer and sarcoma or a blood
cancer, including but not limited to, T-cell malignancies, T-cell
leukemia, T-cell lymphoma, T-cell acute lymphoblastic leukemia,
multiple myeloma, B-cell malignancies, myeloid malignancies, acute
myeloid leukemia and chronic myeloid leukemiain which Notch3 is
expressed, and other cancers yet to be determined in which Notch3
is expressed predominantly.
[0131] An embodiment of the invention is a method of treating a
Notch3 expressing cancer, the method comprising: determining the
level of Notch3 in a biological sample comprising the steps of:
contacting a sample obtained from a subject suspected to have
cancer with an anit-Notch3 antibody, or antigen-binding fragment
thereof; determining the cell surface levels of Notch3 in the
sample; comparing the cell surface levels of Notch3 with that of a
reference subject or standard; and administering an antibody-drug
conjugate of the present invention to the subject. The method may
optionally comprise a step of obtaining the sample from a subject
suspected to have cancer;
[0132] Another embodiment of the invention is a method of treating
a Notch3 expressing cancer the method comprising: determining the
level of Notch3 in a biological sample comprising the steps of:
subjecting a sample obtained from a subject suspected to have
cancer to in-situ hybridization (ISH); determining the level of
Notch3 mRNA in the sample; comparing the levels of Notch3 mRNA with
that of a reference subject or standard; and administering an
antibody-drug conjugate of the present invention to the subject.
The method may optionally comprise a step of obtaining the sample
from a subject suspected to have cancer.
[0133] The present invention further provides for monoclonal
antibodies, humanized antibodies and epitope-binding fragments
thereof that are further labeled for use in research or diagnostic
applications. In some embodiments, the label is a radiolabel, a
fluorophore, a chromophore, an imaging agent or a metal ion.
[0134] A method for diagnosis is also provided in which the labeled
antibodies or epitope-binding fragments thereof are administered to
a subject suspected of having a cancer, and the distribution of the
label within the body of the subject is measured or monitored.
Kit
[0135] The present invention also includes kits, e. g. comprising a
described cytotoxic conjugate and instructions for the use of the
cytotoxic conjugate for killing of particular cell types. The
instructions may include directions for using the cytotoxic
conjugates in vitro, in vivo or ex vivo. Typically, the kit will
have a compartment containing the cytotoxic conjugate. The
cytotoxic conjugate may be in a lyophilized form, liquid form, or
other form amendable to being included in a kit. The kit may also
contain additional elements needed to practice the method described
on the instructions in the kit, such a sterilized solution for
reconstituting a lyophilized powder, additional agents for
combining with the cytotoxic conjugate prior to administering to a
patient, and tools that aid in administering the conjugate to a
patient.
[0136] All publications and patent documents cited above or in the
following Examples are hereby incorporated by reference in their
entirety for all purposes to the same extent as if each were so
individually denoted.
[0137] The invention will be further described with reference to
the following examples; however, it is to be understood that the
invention is not limited to such examples.
[0138] The following examples of specific aspects for carrying out
the present invention are offered for illustrative purposes only,
and are not intended to limit the scope of the present invention in
any way.
Example 1
Generation of Recombinant Notch3 Protein Immunogens
[0139] cDNA constructs encoding the human Notch3 NRR region with a
signal peptide at N-terminus, and Avi and His6 tag at C-terminus,
were cloned into the expression vector pSMED2. These constructs
were transiently transfected into Chinese hamster ovary (CHO) cells
and the secreted proteins in conditioned media were analyzed on
SDS-PAGE. After processing at the S1 cleavage site, the N-terminal
.about.26 kDa (LNR-A, B, C and HD1) and C-terminal .about.12 kDa
(HD2 and Avi_His tag) halves of the Notch3 NRR domain remain
associated through non-covalent interactions to form a
heterodimeric complex, as shown in FIG. 1. S1 processing of the
Notch3 NRR was determined to be about 50% or less in samples
prepared from CHO cells. To enhance processing at the S1 cleavage
site, the Notch3 NRR expression construct was transfected into
CHO-PACE cells (Harrison et, al, Semin Hematol. 1998 April; 35(2
Suppl 2):4-10) and stable cell lines with the highest expression
and complete processing of Notch3 NRR were selected. Culture of
these cell lines was scaled up for the collection of conditioned
media from which Notch3 NRR proteins were purified.
[0140] The amino acid and nucleotide sequences of purified human
and mouse Notch3 NRR_Avi_His tag proteins (hereinafter "Notch3 NRR
recombinant proteins") are provided in FIG. 2. Lower case type
represents the Avi_His tag. SDS-PAGE analysis showed that >90%
of the purified protein was correctly cleaved into the predicted
Notch3 NRR N-terminal and C-terminal peptide sizes (data not
shown).
Example 2
Generation and Cloning of Rat Anti-Notch3 Antibodies
A. Immunization and Hybridoma Generation
[0141] Sprague-Dawley rats were immunized by subcutaneous
injections with a mixture containing 20 .mu.g each of human (SEQ ID
NO. 1) and mouse (SEQ ID NO. 3) Notch3 recombinant proteins in
Freund's complete adjuvant. Immunizations were repeated at 2-week
intervals for 12 weeks. Collected sera samples at day 0, 35, 49,
and 63 after the first injection were tested for circulating
anti-Notch3 antibody titer activity by enzyme-linked immunosorbent
assay (ELISA). When optimal titers were reached, a final dose of
the protein mixture was injected intravenously (tail vein) into a
rat having optimal antibody titer 4 days before it was to be
sacrificed for splenocyte collection. Total splenocytes
(2.times.10.sup.8) from the rat were fused with mouse myeloma cell
line P3X63.Ag8.653 (2.5.times.10.sup.7) using PEG 1500. Fused cells
were plated out in 96-well plates (0.2 mL/well) and subjected to
HAT selection (RPMI 1640 containing 5.times.10.sup.-4 M
Hypoxanthine, 1.6.times.10.sup.-5 M Thymidine, 4.times.10.sup.-4 M
Aminopterin, and 20% Heat Inactivated FCS).
[0142] Fourteen days post fusion, hybridoma supernatants were
harvested and tested for the presence of Notch3 binding activities.
Two parental rat clones 28 and 75 (hereinafter r28 and r75,
respectively) exhibited binding activity to human Notch3 NRR
recombinant protein by ELISA and to the cell surface of U-2 OS
cells stably over-expressing human full length Notch3 by a
cell-based ELISA with an O.D. 450 nM value of 1 or above at 0.1-1
nM antibody concentrations. Further, r28, but not r75, exhibited
binding activity to mouse Notch3 NRR recombinant protein by ELISA
and to the cell surface of U-2 OS cells stably over-expressing
mouse full length Notch3 by a cell-based ELISA with an O.D. 450 nM
value of 1 or above at 0.1-1 nM antibody concentrations.
B. Cloning and Sequencing of Anti-Notch3 Antibodies
[0143] r28 and r75 variable regions were subcloned for further
analysis. RNAs from the subclones were extracted and the variable
region DNA sequences from the expressed antibodies were obtained
via RT-PCR cloning. One to five million of the subcloned hybridoma
cells were homogenized for total RNA isolation with QIAGEN RNAeasy
Mini kit. First strand cDNA was then produced using SuperScript III
RT kit (Invitrogen). Double stranded cDNAs for variable regions of
anti-Notch3 IgGs were subsequently generated and amplified by PCR
using primers from the rat IgG heavy chain (IgG1, 2a, 2b) and light
chain (kappa or lambda) constant regions, as described below. PCR
cycling conditions: 1 cycle at 95.degree. C. for 1 min; 25 cycles
at 95.degree. C. for 1 min, 63.degree. C. for 1 min and 72.degree.
C. for 1 min. The resulting RT-PCR products were cloned into
TOPO-Blunt cloning vector (Invitrogen) and sequenced by
conventional methods. The amino acid and nucleotide sequences of
r28 and r75 variable regions are provided in FIG. 3. CDRs are
underlined.
Example 3
Generation and Characterization of Chimeric Anti-Notch3
Antibodies
[0144] Variable region cDNAs from r28 and r75 were subcloned into
mammalian expression vectors where rat variable heavy chain (VH)
were fused in frame with human IgG1 (hlgG1) and rat variable light
chain (VL) were fused with human kappa. Rat-human chimeric
antibodies 28 and 75 (hereinafter ch28 and ch75, respectively) were
generated from these constructs by transient transfection in HEK293
cells and further analyzed.
[0145] ch28 and ch75 were tested for binding activity in
recombinant protein ELISA. Purified human or mouse Notch3 NRR
recombinant proteins were coated on CoStar hi-bound 96-well ELISA
plates in 100 .mu.l of PBS with Mg/Ca at a concentration of 1
.mu.g/ml overnight. The plates were washed with PBS-Mg/Ca and
blocked for 1 hour with 1% BSA in PBS-Mg/Ca. Blocking solution was
decanted from the plate and 1:3 serial dilutions of antibodies in
blocking solutions were applied to the plate. After incubation at
room temperature for 1 hour, plates were washed again with
PBS-Mg/Can before HRP (horse radish peroxidase)-conjugated
secondary antibody diluted (1:20,000) in blocking buffer was
applied. When the primary antibody tested was rat IgG, the
secondary antibody was goat anti-rat IgG Fc (Bethyl Biotech); and
when the primary antibody was human IgG, the secondary antibody was
goat anti-human IgG Fc (Southern Biotech).
[0146] After 1 hour incubation with the secondary antibody, plates
were washed again, as described above, and TMB substrate solution
was added. The developing reaction was allowed for 10 minutes
before the stopping solution, 0.18M H.sub.2SO.sub.4, was added.
Absorbance at O. D. 450 nM was measured and data was plotted and
analyzed with Microsoft Excel and Graphpad-Prism software. ch28 and
ch75 exhibited strong binding activity to human Notch3 NRR
recombinant protein, having an EC50 of 1 nM or lower, and ch28, but
not ch75, also exhibited strong binding activity to mouse Notch3
NRR recombinant with an EC50 of 1 nM or below. ch28 and ch75 were
selected for further cell based ELISA, as described below.
Example 4
Humanization
[0147] r28 and r75 were selected to be humanized for further
development. Humanization was performed by CDR grafting onto human
acceptor frameworks, DP-54 for heavy chain and DPK9 for light
chain, followed by selected back mutations in human acceptor
framework to recover full activity of parental rat antibodies.
Table 1 shows selected back mutations in the human acceptor
framework for different variants.
TABLE-US-00001 TABLE 1 Selected back mutations for humanized
anti-Notch3 antibody variants. Variant Back Mutations (Kabat) VH
hu28 VH1.0 none hu28 VH1.1 A93T, V37I VL hu28 VL1.0 none hu28 VL1.1
Y36F VH hu75 VH1.8 R71V hu75 VH 1.9 V34M (CDR1), R71V VL hu75 VL
1.1 S60D hu75 VL 1.2 S60D, T85F hu75 VL 1.3 S67Y
[0148] cDNAs containing grafted CDR donor sequences of r28 and r75
onto human acceptor frameworks, DP-54 and DPK9, with selected back
mutations were synthesized by Genewiz. Synthesized cDNA products
were subcloned and fused in frame with human IgG1 heavy chain
constant region for the heavy chain and human kappa for the light
chain in mammalian expression vectors pSMED2 and pSMEN3,
respectively.
[0149] Humanized r28 variant VH1.0/VL1.0 (hereinafter hu28) fully
retained the antigen binding epitope and affinity of ch28 and
lacked potent signaling inhibition activity observed in ch28. The
CDRs of the VH and VL regions of hu28 are the same as the CDRs of
the VH and VL regions of r28, respectively. Humanized r75 variant
VH1.9/VL1.3 (hereinafter hu75) fully retained the antigen binding
epitope and affinity of ch75 and potent signaling inhibition
activity observed in ch75. CDR2 and CDR3 of the VH region of hu75
are the same as CDR2 and CDR3 of the VH region of r75,
respectively. CDR1 of the VH region of hu75 contains one back
mutation V34M. The CDRs of the VL region of hu75 are the same as
the CDRs of the VL region of r28. FIGS. 4A-4C provide amino acid
and nucleotide sequences of hu28 with CDRs identified (Kabat and
Chothia), and FIGS. 5A-5C provide amino acid and nucleotide
sequences of hu75 with CDRs identified (Kabat and Chothia).
[0150] Alignment of the VH and VL regions of the human acceptor
framework for r28 and hu28, and r75 and hu75 are shown in Table 2.
The Kabat CDRs are underlined. The differences in residues of the
framework between rat and humanized sequences are indicated by
lower case text. The homology between the human acceptor framework
for r28 and hu28 variable regions are 73% for VH and 76% for VL.
The homology between the human acceptor framework for r75 and hu75
variable regions are 46% for VH and 64% for VL. Kabat CDRs are
underlined.
TABLE-US-00002 TABLE 2 Alignment of human acceptor frameworks for
anti-Notch3 antibodies. r28 VH: DP54_JH4
EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYWMS WVRQAPGKGLEWVA
NIKQDGSEKYYVDSVKG RFTISRDNAKNSLY r28VH
EVQLVESGGGLVQPGRSLTLSCVASGFTFR DYGMT WIRQAPGKGLTWVA
YISSGSNYIYYAEAVKG RFTISRDNAKNTLY hu28VH1.0
EVQLVESGGGLVQPGGSLRLSCAASGFTFR DYGMT WVRQAPGKGLEWVA
YISSGSNYIYYAEAVKG RFTISRDNAKNSLY DP54_JH4 LQMNSLRAEDTAVYYCAR
----YFDY WGQGTLVTVSS (SEQ ID NO: 67) r28VH LQMTSLRSEDTALYFCTR
RGPFVLDA WGQGASVTVSS (SEQ ID NO: 5) hu28VH1.0 LQMNSLRAEDTAVYYCAR
RGPFVLDA WGQGTLVTVSS (SEQ ID NO: 13) r28 VL: DPK9_Jk4
DIQMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS
GVPSRFSGSGSGTDFTLTISSLQP r28VL DIQMTQSPSFLSASVGDRVTINC KASQSINRYLH
WFQQKLGEAPKLLIY NANGLQT GIPSRFSGSGSGTDFTLTISSLQS hu28VL1.0
DIQMTQSPSSLSASVGDRVTITC KASQSINRYLH WYQQKPGKAPKLLIY NANGLQT
GVPSRFSGSGSGTDFTLTISSLQP DPK9_Jk4 EDFATYYC QQSYSTPLT FGGGTKVEIK
(SEQ ID NO: 68) r28VL EDVATYFC LQHNTWPDT FGAGTKLELK (SEQ ID NO: 7)
hu28VL1.0 EDFATYYC LQHNTWPDT FGGGTKVEIK (SEQ ID NO: 25) r75 VH:
DP54_JH4 EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYWmS
WVRQAPGKGLEWVANIKQDGSEKYYVDSVKG RFTISrDNAKNSLY r75VH
QVKLLQSGAALVKPGASVKMSCKASGYAFT DYWvT
WVKQSHGKSLEWIGEISPNSGGTNFNEKFKG KATLTvDKSTSTAY hu75VH1.9
EVQLVESGGGLVQPGGSLRLSCAASGYAFT DYWmT
WVRQAPGKGLEWVAEISPNSGGTNFNEKFKG RFTISvDNAKNSLY DP54_JH4
LQMNSLRAEDTAVYYCAR ------YFDY WGQGTLVTVSS (SEQ ID NO: 67) r75VH
MELSRLTSEDSAIYYCTR GEIRYNWFAY WGQGTLVTVSS (SEQ ID NO: 9) hu75VH1.9
LQMNSLRAEDTAVYYCAR GEIRYNWFAY WGQGTLVTVSS (SEQ ID NO: 37) r75 VL:
DPK9_Jk4 DIQMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY
AASSLQS GVPSRFSGSGsGTDFTLTISSLQP r75VL NIVMTQSPKSMSISVGDRVTMNC
KASQNVGNNIA WYQQKPGQSPKLLIY YASNRYT GVPDRFTGGGyGTDFTLTINSMQA
hu75VL1.3 DIQMTQSPSSLSASVGDRVTITC KASQNVGNNIA WYQQKPGKAPKLLIY
YASNRYT GVPSRFSGSGyGTDFTLTISSLQP DPK9_Jk4 EDFATYYC QQSYSTPLT
FGGGTKVEIK (SEQ ID NO: 68) r75VL EDAAFYYC QRLYNSPFT FGSGTKLEIK (SEQ
ID NO: 11) hu75VL1.3 EDFATYYC QRLYNSPFT FGGGTKVEIK (SEQ ID NO:
49)
Example 5
Characterization of Hu28 and Hu75
A. Cell-Based ELISA
[0151] Humanized hu28 and hu75, and rat-human chimeric, ch28 and
ch75, anti-Notch3 antibodies were screened for cell surface Notch3
binding in a cell-based ELISA. U-2 OS cells stably over-expressing
human or mouse full length Notch3 protein on cell surface
(hereinafter U2OS/hNotch3 and U2OS/mNotch3, respectively) were
plated at 50,000 cells/well in 96 well plates (white opaque,
BD/VWR) the day before ELISA assay. On the day of the ELISA,
culture media was removed from wells and serially diluted (1:3 in
blocking buffer) antibody solutions were applied to the plate.
Plates were incubated at room temperature for 2 hours before being
washed with PBS-Mg/Ca. HRP-conjugated secondary antibody was then
applied and incubated with cells for 1 hour as described above for
recombinant protein ELISA. Plates were washed with PBS-Mg/Ca before
being developed with Pico-Chemiluminescent developing kit (Thermal
Scientific), and chemiluminescence measurements were performed per
manufacturer's instructions. Data plotting and analyses were
performed with Microsoft Excel and Graphpad-Prism software.
EC.sub.50 (nM) values were calculated from cell surface Notch3
binding ELISAs and provided in Table 3.
[0152] The data demonstrates that hu28 is similar to ch28 in
binding to full-length human Notch3 expressed on the cell surface
U2OS/hNotch3 cells. Further, the data demonstrates that hu28 fully
retained the cross-reactivity of ch28 to mouse Notch3 expressed on
the cell surface of U2OS/mNotch3 cells. The data also demonstrates
that hu75 is similar to ch75 in binding to full-length human Notch3
expressed on the cell surface of U2OS/hNotch3 cells. Further, the
data demonstrates that hu75 fully retained specificity of ch75 to
human Notch3; no cross-reactivity was observed to mouse Notch3
expressed on the cell surface of U2OS/mNotch3 cells. N/B represents
non-binding.
TABLE-US-00003 TABLE 3 EC.sub.50 (nM) values of cell surface Notch3
binding ELISAs for anti-Notch3 antibodies. EC.sub.50 (nM) hu28 hu75
Antibody ch28 VH1.0/VL1.0 ch75 VH1.9/VL1.3 Full-length 0.098 0.093
0.20 0.22 Human Notch3 Full-length 0.13 0.13 N/B N/B Mouse
Notch3
B. Competition ELISA
[0153] Competition ELISAs were performed for hu28 and ch28, and
hu75 and ch75 on cell surface expressed full-length human Notch3.
96 well cell culture plates were seeded with U2OS/hNotch3 cells
(cell culture plate, Co-star). Serially diluted (1:3 in blocking
buffer) antibody solutions, in the presence of 0.8 nM of ch28
(biotinylated) or ch75 (biotinylated) antibodies were applied to
the plate. After incubation for 2 hours, the plates were washed, as
described above, and HRP-conjugated streptavidin (Southern Biotech)
diluted 1:5000 in blocking buffer was applied. After incubation
with streptavidin for 30 minutes, the plates were washed again
before being developed with TMB solution for 10 minutes. The
developing reaction was stopped by adding 0.18M H.sub.2SO.sub.4 and
absorbance at 450 nM was measured. Data plotting and analyses were
performed with Microsoft Excel and Graphpad-Prism software.
[0154] Table 4 shows EC.sub.50 (nM) values from competition ELISAs
for binding to full-length human Notch3 expressed on the cell
surface of U2OS/hNotch3 cells. The data shows that hu28 has a
similar EC.sub.50 value to the unlabelled r28, demonstrating that
hu28 competes as well as unlabelled ch28 with biotinylated ch28 for
binding to full-length human Notch3 expressed on the cell surface
of U2OS/hNotch3 cells. The data indicates that hu28 binds to the
same, or a highly similar, epitope on full-length human Notch3
expressed on the cell surface of U2OS/hNotch3 cells as ch28.
[0155] The data further shows that hu75 has a similar EC.sub.50
value to unlabelled ch75, demonstrating that hu75 competes as well
as unlabelled ch75 with biotinylated ch75 for binding to
full-length human Notch3 expressed on the cell surface of
U2OS/hNotch3 cells. The data indicates that hu75 binds to the same,
or highly similar, epitope on full-length human Notch3 expressed on
the cell surface of U2OS/hNotch3 cells as ch75.
TABLE-US-00004 TABLE 4 EC.sub.50 (nM) values for competition ELISAs
of anti-Notch3 antibodies. EC.sub.50 (nM) hu28 hu75 Anti- VH1.0/
VH1.9/ E. Tenella Antibody ch28 VL1.0 ch75 VL1.3 Control
Full-length 3.0 3.1 1.3 1.7 Non- Human Notch3 Competing
C. Specificity of Binding to Other Human Notch Homologues
[0156] Other members of the Notch receptor family play important
roles in biological processes. For example, a Notch1 or Notch2
deficiency leads to embryonic death in mouse models. In contrast, a
Notch4 deficiency results in no detectable phenotype in mouse
models. The closest homologues of the Notch3 NRR region are Notch1
and Notch2 (.about.50% homology), and Notch4 is a more distant
homologue (.about.30% homology). Cross-reactivity of anti-Notch3
antibodies to other members of the Notch family, especially Notch1
and 2, may lead to undesired effects in patients. Therefore, the
potential cross-reactivity of r28 and hu28, along with r75 and hu75
to other Notch family members were assessed.
[0157] Expression constructs encoding human Notch1 and Notch2 NRR
regions, fused with human IgG1 Fc fragments were stably introduced
into CHO-PACE cells. Conditioned media from these cells expressing
NRR-Fc fusions were collected. Human Notch2 NRR-Fc and human Notch1
NRR-Fc were purified by protein A affinity followed by size
exclusion chromatography (SEC). Purified preparations were dialysed
into TBS with 1 mM CaCl.sub.2 and analyzed on analytical SEC to be
>99% in purity.
[0158] As shown in Table 5, r28 and hu28, along with r75 and hu75
all lacked detectable binding to human Notch2 NRR-Fc fusion
proteins. Further, hu28 and hu75 also lacked detectable binding to
full-length human Notch1 NRR-Fc. This demonstrates that hu28 and
hu75 do not cross-react with Notch1 or Notch2. N/B represents
non-binding.
TABLE-US-00005 TABLE 5 Binding of anti-Notch3 antibodies to Notch2
NRR-Fc and Notch1 NRR-Fc. Antibody Binding Notch2 ch28 N/B NRR-Fc
hu28 VH1.0/V1.0 N/B ch75 N/B hu75 VH1.9/V1.3 N/B Notch1 ch28 N/B
NRR-Fc hu28 VH1.0/V1.0 N/B ch75 N/B hu75 VH1.9/V1.3 N/B
D. Binding Affinity to Human Notch3 NRR
[0159] The kinetic constants of the anti-Notch3 NRR interactions
were determined by surface plasmon resonance (Biacore.RTM. T100,
Biacore Inc., Piscataway, N.J.). Flow cells of a CM5 chip were
immobilized with approximately 10,000 response units (RU) of
anti-human IgG-Fc (Biacore.RTM.) in 10 mM Glycine, pH 5.0 at 10
.mu.l/min for 600 seconds. 10 .mu.g/ml of anti-Notch3 antibodies
ch28 and hu28, and ch75 and hu75 were diluted in TBS with 1 mM
CaCl.sub.2 were captured at 10 .mu.l/min. Association of four
concentrations of human Notch3 NRR recombinant protein (from
3.7-100 nM) and a zero concentration (running buffer) at 100
.mu.l/min were recorded for 3 minutes in TBS with 1 mM CaCl.sub.2.
Dissociation of the complexes was measured for 10 minutes. The
surface of the chip was regenerated by injecting 3M MgCl.sub.2 with
3 mM EGTA for 60 seconds at 10 .mu.l/min. Curves obtained after
subtraction of the reference and buffer signals were fitted to a
1:1 Langmuir binding model with Biacore.RTM. T100 Evaluation
Software (Biacore.RTM.).
[0160] K.sub.a, K.sub.d and K.sub.D are shown in Table 6. Kinetic
analysis shows similar ka (on) and kd (off) rates for ch28 and
hu28. Further, a higher k.sub.a (on) and lower k.sub.d (off) rates
for hu75 than ch75 were observed, resulting in a lower K.sub.D
value for hu75.
TABLE-US-00006 TABLE 6 Kinetic analysis of anti-Notch3 antibodies.
Antibody k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (nM) ch28 4.09E+05
2.49E-04 0.61 hu28 VH1.0/VL1.0 3.86E+05 3.47E-04 0.90 ch75 5.70E+04
9.92E-04 17.40 hu75 VH1.9/VL1.3 3.48E+04 3.40E-04 9.76
D. Thermal Stability
[0161] There is a positive correlation between the thermal
stability of a protein or protein domain with the overall stability
of the protein or protein domain. A higher melting point of a
protein or protein domain often provides improved manufacturability
and longer shelf life. Differential scanning calorimetry (DSC) was
used to assess the thermal stability of hu28 and hu75 versus ch28
and ch75, respectively. Protein samples were diluted in PBS to 0.3
mg/ml in a volume of 250 .mu.l. The corresponding formulation
buffer blank was used for the reference sample. Both samples were
thoroughly degassed using a MicroCal ThermoVac Sample Degassing and
Thermostat (Microcal, Inc., Northampton, Mass.) set to 8.degree. C.
Samples were dispensed into the appropriate cells of a MicroCal
VP-DSC Capillary Cell MicroCalorimter (MicroCal, Inc., Northampton,
Mass.). Samples were equilibrated for 4 minutes at 15.degree. C.
and then scanned up to 100.degree. C. at a rate of 100.degree. C.
per hour. A filtering period of 20 seconds was selected. Raw data
was baseline corrected and the protein concentration was
normalized. Origin Software (OriginLab Corporation, Northampton,
Mass.) was used to fit the data to an MN2-State Model with an
appropriate number of transitions.
[0162] As shown in Table 7 below, both hu28 and hu75 had higher
thermostability, as displayed by higher melting point, in their Fab
region (all above 770C) compared to ch28 and ch75,
respectively.
TABLE-US-00007 TABLE 7 Thermal Stability (DSC) analysis of
anti-Notch3 antibodies. Tm (.degree. C.) .+-. Standard Deviation
Antibody CH2 Fab CH3 ch28 70.20 .+-. 0.05 73.22 .+-. 0.73 84.24
.+-. 0.07 hu28 VH1.0/VL1.0 72.63 .+-. 0.03 83.45 .+-. 0.17 ch75
73.32 .+-. 0.01 84.59 .+-. 0.03 hu75 VH1.9/VL1.3 70.46 .+-. 0.02
74.63 .+-. 0.17 84.17 .+-. 0.05
Example 6
Domain Swap Chimeric Constructs
[0163] As described in Example 3, both hu28 and hu75 lacked
cross-reactivity with the Notch1 protein. Domain swap chimeric
constructs for the Notch1 and Notch3 NRR were prepared for epitope
mapping of the anti-Notch3 ch28 and ch75 antibodies. Expression
constructs encoding human Notch1-Notch3 (hereinafter Notch 1-3) NRR
region domain swap chimera with C-terminal Fc fusion (human IgG1 Fc
fragment) were individually transfected into CHO-PACE and stable
pools expressing each chimera were established. Conditioned media
from each stable pool were applied to protein A affinity
chromatography, followed by size exclusion chromatography (SEC) for
the purification of the chimeric fusion protein. Purified
preparations were dialysed into TBS with 1 mM CaCl.sub.2 and
analyzed on analytical SEC. FIG. 6 shows the recombinant NRR
chimeric proteins consist of various Notch1 (shown in grey) and
Notch3 (shown in black) domains fused to human Fc (not shown).
[0164] Relative binding capacities of ch28 and ch75 to Notch 1-3
NRR domain swap chimeras were tested in ELISAs as described in
Examples 3 and 5. The Notch 1-3 NRR domain swap chimera were coated
on ELISA plates at 1 ug/ml, followed by blocking with 1% BSA in PBS
with 0.9 mM Mg.sup.2+ and Ca.sup.2+. ch28 and ch75 were then
applied to the blocked plates at 5 ug/ml diluted in the blocking
buffer. After 1 hour of incubation, plates were washed with PBS
with 0.9 mM Mg.sup.2+ and Ca.sup.2+, before secondary goat
anti-human Fc antibody conjugated with HRP were applied and
incubated on the plates. After washing again, the plates were
developed with TMB and the developing reactions were stopped by
adding 0.18M H.sub.2SO.sub.4. O.D. 450 nM were read on plate reader
and relative binding capacities indicated by O.D. values.
[0165] As shown in FIG. 6, the epitope binding profile of ch28 and
ch75 to domains of the Notch3 NRR are distinct. More specifically,
the binding of ch28 to the Notch3 NRR was more dependent on the
LNR-C and HD-1 domains, while ch75 was more dependent on the LNR-A
and both the HD-1 and HD-2 domains. The observed binding profile
for ch28 and ch75 was consistent with the signaling inhibition
activities of the antibodies. In particular, ch28 only interacted
with the N-terminus of the Notch3 NRR heterodimer (HD-1) which
would not maintain the Notch3 NRR region in the auto-inhibitory
conformation that is required for inhibiting the activation of
Notch3 signaling. In contrast, ch75 interacted with both the HD-1
and HD-2 domains of the Notch3 NRR heterodimer which would maintain
the Notch3 NRR region in the auto-inhibitory conformation, thereby
inhibiting the activation of Notch3 signaling.
Example 7
Identification of Notch3 Expressing Cancer Cell Lines
[0166] Expression of Notch3 was determined in a panel of cancer
cell lines by western blot analysis to identify Notch3 positive
cells for further analysis and testing of anti-Notch3 antibodies
and anti-Notch3 antibody-drug conjugates. The panel included
HCC2429 lung cancer cell line, OVCAR3 ovarian cancer cell line,
MDA-MB-468 breast cancer cell line, N87 gastric cancer cell line,
along with cell lines engineered to over-express human Notch3
including U-2 OS and MDA-MB-468 cells, hereinafter referred to as
U2OS/hNotch3 and MDAMB468/hNotch3, respectively. Notch3 was
detected with a rabbit monoclonal anti-Notch3 antibody D11B8 (Cell
Signaling Technologies) or a mouse monoclonal 1G5 (Abnova) using
standard western blot procedures.
[0167] The D11B8 anti-Notch3 antibody binds to an epitope
surrounding Glu2312 within the C-terminal tail of the human Notch3
protein and recognizes both uncleaved, full-length Notch3
(.about.270 kDa) and cleaved, C-terminal domain containing Notch3
protein fragments at (.about.80-90 kDa). Notch3 bands at the
.about.80-90 kDa molecular weights represent the TMIC
(Transmembrane and intracellular domain) and/or NEXT (Notch
extracellular truncation) proteolytic fragments, as shown in FIG.
7. The 1 G5 antibodies bind an epitope within amino acids 47-156 of
the Notch3 extracellular domain (ECD) and recognize both the
uncleaved, full-length (.about.270 kDa) and the cleaved, N-terminal
fragment (.about.210 kDa) (data not shown).
[0168] Both the Notch3-ECD (data not shown) and the cleaved
C-terminal domain containing Notch3 protein fragments were detected
in HCC2429, OVCAR3, MDA-MB-468, N87, MDAMB468/hNotch3 and
U2OS/hNotch3, as shown in FIG. 7. Further, Notch3 was not detected
in the SW900 lung cancer cell line and thus represents a Notch3
negative control cell line.
Example 8
Internalization and Trafficking of Anti-Notch3 Antibodies
[0169] Anti-Notch3 antibody-drug conjugates consist of a peptide
cleavable
maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl (vc)
linker or a non-cleavable, thioether-based maleimidocaproyl (mc)
linker conjugated to a series of cytotoxic agents. Release of
payloads from the antibody requires trafficking and localization of
the antibody-drug conjugate to lysosomes that possess proteolytic
enzymes, such as cathepsin B, to cleave the vc-type linker or late
lysosomes for complete catabolism of the antibody in order to
release the mc-linked payloads. The internalization and
intracellular trafficking of anti-Notch3 antibodies to lysosomal
vesicles were monitored by indirect and direct immunofluoresence
microscopy-based assays. To directly visual intracellular
trafficking of anti-Notch3 antibodies, they were conjugated to
fluorescent dyes and incubated with live cells in the presence of
pHrodo.TM. red dextran (Life Technologies) to stain acidic vesicles
such as lysosomes. Live cell imaging was performed to identify
co-localization of the fluorescence-labeled antibody with lysosomes
and other acidic vesicles. Further, indirect immunofluorescence
microscopy-based assays were performed on cells to confirm
co-localization of anti-Notch3 antibodies and LAMP1, a
lysosomal-associated protein.
A. Internalization and Lysosomal Trafficking
[0170] HCC2429 or MDA-MB-468 cells were cultured in a Lab Tec II 4
chambered coverglass with cover #1.5 borosilicate sterile slides
(Thermo Fisher Scientific Inc.). On day 1, pHrodo.TM. red dextran
(Life Technologies) was added to the medium at 10 .mu.g/ml
concentration and incubated for 16 hours in order to stain acidic
vesicles such as lysosomes. On day 2, cells were washed twice with
HBSS++(Gibco Life Technologies). Anti-Notch3 antibody hu75 was
conjugated to Alexa Fluor 488 (hereinafter "hu75-Alexa488") (Life
Technologies Protein Labeling kit) and hu28 was directly conjugated
to the DyLight650 maleimide reagent (hereinafter "hu28-DyLight650")
(Thermo Scientific) according to manufacturer's instructions.
Hu75-Alexa488 or hu28-DyLight650 labeled antibodies were added to
the cells at a concentration 5 .mu.g/ml in 2% bovine serum albumin
(BSA)/HBSS++ for 25 minutes on wet ice. Cells were washed twice
with ice cold HBSS++ on ice, placed in 2% BSA/HBSS++ and imaged on
a spinning disk CSU-X1M 5000 microscope (Yokogawa) equipped with a
eXcelon Evolve 512 camera (Photometrics) and a XL S Series chamber
(Zeiss) for temperature, humidity and 5% CO.sub.2 control, from 5
minutes to 12-18 hours. Images were captured every 5 minutes and
combined using the Zen CZI file format (Zeiss). A Pearson's
correlation coefficient was calculated using the Volocity v6.3
software (PerkinElmer) to determine the degree of colocalization
between hu28-DyLight650 and pHrodo.TM. red dextran.
[0171] Once cells were prepared for live cell imaging, the earliest
time point that could be assessed was 10 minutes due to set up and
optimization of instrument setting for image acquisition.
Approximately 10 minutes after cells were place in the humidified,
37.degree. C., 5% CO.sub.2 chamber, hu75-Alexa488 could be observed
at the cell surface as well as in several punctuate-like structures
inside the cells, as shown in FIG. 8A. This result indicates that
anti-Notch3 antibody hu75 rapidly underwent internalization upon
binding the Notch3 receptor at the cell membrane. At the early time
points (10 minutes to about 65 minutes), intracellular anti-Notch3
antibody hu75-Alexa488 and acidic vesicles that were stained with
pHrodo.TM. red dextran appeared as discrete puncta that did not
co-localize. From time about 70 minutes and later, anti-Notch3
antibody hu75 and pHrodo.TM. red dextran-labeled vesicles
co-localized to the same discrete punctate-like structures, as
shown by arrows in FIG. 8B. This data suggests that anti-Notch3
antibody hu75 internalized and trafficked to acidic vesicles such
as lysosomes in Notch3 expressing HCC2429 (data not shown) and
MDA-MB-468 cells, as shown in FIG. 8B.
[0172] About 10 minutes after cells were place in the humidified,
37.degree. C., 5% CO.sub.2 chamber, hu28-DyLight650 could be
observed at the cell surface, as shown in FIG. 8C. Intracellular
anti-Notch3 antibody hu28-DyLight650 and pHrodo.TM. red
dextran-stained acidic vesicles appeared as discrete puncta and
gradually co-localized over time, as shown by arrows in FIG. 8D. A
Pearson's correlation coefficient (PCC) was calculated to determine
the degree of overlap or co-localization between hu28-DyLight650
and pHrodo.TM. red dextran fluorescent labels in MDA-MB-468 cells
over time. The higher the PCC number, the greater degree of
co-localization between the hu28-DyLight650 and pHrodo.TM. red
dextran fluorescence labels. FIG. 8E demonstrates a steady increase
in co-localization up to about 360 minutes and a maximal
colocalization between hu28-DyLight650 and pHrodo.TM. red dextran
occurs at about 420 minutes and then plateaus. The data suggests
that anti-Notch3 antibody hu28-DyLight650 internalized and
trafficked to acidic vesicles such as lysosomes in Notch3
expressing MDA-MB-468 cells.
B. Co-Localization with Lysosomal-Associated Membrane Protein 1
(LAMP1)
[0173] HCC2429 or MDA-MB-468 cells were cultured in a Lab Tec II 4
chambered coverglass with cover #1.5 borosilicate sterile slides
(Thermo Fisher Scientific Inc.). For binding and internalization
assays, cells were washed twice with HBSS++ and unconjugated
anti-Notch3 antibodies, hu75 and hu28, were added to the cells at a
concentration 10 .mu.g/ml in 2% bovine serum albumin (BSA)/HBSS++
for 25 minutes on ice. To visual membrane binding at time 0
minutes, control cells were washed with ice cold HBSS++ and then
fixed in 4% paraformaldehyde in PBS for 10 minutes. For antibody
internalization, cells were washed twice with ice cold HBSS++ and
then pre-warmed complete growth media was added to the cells and
placed inside a humidified 37.degree. C., 5% CO.sub.2 incubator.
Cells were removed from the incubator, washed and fixed as before
at multiple time points from 5 minutes to 18 hours. Cells were then
washed 3 times with PBS, and permeabilized for 10 minutes with 0.3%
Triton X-100 in PBS. Cells were washed 3 times with PBS for 5
minutes each time and then blocked for 1 hour in 3% BSA/PBS.
Anti-LAMP-1 mouse monoclonal antibody (H4A3, Abcam) was added at
1:100 in 2% BSA/PBS for overnight incubation at 4.degree. C. Cells
were washed twice with PBS for 5 minutes each and then secondary
goat anti-human Alexa Fluor 488 and goat anti-mouse Alexa Fluor 555
(Life technologies) were added for 45 minutes in the dark. Cells
were washed three times with PBS and were imaged on Zeiss LSM510
confocal microscope or a CSU-X1M 5000 (Yokogawa) spinning disk
confocal microscope.
[0174] In control cells incubated on ice with anti-Notch3
antibodies and then immediately fixed, both hu28 and hu75 were
localized at the cell surface of Notch3 expressing cells and no
staining was observed inside the cells. In control cells, lysosomes
were stained with an anti-LAMP1 antibody and appeared as discrete
punctuate-like structures inside the cells that did not co-localize
with anti-Notch3 antibodies hu28 and hu75. After incubation at
37.degree. C. for 90 minutes, anti-Notch3 antibodies hu28 and hu75
were observed in punctuate-like structures inside the cells that
co-localized with anti-LAMP1 antibodies. After incubation at
37.degree. C., cell membrane staining of anti-Notch3 antibodies
hu28 and hu75 was reduced and eventually became undetectable. This
data suggested that anti-Notch3 antibodies hu28 and hu75 bound the
cell surface after incubation with cells on ice and then underwent
temperature-dependent internalization at 37.degree. C. Once
internalized, anti-Notch3 antibodies hu28 and hu75 co-localized
with the LAMP1 protein indicating they trafficked specifically to
the lysosome (data not shown).
Example 9
Effects of Anti-Notch3 Antibodies on Notch3 Signaling in Cell-Based
Assays
[0175] Notch3 signaling is initiated by ligand-induced proteolysis.
The mature Notch3 heterodimer after furin-like protease cleavage at
site S1 is held in an auto-inhibited state by the juxtamembrane
negative regulatory region (NRR). Binding of ligands, such as DLL4
or Jagged1, to the Notch3-ECD induces two successive additional
cleavages at sites S2 and S3 that are catalyzed by ADAM-type
metalloproteinase and gamma-secretase, respectively. The latter
cleavage releases the intracellular domain of Notch3 (NICD3),
permitting it to translocate to the nucleus and activate the
transcription of target genes containing consensus DNA binding site
motifs for the CSL protein.
[0176] The ability of anti-Notch3 chimeric antibodies, ch28-hulgG1
and ch75-hulgG1, and humanized antibodies, hu28 and hu75, to
inhibit Notch3 signaling were tested in a Notch3-dependent reporter
gene co-culture assay. Anti-Notch3 antibodies were pre-incubated
with Notch3 reporter cells and then co-cultured with DLL4-HEK293
cells to activate Notch3 signaling or with parental HEK293 cells as
a control.
A. Cell Line Construction for Notch3 Reporter Gene Co-Culture
Assay
[0177] To generate the Notch3 reporter cell line, a series of three
sequential, stable transfections were performed in the U-2 OS human
osteosarcoma cell line (ATCC, Manassas, Va.). The first
transfection used a vector for expression of full-length human
Notch3 based on the pCMV6-Entry-Myc-Flag backbone (Origene), and
the correct DNA sequence of the Notch3 insert was confirmed.
Following transfection with the TransIT-LT1 transfection reagent
(Mirus, Madison, Wis.), U-2 OS cells were selected in G418 and
clonal lines were isolated. Second, stable Notch3-expressing U-2 OS
clones were re-transfected with the pGL4.27 [luc2P/minP/Hygro]
vector (Promega, Madison, Wis.) containing eight tandem copies of
the CSL enhancer sequence (CGTGGGAAAAT), selected in Hygromycin B
plus G418 and clonal lines were isolated. The 8.times.CSL
Firefly-luciferase reporter construct is responsive to activated
Notch signaling (for example, see, Jeffries et al., Mol. Cell.
Biol. 22(11):3927-3941, 2002). Thirdly, the human Notch3
8.times.CSL Firefly-Luciferase U-2 OS cells were transduced with
Cignal Lenti Renilla Control (luc) (Qiagen, Calif.) lentiviral
particles, selected in Puromycin, Hygromycin B and G418, and clonal
lines were isolated. The Cignal Lenti Renilla control (luc) vector
encoded the Renilla-luciferase gene that is constitutively
expressed from a CMV promoter and served as an internal control.
The triple stable transfected U-2 OS line (hereinafter termed
"Notch3 reporter cells") was maintained in McCoy's 5A medium
(Gibco, Grand Island, N.Y.) containing 10% FBS, 1.times.
Penicillin/Streptomycin/L-Glutamine (Gibco), 0.25 mg/ml G418
sulfate, 0.3 mg/ml Hygromycin B and 0.001 mg/ml Puromycin.
[0178] To generate the ligand-expressing cells, HEK293 cells (ATCC)
were transfected with a vector for expression of human DLL4. The
vector was based on the pCMV6-AC-HA-His backbone (Origene,
Rockville, Md.), and the correct DNA sequence of the DLL4 insert
was confirmed. Following transfection, HEK293 cells were selected
in 0.5 mg/ml G418, and clonal lines were isolated, expanded and
analyzed for DLL4 expression. Clones with high DLL4 expression and
high induction of Notch3 reporter activity in the U-2 OS cells were
used to assess the inhibitory effect of anti-Notch3 antibodies.
[0179] The luminescent readings from Firefly-luciferase were
divided by the internal control Renilla-luciferase reading to
normalize the signals (termed hereinafter "F/R ratio"). To
calculate the fold-induction of Notch3 signaling, the F/R ratios
generated from the DLL4-HEK293 co-culture reporter assays were
divided by the F/R ratios from the parental HEK293 co-cultures and
termed relative luciferase unit (RLU) or activity.
B. Reporter Gene Co-Culture Assays
[0180] Human Notch3 reporter cells were trypinized and harvested
from culture plate in assay medium which consisted 50% complete
McCoy's 5A media (McCoy's 5A with 10% FBS and penicillin,
streptomycin, Invitrogen) and 50% of complete MEM media (MEM with
10% FBS and penicillin, streptomycin, Invitrogen) and counted.
Appropriate dilutions of cells were made with the same medium to
allow for 10,000 cells/well in a total volume of 45 .mu.l/well on a
96 well culture plate (white opaque, BD/VWR), in the presence of
serially diluted (1:3 in complete McCoy's 5A media) antibody
solutions or hybridoma culture supernatants. The mixture of cells
and antibody dilutions were incubated on the plates in a sterile
hood at room temperature for 1 hr before 30,000/45 .mu.l of human
DLL4-HEK293 cells were added to each well. After addition of
hDLL4-HEK293 cells, the plates were further incubated for 20 hrs in
the incubator and Dual-Glo Luciferase assay system (Promega) was
used to measure the firefly luciferase and internal control Renilla
luciferase activity per manufacturer's instructions. Data was
plotted and analyzed using Microsoft Excel and Graphpad-Prism
software.
[0181] A titration of ch75-hulgG1 and hu75 in the human Notch3
reporter co-culture assay demonstrated potent inhibition of Notch3
signaling in a dose-dependent manner. Table 8 shows the inhibitory
activities of ch75-hulgG1 and hu75 antibodies against Notch3
dependent signaling in human Notch3 reporter cells. ch75-hulgG1 and
hu75 showed similar neutralization activities in human Notch3
dependent signaling reporter assays. Therefore, hu75 fully retained
the inhibitory activity of ch75-hulgG1. Both ch28-hulgG1 and hu28
weakly inhibited Notch3 signaling at a level similar to control
ch2H6-hulgG1 and huNeg8.8 antibodies. This indicates that the
inhibitory activity of ch28-hulgG1 and hu28 were non-specific.
Further, the data demonstrates that anti-Notch3 antibodies
ch28-hulgG1 and hu28 do not inhibit Notch3 signaling and therefore
are functionally distinct from ch75-hulgG1 and hu75.
TABLE-US-00008 TABLE 8 Dose-dependent inhibitory activity of
humanized and chimeric antibodies. % inhibition (SD) hu28 VH1.0/
hu75 VH1.9/ huNeg- ch28- ch75- ch2H6- nM VL1.0 VL1.3 8.8 hIgG1
hIgG1 hIgG1 0 100 100 100 100 100 100 (0) (0) (0) (0) (0) (0) 0.27
99.9 104.4 84.5 102.8 88.4 95.7 (6.2) (4.9) (8.6) (6.1) (8.7) (6.9)
0.82 88.6 103.0 88.2 83.2 70.6 93.1 (11.8) (17.8) (9.0) (11.7)
(4.1) (11.0) 2.47 77.7 65.6 84.6 75.0 48.4 92.4 (9.5) (12.3) (7.5)
(5.2) (4.2) (9.0) 7.41 71.9 40.5 79.3 74.0 26.8 84.8 (4.7) (4.1)
(10.4) (2.9) (1.9) (9.4) 22.2 68.6 22.7 83.5 76.5 20.3 94.8 (7.9)
(3.2) (8.8) (6.2) (2.1) (8.6) 66.7 66.8 14.2 81.4 72.3 13.2 83.1
(2.0) (1.1) (4.3) (8.9) (1.0) (2.8) 200 71.1 13.7 75.7 70.3 13.9
78.2 (10.3) (0.9) (5.1) (4.1) (1.4) (5.1)
[0182] The IC.sub.50 (nM) values of ch75-hulgG1 and hu75 variants
were calculated from the inhibition of Notch3-dependent signaling
of the Notch3 reporter gene co-culture assays. IC.sub.50 (nM) data
from two or three independent experiments were averaged, as
provided in Table 9. Both hu75 and ch75-hulgG1 have low IC.sub.50
values indicating they are potent inhibitors of Notch3
signaling.
TABLE-US-00009 TABLE 9 IC.sub.50 values (nM) for inhibitory
activity of hu75 and ch75-hlgG1. Antibody IC.sub.50 (nM) SD hu75
VH1.9/VL1.3 4.43 2.36 ch75-hlgG1 2.10 0.66
C. Effect of Anti-Notch3 Antibodies on Site 2 Cleavage by
Metalloprotease
[0183] To confirm that binding of anti-Notch3 antibody hu75, but
not hu28, to the NRR domain of Notch3 is accompanied by a decrease
in S2-cleavage, western blot analysis was performed. The rabbit
monoclonal anti-Notch3 antibody D11B8 (Cell Signaling Technologies)
recognizes the Notch3 C-terminal fragments that are products of S1
and S2 proteolytic cleavage events. As demonstrated by NRR domain
swap experiments, the anti-Notch3 antibody hu75 bound
simultaneously to LNR-A, HD-1 and HD-2 domains located on two
non-covalently linked regions of the NRR that are separated by
furin-cleavage at site 1 (S1). The anti-Notch3 antibody hu28 bound
to LNR-C and HD1 domains that are located on a linear, covalently
linked region of the NRR domain that is N-terminal to the S1-site.
An inhibitory antibody is expected to decrease the detection of
S2-cleaved Notch3 by stabilizing the NRR domain in an
auto-inhibitory conformation thus preventing S2-cleavage, while a
non-inhibitory antibody is not expected to have an effect on
S2-cleavage.
[0184] Site 2 (S2)-cleavage of the Notch3 receptor was assessed by
western blot analysis of the protein using the D11B8 (Cell
Signaling Technologies) antibody which recognizes an epitope
surrounding Glu2312 in the C-terminal domain. HCC2429 and
MDA-MB-468 breast cancer cells were used to examine the effects of
inhibitory anti-Notch3 antibody hu75 and non-inhibitory anti-Notch3
antibody hu28 on S2-cleavage. For the assay, 1-2.5.times.10.sup.6
cells were plated in complete growth medium. Hu75, hu28 and
huNeg8.8 control antibody were added at a concentration of 5
.mu.g/ml. Cells were incubated at 37.degree. C. in a 5% CO.sub.2
incubator for 24 hours and then directly lysed in buffer. Extracts
were resolved by denaturing SDS-PAGE on a 7.5% polyacrylamide gel
(Bio-Rad criterion gel) and transferred to nitrocellulose paper
using an iBlot Gel transfer system (Invitrogen). Notch3 was
detected with D11B8 antibody and, as a loading control, anti-GAPDH
(Sigma) using standard western blot procedures.
[0185] FIG. 9 shows a Western blot analysis of the S2-cleavage
assay. In untreated and control huNeg8.8-treated HCC2429 and
MDA-MB-468 cells for 24 hours, both S1- and S2-cleaved C-terminal
Notch3 protein fragments could be detected by western blot analysis
with D11B8 antibody, as expected. Further, in cells treated with
inhibitory anti-Notch3 antibody hu75, the S1-cleaved Notch3
C-terminal fragment was detected, but the S2-cleaved fragment was
not detected indicating that metalloprotease cleavage was blocked
by hu75. Furthermore, in cells treated with non-inhibitory
anit-Notch3 antibody hu28, both S1- and S2-cleaved Notch3
C-terminal fragments were detected indicating that hu28 does not
inhibit proteolysis of the receptor. Therefore, hu28 binds with
high affinity to the Notch3-NRR as demonstrated in Example 5 and
had a high binding activity to full-length Notch3 expressed on the
cell surface, but does not inhibit Notch3 signaling indicating it
is functionally distinct from inhibitory anti-Notch3-NRR
antibodies, including hu75.
Example 10
Synthesis of Compounds
[0186] Compounds 0101, 6780, 0131, 3377 and 8261 were prepared
according to the methods described in International Publication No.
WO/2013/072813, which is incorporated herein by reference.
A. Experimental for Compound 0101 (#54 in the Schematic)
Preparation of
2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-met-
hyl-3-oxo-3-{[(1
S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-met-
hyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#54)
##STR00003##
[0188] Step 1.
[0189] Synthesis of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-3-metho-
xy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1
S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-met-
hyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#53). According to
general procedure D (below), from #32 (2.05 g, 2.83 mmol, 1 eq.) in
dichloromethane (20 mL, 0.1 M) and N,N-dimethylformamide (3 mL),
the amine #19 (2.5 g, 3.4 mmol, 1.2 eq.), HATU (1.29 g, 3.38 mmol,
1.2 eq.)
##STR00004##
and triethylamine (1.57 mL, 11.3 mmol, 4 eq.) was synthesized the
crude desired material, which was purified by silica gel
chromatography (Gradient: 0% to 55% acetone in heptane), producing
#53 (2.42 g, 74%) as a solid. LC-MS: m/z 965.7 [M+H.sup.+], 987.6
[M+Na.sup.+], retention time=1.04 minutes; HPLC (Protocol A): m/z
965.4 [M+H.sup.+], retention time=11.344 minutes (purity >97%);
.sup.1H NMR (400 MHz, DMSO-d.sub.6), presumed to be a mixture of
rotamers, characteristic signals: .delta. 7.86-7.91 (m, 2H), [7.77
(d, J=3.3 Hz) and 7.79 (d, J=3.2 Hz), total 1H], 7.67-7.74 (m, 2H),
[7.63 (d, J=3.2 Hz) and 7.65 (d, J=3.2 Hz), total 1H], 7.38-7.44
(m, 2H), 7.30-7.36 (m, 2H), 7.11-7.30 (m, 5H), [5.39 (ddd, J=11.4,
8.4, 4.1 Hz) and 5.52 (ddd, J=11.7, 8.8, 4.2 Hz), total 1H], [4.49
(dd, J=8.6, 7.6 Hz) and 4.59 (dd, J=8.6, 6.8 Hz), total 1H], 3.13,
3.17, 3.18 and 3.24 (4 s, total 6H), 2.90 and 3.00 (2 br s, total
3H), 1.31 and 1.36 (2 br s, total 6H), [1.05 (d, J=6.7 Hz) and 1.09
(d, J=6.7 Hz), total 3H].
[0190] Step 2.
[0191] Synthesis of
2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-met-
hyl-3-oxo-3-{[(1
S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-met-
hyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#54). According to
general procedure A (below), from #53 (701 mg, 0.726 mmol) in
dichloromethane (10 mL, 0.07 M) was synthesized the crude desired
material, which was purified by silica gel chromatography
(Gradient: 0% to 10% methanol in dichloromethane). The residue was
diluted with diethyl ether and heptane and was concentrated in
vacuo to afford #54 (406 mg, 75%) as a white solid. LC-MS: m/z
743.6 [M+H.sup.+], retention time=0.70 minutes; HPLC (Protocol A):
m/z 743.4 [M+H.sup.+], retention time=6.903 minutes, (purity
>97%); .sup.1H NMR (400 MHz, DMSO-d.sub.6), presumed to be a
mixture of rotamers, characteristic signals: .delta. [8.64 (br d,
J=8.5 Hz) and 8.86 (br d, J=8.7 Hz), total 1H], [8.04 (br d, J=9.3
Hz) and 8.08 (br d, J=9.3 Hz), total 1H], [7.77 (d, J=3.3 Hz) and
7.80 (d, J=3.2 Hz), total 1H], [7.63 (d, J=3.3 Hz) and 7.66 (d,
J=3.2 Hz), total 1H], 7.13-7.31 (m, 5H), [5.39 (ddd, J=11, 8.5, 4
Hz) and 5.53 (ddd, J=12, 9, 4 Hz), total 1H], [4.49 (dd, J=9, 8 Hz)
and 4.60 (dd, J=9, 7 Hz), total 1H], 3.16, 3.20, 3.21 and 3.25 (4
s, total 6H), 2.93 and 3.02 (2 br s, total 3H), 1.21 (s, 3H), 1.13
and 1.13 (2 s, total 3H), [1.05 (d, J=6.7 Hz) and 1.10 (d, J=6.7
Hz), total 3H], 0.73-0.80 (m, 3H).
B. Experimental for Compound 6780 (#112 in the Schematic)
Preparation of
2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1
S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl-
]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinami-
de (#112)
##STR00005##
[0193] Step 1.
[0194] Synthesis of tert-butyl (2S)-2-[(1R,2R)-3-{[(1
S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl-
]pyrrolidine-1-carboxylate (#109). To a solution of #11 (2.00 g,
6.96 mmol, 1 eq.) in dichloromethane (21 mL, 0.3 M) and
N,N-dimethylformamide (3 mL) was added HATU (3270 mg, 8.35 mmol,
1.2 eq.). After two minutes, the amine (1R,2S)-(+)-norephedrine
(1.07 mg, 6.96 mmol, 1 eq.) and triethylamine (1.94 mL, 13.9 mmol,
2 eq.) were added. After two hours, the reaction mixture was
diluted with ethyl acetate (100 mL), washed with a 1 M aqueous
solution of hydrochloric acid and with brine, dried over sodium
sulfate, filtered, concentrated in vacuo, and purified by silica
gel chromatography (Gradient: 0% to 60% ethyl acetate in heptane)
to provide #109 (2.18 g, 74%) as a white solid. LC-MS: m/z 321.3
[(M-Boc)+H.sup.+], retention time=3.14 minutes; .sup.1H NMR (400
MHz, DMSO-d.sub.6), presumed to be a mixture of rotamers,
characteristic signals: .delta. 7.64 (d, J=8.6 Hz, 1H), 7.24-7.33
(m, 4H), 7.15-7.21 (m, 1H), 5.35 (br d, J=5 Hz, 1H), 4.45 (br dd,
J=5, 5 Hz, 1H), 3.91-4.00 (m, 1H), 3.30-3.39 (m, 1H), 3.26 (s, 3H),
2.94-3.07 (m, 1H), 2.04-2.14 (m, 1H), 1.46-1.78 (m, 4H), 1.40 (s,
9H), 0.97-1.04 (m, 6H).
[0195] Step 2.
[0196] Synthesis of (2R,3R)--N-[(1
S,2R)-1-hydroxy-1-phenylpropan-2-yl]-3-methoxy-2-methyl-3-[(2S)-pyrrolidi-
n-2-yl]propanamide, trifluoroacetic acid salt (#110)
[0197] According to general procedure C (below), at 0.degree. C.
from #109 (414 mg, 0.984 mmol, 1 eq.), dioxane (5 mL, 0.2 M) and a
4 M solution of hydrogen chloride in dioxane (15 mL, 60 mmol, 60
eq.) was synthesized the crude desired compound, which was purified
by reverse phase chromatography (Method C) to give #110 (120 mg,
34%) as a viscous liquid. LC-MS: m/z 321.1 [M+H.sup.+], retention
time=0.55 minutes; .sup.1H NMR (400 MHz, DMSO-d.sub.6),
characteristic signals: .delta. 7.90 (d, J=8.6 Hz, 1H), 7.28-7.36
(m, 4H), 7.20-7.27 (m, 1H), 4.46 (d, J=6.2 Hz, 1H), 3.48 (dd,
J=8.6, 2.3 Hz, 1H), 3.38 (s, 3H), 2.92-3.16 (m, 3H), 2.24-2.35 (m,
1H), 1.49-1.88 (m, 4H), 1.09 (d, J=6.6 Hz, 3H), 1.01 (d, J=6.6 Hz,
3H).
[0198] Step 3.
[0199] Synthesis of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-
-2-[(1R,2R)-3-{[(1
S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl-
]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinami-
de (#111) .According to general procedure D (below), from #32 (140
mg, 0.230 mmol, 1 eq.),
##STR00006##
#110 (110 mg, 0.253 mmol, 1.1 eq.), dichloromethane (3 mL, 0.08 M),
N,N-dimethylformamide (0.5 mL), HATU (96.2 mg, 0.253 mmol, 1.1 eq)
and triethylamine (96 .mu.L, 0.69 mmol, 3 eq.) was synthesized the
crude desired product, which was purified by silica gel
chromatography (Gradient: 0% to 40% acetone in heptane) to give 20
#111 (220 mg, 95%). LC-MS: m/z 912.4 [M+H.sup.+], 935.4
[M+Na.sup.+], retention time=2.15 minutes; HPLC (Protocol B): m/z
912.5 [M+H.sup.+], 934.5 [M+Na.sup.+], retention time=10.138
minutes (purity >94%); .sup.1H NMR (400 MHz, DMSO-d.sub.6),
presumed to be a mixture of rotamers, characteristic signals:
.delta. 7.89 (d, J=7.8 Hz, 2H), 7.66-7.75 (m, 2H), 7.41 (dd, J=7.4,
7.4 Hz, 2H), 7.12-7.20 (m, 1H), [5.33 (d, J=4.7 Hz) and 5.38 (d,
J=4.7 Hz), total 1H], 3.15, 3.18, 3.22 and 3.23 (4 s, total 6H),
1.30, 1.33, 1.36 and 1.39 (4 s, total 6H), 0.95-1.06 (m, 6H).
[0200] Step 4.
[0201] Synthesis of
2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1
S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl-
]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinami-
de (#112). According to general procedure A (below), from #111 (210
mg, 0.230 mmol) in dichloromethane (5 mL, 0.05 M) and diethylamine
(5 mL) was synthesized the crude desired material, which was
purified by silica gel chromatography (Gradient: 0% to 10% methanol
in dichloromethane) to give a mixture of an oil and solid. Diethyl
ether and heptane were added and the mixture was concentrated in
vacuo, producing #112 (81 mg, 51%) as a white solid. LC-MS: m/z
690.4 [M+H.sup.+], retention time=1.10 minutes; HPLC (Protocol A):
m/z 690.5 [M+H*], 712.4 [M+Na.sup.+], retention time=7.229 minutes
(purity >90%); .sup.1H NMR (400 MHz, DMSO-d.sub.6), presumed to
be a mixture of rotamers, characteristic signals: .delta. [7.62 (br
d, J=8 Hz), 7.88 (br d, J=8 Hz), 8.07 (br d, J=9 Hz) and 8.11 (br
d, J=9 Hz), total 2H], 7.15-7.34 (m, 5H), [5.34 (d, J=4 Hz) and
5.41 (d, J=5 Hz), total 1H], 3.18, 3.21, 3.23 and 3.25 (4 s, total
6H), 2.93 and 3.08 (2 br s, total 3H), 1.15, 1.18, 1.21 and 1.25 (4
s, total 6H).
C. General Procedures
[0202] General Procedure A:
[0203] Fmoc removal using diethylamine or piperidine. To a solution
of the Fmoc-containing compound in dichloromethane or
N,N-dimethylformamide (also referred to as DMF), was added an equal
volume of diethylamine or piperidine. Reaction progress was
monitored by LC-MS (or HPLC or TLC). Solvents were removed in
vacuo, and in some cases the residue was azeotroped one to four
times with heptane. Residue was usually diluted with
dichloromethane and a small amount of methanol before being reduced
down onto silica and purified by chromatography on silica gel,
eluting with methanol in dichloromethane (or other appropriate
mixture of solvents) to afford the desired material (or crude
material was used as is).
[0204] General Procedure C:
[0205] Boc removal or tert-butyl ester (also refers to t-Bu ester)
cleavage using hydrochloric acid in dioxane. To either a solution
of Boc-containing compound or tert-butyl ester-containing compound
in dioxane (or in some cases no solution, or other relevant
solvent) was added a 4 M solution of hydrochloric acid in dioxane.
Reaction progress was monitored by LC-MS (or HPLC or TLC). The
reaction was concentrated in vacuo and in some cases azeotroped one
to four time with heptanes.
[0206] General Procedure D:
[0207] coupling with
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU). To a stirring solution of the amine
(1.0 eq.) and acid (1.0-2.0 eq.) in dichloromethane,
N,N-dimethylformamide (also referred to as DMF), or a mixture of
both, HATU (1.0-2.0 eq.) was added followed by triethylamine
(2.0-4.0 eq.) or diisopropylethylamine (2.0-4.0 eq., also referred
to as Hunig's base). Reaction progress was monitored by LC-MS (or
HPLC or TLC); the reaction was usually completed within three
hours. Solvents were removed in vacuo. The residue was purified by
silica gel or reverse phase chromatography or in some cases
azeotroped three times with heptanes, diluted with a small amount
of ethyl acetate before being reduced down onto silica or C18
bonded silica and purified by silica gel or reverse phase
chromatography.
D. Other Compounds
[0208] Further compounds used in the present invention are
described in International Publication No. WO/2013/072813 and shown
below.
[0209] As used herein, compound 0131 or
2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1
S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolid-
in-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,
trifluoroacetic acid salt (#118) has the formula:
##STR00007##
[0210] As used herein, compound 3377 or
N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1
S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolid-
in-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinami-
de, trifluoroacetic acid salt (#115) has the formula:
##STR00008##
[0211] As used herein, compound 8261 or
2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1
S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolid-
in-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
(#69) has the formula:
##STR00009##
Example 11
Preparation of Anti-Notch3 Antibody-Drug Conjugates (ADCs)
[0212] The ADCs of the present invention were prepared using a
section of a linker having a reactive site for binding to a
chemical compound and introducing another section of the linker
unit having a reactive site for an antibody. In one aspect, the
linker unit has a reactive site which has an electrophilic group
that is reactive with a nucleophilic group present on an antibody
unit, such as an antibody. Useful nucleophilic groups on an
antibody include but are not limited to, sulfhydryl, hydroxyl and
amino groups. The heteroatom of the nucleophilic group of an
antibody is reactive to an electrophilic group on a linker unit and
forms a covalent bond to a linker unit. Useful electrophilic groups
on the linker include, but are not limited to, maleimide and
haloacetamide groups.
[0213] The linker unit has a reactive site which has a nucleophilic
group that is reactive with an electrophilic group present on an
antibody unit. The electrophilic group on an antibody provides a
convenient site for attachment to a linker unit. Useful
electrophilic groups on an antibody include, but are not limited
to, aldehyde and ketone carbonyl groups. The heteroatom of a
nucleophilic group of a linker unit can react with an electrophilic
group on an antibody and form a covalent bond to the antibody.
Useful nucleophilic groups on a linker unit include, but are not
limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,
hydrazine carboxylate, and arylhydrazide.
[0214] As used herein, "mc-" also known as "MalC-" refers to:
##STR00010##
[0215] As used herein, "vc-", also known as "mcValCitPABC-" or
"MalCValCitPABC-" refers to:
##STR00011##
[0216] As used herein, "me-" refers to
##STR00012##
[0217] As used here, "MalPeg6C2-" refers to "MalPegXC2-" shown
below, wherein X=6:
##STR00013##
[0218] The ADCs of the present invention were prepared via partial
reduction of the antibody with tris(2-carboxyethyl)phosphine (TCEP)
followed by a reaction of reduced cysteine residues with the
desired maleimide terminated linker-payload. Specifically, the
antibodies were partially reduced via addition of about
2.3-3.0-fold molar excess of tris(2-carboxyethyl)phosphine (TCEP)
in 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
buffer), pH 7.0 and 1 mM diethylenetriaminepentaacetic acid (DTPA)
for 2 hours at 37.degree. C. The desired linker-payload was then
added to the reaction mixture at a linker-payload/antibody molar
ratio of about 7-8 and reacted for an additional 1 hour at
25.degree. C. in the presence of 15% v/v of dimethylacetamide
(DMA). After the 1 hour incubation period, 3-fold excess of
N-ethylmaleimide was added to cap the unreacted thiols and was
allowed to react for 15 minutes, followed by addition of 6-fold
excess L-Cys to quench any unreacted linker-payload. The reaction
mixture was dialyzed overnight at 4.degree. C. in phosphate
buffered saline (PBS), pH 7.4, and purified via SEC (AKTA explorer,
Superdex 200). The hydrolysis of the succinimide ring was further
achieved via incubating the purified ADC in a 100 mM borate, pH 9.2
buffer for 24-72 hours at 37.degree. C. The ring opening was
monitored via liquid chromatography electrospray ionization tandem
mass spectrometry (LC-ESI MS) and purified via size exclusion
chromatography (SEC). The ADC was further characterized via SEC for
purity, hydrophobic interaction chromatography (HIC), and liquid
chromatography electrospray ionization tandem mass spectrometry
(LC-ESI MS) to calculate drug-antibody ratio (loading). The protein
concentration was determined via UV spectrophotometer.
[0219] vc0101 (shows conjugation to antibody X through cysteine
residue)
##STR00014##
[0220] vc6780 (shows conjugation to antibody X through cysteine
residue)
##STR00015##
[0221] Humanized anti-Notch antibodies, hu28 and hu75, and
rat-human chimeric anti-Notch antibodies, ch28 and ch75, were
conjugated to various linker-payload combinations as provided in
Table 10. The ADCs and elements thereof were prepared according to
the methods of the present invention and according to International
Publication No. WO/2013/072813.
TABLE-US-00010 TABLE 10 Anti-Notch3 ADC nomenclature. Corresponding
ADC Nomenclature ADC Linker-Payload # hu28-vc0101
Notch3-28-v1010-hG1- (C)_mcValCitPABC-#54 hu28-vc6780
Notch3-28-v1010-hG1- (C)_mcValCitPABC-#112 hu75-vc0101
Notch3-75-v1913-hG1- (C)_mcValCitPABC-#54 hu75-vc6780
Notch3-75-v1913-hG1- (C)_mcValCitPABC-#112 ch28-vc0101
Notch3-28-cG1-(C)_mcValCitPABC-#54 ch28-vc6780
Notch3-28-cG1-(C)_mcValCitPABC-#112 ch28-mc0101
Notch3-28-cG1-(C)_mc-#54 ch28-mc0131 Notch3-28-cG1-(C)_mc-0#118
ch28-mc3377 Notch3-28-cG1-(C)_mc-#115 ch28-mc8261
Notch3-28-cG1-(C)_mc-#69 ch28-MalPeg6C2-0131
Notch3-28-cG1-(C)_MalPeg6C2-0#118 ch28-MalPeg6C2-8261
Notch3-28-cG1-(C)_MalPeg6C2-#69 ch28-me0131
Notch3-28-cG1-(C)_me-0#118 ch28-m(H2O)c-0131
Notch3-28-cG1-(C)_m(H2O)c-0#118 ch75-vc0101
Notch3-75-cG1-(C)_mcValCitPABC-#54 ch75-vc6780
Notch3-75-cG1-(C)_mcValCitPABC-#112 ch75-mc0131
Notch3-75-cG1-(C)_mc-0#118 ch75-mc3377 Notch3-75-cG1-(C)_mc-#115
ch75-MalPegC2-0131 Notch3-75-cG1-(C)_MalPeg6C2-0#118
ch75-MalPeg6C2-8261 Notch3-75-cG1-(C)_MalPeg6C2-#69 ch75-me0131
Notch3-75-cG1-(C)_me-0#118 ch75-m(H2O)c-0131
Notch3-75-cG1-(C)_m(H2O)c-0#118 huNeg8.8-vc0101
huNeg8.8-(C)_mcValCitPABC-#54 huNeg8.8-vc6780
huNeg8.8-(C)_mcValCitPABC-#112 huNeg8.8-mc0131
huNeg8.8-(C)_mc-0#118 huNeg8.8-mc3377 huNeg8.8-(C)_mc-#115
huNeg8.8-me0131 huNeg8.8-(C)_me-0#118 huNeg8.8-MalPeg6C2-8261
huNeg8.8-(C)_MalPeg6C2-#69 ch2H6-mc8261 ch2H6-(C)_mc-#69
Example 12
Binding Activity and Specificity of Anti-Notch3 Antibodies and
ADCs
A. Cell-Based ELISA
[0222] Unconjugated anti-Notch3 antibodies, anti-Notch3 ADCs and a
negative control antibody (huNeg8.8) were screened for cell surface
binding activity to Notch3 expressing cell lines in a cell-based
ELISA. Over-expressing Notch3 cell line, U2OS/hNotch3, and
endogenous Notch3 expressing cell lines, HCC2429 and MDA-MB-468,
were plated at 50,000, 200,000 and 100,000 cells/well,
respectively, in 96 well plates (white opaque, BD/VWR) the day
before ELISA assay. On the day of the ELISA, culture media was
removed from wells and serially diluted (1:3 in DPBS with calcium
chloride and magnesium chloride (Ca/Mg) and 1% BSA) antibody and
ADC solutions were applied to the plate. Plates were incubated at
room temperature for 2 hours before washed with DPBS with Ca/Mg and
1% BSA. HRP-conjugated secondary antibody was then applied and
incubated with cells for 1 hour. Plates were washed with DPBS with
Ca/Mg and 1% BSA before being developed with Pico-Chemiluminescent
developing kit (Thermal Scientific), and chemiluminescence
measurements were performed per manufacturer's instructions. Data
plotting and analyses were performed with Microsoft Excel and
Graphpad-Prism software.
[0223] Table 11 shows EC.sub.50 (nM) values and standard deviations
(SD) calculated for two to four independent experiments from cell
surface Notch3 binding ELISAs for unconjugated anti-Notch3
antibodies, hu28 and hu75, and anti-Notch3 ADCs, hu28-vc0101,
hu28-vc6780, hu75-vc0101 and hu75-vc6780. The data demonstrates
that hu28-ADCs and hu75-ADCs are similar to unconjugated antibodies
hu28 and hu75, respectively, in binding to full-length human Notch3
expressed on the cell surface of U2OS/hNotch3, HCC2429 and
MDA-MB-468 cells. Further, the data demonstrates that conjugation
of various linker-payloads to the hu28 and hu75 antibodies did not
affect or alter the binding characteristics. Furthermore, the data
demonstrates that hu28 and both hu28-vc0101 and hu28-vc6780 have a
higher binding capacity for cell surface Notch3, as demonstrated by
their lower EC.sub.50 values, than hu75 and hu75-vc0101 and
hu75-vc6780. The negative control, unconjugated huNeg8.8 antibody,
did not bind any of the cell lines tested. For the control antibody
that lacked binding (LB), EC.sub.50 values were not generated as
indicated. (SD=Standard Deviation)
TABLE-US-00011 TABLE 11 EC.sub.50 (nM) values for unconjugated
anti-Notch3 antibodies and anti-Notch3 ADCs. (SD = Standard
Deviation) EC.sub.50 (nM) and (SD) U2OS/ HCC2429 MDA-MB-468 hNotch3
EC50 SD EC50 SD EC50 SD hu28 VH1.0/VL1.0 0.132 0.009 0.192 0.054
0.172 0.077 hu28-vc0101 0.176 0.025 0.273 0.063 0.266 0.029
hu28-vc6780 0.154 0.034 0.242 0.046 0.236 0.008 hu75 VH1.9/VL1.3
0.335 0.053 1.284 0.176 0.585 0.160 hu75-vc0101 0.427 0.115 2.022
0.167 0.798 0.057 hu75-vc6780 0.343 0.063 1.804 0.062 0.723 0.041
huNeg8.8 LB -- LB -- LB --
B. Flow Cytometry
[0224] Unconjugated anti-Notch3 antibodies, hu28 and hu75, were
examined for cell surface binding activity to Notch3 expressing
cell lines by flow cytometry. Fluorescence activated cell sorting
(FACS) analysis was conducted according to standard procedures.
Cells were rinsed in HBSS with calcium chloride and magnesium
chloride (herein termed HBSS++), harvested using trypsin without
EDTA and neutralized with medium containing FBS. Cells were
incubated on ice for 30 minutes with 4 .mu.g/mL anti-Notch3
antibodies, hu28 and hu75, in HBSS++ containing 3% HICS
(heat-inactivated calf serum). Cells were washed three times with
cold HBSS++, 3% HICS buffer. Cells were incubated in
allophycocyanin-conjugated AffiniPure F(ab')2 fragment goat
anti-human IgG, Fc fragment secondary antibody (Jackson
ImmunoResearch Laboratories, Inc., West Grove, Pa.) at 10 .mu.g/mL
for 30 minutes on ice in the dark. Cells were washed once with cold
HBSS++/3% HICS buffer. Cells were resuspended in HBSS++/3% HICS, 25
mM HEPES, 1 mM MgCl.sub.2 and 25 .mu.g/ml DNasel. 7-AAD
(7-Amino-Actinomycin D) was added for exclusion of nonviable cells.
Live cells were analyzed on a BD FACSCalibur flow cytometer (BD
Biosciences, San Jose, Calif.). Mean fluorescence intensity (MFI)
from channel FL-4 was calculated with FlowJo flow cytometry
analysis software (Ashland, Oreg.).
[0225] Table 12 shows the binding activity of unconjugated
anti-Notch3 antibodies hu28 and hu75 by FACS analysis to a panel of
Notch3 expressing cell lines. The U-2 OS cell line was used as a
Notch3 negative/low control cell line. Both hu28 and hu75 had low
levels of binding to U-2 OS that were minimally higher than the
negative control huNeg8.8. Further, the data demonstrates that hu28
and hu75 bound specifically to the Notch3 over-expressing cell,
U2OS/hNotch3 and MDAMB468/hNotch3, and Notch3 endogenously
expressing cells MDA-MB-468, HCC2429 and OVCAR3. Furthermore, the
data demonstrates that the binding activity of hu28 was greater
than hu75 to all Notch3 expressing cell lines similar to the
observation of binding activity by cell-based ELISA.
TABLE-US-00012 TABLE 12 MFI values by FACS analysis for anti-Notch3
antibodies. MFI hu28 VH1.0/ hu75 VH1.9/ huNeg8.8 VL1.0 VL1.3
(negative Cell line (anti-Notch3) (anti-Notch3) control)
U2OS/hNotch3 256 171 6 (positive control) MDA-MB-468 557 274 6
MDA-MB-468/hNotch3 1192 530 6 HCC2429 564 322 3 OVCAR3 116 80 5 U-2
OS 17 18 7 (negative/low control)
Example 13
In Vitro Cytotoxicity Assay
[0226] The effects of anti-Notch3 ADCs were assessed on 1) cell
lines endogenously expressing Notch3 protein: HCC2429 (lung
cancer), OVCAR3 (ovarian cancer) and MDA-MB-468 (breast cancer), 2)
cell lines engineered to over-express full length human Notch3
protein: MDA-MB-468/hNotch3 and U2OS/hNotch3, and 3) a negative
control cell line (SW900) using an MTS cellular viability indicator
(Promega, Madison, Wis.). These cell lines were cultured with
increasing concentrations of anti-Notch3 ADCs comprising rat-human
chimeric anti-Notch3 antibodies, ch28 and ch75, and humanized
anti-Notch3 antibodies, hu28 and hu75, conjugated to various
linker-payload combinations of the present invention. As a
specificity control for the anti-Notch3-ADCs, non-targeted
control-ADCs (huNeg8.8-ADCs or ch2H6-ADCs) were also tested on the
same cell lines. After four days, viability of each culture was
assessed. IC.sub.50 values were calculated by logistic non-linear
regression, model #203 with XL fit v4.2 (IDBS, Guildford, Surry,
UK) and presented as ng Ab/mL. The drug antibody ratio (DAR) is
also provided.
[0227] Table 13 shows IC.sub.50 (ng Ab/mL) values of the rat-human
chimeric anti-Notch3 ADC treatments. For experiments with 2-4
individual repeats, average IC.sub.50 values were calculated along
with standard error of the mean (S.E.M.). The data demonstrates
that the rat-human chimeric anti-Notch3 ADCs with various
linker-payloads were active and induced cell death in the Notch3
expressing and over-expressing cancer cell lines HCC2429, OVCAR3,
MDA-MB-468, MDA-MB-468/hNotch3, U2OS/hNotch3. The non-targeted
control-ADCs either lacked potency (LP) and therefore IC.sub.50
values were not generated as indicated, or were minimally active at
the highest doses tested. Anti-Notch3 ADCs having IC.sub.50 values
equal to or higher than IC.sub.50 values for control-ADCs were
considered to lack potency in vitro and indicted as LP.
TABLE-US-00013 TABLE 13 IC.sub.50 (ng Ab/mL) values of rat-human
chimeric anti-Notch3 ADCs (nd = not determined). IC.sub.50 (ng
Ab/mL) .+-. S.E.M. MDA-MB- U2OS/ ADC DAR HCC2429 OVCAR3 MDA-MB-468
468/hNotch3 hNotch3 ch28-mc8261 3.7 LP nd 12147 .+-. 4806.4 nd nd
ch2H6-mc8261 4.1 LP nd LP nd nd ch28-MalPeg6C2-8261 4.3 LP nd 83
.+-. 35.5 nd nd ch75-MalPeg6C2-8261 3.8 LP nd 4255 .+-. 2375.sup.
nd nd huNeg8.8-MalPeg6C2- 4.1 LP nd LP nd nd 8261 ch28-mc0131 3.4
251 .+-. 77.5 6 .+-. 1.0 35 .+-. 18.5 nd 3 .+-. 0.5 ch75-mc0131 3.3
671 .+-. 406.5 289 8202 .+-. 2773.0 nd 19 huNeg8.8-mc0131 3.9 nd LP
LP nd LP ch28-me0131 3.9 30 8 .+-. 2.0 24 .+-. 14.0 nd 3 .+-. 1.15
ch75-me0131 3.5 nd nd 259 nd nd huNeg8.8-me0131 3.7 nd LP LP nd LP
ch28-mc3377 3.7 LP 14 .+-. 5.5 27 .+-. 11.3 nd 3 .+-. 0.5
ch75-mc3377 3.7 nd nd 560 nd nd huNeg8.8-mc3377 3.6 nd LP LP nd LP
ch28-MalPeg6C2-0131 4.1 LP 10 .+-. 2.0 10 .+-. 1.0 nd 3 .+-. 0.85
ch28-vc0101 3.8 3230 .+-. 1116.5 635 5443 .+-. 2630.9 4 .+-. 0.5 95
.+-. 18.2 ch75-vc0101 2.7 2112 .+-. 826.0 LP 4064 .+-. 1793.9 24
.+-. 4.0 LP huNeg8.8-vc0101 3.7 15341 LP 4523 .+-. 2789.5 8833 LP
ch28-vc6780 4.1 324 .+-. 78.9 90 .+-. 48.5 4407 .+-. 2128.2 3 .+-.
0.5 LP ch75-vc6780 2.8 1004 .+-. 177.0 922 6873 .+-. 4230.0 21 .+-.
3.5 LP huNeg8.8-vc6780 4.1 LP LP LP LP LP
[0228] Table 14 shows IC.sub.50 (ng Ab/mL) values of the humanized
anti-Notch3 ADC treatments. HCC2429 and MDA-MB-468/hNotch3 cell
lines had two individual repeats. The data demonstrates that the
humanized anti-Notch3 ADCs with various linker-payloads were active
and induced cell death in the Notch3 expressing and over-expressing
cancer cell lines HCC2429, OVCAR3, MDA-MB-468, MDA-MB-468/hNotch3,
U2OS/hNotch3, but not in the negative control cell line SW900
lacking Notch3 expression. The non-targeted control-ADCs either
lacked potency (LP) and therefore IC.sub.50 values were not
generated as indicated, or were minimally active at the highest
doses tested. Anti-Notch3 ADCs having IC.sub.50 values equal to or
higher than IC.sub.50 values for control-ADCs were considered to
lack potency in vitro and indicted as LP. Unconjugated humanized
anti-Notch3 antibodies hu28 and hu75 did not affect the viability
of HCC2429 or MDAMB468/hNotch3, indicating cytotoxicity can be
specifically attributed to payloads (data not shown).
TABLE-US-00014 TABLE 14 IC.sub.50 (ng Ab/mL) values of humanized
anti-Notch3 ADCs. IC.sub.50 (ng Ab/mL) MDA- MDA-MB-468/ U2OS/ ADC
DAR HCC2429 OVCAR3 MB-468 hNotch3 hNotch3 SW900 hu28-vc0101 3.9 473
2940 306 6545 3.2 3 1330 LP hu75-vc0101 3.8 611 3295 515 7001 37 36
523 LP huNeg8.8-vc0101 3.7 18417 23978 3770 LP 5122 LP LP 23379
hu28-vc6780 3.9 148 2050 17 LP 1.3 3 LP LP hu75-vc6780 4.2 214 630
254 LP 26 25 LP LP huNeg8.8-vc6780 4.2 LP LP 9238 LP LP LP LP
LP
[0229] Notch3 knockdown with siRNAs was used to confirm that the in
vitro cytotoxicity of anti-Notch3 ADCs was dependent on the
expression of the Notch3 protein. siRNA transfections were
generated using ON-TARGET plus SMART pool human Notch3
(L-011093-00), ON-TARGET plus Control Non-Targeting pool
(D-001810-10) (Thermo Scientific Dharmacon) and Lipofectamine
RNAiMAX Reagent (Invitrogen). Two to 2.5.times.10.sup.6 cells of
HCC2429, OVCAR3 and MDA-MB-468/hNotch3 cells were plated in 10 cm
dishes in growth medium without antibiotics the day before
transfection. The next day fresh medium without antibiotics was
added. The siRNAs and the Lipofectamine RNAiMAX were diluted in
OPTI-MEM media and used as per the manufacturer's specifications.
Each of the cell lines was transfected with control and Notch3
siRNAs. The cells were incubated with the transfection mixture for
24 hours in a humidified, 37.degree. C., 5% CO.sub.2 incubator.
After 24 hours, cells were trypsinized and plated for assessment
using MTS cellular viability indicator (Promega, Madison,
Wis.).
[0230] Depending on the cell line, cells were then seeded at a
density of 2,500 to 5,000 cells per well 24 hours before treatment.
Cells were treated with 3-fold serially diluted humanized
anti-Notch3 ADCs in triplicates at 10 concentrations (range 0-30
.mu.g Ab/ml). Relative cell viability was determined as percentage
of untreated control 96 hours after treatment IC.sub.50 values were
calculated by logistic non-linear regression, model #203 with XL
fit v4.2 (IDBS, Guildford, Surry, UK) and presented as ng Ab/mL.
For experiments with 2 individual repeats, average IC.sub.50 values
were calculated along with standard error of the mean (S.E.M.).
Western blot analysis on extracts prepared from control and Notch3
siRNA-treated cells was performed to confirm that Notch3 knockdown
occurred for up to 144 hours (data not shown). Cell lines that
expressed Notch3 (herein termed control siRNA) or had reduced
Notch3 expression after siRNA knockdown (herein termed Notch3
siRNA) were cultured with increasing concentrations of humanized
anti Notch3 ADCs. As a specificity control for the anti-Notch3
ADCs, non-targeted control-ADCs (huNeg8.8-ADCs) were also tested on
the same cell lines. After four days, viability of each culture was
assessed. IC.sub.50 values were calculated by logistic non-linear
regression, model #203 with XL fit v4.2 (IDBS, Guildford, Surry,
UK) and presented as ng Ab/mL. The drug antibody ratio (DAR) is
also provided.
[0231] Table 15 shows IC.sub.50 (ng Ab/mL) values of humanized
anti-Notch3 ADC treatments of a panel of control siRNA or Notch3
siRNA treated cancer cell lines. The data demonstrates that
humanized anti-Notch3 ADCs with various linker-payloads were active
and induced cell death in the Notch3-expressing cancer cell lines
(control siRNA). In cells that had reduced Notch3 expression after
siRNA knockdown (Notch3 siRNA), IC.sub.50 values were greater than
control siRNA indicating that a reduction in Notch3 expression was
accompanied by a reduction in cytotoxicity of anti-Notch3 ADCs.
Control-ADCs lacked potency (LP) and therefore IC.sub.50 values
were not generated as indicated, or were minimally active at the
highest doses tested. The data further demonstrates that humanized
anti-Notch3 ADCs specifically induced cell death on Notch3
expressing and over-expressing cancer cell lines. The observed
cytotoxicity was dependent on Notch3 expression because Notch3
siRNA knockdown on the same cells reduced cell death by anti-Notch3
ADCs. Therefore, humanized anti-Notch3 ADCs were dependent on
Notch3 expression for their in vitro cytotoxicity.
TABLE-US-00015 TABLE 15 IC.sub.50 (ng Ab/ml) values of humanized
anti-Notch3 ADCs. IC.sub.50 (ng Ab/mL) .+-. S.E.M. MDA-MB 468/
HCC2429 OVCAR3 hNotch3 Control Notch3 Control Notch3 Control Notch3
ADC DAR siRNA siRNA siRNA siRNA siRNA siRNA hu28-vc0101 3.9 930
.+-. 529 7138 .+-. 827 684 .+-. 183 1994 .+-. 262 3.0 3340
hu75-vc0101 3.8 956 .+-. 259 8972 .+-. 891 1056 .+-. 158 2408 .+-.
27.5 29.0 3187 huNeg8.8-vc0101 3.7 17832 .+-. 2319 21374 .+-. 1614
3751 .+-. 922 2902 .+-. 654 4226 5580 hu28-vc6780 3.9 259 .+-. 179
14120 .+-. 1175 189 .+-. 6.0 6100 .+-. 200 2.0 LP hu75-vc6780 4.2
236 .+-. 30.5 LP 690 .+-. 137 7300 .+-. 147 17.0 7981
huNeg8.8-vc6780 4.2 LP LP 8163 .+-. 1031 7674 .+-. 705 LP LP
Example 14
In Vitro Assessment of Anti-Notch3 ADC Mechanism of Action
A. Anti-Notch3 ADC Disruption of Microtubules
[0232] Following internalization and intracellular release of the
payload from the Notch3-ADC, the presumed mechanism of action of
the released payload is disruption of microtubules that are
required for cell division, thereby leading to cell cycle arrest,
induction of apoptosis and cell death. To confirm this mechanism of
action, OVCAR3 ovarian cancer cells were treated with hu28-vc0101,
huNeg8.8-vc0101 (control ADC) or left untreated, and then subjected
to immunofluorescence analysis by staining with an
anti-alpha-tubulin antibody to mark microtubules and an
anti-phospho-Histone H3 antibody to identify cells undergoing or
arrested in mitosis.
[0233] OVCAR3 cells were seeded in a Lab Tec II 4 chambered
coverglass with cover #1.5 borosilicate sterile slides (Thermo
Fisher Scientific Inc.). Next day, cells were treated with 1.0
.mu.g/ml of hu28-vc0101, control huNeg8.8-vc0101 or left untreated.
Forty-eight hours later, cells were washed twice with HBSS++ and
fixed in 4% paraformaldyhide for 10 minutes. Cells were washed
three times with PBS and permeabilized with 0.5% Triton X-100 for 2
minutes. After washing cells three times with PBS, cells were
blocked with 3% BSA/PBS for 30 minutes at room temperature. Cells
were then incubated with 1:100 of anti-phospho-Histone H3 (Ser10)
(Cell Signaling) and 2 .mu.g/ml of anti-alpha-Tubulin, (Millipore)
in 2% BSA/PBS for 2 hours at room temperature. After 2 hours, cells
were washed twice with PBS and then incubated with a 1:500 dilution
of goat anti-mouse Alexa Fluor 488 and goat anti-rabbit 555 for 45
minutes at room temperature. Cells were washed twice with PBS and
samples were imaged on an LSM710 confocal microscope (Zeiss).
[0234] As shown in FIG. 10, untreated OVCAR3 cells in the mitotic
phase of the cell cycle stain positive for phospho-histone H3 and
contain a normal bipolar spindle apparatus as demonstrated by
anti-alpha-tubulin staining. Treatment with hu28-vc0101, but not
control huNeg8.8-vc0101, disrupted the structure of the mitotic
spindle apparatus as demonstrated by its abnormal morphology in
phospho-Histone H3 stained cells. The data demonstrates that
hu28-vc0101 inhibited cell proliferation by disrupting microtubules
that are required during mitosis to complete cell division.
B. Anti-Notch3 ADC Induction of Apoptosis
[0235] Caspase-3 and caspase-7 (caspase-3/7) proteases are key
members of the programmed cell death machinery responsible for
mediating late apoptotic events in mammalian cells. The activity of
caspase-3/7 was measured in Notch3 expressing cell lines that were
treated with Notch3-ADCs.
[0236] OVCAR3, HCC2429 and MDA-MB-468/hNotch3 cells were seeded
onto opaque white tissue culture plates and incubated overnight.
OVCAR3 and HCC2429 were treated at 1.1 .mu.g/ml and
MDA-MB-468/hNotch3 cells were treated 1 .mu.g/ml of hu28-vc0101 and
huNeg8.8-vc010 (control ADC). After 48 hours incubation, cells were
treated with the Caspase-Glo 3/7 reagent (Promega #G8090) for 2
hours at room temperature. Luminescence was measured with a
luminometer and following subtraction of background, values were
reported as relative luminescence units.
[0237] Table 16 shows that hu28-vc0101 induced the activity of
caspase-3/7 in Notch3 expressing cell lines MDA-MB-468/hNotch3,
HCC2429 and OVCAR3 cells from 2-3 fold over control Neg8.8-vc0101
treated cells. Thus, hu28-vc0101 inhibits cell growth by inducing
apoptosis.
TABLE-US-00016 TABLE 16 Relative luminescence units for anti-Notch3
ADCs. hu28-vc0101 huNeg8.8-vc0101 MDA-MB-468/hNotch3 78,622 .+-.
1160 47,362 .+-. 678 HCC2429 128,518 .+-. 3483 55,255 .+-. 2965
OVCAR3 209,715 .+-. 15572 118,150 .+-. 4280
Example 15
Notch3 Immunohistochemistry
[0238] The effects of anti-Notch3 ADCs were assessed in
pre-clinical models that had detectable levels of Notch3 expression
at the cell membrane of xenografted human tumor cells. To identify
pre-clinical models expressing Notch3, immunohistochemistry
(hereinafter "IHC") using an anti-Notch3 antibody was performed on
a panel of xenograft models including: 37622A1 NSCLC (patient
derived), HCC2429 lung cancer, MDA-MB-468 breast cancer and N87
gastric cancer xenograft models.
[0239] A tissue fragment from each xenograft was formalin-fixed and
paraffin embedded (FFPE) using standard histological procedures.
Five micron FFPE sections were cut, dewaxed and hydrated to
distilled water. Antigens were retrieved in EDTA buffer pH 8.0 in a
pressure cooker. Endogenous peroxidase was blocked with 3.0%
H.sub.2O.sub.2 for 10 minutes. Sections were incubated with DAKO
Protein block for 20 minutes. A 1:2000 dilution of rabbit
anti-Notch3 (D11B8; Cell Signaling Technologies) was applied to the
sections for 1 hour at room temperature. Signalstain Boost
anti-rabbit IgG-HRP polymer (Cell Signaling Technologies) was
applied to the sections for 30 minutes at room temperature. DAB was
used to develop color for 5 minutes. Sections were briefly
counterstained in Mayer's hematoxylin, dehydrated, cleared and
coverslipped. Table 17 shows the staining intensity and staining
distribution grade that were scored on a scale of 0-4, with 0 being
negative and 4 being the highest intensity.
TABLE-US-00017 TABLE 17 Staining intensity and staining
distribution grade. Staining Intensity Staining Distribution Grade
Grade 0 = negative 0 = negative 1 = minimal 1 = 0-25% 2 = mild 2 =
26-50% 3 = moderate 3 = 51-75% 4 = marked 4 = 76-100%
[0240] As shown in Table 18, Notch3 protein was detected on the
cell membrane (Mem) and in the cytoplasm (Cyto) and/or nucleus
(Nuc) of cells from the 37622A1 NSCLC, HCC2429, MDA-MB-468 and N87
xenografts. The data demonstrates that both HCC2429 and N87
xenografts had a homogenous distribution of Notch3 protein at the
cell membrane of 76-100% of cells. Further, the data demonstrates
that 37622A1 NSCLC and MDA-MB-468 xenografts had a heterogenous
distribution of Notch3 protein at the cell membrane in 51-75% of
cells. Furthermore, the detection of the Notch3 C-terminal
intracellular domain in the nucleus of some epithelial tumor cells
with the D11B8 antibody suggests that Notch3 signaling was active
in these cells.
TABLE-US-00018 TABLE 18 Subcellular localization, intensity and
distribution scores of Notch3 immunohistochemistry on a panel of
xenograft models. Tissue Subcellular Intensity Score Distribution
Grade Xenograft Type Cell type Localization (Scale 0-4) (Scale 0-4)
37622A1 Lung Human Cyto/Mem/Nuc Cyto/Mem/Nuc = 1-3 Cyto/Mem/Nuc = 3
NSCLC epithelial HCC2429 Lung Human Cyto/Mem/Nuc Cyto = 2-3
Cyto/Mem = 4 epithelial Mem = 2-4 Nuc = 2 Nuc = 1 MDA-MB-468 Breast
Human Cyto/Mem/Nuc Cyto = 3-4 Mem = 3 epithelial Mem/Nuc = 2
Cyto/Nuc = 4 N87 Gastric Human Cyto/Mem/Nuc Cyto/Mem = 2-4
Cyto/Mem/Nuc = 4 epithelial Nuc = 1-2 OVCAR3 Ovarian Human
Cyto/Mem/Nuc Cyto/Nuc = 1 Cyto/Nuc = 2 epithelial Mem = 1-3 Mem =
4
[0241] As shown in FIG. 11, Western blot analysis confirmed the
Notch3 expression levels in the panel of xenografts that were used
for in vivo efficacy studies. An .about.210 kDa Notch3 protein
fragment of the extracellular domain (hereinafter Notch3-ECD) was
detected in HCC2429, MDA-MB-468, N87 and 37622A1 xenograft extracts
using a mouse monoclonal anti-Notch3 antibody 1G5 (Abnova).
Therefore, the data from the IHC using the anti-Notch3 antibody
D11B8 demonstrated Notch3 at the cell membrane of human epithelial
tumor cells and the Western blot analysis using the anti-Notch3
antibody 1 G5 demonstrated expression of the Notch3-ECD which
contains the NRR domain, the target of anti-Notch3 ADCs.
Example 16
In Vivo Tumor Xenograft Models
[0242] Humanized anti-Notch3 antibodies, hu28 and hu75, and
rat-human chimeric anti-Notch3 antibodies, ch28 and ch75, were
conjugated to various linker-payload combinations and tested in
37622A1 non-small cell lung cancer (NSCLC), HCC2429 lung cancer,
MDA-MB-468 breast cancer and N87 gastric cancer xenograft models.
For each model described below the first dose was given on Day 0.
The tumors were measured at least once a week and their volume was
calculated with the formula: tumor volume
(mm.sup.3)=0.5.times.(tumor width.sup.2)(tumor length). The mean
tumor volumes (.+-.S.E.M.) for each treatment group were calculated
having a maximum of 10 animals and a minimum of 6 animals to be
included.
A. 37622A1 Patient Derived NSCLC Xenografts
[0243] The effects of anti-Notch3 ADCs were examined in
immunodeficient mice on the in vivo growth of human tumor
xenografts that were established from fragments of freshly resected
37622A1 NSCLC tumors obtained in accordance with appropriate
consent procedures (Asterand). The 37622A1 NSCLC patient-derived
xenografts were subcutaneously passaged in vivo as fragments from
animal to animal in nude (Nu/Nu) female mice. When the tumors
reached a volume of 150 to 300 mm.sup.3, they were staged to ensure
uniformity of the tumor size among various treatment groups. The
37622A1 NSCLC patient-derived xenografts model was dosed
intravenously four times every four days (Q4dx4) with PBS vehicle,
humanized anti-Notch3 ADCs, control huNeg-8.8 ADCs and cisplatin at
the doses provided in Table 19. FIG. 12 shows a graph of the data
from Table 19 of the ADCs with vc0101 linker-payloads at 3 mg/kg
dose compared to Cisplatin (5 mg/kg) and PBS vehicle.
[0244] Cisplatin is a platinum-based anti-cancer agent used in the
treatment of cancer and considered a standard-of-care therapy.
Cisplatin cross-links DNA thereby inducing apoptosis and cell
growth inhibition. The data demonstrates that anti-Notch3 ADCs
hu28-vc0101, hu28-vc6780, hu75-vc0101 and hu75-vc6780 inhibited
growth of 37622A1 NSCLC patient-derived xenograft tumors. The 3
mg/kg dose of hu28-vc0101 was the most potent ADC tested in this
study, and by day 84, four out of nine animals still on study
remained tumor-free. Further, the data shows that anti-Notch3 ADCs
inhibited tumor growth more potently than control huNeg8.8-ADCs.
Furthermore, the data shows that anti-Notch3 ADCs inhibited tumor
growth more potently than cisplatin indicating a greater potency
than a platinum-based standard-of-care chemotherapeutic drug (FIG.
12).
TABLE-US-00019 TABLE 19 Efficacy of anti-Notch3 ADCs in 37622A1
NSCLC xenografts. 37622A1 NSCLC patient-derived xenografts, tumor
volume (mm.sup.3 .+-. SEM) hu28- hu28- hu75- hu75- huNeg-8.8-
huNeg-8.8- PBS vc0101 vc6780 vc0101 vc6780 vc0101 vc6780 Cisplatin
Dose mg/kg 0 3 10 3 10 3 10 5 DAY -1 187 .+-. 186 .+-. 182 .+-. 185
.+-. 183 .+-. 184 .+-. 182 .+-. 185 .+-. 10 13 16 17 17 18 17 11
DAY 4 227 .+-. 202 .+-. 176 .+-. 200 .+-. 205 .+-. 225 .+-. 226
.+-. 226 .+-. 19 16 13 16 23 17 26 15 DAY 7 279 .+-. 202 .+-. 176
.+-. 227 .+-. 195 .+-. 274 .+-. 265 .+-. 280 .+-. 24 15 19 16 22 18
28 29 DAY 11 371 .+-. 130 .+-. 122 .+-. 175 .+-. 147 .+-. 309 .+-.
246 .+-. 301 .+-. 42 11 10 20 23 26 30 34 DAY 14 419 .+-. 119 .+-.
95 .+-. 156 .+-. 118 .+-. 303 .+-. 277 .+-. 345 .+-. 49 11 7 19 18
26 41 47 DAY 18 516 .+-. 71 .+-. 65 .+-. 112 .+-. 93 .+-. 298 .+-.
219 .+-. 309 .+-. 63 6 6 16 14 28 31 37 DAY 21 562 .+-. 55 .+-. 56
.+-. 122 .+-. 98 .+-. 320 .+-. 218 .+-. 373 .+-. 65 6 6 27 20 41 42
50 DAY 25 610 .+-. 49 .+-. 51 .+-. 137 .+-. 93 .+-. 315 .+-. 264
.+-. 401 .+-. 78 6 6 33 24 52 52 58 DAY 28 624 .+-. 41 .+-. 51 .+-.
161 .+-. 99 .+-. 358 .+-. 246 .+-. 446 .+-. 94 7 8 53 26 61 51 64
DAY 32 817 .+-. 42 .+-. 72 .+-. 175 .+-. 165 .+-. 398 .+-. 332 .+-.
482 .+-. 99 13 15 52 45 64 77 62 DAY 35 900 .+-. 42 .+-. 92 .+-.
271 .+-. 229 .+-. 487 .+-. 384 .+-. 587 .+-. 104 11 21 79 59 79 94
80 DAY 39 960 .+-. 62 .+-. 120 .+-. 319 .+-. 294 .+-. 569 .+-. 431
.+-. 591 .+-. 117 26 31 103 78 102 114 83 DAY 42 931 .+-. 75 .+-.
151 .+-. 357 .+-. 318 .+-. 590 .+-. 495 .+-. 612 .+-. 108 34 37 113
71 101 128 92 DAY 46 1037 92 .+-. 172 .+-. 431 .+-. 412 .+-. 743
.+-. 610 .+-. 723 .+-. 102 44 47 137 106 133 165 119 DAY 49 1119
.+-. 120 .+-. 248 .+-. 519 .+-. 521 .+-. 810 .+-. 718 .+-. 853 .+-.
120 63 62 135 132 121 202 139 DAY 53 1345 .+-. 144 .+-. 339 .+-.
678 .+-. 629 .+-. 989 .+-. 848 .+-. 970 .+-. 158 67 93 195 162 146
251 193 DAY 56 1485 .+-. 126 .+-. 376 .+-. 818 .+-. 808 .+-. 1149
.+-. 776 .+-. 1215 .+-. 185 51 100 251 196 191 184 231 DAY 60 1691
.+-. 180 .+-. 503 .+-. 710 .+-. 917 .+-. 1287 .+-. 964 .+-. 1428
.+-. 220 85 138 162 209 194 232 273 DAY 63 1736 .+-. 223 .+-. 604
.+-. 824 .+-. 917 .+-. 1503 .+-. 1097 .+-. -- 193 111 160 191 147
227 254 DAY 67 -- 296 .+-. 888 .+-. 938 .+-. 1116 .+-. 1600 .+-.
1167 .+-. -- 152 272 202 173 251 260 DAY 70 -- 312 .+-. 773 .+-.
953 .+-. 1181 .+-. -- 1352 .+-. -- 162 235 209 203 305 DAY 74 --
331 .+-. 881 .+-. -- -- -- -- -- 160 264 DAY 77 -- 422 .+-. 1029
.+-. -- -- -- -- -- 210 325 DAY 81 -- 510 .+-. -- -- -- -- -- --
248 DAY 84 -- 622 .+-. -- -- -- -- -- -- 322
B. HCC2429 Lung Xenografts
[0245] Similar in vivo experiments were performed with the HCC2429
lung cancer cell line as described above. To generate xenografts,
nude (Nu/Nu) female mice were implanted subcutaneously with
3.5.times.10.sup.6 HCC2429 cells in 50% Matrigel (BD Biosciences).
When the tumors reached a volume of 200 to 400 mm.sup.3, the tumors
were staged to ensure uniformity of the tumor mass among various
treatment groups. The HCC2429 lung model was dosed intravenously
Q4dx4 with PBS vehicle, humanized anti-Notch3 ADCs and control
huNeg-8.8 ADCs at the doses provided in Tables 20 and 21. The data
demonstrates that anti-Notch3 ADCs hu28-vc0101, hu28-vc6780,
hu75-vc0101 and hu75-vc6780 inhibited growth of HCC2429 lung
xenografts in a dose-dependent manner. Further, the data shows that
anti-Notch3 ADCs inhibited tumor growth more potently than control
huNeg8.8-ADCs at the 1 and 3 mg/kg doses for anti-Notch3 ADCs with
vc0101 linker-payloads and at the 3 and 10 mg/kg doses for
anti-Notch3 ADCs with vc6780 linker-payloads. Furthermore, the data
demonstrates that a 3 mg/kg dose of hu28-vc0101 was more potent
than a 10 mg/kg dose of hu28-vc6780.
TABLE-US-00020 TABLE 20 Efficacy of anti-Notch3-vc0101 ADCs in
HCC2429 lung xenografts. HCC2429 Lung xenografts, tumor volume
(mm.sup.3 +/- SEM) PBS hu28-vc0101 hu75-vc0101 huNeg-8.8-vc0101
Dose mg/kg 0 3 1 0.3 3 1 0.3 3 1 0.3 DAY -1 245 .+-. 245 .+-. 246
.+-. 246 .+-. 245 .+-. 246 .+-. 247 .+-. 244 .+-. 245 .+-. 246 .+-.
24 23 26 30 28 23 29 30 33 27 DAY 1 529 .+-. 548 .+-. 532 .+-. 528
.+-. 498 .+-. 548 .+-. 524 .+-. 482 .+-. 519 .+-. 514 .+-. 52 52 36
50 39 37 66 59 72 50 DAY 3 742 .+-. 606 .+-. 757 .+-. 733 .+-. 498
.+-. 753 .+-. 713 .+-. 695 .+-. 756 .+-. 724 .+-. 73 78 68 78 44 93
74 91 97 73 DAY 6 1205 .+-. 723 .+-. 1095 .+-. 1112 .+-. 469 .+-.
1096 .+-. 1078 .+-. 1075 .+-. 1144 .+-. 1207 .+-. 120 101 119 132
70 146 74 132 100 100 DAY 8 1720 .+-. 696 .+-. 1324 .+-. 1617 .+-.
407 .+-. 1428 .+-. 1499 .+-. 1404 .+-. 1598 .+-. 1683 .+-. 181 100
173 172 71 200 115 183 133 165 DAY 10 2312 .+-. 620 .+-. 1606 .+-.
2027 .+-. 370 .+-. 1611 .+-. 1830 .+-. 1735 .+-. 1974 .+-. 2163
.+-. 197 90 250 233 81 189 120 253 185 260 DAY 13 3235 .+-. 543
.+-. 1717 .+-. 2642 .+-. 273 .+-. 1803 .+-. 2408 .+-. 2162 .+-.
2676 .+-. 2589 .+-. 120 92 223 297 69 208 226 376 346 287 DAY 15 --
512 .+-. 1865 .+-. -- 298 .+-. 1871 .+-. -- -- -- -- 111 263 88 232
DAY 17 -- 442 .+-. 2228 .+-. -- 250 .+-. 1948 .+-. -- -- -- -- 114
333 77 228 DAY 20 -- 428 .+-. -- -- 177 .+-. -- -- -- -- -- 144 44
DAY 23 -- 405 .+-. -- -- 160 .+-. -- -- -- -- -- 149 35 DAY 27 --
422 .+-. -- -- 174 .+-. -- -- -- -- -- 164 51 DAY 30 -- 394 .+-. --
-- 196 .+-. -- -- -- -- -- 182 72 DAY 34 -- 505 .+-. -- -- 295 .+-.
-- -- -- -- -- 236 121 DAY 37 -- 606 .+-. -- -- 433 .+-. -- -- --
-- -- 283 179 DAY 41 -- 750 .+-. -- -- 606 .+-. -- -- -- -- -- 361
259 DAY 45 -- 872 .+-. -- -- 836 .+-. -- -- -- -- -- 415 359 DAY 49
-- 558 .+-. -- -- 732 .+-. -- -- -- -- -- 303 350 DAY 52 -- 571
.+-. -- -- -- -- -- -- -- -- 310 DAY 56 -- 704 .+-. -- -- -- -- --
-- -- -- 399
TABLE-US-00021 TABLE 21 Efficacy of anti-Notch3-vc6780 ADCs in
HCC2429 lung xenografts. HCC2429 Lung xenografts, tumor volume
(mm.sup.3 .+-. SEM) PBS hu28-vc6780 hu75-vc6780 huNeg-8.8-vc6780
Dose mg/kg 0 10 3 1 10 3 1 10 3 1 DAY -1 245 .+-. 244 .+-. 245 .+-.
245 .+-. 244 .+-. 246 .+-. 245 .+-. 244 .+-. 244 .+-. 245 .+-. 28
22 24 27 19 30 16 22 26 20 DAY 1 398 .+-. 369 .+-. 379 .+-. 400
.+-. 407 .+-. 403 .+-. 418 .+-. 429 .+-. 427 .+-. 402 .+-. 50 31 45
66 43 51 34 56 49 53 DAY 3 701 .+-. 318 .+-. 493 .+-. 579 .+-. 339
.+-. 526 .+-. 629 .+-. 619 .+-. 689 .+-. 655 .+-. 102 31 65 113 36
74 65 62 83 97 DAY 5 949 .+-. 228 .+-. 609 .+-. 826 .+-. 251 .+-.
615 .+-. 916 .+-. 808 .+-. 965 .+-. 837 .+-. 140 28 82 191 33 98 97
101 114 117 DAY 7 1345 .+-. 172 .+-. 638 .+-. 1023 .+-. 225 .+-.
615 .+-. 1164 .+-. 1072 .+-. 1380 .+-. 1099 .+-. 200 22 86 259 24
115 131 154 136 172 DAY 10 2045 .+-. 143 .+-. 784 .+-. 1439 .+-.
198 .+-. 717 .+-. 1705 .+-. 1452 .+-. 2082 .+-. 1722 .+-. 356 22
115 398 24 129 184 210 192 363 DAY 12 -- 134 .+-. 883 .+-. 1442
.+-. 166 .+-. 807 .+-. 2029 .+-. 1673 .+-. 2701 .+-. 1586 .+-. 20
132 487 22 130 270 290 228 337 DAY 14 -- 115 .+-. 895 .+-. -- 150
.+-. 831 .+-. 2294 .+-. 1809 .+-. -- -- 16 175 22 145 287 314 DAY
17 -- 127 .+-. 1105 .+-. -- 158 .+-. 1017 .+-. -- -- -- -- 18 253
32 178 DAY 20 -- 149 .+-. 1219 .+-. -- 164 .+-. 1297 .+-. -- -- --
-- 27 311 48 231 DAY 24 -- 206 .+-. 1618 .+-. -- 261 .+-. 1813 .+-.
-- -- -- -- 60 468 89 343 DAY 27 -- 290 .+-. -- -- 316 .+-. 1970
.+-. -- -- -- -- 100 135 462 DAY 31 -- 378 .+-. -- -- 438 .+-. --
-- -- -- -- 150 201 DAY 34 -- 551 .+-. -- -- 423 .+-. -- -- -- --
-- 244 177 DAY 38 -- 718 .+-. -- -- 504 .+-. -- -- -- -- -- 332 203
DAY 42 -- 1011 .+-. -- -- 655 .+-. -- -- -- -- -- 504 266 DAY 46 --
-- -- -- 793 .+-. -- -- -- -- -- 320 DAY 49 -- -- -- -- 901 .+-. --
-- -- -- -- 351 DAY 53 -- -- -- -- 1228 .+-. -- -- -- -- -- 472
[0246] The HCC2429 lung model was also dosed intravenously Q4dx4
with PBS vehicle, rat-human chimeric anti-Notch3 ADCs and control
huNeg-8.8 ADCs, at a dose of 5 mg/kg as provided in FIG. 16A. The
data demonstrates that anti-Notch3 ADCs with non-cleavable (mc) and
cleavable (vc) linkers and various payload combinations inhibited
growth of HCC2429 lung xenografts. Further, the data shows that
rat-human chimeric anti-Notch3 ADCs inhibited tumor growth more
potently than control huNeg8.8-ADCs. Furthermore, the data
demonstrates that rat-human chimeric anti-Notch3 ADCs with vc0101
linker-payloads were more potent than the other anti-Notch3 ADCs
tested.
[0247] Additional in vivo experiments were performed with the
HCC2429 lung cancer cell line using an unconjugated rat-human
chimeric anti-Notch3 antibody, ch75-hlgG1, to determine whether
ch75-hlgG1's Notch3 signaling inhibition contributed to the
observed potency of the anti-Notch3 hu75-ADCs. To generate
xenografts, nude (Nu/Nu) female mice were implanted subcutaneously
with 3.5.times.10.sup.6 HCC2429 cells in 50% Matrigel (BD
Biosciences). When the tumors reached a volume of 75 to 200
mm.sup.3 the tumors were staged to ensure uniformity of the tumor
mass among various treatment groups.
[0248] The HCC2429 lung model was dosed intravenously Q4dx4 with
PBS vehicle, rat-human chimeric anti-Notch3 antibody, ch75-hlgG1,
and humanized anti-Notch1 antibody, hu438 VH1.1/VL1.8, at the does
provided in Table 22. From 8 animals, mean tumor masses (.+-.SEM)
for each treatment group were calculated and compared to the
control PBS vehicle group. P-values based on analysis of variance
(ANOVA) were calculated to determine statistical significance of
observed growth inhibition of anti-Notch treatments versus control
PBS using Excel built-in statistical functions. Percent (%) growth
inhibition values were calculated from measurements on the final
day of the study for drug-treated compared with vehicle-treated
mice with the formula 100*{1-[(Treated.sub.Day 14-Treated.sub.Day
0)/(Control.sub.Day 14-Control.sub.Day 0)]}.
[0249] The data demonstrates that anti-Notch1 humanized antibody
hu438 VH1.1/VL1.8 inhibited tumor growth by 57% and unconjugated
rat-human chimeric anti-Notch3 antibody ch75-hlgG1 did not inhibit
tumor growth compared to PBS vehicle treated tumors. Further, the
data demonstrates that the observed tumor growth inhibition
reported in Tables 20, 21 and FIG. 16A in the HCC2429 xenografts
with Notch3 ADCs generated with the ch75 antibodies were not due to
signaling inhibition.
TABLE-US-00022 TABLE 22 Effects of inhibitory anti-Notch3 and
anti-Notch1 antibodies in HCC2429 lung xenografts. HCC2429 Lung
xenografts, tumor volume (mm.sup.3 .+-. SEM) p-value hu438 p-value
ch75- (ch75 VH1.1/ (hu438 hlgG1 ch75- VL1.8 VH1.1/ PBS (anti- hlgG1
(anti- VL1.8 control Notch3) vs. PBS) Notch1) vs. PBS) DAY 110 .+-.
7 110 .+-. 5 0.54 112 .+-. 11 0.504757 0 DAY 218 .+-. 24 211 .+-.
24 0.39 173 .+-. 26 0.036706 3 DAY 397 .+-. 41 356 .+-. 41 0.22 254
.+-. 36 0.001177 5 DAY 665 .+-. 61 574 .+-. 59 0.19 311 .+-. 60
0.000001 7 DAY 1267 .+-. 139 1054 .+-. 104 0.19 518 .+-. 92
0.000001 10 DAY 1615 .+-. 187 1441 .+-. 139 0.34 701 .+-. 132
0.000006 12 DAY 1985 .+-. 234 1821 .+-. 175 0.42 909 .+-. 172
0.000048 14
C. MDA-MB-468 Breast Xenografts
[0250] Similar in vivo experiments were performed with the
MDA-MB-468 breast cancer cell line as described above. MDA-MB-468
cells are classified as a triple-negative breast cancer (TNBC)
basal-like subtype since they lack expression of the estrogen
receptor, progesterone receptor and human epidermal growth factor
receptor 2 (HER2) (Lehmann, B D, et al, J Clin Invest. 2011;
121(7):2750-2767). To generate xenografts, female SCID Hairless
Outbred (SHO) mice were orthotopically implanted with
10.times.10.sup.6 MDA-MB-468 cells containing 50% Matrigel (BD
Biosciences) in the mammary fat pad. When the tumors reached a
volume of 250 to 450 mm.sup.3, the tumors were staged to ensure
uniformity of the tumor mass among various treatment groups. The
MDA-MB-468 breast model was dosed intravenously Q4dx4 with PBS
vehicle, humanized anti-Notch3 ADCs and control huNeg-8.8 ADCs at
the doses provided in Tables 23 and 24. FIGS. 13A and 13B show
graphs of the data from Table 23 of anti-Notch3 ADCs with vc0101
linker-payloads compared to control huNeg-8.8 ADCs and PBS vehicle.
FIGS. 14A and 14B show graphs of the data from Table 24 of the
anti-Notch3 ADCs with vc6780 linker-payloads compared to control
huNeg-8.8 ADC and PBS vehicle.
[0251] The data demonstrates that anti-Notch3 ADCs hu28-vc0101,
hu28-vc6780, hu75-vc0101 and hu75-vc6780 inhibited growth of
MDA-MB-468 breast xenografts in a dose-dependent manner. Further,
the data shows that anti-Notch3 ADCs inhibited tumor growth more
potently than control huNeg8.8-ADCs at the 1 and 3 mg/kg doses for
ADCs with vc0101 linker-payloads and 1, 3 and 10 mg/kg doses for
ADC with vc6780 linker-payloads. Furthermore, the data demonstrates
that a 1 mg/kg dose of anti-Notch3 ADCs with vc0101 linker-payloads
were more potent than a 3 mg/kg dose of anti-Notch3 ADCs with
vc6780 linker-payloads.
TABLE-US-00023 TABLE 23 Efficacy of anti-Notch3-vc0101 ADCs in
MDA-MB-468 breast xenografts. MDA-MB-468 Breast xenografts, tumor
volume (mm.sup.3 .+-. SEM) PBS hu28-vc0101 hu75-vc0101
huNeg-8.8-vc0101 Dose mg/kg 0 3 1 0.3 3 1 0.3 3 1 0.3 DAY 0 343
.+-. 347 .+-. 348 .+-. 336 .+-. 347 .+-. 347 .+-. 348 .+-. 334 .+-.
346 .+-. 344 .+-. 12 15 22 19 20 22 21 23 16 19 DAY 4 441 .+-. 359
.+-. 439 .+-. 403 .+-. 444 .+-. 410 .+-. 439 .+-. 424 .+-. 447 .+-.
442 .+-. 24 24 21 28 28 32 31 29 32 19 DAY 7 469 .+-. 326 .+-. 415
.+-. 395 .+-. 338 .+-. 383 .+-. 435 .+-. 411 .+-. 449 .+-. 438 .+-.
32 27 25 38 20 33 27 26 20 23 DAY 11 495 .+-. 227 .+-. 372 .+-. 412
.+-. 277 .+-. 373 .+-. 504 .+-. 439 .+-. 538 .+-. 496 .+-. 28 27 34
42 22 33 38 36 23 37 DAY 14 581 .+-. 147 .+-. 314 .+-. 488 .+-. 181
.+-. 350 .+-. 507 .+-. 445 .+-. 592 .+-. 560 .+-. 35 20 27 45 19 40
30 29 47 36 DAY 18 639 .+-. 77 .+-. 261 .+-. 497 .+-. 90 .+-. 296
.+-. 587 .+-. 479 .+-. 619 .+-. 578 .+-. 43 10 33 55 12 33 44 42 42
36 DAY 21 638 .+-. 16 .+-. 219 .+-. 509 .+-. 60 .+-. 260 .+-. 590
.+-. 481 .+-. 676 .+-. 627 .+-. 46 8 41 60 9 49 55 34 46 30 DAY 26
707 .+-. 0 .+-. 253 .+-. 590 .+-. 16 .+-. 267 .+-. 652 .+-. 548
.+-. 793 .+-. 671 .+-. 41 0 61 66 10 59 64 41 54 56 DAY 29 749 .+-.
0 .+-. 238 .+-. -- 8 .+-. 261 .+-. 675 .+-. -- 819 .+-. 669 .+-. 59
0 64 8 62 63 73 37 DAY 32 812 .+-. 0 .+-. 266 .+-. -- 7 .+-. 264
.+-. 738 .+-. -- 913 .+-. 758 .+-. 80 0 67 7 67 70 72 44 DAY 35 891
.+-. 0 .+-. 271 .+-. -- 0 .+-. 326 .+-. 821 .+-. -- 1023 .+-. 848
.+-. 79 0 73 0 86 69 96 58 DAY 39 892 .+-. 0 .+-. 310 .+-. -- 0
.+-. 324 .+-. 864 .+-. -- -- 884 .+-. 84 0 88 0 81 74 64 DAY 42
1037 .+-. 0 .+-. 349 .+-. -- 0 .+-. 381 .+-. 997 .+-. -- -- 1002
.+-. 104 0 95 0 94 84 55 DAY 47 1173 .+-. 0 .+-. 394 .+-. -- 0 .+-.
442 .+-. -- -- -- 1145 .+-. 134 0 123 0 69 78 DAY 50 -- 0 .+-. 377
.+-. -- 0 .+-. 484 .+-. -- -- -- 1120 .+-. 0 118 0 89 67 DAY 53 --
0 .+-. 414 .+-. -- 0 .+-. 452 .+-. -- -- -- 1229 .+-. 0 127 0 78
100 DAY 56 -- 0 .+-. 470 .+-. -- 0 .+-. 535 .+-. -- -- -- 1314 .+-.
0 128 0 93 120 DAY 60 -- 0 .+-. 532 .+-. -- 0 .+-. 603 .+-. -- --
-- -- 0 140 0 98 DAY 63 -- 0 .+-. 509 .+-. -- 0 .+-. -- -- -- -- --
0 117 0 DAY 67 -- 0 .+-. 611 .+-. -- 0 .+-. -- -- -- -- -- 0 148 0
DAY 70 -- 0 .+-. -- -- 0 .+-. -- -- -- -- -- 0 0 DAY 76 -- 0 .+-.
-- -- 0 .+-. -- -- -- -- -- 0 0 DAY 83 -- 0 .+-. -- -- 0 .+-. -- --
-- -- -- 0 0 DAY 90 -- 0 .+-. -- -- 0 .+-. -- -- -- -- -- 0 0 DAY
97 -- 0 .+-. -- -- 0 .+-. -- -- -- -- -- 0 0 DAY 103 -- 0 .+-. --
-- 0 .+-. -- -- -- -- -- 0 0 DAY 110 -- 0 .+-. -- -- 0 .+-. -- --
-- -- -- 0 0 DAY 118 -- 0 .+-. -- -- 0 .+-. -- -- -- -- -- 0 0 DAY
125 -- 0 .+-. -- -- 0 .+-. -- -- -- -- -- 0 0
TABLE-US-00024 TABLE 24 Efficacy of anti-Notch3-vc6780 ADCs in
MDA-MB-468 breast xenografts. MDA-MB-468 Breast xenografts, tumor
volume (mm.sup.3 .+-. SEM) PBS hu28-vc6780 hu75-vc6780
huNeg-8.8-vc6780 Dose mg/kg 0 10 3 1 10 3 1 10 3 1 DAY 0 342 .+-.
335 .+-. 342 .+-. 342 .+-. 343 .+-. 344 .+-. 340 .+-. 339 .+-. 341
.+-. 346 .+-. 9 9 18 16 10 11 14 18 12 16 DAY 4 466 .+-. 395 .+-.
394 .+-. 462 .+-. 418 .+-. 406 .+-. 423 .+-. 432 .+-. 457 .+-. 466
.+-. 20 19 33 22 15 22 27 45 23 29 DAY 7 481 .+-. 350 .+-. 399 .+-.
452 .+-. 370 .+-. 378 .+-. 434 .+-. 449 .+-. 529 .+-. 528 .+-. 17
19 24 30 18 21 29 45 24 25 DAY 11 611 .+-. 248 .+-. 380 .+-. 512
.+-. 302 .+-. 403 .+-. 471 .+-. 504 .+-. 599 .+-. 621 .+-. 44 26 25
35 21 21 39 38 23 43 DAY 14 610 .+-. 154 .+-. 401 .+-. 507 .+-. 228
.+-. 370 .+-. 470 .+-. 503 .+-. 622 .+-. 639 .+-. 19 23 30 38 19 28
44 64 31 48 DAY 19 707 .+-. 65 .+-. 438 .+-. 538 .+-. 112 .+-. 339
.+-. 536 .+-. 437 .+-. 697 .+-. 713 .+-. 34 17 39 47 23 19 49 54 36
48 DAY 22 -- 25 .+-. 414 .+-. 551 .+-. 52 .+-. 360 .+-. 552 .+-.
415 .+-. 719 .+-. -- 16 41 48 21 17 44 54 40 DAY 25 -- 26 .+-. 491
.+-. 575 .+-. 63 .+-. 381 .+-. 597 .+-. 421 .+-. 773 .+-. -- 19 37
55 25 23 48 76 39 DAY 28 -- 15 .+-. 497 .+-. 654 .+-. 64 .+-. 443
.+-. 660 .+-. 451 .+-. 808 .+-. -- 15 68 74 26 33 53 84 58 DAY 32
-- 0 .+-. 524 .+-. 653 .+-. 71 .+-. 437 .+-. 634 .+-. 456 .+-. --
-- 0 69 82 31 28 74 94 DAY 35 -- 0 .+-. -- 734 .+-. 85 .+-. 495
.+-. 742 .+-. 541 .+-. -- -- 0 89 38 33 80 108 DAY 40 -- 0 .+-. --
761 .+-. 125 .+-. 535 .+-. 794 .+-. 563 .+-. -- -- 0 99 44 41 87
109 DAY 43 -- 0 .+-. -- 816 .+-. 134 .+-. 619 .+-. 832 .+-. 581
.+-. -- -- 0 122 42 47 82 120 DAY 46 -- 0 .+-. -- 859 .+-. 143 .+-.
636 .+-. 868 .+-. 617 .+-. -- -- 0 126 42 38 99 116 DAY 49 -- 0
.+-. -- 948 .+-. 159 .+-. 723 .+-. 996 .+-. 733 .+-. -- -- 0 178 44
71 109 129 DAY 53 -- 0 .+-. -- 1008 .+-. 201 .+-. 795 .+-. -- 758
.+-. -- -- 0 192 63 67 163 DAY 56 -- 0 .+-. -- -- 211 .+-. 819 .+-.
-- -- -- -- 0 63 77 DAY 60 -- 0 .+-. -- -- 240 .+-. 976 .+-. -- --
-- -- 0 63 115 DAY 63 -- 0 .+-. -- -- 201 .+-. -- -- -- -- -- 0 57
DAY 67 -- 0 .+-. -- -- -- -- -- -- -- -- 0 DAY 70 -- 0 .+-. -- --
-- -- -- -- -- -- 0 DAY 76 -- 0 .+-. -- -- -- -- -- -- -- -- 0 DAY
83 -- 0 .+-. -- -- -- -- -- -- -- -- 0 DAY 90 -- 0 .+-. -- -- -- --
-- -- -- -- 0 DAY 96 -- 0 .+-. -- -- -- -- -- -- -- -- 0 DAY 103 --
0 .+-. -- -- -- -- -- -- -- -- 0 DAY 111 -- 0 .+-. -- -- -- -- --
-- -- -- 0 DAY 118 -- 0 .+-. -- -- -- -- -- -- -- -- 0 DAY 124 -- 0
.+-. -- -- -- -- -- -- -- -- 0
[0252] The MDA-MB-468 breast cancer model was also dosed
intravenously Q4dx4 with PBS vehicle, rat-human chimeric
anti-Notch3 ADCs and control huNeg-8.8 ADCs, at a dose of 5 mg/kg
as provided in FIGS. 16B and 16C. The data demonstrates that
rat-human chimeric anti-Notch3 ADCs with non-cleavable (mc) and
cleavable (vc) linkers and various payload combinations inhibited
growth of MDA-MB-468 breast xenografts. Further, the data shows
that rat-human chimeric anti-Notch3 ADCs inhibited tumor growth
more potently than control huNeg8.8-ADCs. Furthermore, the data
demonstrates that rat-human chimeric anti-Notch3 ADCs with vc0101
linker-payloads were more potent than the other rat-human chimeric
anti-Notch3 ADCs tested.
[0253] The MDA-MB-468 breast cancer model was used to examine the
in vivo mechanism of action of hu28-vc0101. The pharmacodynamics of
hu28-vc0101 was visualized at the cellular level by staining with
the mitotic marker phospho-Histone H3 in ADC treated xenografts.
Histone H3 is phosphorylated on Ser-10 residues (hereinafter
"pHH3") within chromatin during the mitotic phase of the cell
cycle.
[0254] Mice bearing MDA-MB-468 breast xenografts were given a
single 3 mg/kg dose intravenously with anti-Notch3 ADC hu28-vc0101,
huNeg-8.8-vc0101 (control ADC) or PBS. Three xenografts were
harvested after 6, 24 and 96 hours and processed for standard
immunohistochemistry. Five micrometer thick formalin fixed,
paraffin embedded tissue sections were deparaffinized in xylene
substitute, rehydrated with graded alcohols to distilled water. To
expose antigenic sites, tissue sections were heated in 10 mM
citrate buffer pH 6.0 (Labvision) in a pressure cooker (Retriever;
Electron Microscopy Sciences) and cooled to room temperature.
Endogenous peroxidase activity was inactivated with 3% hydrogen
peroxide for 15 min. Non specific protein interactions were blocked
by a 10 minute incubation with UV Block (Labvision). Tissue
sections were incubated with anti-pHH3 antibody for one hour,
detected with Signalstain Boost Reagent (8114, Cell Signaling
Technologies) for 30 minutes and color was developed with DAB+
(DAKO) for 5 minutes. All sections were counterstained with
Hematoxylin QS (Vector Laboratories), washed in tap water,
dehydrated in graded alcohols, cleared in xylene substitute, and
mounted with Permount Mounting Medium (FisherChemicals, Fair Lawn,
N.J.).
[0255] Immunohistochemically stained slides were evaluated by image
analysis. Slides were imaged at 20.times. using a Hamamatsu
NanoZoomer automated slide scanner. Once digitized, the virtual
slides were analyzed using Definiens Tissue Studio software. Each
xenograft section was regionally segmented and classified based on
cellularity and morphology. Individual nuclei were identified
within the viable regions of the xenograft and the positivity was
determined using the average brown chromogen intensity of each
nuclei. Data is presented as percent (%) pHH3 positivity which is
calculated using the following equation: (Number of pHH3 positive
nuclei (viable region)/Total number of nuclei (viable region))*100.
Determination of statistical significance was determined for each
treatment group and time point compared to PBS control at 24 hour
time point with Graph Pad Prism using a 2 tailed T test.
[0256] ADCs generated with microtubule inhibitors like hu28-vc0101
are expected to arrest proliferating cells in the mitotic phase of
the cell cycle when histone H3 is phosphorylated on Ser-10 residues
within chromatin. An accumulation of pHH3 staining in ADC treated
xenografts indicates that cancer cells were arrested in mitosis.
Table 25 contains percentages of pHH3 stained cells in anti-Notch3
hu28-vc0101 and huNeg-8.8-vc0101 (control ADC) treated MDA-MB-468
xenografts and untreated MDA-MB-468 xenografts. The data
demonstrates that hu28-vc0101, but not control huNeg8.8-vc0101
resulted in a statistically significant 2 fold increase in the
percentage of pHH3 stained nuclei in MDA-MD-468 cancer cells at 24
and 96 hours post-treatment compared to the PBS control. The data
indicates that anti-Notch3 hu28-vc0101 inhibits in vivo tumor
growth, at least in part, by arresting cells in the mitotic phase
of the cell cycle, thus preventing proliferation.
TABLE-US-00025 TABLE 25 Percentage of pHH3 stained nuclei in Notch3
ADC treated MDA- MB-468 breast cancer xenografts (n.d. = not
determined) 6 hr 24 hr 96 hr p- p- p- % pHH3 value % pHH3 value %
pHH3 value PBS n.d n.d. 2.62 n.d. n.d. n.d. hu28- 2.54 0.84 5.37
0.0046 5.19 0.0003 vc0101 huNeg8.8- 2.74 0.78 3.67 0.1025 3.01
0.0730 vc0101
D. N87 Gastric Xenografts
[0257] Similar in vivo experiments were performed with the N87
gastric cancer cell line as described above. To generate
xenografts, nude (Nu/Nu) female mice were implanted subcutaneously
with 8.times.10.sup.6 N87 cells in 50% Matrigel (BD Biosciences).
When the tumors reached a volume of 250 to 450 mm.sup.3, the tumors
were staged to ensure uniformity of the tumor mass among various
treatment groups. The N87 gastric model was dosed intravenously
Q4dx4 with PBS vehicle, humanized anti-Notch3 ADCs, control
huNeg-8.8 ADCs and cisplatin at the doses provided in Tables 26 and
27. The data demonstrates that anti-Notch3 ADCs hu28-vc0101,
hu28-vc6780, hu75-vc0101 and hu75-vc6780 inhibited growth of N87
gastric xenografts in a dose-dependent manner. Further, the data
shows that anti-Notch3 ADCs inhibited tumor growth more potently
than control huNeg8.8-ADCs at the 1, 3, 5 mg/kg doses for ADCs with
vc0101 linker-payloads and 3 and 10 mg/kg doses for ADCs with
vc6780 linker-payloads. By day 133, the 5 mg/kg dose group of
hu28-vc0101 contained 6 out of 9 animals that were tumor-free,
hu75-vc0101 had 4 out of 9 animals and control huNeg8-8-vc0101 had
1 out of 8 animals that were tumor-free. Furthermore, the data
demonstrates that ADCs with vc0101 linker-payloads were in general
more potent than cisplatin standard-of-care therapy and ADCs with
vc6780 linker-payloads.
TABLE-US-00026 TABLE 26 Efficacy of anti-Notch3-vc0101 ADCs in N87
gastric xenografts. N87 Gastric xenografts, tumor volume (mm.sup.3
.+-. SEM) PBS hu28-vc0101 hu75-vc0101 huNeg-8.8-vc0101 Cisplatin
Dose mg/kg 0 1 3 5 1 3 5 1 3 5 5 DAY 0 327 .+-. 321 .+-. 326 .+-.
321 .+-. 321 .+-. 324 .+-. 320 .+-. 327 .+-. 324 .+-. 321 .+-. 328
.+-. 11 21 13 8 9 19 11 18 11 16 20 DAY 4 526 .+-. 369 .+-. 339
.+-. 344 .+-. 392 .+-. 362 .+-. 315 .+-. 437 .+-. 478 .+-. 423 .+-.
414 .+-. 19 18 11 14 15 35 15 34 19 32 27 DAY 7 706 .+-. 429 .+-.
302 .+-. 272 .+-. 417 .+-. 303 .+-. 246 .+-. 584 .+-. 625 .+-. 512
.+-. 520 .+-. 27 43 10 7 25 21 12 54 34 34 26 DAY 11 854 .+-. 304
.+-. 182 .+-. 152 .+-. 331 .+-. 174 .+-. 156 .+-. 702 .+-. 716 .+-.
501 .+-. 501 .+-. 36 30 14 13 21 14 10 60 53 38 29 DAY 14 887 .+-.
282 .+-. 191 .+-. 155 .+-. 305 .+-. 172 .+-. 151 .+-. 822 .+-. 823
.+-. 549 .+-. 637 .+-. 45 25 5 13 17 10 7 65 42 37 31 DAY 18 1045
.+-. 263 .+-. 161 .+-. 138 .+-. 267 .+-. 151 .+-. 128 .+-. 823 .+-.
789 .+-. 491 .+-. -- 68 24 7 11 17 10 6 73 33 51 DAY 21 1072 .+-.
227 .+-. 123 .+-. 110 .+-. 218 .+-. 130 .+-. 115 .+-. 857 .+-. 785
.+-. 413 .+-. -- 76 23 15 9 23 5 7 78 35 50 DAY 26 1303 .+-. 205
.+-. 108 .+-. 69 .+-. 185 .+-. 92 .+-. 82 .+-. 895 .+-. 825 .+-.
343 .+-. -- 140 32 16 16 24 14 10 126 62 63 DAY 29 1276 .+-. 180
.+-. 99 .+-. 50 .+-. 211 .+-. 104 .+-. 75 .+-. 957 .+-. 879 .+-.
411 .+-. -- 139 30 14 13 37 16 12 126 72 89 DAY 33 1480 .+-. 211
.+-. 106 .+-. 43 .+-. 251 .+-. 91 .+-. 73 .+-. 988 .+-. 966 .+-.
411 .+-. -- 183 43 17 14 53 18 12 180 98 89 DAY 36 -- 215 .+-. 122
.+-. 52 .+-. 272 .+-. 86 .+-. 85 .+-. 884 .+-. 1023 .+-. 481 .+-.
-- 42 22 16 59 18 9 143 106 86 DAY 39 -- 261 .+-. 128 .+-. 45 .+-.
304 .+-. 59 .+-. 72 .+-. 937 .+-. 1142 .+-. 535 .+-. -- 54 23 14 72
16 13 167 121 128 DAY 42 -- 283 .+-. 149 .+-. 34 .+-. 314 .+-. 81
.+-. 74 .+-. 1008 .+-. 1240 .+-. 596 .+-. -- 52 25 15 73 22 13 179
143 119 DAY 47 -- 262 .+-. 105 .+-. 25 .+-. 334 .+-. 80 .+-. 36
.+-. 1061 .+-. 1380 .+-. 621 .+-. -- 64 19 14 95 25 8 210 153 137
DAY 53 -- 302 .+-. 104 .+-. 29 .+-. 393 .+-. 86 .+-. 69 .+-. -- --
757 .+-. -- 75 30 16 115 24 13 189 DAY 62 -- 415 .+-. 116 .+-. 33
.+-. 463 .+-. 106 .+-. 50 .+-. -- -- 690 .+-. -- 111 47 18 155 35
15 122 DAY 70 -- 521 .+-. 139 .+-. 58 .+-. 658 .+-. 148 .+-. 76
.+-. -- -- 852 .+-. -- 135 54 30 241 54 22 150 DAY 78 -- 622 .+-.
172 .+-. 67 .+-. 531 .+-. 161 .+-. 90 .+-. -- -- 937 .+-. -- 169 69
40 152 60 25 168 DAY 84 -- 709 .+-. 149 .+-. 85 .+-. 602 .+-. 200
.+-. 96 .+-. -- -- 1178 .+-. -- 200 73 49 185 76 37 222 DAY 91 --
848 .+-. 178 .+-. 104 .+-. 732 .+-. 225 .+-. 109 .+-. -- -- -- --
236 94 63 246 92 46 DAY 99 -- -- 214 .+-. 118 .+-. -- 170 .+-. 130
.+-. -- -- -- -- 108 72 75 53 DAY 103 -- -- 123 .+-. 135 .+-. --
184 .+-. 142 -- -- -- -- 43 90 82 62 DAY 112 -- -- 126 .+-. 166
.+-. -- 222 .+-. 174 .+-. -- -- -- -- 50 105 108 75 DAY 120 -- --
128 .+-. 226 .+-. -- 240 .+-. 221 .+-. -- -- -- -- 44 140 116 103
DAY 133 -- -- 175 .+-. 258 .+-. -- -- 295 .+-. -- -- -- -- 74 157
139
TABLE-US-00027 TABLE 27 Efficacy of anti-Notch3-vc6780 ADCs in N87
gastric xenografts. N87 Gastric xenografts, tumor volume (mm.sup.3
.+-. SEM) huNeg8.8- PBS hu28-vc6780 hu75-vc6780 vc6780 Dose mg/kg 0
10 3 1 10 3 1 10 3 DAY 0 345 .+-. 350 .+-. 349 .+-. 348 .+-. 349
.+-. 351 .+-. 357 .+-. 356 .+-. 344 .+-. 14 14 10 13 8 20 14 20 14
DAY 4 600 .+-. 434 .+-. 552 .+-. 560 .+-. 468 .+-. 545 .+-. 546
.+-. 581 .+-. 537 .+-. 16 24 24 26 18 37 35 60 36 DAY 8 675 .+-.
379 .+-. 545 .+-. 592 .+-. 351 .+-. 511 .+-. 563 .+-. 605 .+-. 670
.+-. 20 12 37 44 24 31 55 67 45 DAY 11 763 .+-. 315 .+-. 511 .+-.
617 .+-. 316 .+-. 544 .+-. 582 .+-. 636 .+-. 706 .+-. 54 18 25 48
25 43 56 79 38 DAY 14 886 .+-. 292 .+-. 564 .+-. 782 .+-. 269 .+-.
558 .+-. 667 .+-. 717 .+-. 917 .+-. 72 24 29 60 27 36 68 118 36 DAY
18 997 .+-. 199 .+-. 479 .+-. 797 .+-. 224 .+-. 494 .+-. 638 .+-.
665 .+-. 958 .+-. 93 18 29 88 26 41 80 112 57 DAY 21 1041 .+-. 194
.+-. 499 .+-. 839 .+-. 192 .+-. 534 .+-. 709 .+-. 637 .+-. 1002
.+-. 107 20 34 93 19 41 103 119 59 DAY 25 1151 .+-. 181 .+-. 588
.+-. 878 .+-. 227 .+-. 628 .+-. 750 .+-. 647 .+-. 1075 .+-. 144 21
40 105 32 58 122 134 82 DAY 28 1200 .+-. 204 .+-. 672 .+-. 904 .+-.
244 .+-. 645 .+-. 763 .+-. 674 .+-. 1148 .+-. 155 16 48 123 35 57
145 146 77 DAY 33 1481 .+-. 196 .+-. 786 .+-. 1043 .+-. 267 .+-.
730 .+-. 991 .+-. 733 .+-. 1290 .+-. 206 27 65 152 52 66 239 195
128 DAY 36 -- 189 .+-. 827 .+-. 1108 .+-. 300 .+-. 850 .+-. -- 817
.+-. 1265 .+-. 37 69 185 64 74 222 111 DAY 39 -- 228 .+-. 847 .+-.
1204 .+-. 323 .+-. 881 .+-. -- 880 .+-. 1429 .+-. 44 77 209 69 88
247 121 DAY 42 -- 257 .+-. 959 .+-. -- 350 .+-. 1020 .+-. -- 797
.+-. -- 60 81 78 99 244 DAY 46 -- 253 .+-. 1018 .+-. -- 380 .+-.
1097 .+-. -- 874 .+-. -- 59 94 78 129 267 DAY 50 -- 253 .+-. 1111
.+-. -- 415 .+-. 1162 .+-. -- -- -- 67 95 77 134 DAY 56 -- 298 .+-.
1279 .+-. -- 504 .+-. 1331 .+-. -- -- -- 85 108 111 187 DAY 63 --
345 .+-. 1368 .+-. -- 581 .+-. -- -- -- -- 93 133 121 DAY 70 -- 376
.+-. 1483 .+-. -- 726 .+-. -- -- -- -- 117 154 163 DAY 77 -- 388
.+-. -- -- 797 .+-. -- -- -- -- 123 184 DAY 84 -- 463 .+-. -- --
995 .+-. -- -- -- -- 149 239 DAY 91 -- 523 .+-. -- -- -- -- -- --
-- 171 DAY 98 -- 701 .+-. -- -- -- -- -- -- -- 251
[0258] The N87 gastric model was also dosed intravenously Q4dx4
with PBS vehicle, rat-human chimeric anti-Notch3 ADCs and control
huNeg-8.8 ADCs, at a dose of 5 mg/kg as provided in FIG. 16D. The
data demonstrates that rat-human chimeric anti-Notch3 ADCs with
non-cleavable (mc) and cleavable (vc) linkers and various payload
combinations inhibited growth of N87 gastric xenografts. Further,
the data shows that rat-human chimeric anti-Notch3 ADCs inhibited
tumor growth more potently than control huNeg8.8-ADCs. Furthermore,
the data demonstrates that rat-human chimeric anti-Notch3 ADCs with
vc0101 linker-payloads were more potent than the other anti-Notch3
ADCs tested.
[0259] The N87 gastric model was also dosed intravenously Q4dx4
with PBS vehicle and rat-human chimeric anti-Notch3 ADCs
ch28-mc0131, ch75-mc0131, ch28-m(H2O)c-0131 and ch75-m(H2O)c-0131
at a dose of 5 mg/kg as provided in FIG. 16E. The data demonstrates
that rat-human chimeric anti-Notch3 ADCs having mc0131 and
m(H2O)c-0131 linker-payloads inhibited growth of N87 gastric
xenografts. Further, the data demonstrates that rat-human chimeric
anti-Notch3 ADCs having m(H2O)c-0131 linker-payloads were more
potent than rat-human chimeric anti-Notch3 ADCs having mc0131
linker-payloads.
E. OVCAR3 Ovarian Xenografts
[0260] Similar in vivo experiments were performed with the OVCAR3
ovarian cell line as described above. To generate xenografts, SCID
Hairless Outbred (SHO) female mice were implanted subcutaneously
with 5.times.10.sup.6 OVCAR3 cells in 50% Matrigel (BD
Biosciences). When the tumors reached a volume of 120 to 290
mm.sup.3, the tumors were staged to ensure uniformity of the tumor
mass among various treatment groups. The OVCAR3 model was dosed
intravenously Q4dx4 with PBS vehicle, humanized anti-Notch3 ADC
hu28-vc0101, and control ADC huNeg-8.8-vc0101; and dosed
intraperitoneally Q7dx4 with paclitaxel (30 mg/kg twice a day) and
Q7dx8 with carboplatin at the doses provided in Table 28. FIG. 15
and Table 28 demonstrate that hu28-vc0101 inhibited growth of
OVCAR3 ovarian xenografts in a dose-dependent manner. Further, the
data shows that hu28-vc0101 inhibited tumor growth more potently
than control huNeg8.8-vc0101 and carboplatin standard of care
chemotherapy. Furthermore, the data demonstrates that hu28-vc0101
dosed at 3 mg/kg Q4dx4 had similar potency to paclitaxel dosed at
60 mg/kg, Q7dx4 which is the maximum tolerated dose of paclitaxel
in SHO mice.
TABLE-US-00028 TABLE 28 Efficacy of anti-Notch3 ADC hu28-vc0101 in
OVCAR3 ovarian xenografts. OVCAR3 Ovarian Xenografts, tumor volume
(mm3 .+-. S,E.M.) PBS hu28-vc0101 huNeg8.8-vc0101 Carboplatin
Paclitaxel Dose mg/kg 0 0.3 1 3 1 3 20 60 DAY 0 170 .+-. 168 .+-.
169 .+-. 169 .+-. 171 .+-. 168 .+-. 167 .+-. 169 .+-. 14 18 20 18
21 14 13 13 DAY 3 255 .+-. 291 .+-. 255 .+-. 253 .+-. 261 .+-. 286
.+-. 242 .+-. 225 .+-. 18 29 31 35 36 18 20 29 DAY 6 277 .+-. 302
.+-. 226 .+-. 180 .+-. 267 .+-. 287 .+-. 246 .+-. 139 .+-. 25 35 33
19 38 30 17 13 DAY 11 378 .+-. 394 .+-. 216 .+-. 113 .+-. 369 .+-.
329 .+-. 311 .+-. 85 .+-. 18 40 35 9 68 27 25 8 DAY 14 501 .+-. 510
.+-. 242 .+-. 104 .+-. 467 .+-. 437 .+-. 426 .+-. 84 .+-. 33 50 39
9 75 23 27 7 DAY 18 578 .+-. 601 .+-. 212 .+-. 72 .+-. 516 .+-. 431
.+-. 473 .+-. 61 .+-. 43 57 38 9 101 26 29 5 DAY 21 693 .+-. 685
.+-. 190 .+-. 57 .+-. 587 .+-. 407 .+-. 475 .+-. 50 .+-. 48 83 38 6
123 26 26 4 DAY 25 806 .+-. 767 .+-. 216 .+-. 49 .+-. 638 .+-. 447
.+-. 552 .+-. 41 .+-. 59 84 52 5 131 27 25 2 DAY 28 912 .+-. 841
.+-. 282 .+-. 47 .+-. 772 .+-. 518 .+-. 640 .+-. 44 .+-. 71 112 69
6 171 35 26 4 DAY 31 970 .+-. 926 .+-. 292 .+-. 48 .+-. 854 .+-.
564 .+-. 692 .+-. 43 .+-. 68 118 74 4 176 48 28 3 DAY 34 1105 .+-.
1038 .+-. 350 .+-. 48 .+-. 1002 .+-. 665 .+-. 811 .+-. 50 .+-. 83
148 95 6 216 41 44 3 DAY 38 1166 .+-. 1191 .+-. 497 .+-. 49 .+-.
903 .+-. 751 .+-. 869 .+-. 55 .+-. 83 156 124 4 88 56 44 6 DAY 41
1285 .+-. 1254 .+-. 559 .+-. 40 .+-. 979 .+-. 786 .+-. 906 .+-. 38
.+-. 89 188 137 6 95 56 44 3 DAY 45 1616 .+-. 1486 .+-. 750 .+-. 39
.+-. 1109 .+-. 904 .+-. 1009 .+-. 39 .+-. 126 203 160 3 115 67 62 3
DAY 49 1836 .+-. 1479 .+-. 781 .+-. 33 .+-. 1289 .+-. 994 .+-. 1149
.+-. 40 .+-. 142 236 178 4 129 95 52 4 DAY 53 -- -- 934 .+-. 34
.+-. 1486 .+-. 1127 .+-. 1290 .+-. 40 .+-. 200 3 138 129 65 5 DAY
56 -- -- 991 .+-. 30 .+-. 1481 .+-. 1224 .+-. 1370 .+-. 39 .+-. 201
2 131 141 70 4 DAY 59 -- -- 1149 .+-. 33 .+-. 1739 .+-. 1325 .+-.
1514 .+-. 37 .+-. 228 4 182 163 81 3 DAY 63 -- -- 1421 .+-. 35 .+-.
-- 1510 .+-. 1783 .+-. 42 .+-. 328 3 258 128 4 DAY 67 -- -- -- 32
.+-. -- -- 1844 .+-. 33 .+-. 3 112 2 DAY 70 -- -- -- 33 .+-. -- --
-- 28 .+-. 5 2 DAY 74 -- -- -- 41 .+-. -- -- -- 32 .+-. 8 2 DAY 77
-- -- -- 50 .+-. -- -- -- 31 .+-. 10 3 DAY 80 -- -- -- 56 .+-. --
-- -- 31 .+-. 15 2 DAY 83 -- -- -- 63 .+-. -- -- -- 38 .+-. 16 4
DAY 87 -- -- -- 87 .+-. -- -- -- 28 .+-. 26 1 DAY 90 -- -- -- 84
.+-. -- -- -- 32 .+-. 26 2 DAY 94 -- -- -- 137 .+-. -- -- -- 30
.+-. 49 2 DAY 97 -- -- -- 129 .+-. -- -- -- 31 .+-. 47 3 DAY 102 --
-- -- 153 .+-. -- -- -- 26 .+-. 55 2 DAY 105 -- -- -- 176 .+-. --
-- -- 31 .+-. 66 2 DAY 108 -- -- -- 232 .+-. -- -- -- 28 .+-. 93 3
DAY 111 -- -- -- 271 .+-. -- -- -- 26 .+-. 102 2 DAY 115 -- -- --
323 .+-. -- -- -- 31 .+-. 134 3 DAY 118 -- -- -- 318 .+-. -- -- --
26 .+-. 137 4 DAY 122 -- -- -- 399 .+-. -- -- -- 31 .+-. 176 4
Example 17
Cysteine Mutant Generation for Site-Specific Conjugation
[0261] Anti-Notch3 ADCs described in the previous Examples were
generated through conventional, non-specific conjugation of
linker-payloads to cysteine amino acid residues of the target
antibody. Site-specific conjugation of linker-payloads to
antibodies was conducted to facilitate homogeneous drug loading and
avoid ADC subpopulations with potentially altered antigen-binding
or pharmacokinetics properties, which may be observed in some ADCs
generated by conventional conjugation methods. One such
site-specific conjugation strategy has been designed to introduce
cysteine residues at specific sites in the amino acid sequence of
the target antibody. A number of amino acid positions in the
constant heavy chain and constant light chain were identified, as
described in International Publication No. WO/2013/093809, and
substituted with a cysteine residue at the specific amino acid
position in the hu28 antibody.
[0262] Site-specific cysteine substitutions were engineered into
the Fc polypeptide or human IgG1 heavy chain constant domain (Cy)
at positions L443 and K392 (herein L443C and K392C). Site-specific
cysteine substitutions were engineered into the human kappa
(.kappa.) light chain constant domain (CK) polypeptide at position
K183 (herein .kappa.K183C). CDRs of cysteine mutants remain the
same as parental wildtype hu28. FIGS. 17A and 17B provide the amino
acid and nucleotide sequences of the hu28 antibody with the
site-specific cysteine substitutions described. Cysteine
substitutions in the hu28 antibody resulted in the generation of
the following antibodies: hu28-(L443C), hu28-(L443C/K392C) and
hu28-(L443C/.kappa.K183C). All cysteine mutations were constructed
by site-directed mutagenesis, as described in International
Publication No. WO/2013/093809. Expression constructs for these
mutant antibodies were generated in the site-specific integration
(SSI) expression vector (Lonza) for stable expression in CHO cells.
Stable pools of transfected CHO cells expressing the cysteine
mutant antibodies were established and conditioned media generated.
Standard Protein A affinity purification followed by size exclusion
chromatography were employed to purify the anti-Notch3 cysteine
mutant antibodies from conditioned media, as described in previous
Examples.
Example 18
Characterization of Cysteine Mutants
[0263] Unconjugated cysteine mutant humanized anti-Notch3
antibodies were screened for cell surface binding activity to
Notch3 expressing cell lines in a cell-based ELISA, as described in
previous Examples. Table 29 shows EC.sub.50 (nM) values calculated
from cell surface Notch3 binding ELISAs for unconjugated cysteine
mutants, hu28-(L443C), hu28-(L443/K382) and
hu28-(L443/.kappa.K183C). The data demonstrates that the cysteine
mutant antibodies have EC.sub.50 values that are similar to the
parental wildtype hu28 antibody in binding to full-length human
Notch3 expressed on the cell surface of U2OS/hNotch3, HCC2429 and
OVCAR3 cells. Further, the data demonstrates that substitution of
cysteine amino acids into the constant regions of the heavy chain
or both the heavy and light chains of the humanized hu28 antibody
did not affect or alter their binding characteristics. The negative
control huNeg8.8 antibody did not bind any of the cell lines
tested. For the control antibody that lacked binding (LB),
EC.sub.50 values were not generated as indicated.
TABLE-US-00029 TABLE 29 EC.sub.50 (nM) values for unconjugated
cysteine mutant anti-Notch3 antibodies EC.sub.50 (nM Ab/ml)
Antibody U2OS/hNotch3 HCC2429 OVCAR3 hu28 VH1.0/VL1.0 0.3 0.26 0.10
0.46 hu28-(L443C) 0.28 0.30 0.08 0.20 hu28-(L443C/.kappa.K183C)
0.38 0.27 0.11 0.20 hu28-(L443C/K392C) 0.31 0.29 0.11 0.31 huNeg8.8
LB -- LB LB
Example 19
In Vitro Cytotoxicity Assay of Cysteine Mutant ADCs
[0264] The conjugation of maleimide functionalized linker-payloads
to the anti-Notch3 hu28 cysteine mutant antibodies was achieved via
complete reduction of the antibodies with 100-fold molar excess of
tris(2-carboxyethyl)phosphine (TCEP) in 100 mM HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer), pH 7.0
and 1 mM diethylenetriaminepentaacetic acid (DTPA) for 2 hours at
37.degree. C. followed by desalting to remove excess TCEP. The
reduced antibodies were incubated in 2 mM dehydro ascorbic acid
(DHA), 100 mM HEPES, pH 7.0 and 1 mM DTPA for 16 hours at 4.degree.
C. to reform the inter-chain disulfide bonds. After desalting, the
vc0101 linker-payload was added to the reaction mixture at a
linker-payload/antibody molar ratio of about 7 and reacted for an
additional 1 hour at 25.degree. C. in the presence of 15% v/v of
dimethylacetamide (DMA). After the 1 hour incubation period, 6-fold
excess L-Cys was added to quench any unreacted linker-payload. The
reaction mixture was dialyzed overnight at 4.degree. C. in
phosphate buffered saline (PBS), pH 7.4, and purified via SEC (AKTA
explorer, Superdex 200). The ADC was further characterized via SEC
for purity, hydrophobic interaction chromatography (HIC), and
liquid chromatography electrospray ionization tandem mass
spectrometry (LC-ESI MS) to calculate drug-antibody ratio
(loading). The protein concentration was determined via UV
spectrophotometer. Additional conjugation techniques are described
in International Publication No. WO/2013/093809.
[0265] This process generated the anti-Notch3 cysteine mutant ADCs:
hu28-(L443C)-vc0101 and hu28-(L443C/.kappa.K183C)-vc0101. The
activity of the anti-Notch3 cysteine mutant ADCs were assessed on
cell lines endogenously expressing Notch3 protein: HCC2429 (lung
cancer) and OVCAR3 (ovarian cancer) using an MTS cellular viability
indicator as described in previous Examples.
[0266] Table 30 shows IC.sub.50 (ng Ab/mL) values of the
anti-Notch3 cysteine mutant ADC treatments. The data demonstrate
that hu28-(L443C/.kappa.K183C)-vc0101 with a DAR=3.9 was active and
had similar potency to the wildtype hu28-vc0101 ADC with DAR=3.9,
and both induced cell death in the Notch3 expressing cancer cell
lines HCC2429 and OVCAR3. The data further demonstrates that the
single cysteine mutant hu28-(L443)-vc0101 with a DAR=2 and the
non-targeted control huNeg8.8 ADC with DAR=4 were minimally active
at the highest doses tested.
TABLE-US-00030 TABLE 30 IC.sub.50 (ng Ab/mL) values of cysteine
mutant humanized anti-Notch3 ADCs. IC.sub.50 (ng Ab/ml) ADC DAR
HCC2429 OVCAR3 hu28-vc0101 3.9 124 463 145 404 1450 1173
hu28-(L443C)-vc0101 2 20308 16879 14324 13129 15840 12189
hu28-(L443C/ 3.9 219 352 10 3020 3673 1250 .kappa.K183C)-vc0101
huNeg8.8-vc0101 4.0 18850 13944 18302 9944 9826 7564
Example 20
In Vivo Efficacy of Cysteine Mutant ADCs
[0267] Similar in vivo experiments were performed with the
anti-Notch3 cysteine mutant ADCs using the OVCAR3 ovarian cell line
as described in previous Examples. The OVCAR3 model was dosed
intravenously Q4dx4 with PBS vehicle, hu28-vc0101,
anti-hu28-(L443C)-vc0101, hu28-(L443C/.kappa.K183C)-vc0101, and
control huNeg-8.8 ADC at the doses provided in Table 31. The data
demonstrates that the double cysteine mutant
hu28-(L443C/.kappa.K183C)-vc0101 inhibited growth of OVCAR3 ovarian
xenografts with activity similar to wildtype hu28-vc0101 at the 3
mg/kg dose, but less at the 1 mg/kg dose. The data further
demonstrates that the single cysteine mutant hu28-(L443C)-vc0101
with DAR=2 was efficacious in vivo, but was less active than the
wildtype hu28-vc0101 and hu28-(L443C/.kappa.K183C)-vc0101 at both
the 3 1 mg/kg and 3 mg/kg doses. The data indicates that
anti-Notch3 cysteine mutant ADCs generated by site-specific
conjugation are efficacious in vivo and inhibit tumor growth.
TABLE-US-00031 TABLE 31 In vivo efficacy of anti-Notch3 cysteine
mutant ADCs in OVCAR3 ovarian xenograft OVCAR3 Ovarian Xenografts,
tumor volume (mm3 .+-. S,E.M.) hu28- hu28-(L443C)-
(L443C/.kappa.K183C)- PBS hu28-vc0101 vc0101 vc0101 huNeg8.8-vc0101
0 1 3 1 3 1 3 1 3 DAY -2 161 .+-. 160 .+-. 160 .+-. 160 .+-. 160
.+-. 160 .+-. 159 .+-. 160 .+-. 160 .+-. 10 13 10 10 7 11 12 11 10
DAY 2 248 .+-. 249 .+-. 246 .+-. 235 .+-. 244 .+-. 247 .+-. 247
.+-. 245 .+-. 265 .+-. 27 28 21 17 16 31 24 34 18 DAY 6 312 .+-.
257 .+-. 219 .+-. 295 .+-. 259 .+-. 281 .+-. 223 .+-. 323 .+-. 325
.+-. 32 30 19 23 17 39 24 43 31 DAY 9 404 .+-. 282 .+-. 192 .+-.
346 .+-. 240 .+-. 322 .+-. 169 .+-. 394 .+-. 386 .+-. 42 29 13 26
20 35 25 51 26 DAY 12 477 .+-. 257 .+-. 133 .+-. 359 .+-. 198 .+-.
325 .+-. 133 .+-. 454 .+-. 400 .+-. 61 28 10 35 23 41 21 60 36 DAY
15 590 .+-. 263 .+-. 99 .+-. 349 .+-. 142 .+-. 331 .+-. 98 .+-. 524
.+-. 433 .+-. 71 24 9 49 15 51 11 72 43 DAY 19 655 .+-. 236 .+-. 73
.+-. 383 .+-. 115 .+-. 319 .+-. 76 .+-. 585 .+-. 466 .+-. 95 34 5
63 13 62 5 89 42 DAY 22 742 .+-. 251 .+-. 63 .+-. 427 .+-. 94 .+-.
363 .+-. 68 .+-. 666 .+-. 452 .+-. 99 40 5 77 9 89 5 101 45 DAY 26
831 .+-. 303 .+-. 63 .+-. 519 .+-. 85 .+-. 473 .+-. 63 .+-. 780
.+-. 497 .+-. 135 49 5 101 12 123 4 108 70 DAY 29 1008 .+-. 396
.+-. 56 .+-. 624 .+-. 97 .+-. 583 .+-. 54 .+-. 834 .+-. 555 .+-.
171 69 4 118 14 167 3 124 80 DAY 33 1168 .+-. 555 .+-. 58 .+-. 789
.+-. 114 .+-. 691 .+-. 58 .+-. 1007 .+-. 695 .+-. 201 96 4 138 28
194 4 155 88 DAY 36 1207 .+-. 683 .+-. 56 .+-. 942 .+-. 146 .+-.
872 .+-. 48 .+-. 1152 .+-. 782 .+-. 81 125 6 164 39 246 2 155 93
DAY 40 1330 .+-. 811 .+-. 58 .+-. 1046 .+-. 205 .+-. 1128 .+-. 46
.+-. 1328 .+-. 872 .+-. 91 144 8 191 56 311 3 220 131 DAY 42 1363
.+-. 850 .+-. 55 .+-. 1130 .+-. 246 .+-. 955 .+-. 51 .+-. 1307 .+-.
910 .+-. 111 157 10 190 71 247 5 200 130 DAY 47 1656 .+-. 1121 .+-.
76 .+-. 1493 .+-. 378 .+-. 1140 .+-. 44 .+-. 1470 .+-. 1032 .+-. 98
197 23 243 103 255 2 223 116 DAY 50 1661 .+-. 1107 .+-. 74 .+-. --
452 .+-. 1152 .+-. 40 .+-. -- 1101 .+-. 95 204 25 131 248 2 110 DAY
54 2005 .+-. 1322 .+-. 102 .+-. -- 604 .+-. 1463 .+-. 48 .+-. --
1272 .+-. 140 228 40 184 317 4 161 DAY 57 -- 1286 .+-. 117 .+-. --
826 .+-. -- 43 .+-. -- 1270 .+-. 235 51 264 4 120 DAY 61 -- -- 152
.+-. -- 820 .+-. -- 63 .+-. -- 1610 .+-. 69 248 10 160 DAY 64 -- --
182 .+-. -- -- -- 55 .+-. -- -- 88 9 DAY 68 -- -- 217 .+-. -- -- --
67 .+-. -- -- 102 15 DAY 71 -- -- 242 .+-. -- -- -- 73 .+-. -- --
120 20 DAY 75 -- -- 286 .+-. -- -- -- 84 .+-. -- -- 146 24 DAY 78
-- -- -- -- -- -- 107 .+-. -- -- 32 DAY 82 -- -- -- -- -- -- 99
.+-. -- -- 36 DAY 85 -- -- -- -- -- -- 119 .+-. -- -- 50 DAY 89 --
-- -- -- -- -- 173 .+-. -- -- 63 DAY 99 -- -- -- -- -- -- 328 .+-.
-- -- 130 DAY 111 -- -- -- -- -- -- 525 .+-. -- -- 203
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 68 <210> SEQ ID NO 1 <211> LENGTH: 296 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 1 Ala Pro Glu Val Ser Glu Glu Pro Arg Cys Pro
Arg Ala Ala Cys Gln 1 5 10 15 Ala Lys Arg Gly Asp Gln Arg Cys Asp
Arg Glu Cys Asn Ser Pro Gly 20 25 30 Cys Gly Trp Asp Gly Gly Asp
Cys Ser Leu Ser Val Gly Asp Pro Trp 35 40 45 Arg Gln Cys Glu Ala
Leu Gln Cys Trp Arg Leu Phe Asn Asn Ser Arg 50 55 60 Cys Asp Pro
Ala Cys Ser Ser Pro Ala Cys Leu Tyr Asp Asn Phe Asp 65 70 75 80 Cys
His Ala Gly Gly Arg Glu Arg Thr Cys Asn Pro Val Tyr Glu Lys 85 90
95 Tyr Cys Ala Asp His Phe Ala Asp Gly Arg Cys Asp Gln Gly Cys Asn
100 105 110 Thr Glu Glu Cys Gly Trp Asp Gly Leu Asp Cys Ala Ser Glu
Val Pro 115 120 125 Ala Leu Leu Ala Arg Gly Val Leu Val Leu Thr Val
Leu Leu Pro Pro 130 135 140 Glu Glu Leu Leu Arg Ser Ser Ala Asp Phe
Leu Gln Arg Leu Ser Ala 145 150 155 160 Ile Leu Arg Thr Ser Leu Arg
Phe Arg Leu Asp Ala His Gly Gln Ala 165 170 175 Met Val Phe Pro Tyr
His Arg Pro Ser Pro Gly Ser Glu Pro Arg Ala 180 185 190 Arg Arg Glu
Leu Ala Pro Glu Val Ile Gly Ser Val Val Met Leu Glu 195 200 205 Ile
Asp Asn Arg Leu Cys Leu Gln Ser Pro Glu Asn Asp His Cys Phe 210 215
220 Pro Asp Ala Gln Ser Ala Ala Asp Tyr Leu Gly Ala Leu Ser Ala Val
225 230 235 240 Glu Arg Leu Asp Phe Pro Tyr Pro Leu Arg Asp Val Arg
Gly Glu Pro 245 250 255 Leu Glu Pro Pro Glu Pro Ser Val Pro Gly Gly
Gly Ser Gly Gly Gly 260 265 270 Leu Asn Asp Ile Phe Glu Ala Gln Lys
Ile Glu Trp His Glu Gly Gly 275 280 285 Pro Pro His His His His His
His 290 295 <210> SEQ ID NO 2 <211> LENGTH: 888
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 2 gcacccgagg tctcggagga
gccgcggtgc ccgcgcgccg cctgccaggc caagcgcggg 60 gaccagcgct
gcgaccgcga gtgcaacagc ccaggctgcg gctgggacgg cggcgactgc 120
tcgctgagcg tgggcgaccc ctggcggcaa tgcgaggcgc tgcagtgctg gcgcctcttc
180 aacaacagcc gctgcgaccc cgcctgcagc tcgcccgcct gcctctacga
caacttcgac 240 tgccacgccg gtggccgcga gcgcacttgc aacccggtgt
acgagaagta ctgcgccgac 300 cactttgccg acggccgctg cgaccagggc
tgcaacacgg aggagtgcgg ctgggatggg 360 ctggattgtg ccagcgaggt
gccggccctg ctggcccgcg gcgtgctggt gctcacagtg 420 ctgctgccgc
cggaggagct actgcgttcc agcgccgact ttctgcagcg gctcagcgcc 480
atcctgcgca cctcgctgcg cttccgcctg gacgcgcacg gccaggccat ggtcttccct
540 taccaccggc ctagtcctgg ctccgaaccc cgggcccgtc gggagctggc
ccccgaggtg 600 atcggctcgg tagtaatgct ggagattgac aaccggctct
gcctgcagtc gcctgagaat 660 gatcactgct tccccgatgc ccagagcgcc
gctgactacc tgggagcgtt gtcagcggtg 720 gagcgcctgg acttcccgta
cccactgcgg gacgtgcggg gggagccgct ggagcctcca 780 gaacccagcg
tcccgggagg gggaagcgga ggcggactga acgacatctt cgaggctcag 840
aaaatcgaat ggcacgaagg tggcccacca catcatcatc atcatcac 888
<210> SEQ ID NO 3 <211> LENGTH: 296 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 3 Ala Pro Glu Val Pro Glu Glu Pro Arg Cys Pro
Arg Ala Ala Cys Gln 1 5 10 15 Ala Lys Arg Gly Asp Gln Asn Cys Asp
Arg Glu Cys Asn Thr Pro Gly 20 25 30 Cys Gly Trp Asp Gly Gly Asp
Cys Ser Leu Asn Val Asp Asp Pro Trp 35 40 45 Arg Gln Cys Glu Ala
Leu Gln Cys Trp Arg Leu Phe Asn Asn Ser Arg 50 55 60 Cys Asp Pro
Ala Cys Ser Ser Pro Ala Cys Leu Tyr Asp Asn Phe Asp 65 70 75 80 Cys
Tyr Ser Gly Gly Arg Asp Arg Thr Cys Asn Pro Val Tyr Glu Lys 85 90
95 Tyr Cys Ala Asp His Phe Ala Asp Gly Arg Cys Asp Gln Gly Cys Asn
100 105 110 Thr Glu Glu Cys Gly Trp Asp Gly Leu Asp Cys Ala Ser Glu
Val Pro 115 120 125 Ala Leu Leu Ala Arg Gly Val Leu Val Leu Thr Val
Leu Leu Pro Pro 130 135 140 Glu Glu Leu Leu Arg Ser Ser Ala Asp Phe
Leu Gln Arg Leu Ser Ala 145 150 155 160 Ile Leu Arg Thr Ser Leu Arg
Phe Arg Leu Asp Ala Arg Gly Gln Ala 165 170 175 Met Val Phe Pro Tyr
His Arg Pro Ser Pro Gly Ser Glu Ser Arg Val 180 185 190 Arg Arg Glu
Leu Gly Pro Glu Val Ile Gly Ser Val Val Met Leu Glu 195 200 205 Ile
Asp Asn Arg Leu Cys Leu Gln Ser Ala Glu Asn Asp His Cys Phe 210 215
220 Pro Asp Ala Gln Ser Ala Ala Asp Tyr Leu Gly Ala Leu Ser Ala Val
225 230 235 240 Glu Arg Leu Asp Phe Pro Tyr Pro Leu Arg Asp Val Arg
Gly Glu Pro 245 250 255 Leu Glu Ala Pro Glu Gln Ser Val Pro Gly Gly
Gly Ser Gly Gly Gly 260 265 270 Leu Asn Asp Ile Phe Glu Ala Gln Lys
Ile Glu Trp His Glu Gly Gly 275 280 285 Pro Pro His His His His His
His 290 295 <210> SEQ ID NO 4 <211> LENGTH: 888
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 4 gcccctgagg tccccgagga
gccacggtgc ccgcgagcgg cttgccaggc caagcgaggg 60 gaccagaact
gcgatcgtga gtgcaacacc ccaggctgtg gctgggatgg cggtgactgc 120
tcactgaacg tggacgaccc ctggaggcag tgtgaggcac tgcagtgctg gcgtctcttc
180 aacaacagcc ggtgtgaccc ggcctgcagc tctccagcct gcctctatga
caactttgac 240 tgctactctg gtggccgcga ccgcacctgc aaccctgttt
atgagaagta ctgcgccgac 300 cactttgcag atggccgttg tgaccagggc
tgcaacactg aggaatgcgg ctgggatggg 360 ctggactgtg ccagcgaggt
cccggccctt ttggcccgag gggttctggt cctcacagtt 420 cttctgcctc
ctgaagagtt gctgcgctcc agtgccgact ttctgcagcg actcagcgct 480
attctgcgca cctcactgcg cttccgcttg gacgcacgtg gccaggccat ggtcttcccc
540 tatcaccggc caagccctgg ctctgaatcc cgggtccgtc gtgagctggg
tcctgaggtg 600 atcggctctg tggtgatgct ggagattgac aaccggctct
gtctgcagtc agctgagaat 660 gaccactgct tccctgatgc ccagagtgct
gctgactacc tgggagcctt gtcagcagtg 720 gagcgacttg atttcccata
cccacttcgg gatgtgcgag gagagccgct ggaggcccca 780 gagcagagcg
tgccaggagg gggaagcgga ggcggactga acgacatctt cgaggctcag 840
aaaatcgaat ggcacgaagg tggcccacca catcatcatc atcatcac 888
<210> SEQ ID NO 5 <211> LENGTH: 117 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Thr Leu Ser Cys Val Ala Ser
Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Gly Met Thr Trp Ile Arg Gln
Ala Pro Gly Lys Gly Leu Thr Trp Val 35 40 45 Ala Tyr Ile Ser Ser
Gly Ser Asn Tyr Ile Tyr Tyr Ala Glu Ala Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Phe Cys 85 90
95 Thr Arg Arg Gly Pro Phe Val Leu Asp Ala Trp Gly Gln Gly Ala Ser
100 105 110 Val Thr Val Ser Ser 115 <210> SEQ ID NO 6
<211> LENGTH: 351 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic nucleotide sequence <400> SEQUENCE: 6
gaggtgcaac tggtggagtc tggaggaggc ttagtgcagc ctggaaggtc cctgacactc
60 tcctgtgtag cctctggatt cactttcagg gactatggaa tgacctggat
tcgccaggct 120 ccagggaagg ggctgacatg ggttgcatat attagtagtg
gtagcaatta catctattat 180 gcagaagcgg tgaagggccg attcaccatc
tccagagaca atgccaagaa caccctgtac 240 ctgcaaatga ccagtctgag
gtctgaagac actgccttgt atttttgtac aagacgaggc 300 ccgtttgttt
tggatgcctg gggtcaagga gcttcagtca ctgtctcctc a 351 <210> SEQ
ID NO 7 <211> LENGTH: 107 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic peptide sequence <400> SEQUENCE:
7 Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Asn Cys Lys Ala Ser Gln Ser Ile Asn Arg
Tyr 20 25 30 Leu His Trp Phe Gln Gln Lys Leu Gly Glu Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Asn Ala Asn Gly Leu Gln Thr Gly Ile Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe
Cys Leu Gln His Asn Thr Trp Pro Asp 85 90 95 Thr Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys 100 105 <210> SEQ ID NO 8 <211>
LENGTH: 320 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleotide sequence <400> SEQUENCE: 8 gacatccaga
tgacccagtc tccttcattc ctgtctgcat ctgtgggaga cagagtcact 60
atcaactgca aagcaagtca gagtattaac aggtacttac actggtttca gcagaaactt
120 ggagaagctc ccaaactcct gatatataat gcaaacggtt tgcaaacggg
catcccatca 180 aggttcagtg gcagtggatc tggtactgat ttcacactta
ccatcagcag cctgcagtct 240 gaagatgttg ccacatattt ctgcttgcag
cataatacgt ggccggacac gtttggcgct 300 gggaccaagc tggaactgaa 320
<210> SEQ ID NO 9 <211> LENGTH: 119 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 9 Gln Val Lys Leu Leu Gln Ser Gly Ala Ala Leu
Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser
Gly Tyr Ala Phe Thr Asp Tyr 20 25 30 Trp Val Thr Trp Val Lys Gln
Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Glu Ile Ser Pro
Asn Ser Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Gly Lys
Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90
95 Thr Arg Gly Glu Ile Arg Tyr Asn Trp Phe Ala Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID NO
10 <211> LENGTH: 357 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleotide sequence <400>
SEQUENCE: 10 caggtcaagc tgctgcagtc tggggctgca ctggtgaagc ctggagcctc
tgtgaagatg 60 tcttgcaaag cttctggtta tgcattcact gactactggg
tgacctgggt gaagcagagt 120 catggaaaga gccttgagtg gattggggaa
atttctccta acagtggtgg tactaacttc 180 aatgaaaagt tcaagggcaa
ggccacattg actgtagaca aatccaccag cacagcctat 240 atggagctca
gcagattgac atctgaggac tctgcaatct attactgtac aagaggggaa 300
atccgttaca attggtttgc ttactggggc caaggcactc tggtcactgt ctcctca 357
<210> SEQ ID NO 11 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 11 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Asn Val Gly Asn Asn 20 25 30 Ile Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser
Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Leu Tyr Asn Ser Pro Phe 85
90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
<210> SEQ ID NO 12 <211> LENGTH: 321 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 12 aacattgtga tgacccagtc tcccaaatcc
atgtccatat cagtaggaga cagggtcacc 60 atgaactgca aggccagtca
gaatgtgggt aataatatag cctggtatca acagaaacca 120 gggcagtctc
ctaaactgtt gatctactat gcatctaacc ggtacactgg ggtccctgat 180
cgcttcacag gcggtggata tgggacagat ttcactctca ccatcaatag tatgcaagct
240 gaagatgcag ccttttatta ctgtcagcgt ctttacaatt ctccattcac
gttcggctca 300 gggacgaagt tggaaataaa g 321 <210> SEQ ID NO 13
<211> LENGTH: 117 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic peptide sequence <400> SEQUENCE: 13
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp
Tyr 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Asn Tyr Ile Tyr
Tyr Ala Glu Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Pro
Phe Val Leu Asp Ala Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 <210> SEQ ID NO 14 <211> LENGTH: 351
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 14 gaggtgcagc tggtggagtc
tgggggaggc ttggtccagc ctggggggtc cctgagactc 60 tcctgtgcag
cctctggatt cactttcagg gactatggaa tgacctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtggcctat attagtagtg gtagcaatta catctattat
180 gcagaagcgg tgaagggccg attcaccatc tccagagaca acgccaagaa
ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gagacgaggc 300 ccgtttgttt tggatgcctg gggccaggga
accctggtca ccgtctcctc a 351 <210> SEQ ID NO 15 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide sequence <400> SEQUENCE: 15 Asp Tyr Gly Met
Thr 1 5 <210> SEQ ID NO 16 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 16 Gly Phe Thr Phe Arg Asp Tyr 1 5
<210> SEQ ID NO 17 <211> LENGTH: 15 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 17 gactatggaa tgacc 15 <210> SEQ ID NO
18 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleotide sequence <400>
SEQUENCE: 18 ggattcactt tcagggacta t 21 <210> SEQ ID NO 19
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic peptide sequence <400> SEQUENCE: 19
Tyr Ile Ser Ser Gly Ser Asn Tyr Ile Tyr Tyr Ala Glu Ala Val Lys 1 5
10 15 Gly <210> SEQ ID NO 20 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 20 Ser Ser Gly Ser Asn Tyr 1
5 <210> SEQ ID NO 21 <211> LENGTH: 51 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 21 tatattagta gtggtagcaa ttacatctat
tatgcagaag cggtgaaggg c 51 <210> SEQ ID NO 22 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleotide sequence <400> SEQUENCE: 22 agtagtggta
gcaattac 18 <210> SEQ ID NO 23 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 23 Arg Gly Pro Phe Val Leu
Asp Ala 1 5 <210> SEQ ID NO 24 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 24 cgaggcccgt ttgttttgga
tgcc 24 <210> SEQ ID NO 25 <211> LENGTH: 107
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 25 Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Ser Ile Asn Arg Tyr 20 25 30 Leu His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Asn Ala Asn Gly Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Thr
Trp Pro Asp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 <210> SEQ ID NO 26 <211> LENGTH: 321
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 26 gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgca
aagcaagtca gagtattaac aggtacttac actggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctataat gcaaacggtt tgcaaacggg ggtcccatca
180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240 gaagattttg caacttacta ctgtttgcag cataatacgt
ggccggacac gtttggcgga 300 gggaccaagg tggagatcaa a 321 <210>
SEQ ID NO 27 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 27 Lys Ala Ser Gln Ser Ile Asn Arg Tyr Leu
His 1 5 10 <210> SEQ ID NO 28 <211> LENGTH: 33
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 28 aaagcaagtc agagtattaa
caggtactta cac 33 <210> SEQ ID NO 29 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 29 Asn Ala Asn Gly Leu Gln
Thr 1 5 <210> SEQ ID NO 30 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleotide
sequence <400> SEQUENCE: 30 aatgcaaacg gtttgcaaac g 21
<210> SEQ ID NO 31 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 31 Leu Gln His Asn Thr Trp Pro Asp Thr 1 5
<210> SEQ ID NO 32 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 32 ttgcagcata atacgtggcc ggacacgttt 30
<210> SEQ ID NO 33 <211> LENGTH: 446 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Gly Met Thr Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser
Ser Gly Ser Asn Tyr Ile Tyr Tyr Ala Glu Ala Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Arg Gly Pro Phe Val Leu Asp Ala Trp Gly Gln Gly Thr
Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210
215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330
335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210>
SEQ ID NO 34 <211> LENGTH: 1338 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 34 gaggtgcagc tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt
cactttcagg gactatggaa tgacctgggt ccgccaggct 120 ccagggaagg
ggctggagtg ggtggcctat attagtagtg gtagcaatta catctattat 180
gcagaagcgg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat
240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc
gagacgaggc 300 ccgtttgttt tggatgcctg gggccaggga accctggtca
ccgtctcctc agcgtcgacc 360 aagggcccat cggtcttccc cctggcaccc
tcctccaaga gcacctctgg gggcacagcg 420 gccctgggct gcctggtcaa
ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480 ggcgccctga
ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540
tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
600 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc
caaatcttgt 660 gacaaaactc acacatgccc accgtgccca gcacctgaac
tcctgggggg accgtcagtc 720 ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca 780 tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 840 ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag
960 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc
caaagccaaa 1020 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggagga gatgaccaag 1080 aaccaggtca gcctgacctg cctggtcaaa
ggcttctatc ccagcgacat cgccgtggag 1140 tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200 gacggctcct
tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1260
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc
1320 ctctccctgt ccccgggt 1338 <210> SEQ ID NO 35 <211>
LENGTH: 214 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide sequence <400> SEQUENCE: 35 Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile Asn Arg Tyr 20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Asn Ala Asn Gly Leu Gln Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His
Asn Thr Trp Pro Asp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> SEQ
ID NO 36 <211> LENGTH: 642 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleotide sequence <400>
SEQUENCE: 36 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
cagagtcacc 60 atcacttgca aagcaagtca gagtattaac aggtacttac
actggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctataat
gcaaacggtt tgcaaacggg ggtcccatca 180 aggttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg
caacttacta ctgtttgcag cataatacgt ggccggacac gtttggcgga 300
gggaccaagg tggagatcaa acggaccgtg gccgctcctt ccgtgttcat cttcccccct
360 tccgacgagc agctgaagtc tggcaccgcc tctgtggtgt gtctgctgaa
caacttctac 420 ccccgggagg ccaaggtgca gtggaaggtg gacaacgctc
tgcagtccgg caactcccag 480 gagtctgtga ccgagcagga ctccaaggac
agcacctact ccctgtcctc taccctgacc 540 ctgtccaagg ccgactacga
gaagcacaag gtgtacgcct gtgaggtgac ccaccagggc 600 ctgtcctctc
ctgtgaccaa gtccttcaac cggggcgagt gc 642 <210> SEQ ID NO 37
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic peptide sequence <400> SEQUENCE: 37
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ala Phe Thr Asp
Tyr 20 25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Glu Ile Ser Pro Asn Ser Gly Gly Thr Asn
Phe Asn Glu Lys Phe 50 55 60 Lys Gly Arg Phe Thr Ile Ser Val Asp
Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Glu Ile
Arg Tyr Asn Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 <210> SEQ ID NO 38 <211> LENGTH:
357 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 38 gaggtgcagc tggtggagtc
tgggggaggc ttggtccagc ctggggggtc cctgagactc 60 tcctgtgcag
cctctggtta tgcattcact gactactgga tgacctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtggccgaa atttctccta acagtggtgg tactaacttc
180 aatgaaaagt tcaagggccg attcaccatc tccgttgaca acgccaagaa
ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gagaggggaa 300 atccgttaca attggtttgc ttactggggc
cagggaaccc tggtcaccgt ctcctca 357 <210> SEQ ID NO 39
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic peptide sequence <400> SEQUENCE: 39
Asp Tyr Trp Met Thr 1 5 <210> SEQ ID NO 40 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide sequence <400> SEQUENCE: 40 Gly Tyr Ala Phe
Thr Asp Tyr 1 5 <210> SEQ ID NO 41 <211> LENGTH: 15
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 41 gactactgga tgacc 15
<210> SEQ ID NO 42 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 42 ggttatgcat tcactgacta c 21 <210> SEQ
ID NO 43 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic peptide sequence <400> SEQUENCE:
43 Glu Ile Ser Pro Asn Ser Gly Gly Thr Asn Phe Asn Glu Lys Phe Lys
1 5 10 15 Gly <210> SEQ ID NO 44 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 44 Ser Pro Asn Ser Gly Gly 1
5 <210> SEQ ID NO 45 <211> LENGTH: 51 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 45 gaaatttctc ctaacagtgg tggtactaac
ttcaatgaaa agttcaaggg c 51 <210> SEQ ID NO 46 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleotide sequence <400> SEQUENCE: 46 tctcctaaca
gtggtggt 18 <210> SEQ ID NO 47 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 47 Gly Glu Ile Arg Tyr Asn
Trp Phe Ala Tyr 1 5 10 <210> SEQ ID NO 48 <211> LENGTH:
30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 48 ggggaaatcc gttacaattg
gtttgcttac 30 <210> SEQ ID NO 49 <211> LENGTH: 107
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 49 Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn 20 25 30 Ile Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Leu Tyr Asn
Ser Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 <210> SEQ ID NO 50 <211> LENGTH: 321
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 50 gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgca
aggccagtca gaatgtgggt aataatatag cctggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctattat gcatctaacc ggtacactgg ggtcccatca
180 aggttcagtg gcagtggata tgggacagat ttcactctca ccatcagcag
tctgcaacct 240 gaagattttg caacttacta ctgtcagcgt ctttacaatt
ctccattcac gttcggcgga 300 gggaccaagg tggagatcaa a 321 <210>
SEQ ID NO 51 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 51 Lys Ala Ser Gln Asn Val Gly Asn Asn Ile
Ala 1 5 10 <210> SEQ ID NO 52 <211> LENGTH: 33
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 52 aaggccagtc agaatgtggg
taataatata gcc 33 <210> SEQ ID NO 53 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 53 Tyr Ala Ser Asn Arg Tyr
Thr 1 5 <210> SEQ ID NO 54 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleotide
sequence <400> SEQUENCE: 54 tatgcatcta accggtacac t 21
<210> SEQ ID NO 55 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 55 Gln Arg Leu Tyr Asn Ser Pro Phe Thr 1 5
<210> SEQ ID NO 56 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 56 cagcgtcttt acaattctcc attcacgttc 30
<210> SEQ ID NO 57 <211> LENGTH: 448 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 57 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Tyr Ala Phe Thr Asp Tyr 20 25 30 Trp Met Thr Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu Ile Ser
Pro Asn Ser Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Val Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Gly Glu Ile Arg Tyr Asn Trp Phe Ala Tyr Trp Gly Gln
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210
215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330
335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
<210> SEQ ID NO 58 <211> LENGTH: 1344 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 58 gaggtgcagc tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggtta
tgcattcact gactactgga tgacctgggt ccgccaggct 120 ccagggaagg
ggctggagtg ggtggccgaa atttctccta acagtggtgg tactaacttc 180
aatgaaaagt tcaagggccg attcaccatc tccgttgaca acgccaagaa ctcactgtat
240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc
gagaggggaa 300 atccgttaca attggtttgc ttactggggc cagggaaccc
tggtcaccgt ctcctcagcg 360 tcgaccaagg gcccatcggt cttccccctg
gcaccctcct ccaagagcac ctctgggggc 420 acagcggccc tgggctgcct
ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480 aactcaggcg
ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540
ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac
600 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt
tgagcccaaa 660 tcttgtgaca aaactcacac atgcccaccg tgcccagcac
ctgaactcct ggggggaccg 720 tcagtcttcc tcttcccccc aaaacccaag
gacaccctca tgatctcccg gacccctgag 780 gtcacatgcg tggtggtgga
cgtgagccac gaagaccctg aggtcaagtt caactggtac 840 gtggacggcg
tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag
960 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac
catctccaaa 1020 gccaaagggc agccccgaga accacaggtg tacaccctgc
ccccatcccg ggaggagatg 1080 accaagaacc aggtcagcct gacctgcctg
gtcaaaggct tctatcccag cgacatcgcc 1140 gtggagtggg agagcaatgg
gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200 gactccgacg
gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1260
caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag
1320 aagagcctct ccctgtcccc gggt 1344 <210> SEQ ID NO 59
<211> LENGTH: 214 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic peptide sequence <400> SEQUENCE: 59
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Asn
Asn 20 25 30 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Arg Leu Tyr Asn Ser Pro Phe 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
<210> SEQ ID NO 60 <211> LENGTH: 642 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 60 gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgca aggccagtca
gaatgtgggt aataatatag cctggtatca gcagaaacca 120 gggaaagccc
ctaagctcct gatctattat gcatctaacc ggtacactgg ggtcccatca 180
aggttcagtg gcagtggata tgggacagat ttcactctca ccatcagcag tctgcaacct
240 gaagattttg caacttacta ctgtcagcgt ctttacaatt ctccattcac
gttcggcgga 300 gggaccaagg tggagatcaa acggaccgtg gccgctcctt
ccgtgttcat cttcccccct 360 tccgacgagc agctgaagtc tggcaccgcc
tctgtggtgt gtctgctgaa caacttctac 420 ccccgggagg ccaaggtgca
gtggaaggtg gacaacgctc tgcagtccgg caactcccag 480 gagtctgtga
ccgagcagga ctccaaggac agcacctact ccctgtcctc taccctgacc 540
ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gtgaggtgac ccaccagggc
600 ctgtcctctc ctgtgaccaa gtccttcaac cggggcgagt gc 642 <210>
SEQ ID NO 61 <211> LENGTH: 446 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 61 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Gly Met Thr Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser
Ser Gly Ser Asn Tyr Ile Tyr Tyr Ala Glu Ala Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Arg Gly Pro Phe Val Leu Asp Ala Trp Gly Gln Gly Thr
Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210
215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330
335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His
Tyr Thr Gln Lys Ser Leu Ser Cys Ser Pro Gly 435 440 445 <210>
SEQ ID NO 62 <211> LENGTH: 1338 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 62 gaggtgcagc tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt
cactttcagg gactatggaa tgacctgggt ccgccaggct 120 ccagggaagg
ggctggagtg ggtggcctat attagtagtg gtagcaatta catctattat 180
gcagaagcgg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat
240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc
gagacgaggc 300 ccgtttgttt tggatgcctg gggccaggga accctggtca
ccgtctcctc agcgtcgacc 360 aagggcccat cggtcttccc cctggcaccc
tcctccaaga gcacctctgg gggcacagcg 420 gccctgggct gcctggtcaa
ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480 ggcgccctga
ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540
tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
600 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc
caaatcttgt 660 gacaaaactc acacatgccc accgtgccca gcacctgaac
tcctgggggg accgtcagtc 720 ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca 780 tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 840 ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag
960 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc
caaagccaaa 1020 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggagga gatgaccaag 1080 aaccaggtca gcctgacctg cctggtcaaa
ggcttctatc ccagcgacat cgccgtggag 1140 tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200 gacggctcct
tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1260
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc
1320 ctctcctgct ccccgggt 1338 <210> SEQ ID NO 63 <211>
LENGTH: 214 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide sequence <400> SEQUENCE: 63 Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile Asn Arg Tyr 20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Asn Ala Asn Gly Leu Gln Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His
Asn Thr Trp Pro Asp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175 Ser Thr Leu Thr Leu Ser Cys Ala Asp Tyr Glu Lys His Lys Val
Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> SEQ
ID NO 64 <211> LENGTH: 642 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleotide sequence <400>
SEQUENCE: 64 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
cagagtcacc 60 atcacttgca aagcaagtca gagtattaac aggtacttac
actggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctataat
gcaaacggtt tgcaaacggg ggtcccatca 180 aggttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg
caacttacta ctgtttgcag cataatacgt ggccggacac gtttggcgga 300
gggaccaagg tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca
360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa
taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac
agcacctaca gcctcagcag caccctgacg 540 ctgagctgcg cagactacga
gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc
ccgtcacaaa gagcttcaac aggggagagt gt 642 <210> SEQ ID NO 65
<211> LENGTH: 446 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic peptide sequence <400> SEQUENCE: 65
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp
Tyr 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Asn Tyr Ile Tyr
Tyr Ala Glu Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Pro
Phe Val Leu Asp Ala Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260
265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380
Gly Gln Pro Glu Asn Asn Tyr Cys Thr Thr Pro Pro Val Leu Asp Ser 385
390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser
Cys Ser Pro Gly 435 440 445 <210> SEQ ID NO 66 <211>
LENGTH: 1338 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleotide sequence <400> SEQUENCE: 66 gaggtgcagc
tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cactttcagg gactatggaa tgacctgggt ccgccaggct
120 ccagggaagg ggctggagtg ggtggcctat attagtagtg gtagcaatta
catctattat 180 gcagaagcgg tgaagggccg attcaccatc tccagagaca
acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagacgaggc 300 ccgtttgttt tggatgcctg
gggccaggga accctggtca ccgtctcctc agcgtcgacc 360 aagggcccat
cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420
gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca
480 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc
aggactctac 540 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg
gcacccagac ctacatctgc 600 aacgtgaatc acaagcccag caacaccaag
gtggacaaga aagttgagcc caaatcttgt 660 gacaaaactc acacatgccc
accgtgccca gcacctgaac tcctgggggg accgtcagtc 720 ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac
840 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa
cagcacgtac 900 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc
tgaatggcaa ggagtacaag 960 tgcaaggtct ccaacaaagc cctcccagcc
cccatcgaga aaaccatctc caaagccaaa 1020 gggcagcccc gagaaccaca
ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1080 aaccaggtca
gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140
tgggagagca atgggcagcc ggagaacaac tactgcacca cgcctcccgt gctggactcc
1200 gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg
gcagcagggg 1260 aacgtcttct catgctccgt gatgcatgag gctctgcaca
accactacac gcagaagagc 1320 ctctcctgct ccccgggt 1338 <210> SEQ
ID NO 67 <211> LENGTH: 113 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic peptide sequence <400> SEQUENCE:
67 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys
Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser
<210> SEQ ID NO 68 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 68 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85
90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 68 <210>
SEQ ID NO 1 <211> LENGTH: 296 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 1 Ala Pro Glu Val Ser Glu Glu Pro Arg Cys Pro
Arg Ala Ala Cys Gln 1 5 10 15 Ala Lys Arg Gly Asp Gln Arg Cys Asp
Arg Glu Cys Asn Ser Pro Gly 20 25 30 Cys Gly Trp Asp Gly Gly Asp
Cys Ser Leu Ser Val Gly Asp Pro Trp 35 40 45 Arg Gln Cys Glu Ala
Leu Gln Cys Trp Arg Leu Phe Asn Asn Ser Arg 50 55 60 Cys Asp Pro
Ala Cys Ser Ser Pro Ala Cys Leu Tyr Asp Asn Phe Asp 65 70 75 80 Cys
His Ala Gly Gly Arg Glu Arg Thr Cys Asn Pro Val Tyr Glu Lys 85 90
95 Tyr Cys Ala Asp His Phe Ala Asp Gly Arg Cys Asp Gln Gly Cys Asn
100 105 110 Thr Glu Glu Cys Gly Trp Asp Gly Leu Asp Cys Ala Ser Glu
Val Pro 115 120 125 Ala Leu Leu Ala Arg Gly Val Leu Val Leu Thr Val
Leu Leu Pro Pro 130 135 140 Glu Glu Leu Leu Arg Ser Ser Ala Asp Phe
Leu Gln Arg Leu Ser Ala 145 150 155 160 Ile Leu Arg Thr Ser Leu Arg
Phe Arg Leu Asp Ala His Gly Gln Ala 165 170 175 Met Val Phe Pro Tyr
His Arg Pro Ser Pro Gly Ser Glu Pro Arg Ala 180 185 190 Arg Arg Glu
Leu Ala Pro Glu Val Ile Gly Ser Val Val Met Leu Glu 195 200 205 Ile
Asp Asn Arg Leu Cys Leu Gln Ser Pro Glu Asn Asp His Cys Phe 210 215
220 Pro Asp Ala Gln Ser Ala Ala Asp Tyr Leu Gly Ala Leu Ser Ala Val
225 230 235 240 Glu Arg Leu Asp Phe Pro Tyr Pro Leu Arg Asp Val Arg
Gly Glu Pro 245 250 255 Leu Glu Pro Pro Glu Pro Ser Val Pro Gly Gly
Gly Ser Gly Gly Gly 260 265 270 Leu Asn Asp Ile Phe Glu Ala Gln Lys
Ile Glu Trp His Glu Gly Gly 275 280 285 Pro Pro His His His His His
His 290 295 <210> SEQ ID NO 2 <211> LENGTH: 888
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 2 gcacccgagg tctcggagga
gccgcggtgc ccgcgcgccg cctgccaggc caagcgcggg 60 gaccagcgct
gcgaccgcga gtgcaacagc ccaggctgcg gctgggacgg cggcgactgc 120
tcgctgagcg tgggcgaccc ctggcggcaa tgcgaggcgc tgcagtgctg gcgcctcttc
180 aacaacagcc gctgcgaccc cgcctgcagc tcgcccgcct gcctctacga
caacttcgac 240 tgccacgccg gtggccgcga gcgcacttgc aacccggtgt
acgagaagta ctgcgccgac 300 cactttgccg acggccgctg cgaccagggc
tgcaacacgg aggagtgcgg ctgggatggg 360 ctggattgtg ccagcgaggt
gccggccctg ctggcccgcg gcgtgctggt gctcacagtg 420 ctgctgccgc
cggaggagct actgcgttcc agcgccgact ttctgcagcg gctcagcgcc 480
atcctgcgca cctcgctgcg cttccgcctg gacgcgcacg gccaggccat ggtcttccct
540 taccaccggc ctagtcctgg ctccgaaccc cgggcccgtc gggagctggc
ccccgaggtg 600 atcggctcgg tagtaatgct ggagattgac aaccggctct
gcctgcagtc gcctgagaat 660 gatcactgct tccccgatgc ccagagcgcc
gctgactacc tgggagcgtt gtcagcggtg 720 gagcgcctgg acttcccgta
cccactgcgg gacgtgcggg gggagccgct ggagcctcca 780 gaacccagcg
tcccgggagg gggaagcgga ggcggactga acgacatctt cgaggctcag 840
aaaatcgaat ggcacgaagg tggcccacca catcatcatc atcatcac 888
<210> SEQ ID NO 3 <211> LENGTH: 296 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 3 Ala Pro Glu Val Pro Glu Glu Pro Arg Cys Pro
Arg Ala Ala Cys Gln 1 5 10 15 Ala Lys Arg Gly Asp Gln Asn Cys Asp
Arg Glu Cys Asn Thr Pro Gly 20 25 30 Cys Gly Trp Asp Gly Gly Asp
Cys Ser Leu Asn Val Asp Asp Pro Trp 35 40 45 Arg Gln Cys Glu Ala
Leu Gln Cys Trp Arg Leu Phe Asn Asn Ser Arg 50 55 60 Cys Asp Pro
Ala Cys Ser Ser Pro Ala Cys Leu Tyr Asp Asn Phe Asp 65 70 75 80 Cys
Tyr Ser Gly Gly Arg Asp Arg Thr Cys Asn Pro Val Tyr Glu Lys 85 90
95 Tyr Cys Ala Asp His Phe Ala Asp Gly Arg Cys Asp Gln Gly Cys Asn
100 105 110 Thr Glu Glu Cys Gly Trp Asp Gly Leu Asp Cys Ala Ser Glu
Val Pro 115 120 125 Ala Leu Leu Ala Arg Gly Val Leu Val Leu Thr Val
Leu Leu Pro Pro 130 135 140 Glu Glu Leu Leu Arg Ser Ser Ala Asp Phe
Leu Gln Arg Leu Ser Ala 145 150 155 160 Ile Leu Arg Thr Ser Leu Arg
Phe Arg Leu Asp Ala Arg Gly Gln Ala 165 170 175 Met Val Phe Pro Tyr
His Arg Pro Ser Pro Gly Ser Glu Ser Arg Val 180 185 190 Arg Arg Glu
Leu Gly Pro Glu Val Ile Gly Ser Val Val Met Leu Glu 195 200 205 Ile
Asp Asn Arg Leu Cys Leu Gln Ser Ala Glu Asn Asp His Cys Phe 210 215
220 Pro Asp Ala Gln Ser Ala Ala Asp Tyr Leu Gly Ala Leu Ser Ala Val
225 230 235 240 Glu Arg Leu Asp Phe Pro Tyr Pro Leu Arg Asp Val Arg
Gly Glu Pro 245 250 255 Leu Glu Ala Pro Glu Gln Ser Val Pro Gly Gly
Gly Ser Gly Gly Gly 260 265 270 Leu Asn Asp Ile Phe Glu Ala Gln Lys
Ile Glu Trp His Glu Gly Gly 275 280 285 Pro Pro His His His His His
His 290 295 <210> SEQ ID NO 4 <211> LENGTH: 888
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 4 gcccctgagg tccccgagga
gccacggtgc ccgcgagcgg cttgccaggc caagcgaggg 60 gaccagaact
gcgatcgtga gtgcaacacc ccaggctgtg gctgggatgg cggtgactgc 120
tcactgaacg tggacgaccc ctggaggcag tgtgaggcac tgcagtgctg gcgtctcttc
180 aacaacagcc ggtgtgaccc ggcctgcagc tctccagcct gcctctatga
caactttgac 240 tgctactctg gtggccgcga ccgcacctgc aaccctgttt
atgagaagta ctgcgccgac 300 cactttgcag atggccgttg tgaccagggc
tgcaacactg aggaatgcgg ctgggatggg 360 ctggactgtg ccagcgaggt
cccggccctt ttggcccgag gggttctggt cctcacagtt 420 cttctgcctc
ctgaagagtt gctgcgctcc agtgccgact ttctgcagcg actcagcgct 480
attctgcgca cctcactgcg cttccgcttg gacgcacgtg gccaggccat ggtcttcccc
540 tatcaccggc caagccctgg ctctgaatcc cgggtccgtc gtgagctggg
tcctgaggtg 600 atcggctctg tggtgatgct ggagattgac aaccggctct
gtctgcagtc agctgagaat 660 gaccactgct tccctgatgc ccagagtgct
gctgactacc tgggagcctt gtcagcagtg 720 gagcgacttg atttcccata
cccacttcgg gatgtgcgag gagagccgct ggaggcccca 780 gagcagagcg
tgccaggagg gggaagcgga ggcggactga acgacatctt cgaggctcag 840
aaaatcgaat ggcacgaagg tggcccacca catcatcatc atcatcac 888
<210> SEQ ID NO 5 <211> LENGTH: 117 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Thr Leu Ser Cys Val Ala Ser
Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Gly Met Thr Trp Ile Arg Gln
Ala Pro Gly Lys Gly Leu Thr Trp Val 35 40 45 Ala Tyr Ile Ser Ser
Gly Ser Asn Tyr Ile Tyr Tyr Ala Glu Ala Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Phe Cys 85 90
95 Thr Arg Arg Gly Pro Phe Val Leu Asp Ala Trp Gly Gln Gly Ala Ser
100 105 110
Val Thr Val Ser Ser 115 <210> SEQ ID NO 6 <211> LENGTH:
351 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 6 gaggtgcaac tggtggagtc
tggaggaggc ttagtgcagc ctggaaggtc cctgacactc 60 tcctgtgtag
cctctggatt cactttcagg gactatggaa tgacctggat tcgccaggct 120
ccagggaagg ggctgacatg ggttgcatat attagtagtg gtagcaatta catctattat
180 gcagaagcgg tgaagggccg attcaccatc tccagagaca atgccaagaa
caccctgtac 240 ctgcaaatga ccagtctgag gtctgaagac actgccttgt
atttttgtac aagacgaggc 300 ccgtttgttt tggatgcctg gggtcaagga
gcttcagtca ctgtctcctc a 351 <210> SEQ ID NO 7 <211>
LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide sequence <400> SEQUENCE: 7 Asp Ile Gln Met
Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Asn Cys Lys Ala Ser Gln Ser Ile Asn Arg Tyr 20 25 30
Leu His Trp Phe Gln Gln Lys Leu Gly Glu Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Asn Ala Asn Gly Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Ser 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Leu Gln His
Asn Thr Trp Pro Asp 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu
Leu Lys 100 105 <210> SEQ ID NO 8 <211> LENGTH: 320
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 8 gacatccaga tgacccagtc
tccttcattc ctgtctgcat ctgtgggaga cagagtcact 60 atcaactgca
aagcaagtca gagtattaac aggtacttac actggtttca gcagaaactt 120
ggagaagctc ccaaactcct gatatataat gcaaacggtt tgcaaacggg catcccatca
180 aggttcagtg gcagtggatc tggtactgat ttcacactta ccatcagcag
cctgcagtct 240 gaagatgttg ccacatattt ctgcttgcag cataatacgt
ggccggacac gtttggcgct 300 gggaccaagc tggaactgaa 320 <210> SEQ
ID NO 9 <211> LENGTH: 119 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic peptide sequence <400> SEQUENCE:
9 Gln Val Lys Leu Leu Gln Ser Gly Ala Ala Leu Val Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asp
Tyr 20 25 30 Trp Val Thr Trp Val Lys Gln Ser His Gly Lys Ser Leu
Glu Trp Ile 35 40 45 Gly Glu Ile Ser Pro Asn Ser Gly Gly Thr Asn
Phe Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp
Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Thr
Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Thr Arg Gly Glu Ile
Arg Tyr Asn Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 <210> SEQ ID NO 10 <211> LENGTH:
357 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 10 caggtcaagc tgctgcagtc
tggggctgca ctggtgaagc ctggagcctc tgtgaagatg 60 tcttgcaaag
cttctggtta tgcattcact gactactggg tgacctgggt gaagcagagt 120
catggaaaga gccttgagtg gattggggaa atttctccta acagtggtgg tactaacttc
180 aatgaaaagt tcaagggcaa ggccacattg actgtagaca aatccaccag
cacagcctat 240 atggagctca gcagattgac atctgaggac tctgcaatct
attactgtac aagaggggaa 300 atccgttaca attggtttgc ttactggggc
caaggcactc tggtcactgt ctcctca 357 <210> SEQ ID NO 11
<211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic peptide sequence <400> SEQUENCE: 11
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Asn
Asn 20 25 30 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Arg Leu Tyr Asn Ser Pro Phe 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 <210> SEQ ID NO 12
<211> LENGTH: 321 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic nucleotide sequence <400> SEQUENCE: 12
aacattgtga tgacccagtc tcccaaatcc atgtccatat cagtaggaga cagggtcacc
60 atgaactgca aggccagtca gaatgtgggt aataatatag cctggtatca
acagaaacca 120 gggcagtctc ctaaactgtt gatctactat gcatctaacc
ggtacactgg ggtccctgat 180 cgcttcacag gcggtggata tgggacagat
ttcactctca ccatcaatag tatgcaagct 240 gaagatgcag ccttttatta
ctgtcagcgt ctttacaatt ctccattcac gttcggctca 300 gggacgaagt
tggaaataaa g 321 <210> SEQ ID NO 13 <211> LENGTH: 117
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 13 Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Gly Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Tyr Ile Ser Ser Gly Ser Asn Tyr Ile Tyr Tyr Ala Glu Ala Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Pro Phe Val Leu Asp Ala Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210>
SEQ ID NO 14 <211> LENGTH: 351 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 14 gaggtgcagc tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt
cactttcagg gactatggaa tgacctgggt ccgccaggct 120 ccagggaagg
ggctggagtg ggtggcctat attagtagtg gtagcaatta catctattat 180
gcagaagcgg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat
240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc
gagacgaggc 300 ccgtttgttt tggatgcctg gggccaggga accctggtca
ccgtctcctc a 351 <210> SEQ ID NO 15 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 15
Asp Tyr Gly Met Thr 1 5 <210> SEQ ID NO 16 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide sequence <400> SEQUENCE: 16 Gly Phe Thr Phe
Arg Asp Tyr 1 5 <210> SEQ ID NO 17 <211> LENGTH: 15
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 17 gactatggaa tgacc 15
<210> SEQ ID NO 18 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 18 ggattcactt tcagggacta t 21 <210> SEQ
ID NO 19 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic peptide sequence <400> SEQUENCE:
19 Tyr Ile Ser Ser Gly Ser Asn Tyr Ile Tyr Tyr Ala Glu Ala Val Lys
1 5 10 15 Gly <210> SEQ ID NO 20 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 20 Ser Ser Gly Ser Asn Tyr 1
5 <210> SEQ ID NO 21 <211> LENGTH: 51 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 21 tatattagta gtggtagcaa ttacatctat
tatgcagaag cggtgaaggg c 51 <210> SEQ ID NO 22 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleotide sequence <400> SEQUENCE: 22 agtagtggta
gcaattac 18 <210> SEQ ID NO 23 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 23 Arg Gly Pro Phe Val Leu
Asp Ala 1 5 <210> SEQ ID NO 24 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 24 cgaggcccgt ttgttttgga
tgcc 24 <210> SEQ ID NO 25 <211> LENGTH: 107
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 25 Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Ser Ile Asn Arg Tyr 20 25 30 Leu His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Asn Ala Asn Gly Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Thr
Trp Pro Asp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 <210> SEQ ID NO 26 <211> LENGTH: 321
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 26 gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgca
aagcaagtca gagtattaac aggtacttac actggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctataat gcaaacggtt tgcaaacggg ggtcccatca
180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240 gaagattttg caacttacta ctgtttgcag cataatacgt
ggccggacac gtttggcgga 300 gggaccaagg tggagatcaa a 321 <210>
SEQ ID NO 27 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 27 Lys Ala Ser Gln Ser Ile Asn Arg Tyr Leu
His 1 5 10 <210> SEQ ID NO 28 <211> LENGTH: 33
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 28 aaagcaagtc agagtattaa
caggtactta cac 33 <210> SEQ ID NO 29 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 29 Asn Ala Asn Gly Leu Gln
Thr 1 5 <210> SEQ ID NO 30 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleotide
sequence <400> SEQUENCE: 30 aatgcaaacg gtttgcaaac g 21
<210> SEQ ID NO 31 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 31 Leu Gln His Asn Thr Trp Pro Asp Thr 1 5
<210> SEQ ID NO 32 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 32 ttgcagcata atacgtggcc ggacacgttt 30
<210> SEQ ID NO 33 <211> LENGTH: 446 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 33 Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Gly Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Tyr Ile Ser Ser Gly Ser Asn Tyr Ile Tyr Tyr Ala Glu Ala Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Pro Phe Val Leu Asp Ala Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180
185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305
310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425
430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 <210> SEQ ID NO 34 <211> LENGTH: 1338 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleotide
sequence <400> SEQUENCE: 34 gaggtgcagc tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt
cactttcagg gactatggaa tgacctgggt ccgccaggct 120 ccagggaagg
ggctggagtg ggtggcctat attagtagtg gtagcaatta catctattat 180
gcagaagcgg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat
240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc
gagacgaggc 300 ccgtttgttt tggatgcctg gggccaggga accctggtca
ccgtctcctc agcgtcgacc 360 aagggcccat cggtcttccc cctggcaccc
tcctccaaga gcacctctgg gggcacagcg 420 gccctgggct gcctggtcaa
ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480 ggcgccctga
ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540
tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
600 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc
caaatcttgt 660 gacaaaactc acacatgccc accgtgccca gcacctgaac
tcctgggggg accgtcagtc 720 ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca 780 tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 840 ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag
960 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc
caaagccaaa 1020 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggagga gatgaccaag 1080 aaccaggtca gcctgacctg cctggtcaaa
ggcttctatc ccagcgacat cgccgtggag 1140 tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200 gacggctcct
tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1260
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc
1320 ctctccctgt ccccgggt 1338 <210> SEQ ID NO 35 <211>
LENGTH: 214 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide sequence <400> SEQUENCE: 35 Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile Asn Arg Tyr 20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Asn Ala Asn Gly Leu Gln Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His
Asn Thr Trp Pro Asp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> SEQ
ID NO 36 <211> LENGTH: 642 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleotide sequence <400>
SEQUENCE: 36 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
cagagtcacc 60 atcacttgca aagcaagtca gagtattaac aggtacttac
actggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctataat
gcaaacggtt tgcaaacggg ggtcccatca 180 aggttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg
caacttacta ctgtttgcag cataatacgt ggccggacac gtttggcgga 300
gggaccaagg tggagatcaa acggaccgtg gccgctcctt ccgtgttcat cttcccccct
360 tccgacgagc agctgaagtc tggcaccgcc tctgtggtgt gtctgctgaa
caacttctac 420 ccccgggagg ccaaggtgca gtggaaggtg gacaacgctc
tgcagtccgg caactcccag 480 gagtctgtga ccgagcagga ctccaaggac
agcacctact ccctgtcctc taccctgacc 540 ctgtccaagg ccgactacga
gaagcacaag gtgtacgcct gtgaggtgac ccaccagggc 600 ctgtcctctc
ctgtgaccaa gtccttcaac cggggcgagt gc 642 <210> SEQ ID NO 37
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic peptide sequence <400> SEQUENCE: 37
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ala Phe Thr Asp
Tyr 20 25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Glu Ile Ser Pro Asn Ser Gly Gly Thr Asn
Phe Asn Glu Lys Phe 50 55 60 Lys Gly Arg Phe Thr Ile Ser Val Asp
Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Glu Ile
Arg Tyr Asn Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser
115 <210> SEQ ID NO 38 <211> LENGTH: 357 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleotide
sequence <400> SEQUENCE: 38 gaggtgcagc tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggtta
tgcattcact gactactgga tgacctgggt ccgccaggct 120 ccagggaagg
ggctggagtg ggtggccgaa atttctccta acagtggtgg tactaacttc 180
aatgaaaagt tcaagggccg attcaccatc tccgttgaca acgccaagaa ctcactgtat
240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc
gagaggggaa 300 atccgttaca attggtttgc ttactggggc cagggaaccc
tggtcaccgt ctcctca 357 <210> SEQ ID NO 39 <211> LENGTH:
5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 39 Asp Tyr Trp Met Thr 1 5
<210> SEQ ID NO 40 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 40 Gly Tyr Ala Phe Thr Asp Tyr 1 5
<210> SEQ ID NO 41 <211> LENGTH: 15 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 41 gactactgga tgacc 15 <210> SEQ ID NO
42 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleotide sequence <400>
SEQUENCE: 42 ggttatgcat tcactgacta c 21 <210> SEQ ID NO 43
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic peptide sequence <400> SEQUENCE: 43
Glu Ile Ser Pro Asn Ser Gly Gly Thr Asn Phe Asn Glu Lys Phe Lys 1 5
10 15 Gly <210> SEQ ID NO 44 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 44 Ser Pro Asn Ser Gly Gly 1
5 <210> SEQ ID NO 45 <211> LENGTH: 51 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleotide sequence
<400> SEQUENCE: 45 gaaatttctc ctaacagtgg tggtactaac
ttcaatgaaa agttcaaggg c 51 <210> SEQ ID NO 46 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleotide sequence <400> SEQUENCE: 46 tctcctaaca
gtggtggt 18 <210> SEQ ID NO 47 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 47 Gly Glu Ile Arg Tyr Asn
Trp Phe Ala Tyr 1 5 10 <210> SEQ ID NO 48 <211> LENGTH:
30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 48 ggggaaatcc gttacaattg
gtttgcttac 30 <210> SEQ ID NO 49 <211> LENGTH: 107
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 49 Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn 20 25 30 Ile Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Leu Tyr Asn
Ser Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 <210> SEQ ID NO 50 <211> LENGTH: 321
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 50 gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgca
aggccagtca gaatgtgggt aataatatag cctggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctattat gcatctaacc ggtacactgg ggtcccatca
180 aggttcagtg gcagtggata tgggacagat ttcactctca ccatcagcag
tctgcaacct 240 gaagattttg caacttacta ctgtcagcgt ctttacaatt
ctccattcac gttcggcgga 300 gggaccaagg tggagatcaa a 321 <210>
SEQ ID NO 51 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 51 Lys Ala Ser Gln Asn Val Gly Asn Asn Ile
Ala 1 5 10 <210> SEQ ID NO 52 <211> LENGTH: 33
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleotide sequence <400> SEQUENCE: 52 aaggccagtc agaatgtggg
taataatata gcc 33 <210> SEQ ID NO 53 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 53 Tyr Ala Ser Asn Arg Tyr
Thr 1 5 <210> SEQ ID NO 54 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleotide
sequence <400> SEQUENCE: 54
tatgcatcta accggtacac t 21 <210> SEQ ID NO 55 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide sequence <400> SEQUENCE: 55 Gln Arg Leu Tyr
Asn Ser Pro Phe Thr 1 5 <210> SEQ ID NO 56 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleotide sequence <400> SEQUENCE: 56 cagcgtcttt
acaattctcc attcacgttc 30 <210> SEQ ID NO 57 <211>
LENGTH: 448 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide sequence <400> SEQUENCE: 57 Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ala Phe Thr Asp Tyr 20 25 30
Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Glu Ile Ser Pro Asn Ser Gly Gly Thr Asn Phe Asn Glu Lys
Phe 50 55 60 Lys Gly Arg Phe Thr Ile Ser Val Asp Asn Ala Lys Asn
Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Glu Ile Arg Tyr Asn Trp
Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165
170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290
295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410
415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly 435 440 445 <210> SEQ ID NO 58 <211> LENGTH:
1344 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleotide sequence <400> SEQUENCE: 58 gaggtgcagc
tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggtta tgcattcact gactactgga tgacctgggt ccgccaggct
120 ccagggaagg ggctggagtg ggtggccgaa atttctccta acagtggtgg
tactaacttc 180 aatgaaaagt tcaagggccg attcaccatc tccgttgaca
acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagaggggaa 300 atccgttaca attggtttgc
ttactggggc cagggaaccc tggtcaccgt ctcctcagcg 360 tcgaccaagg
gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420
acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg
480 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca
gtcctcagga 540 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca
gcttgggcac ccagacctac 600 atctgcaacg tgaatcacaa gcccagcaac
accaaggtgg acaagaaagt tgagcccaaa 660 tcttgtgaca aaactcacac
atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720 tcagtcttcc
tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780
gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac
840 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca
gtacaacagc 900 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg
actggctgaa tggcaaggag 960 tacaagtgca aggtctccaa caaagccctc
ccagccccca tcgagaaaac catctccaaa 1020 gccaaagggc agccccgaga
accacaggtg tacaccctgc ccccatcccg ggaggagatg 1080 accaagaacc
aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg
1200 gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag
caggtggcag 1260 caggggaacg tcttctcatg ctccgtgatg catgaggctc
tgcacaacca ctacacgcag 1320 aagagcctct ccctgtcccc gggt 1344
<210> SEQ ID NO 59 <211> LENGTH: 214 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 59 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Asn Val Gly Asn Asn 20 25 30 Ile Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser
Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Leu Tyr Asn Ser Pro Phe 85
90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205
Phe Asn Arg Gly Glu Cys 210 <210> SEQ ID NO 60 <211>
LENGTH: 642 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleotide sequence <400> SEQUENCE: 60 gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgca aggccagtca gaatgtgggt aataatatag cctggtatca gcagaaacca
120 gggaaagccc ctaagctcct gatctattat gcatctaacc ggtacactgg
ggtcccatca 180 aggttcagtg gcagtggata tgggacagat ttcactctca
ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcagcgt
ctttacaatt ctccattcac gttcggcgga 300 gggaccaagg tggagatcaa
acggaccgtg gccgctcctt ccgtgttcat cttcccccct 360 tccgacgagc
agctgaagtc tggcaccgcc tctgtggtgt gtctgctgaa caacttctac 420
ccccgggagg ccaaggtgca gtggaaggtg gacaacgctc tgcagtccgg caactcccag
480 gagtctgtga ccgagcagga ctccaaggac agcacctact ccctgtcctc
taccctgacc 540 ctgtccaagg ccgactacga gaagcacaag gtgtacgcct
gtgaggtgac ccaccagggc 600 ctgtcctctc ctgtgaccaa gtccttcaac
cggggcgagt gc 642 <210> SEQ ID NO 61 <211> LENGTH: 446
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
peptide sequence <400> SEQUENCE: 61 Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Gly Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Tyr Ile Ser Ser Gly Ser Asn Tyr Ile Tyr Tyr Ala Glu Ala Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Pro Phe Val Leu Asp Ala Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180
185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305
310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425
430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Cys Ser Pro Gly 435 440
445 <210> SEQ ID NO 62 <211> LENGTH: 1338 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleotide
sequence <400> SEQUENCE: 62 gaggtgcagc tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt
cactttcagg gactatggaa tgacctgggt ccgccaggct 120 ccagggaagg
ggctggagtg ggtggcctat attagtagtg gtagcaatta catctattat 180
gcagaagcgg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat
240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc
gagacgaggc 300 ccgtttgttt tggatgcctg gggccaggga accctggtca
ccgtctcctc agcgtcgacc 360 aagggcccat cggtcttccc cctggcaccc
tcctccaaga gcacctctgg gggcacagcg 420 gccctgggct gcctggtcaa
ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480 ggcgccctga
ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540
tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
600 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc
caaatcttgt 660 gacaaaactc acacatgccc accgtgccca gcacctgaac
tcctgggggg accgtcagtc 720 ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca 780 tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 840 ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag
960 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc
caaagccaaa 1020 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggagga gatgaccaag 1080 aaccaggtca gcctgacctg cctggtcaaa
ggcttctatc ccagcgacat cgccgtggag 1140 tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200 gacggctcct
tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1260
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc
1320 ctctcctgct ccccgggt 1338 <210> SEQ ID NO 63 <211>
LENGTH: 214 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide sequence <400> SEQUENCE: 63 Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile Asn Arg Tyr 20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Asn Ala Asn Gly Leu Gln Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His
Asn Thr Trp Pro Asp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175 Ser Thr Leu Thr Leu Ser Cys Ala Asp Tyr Glu Lys His Lys Val
Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> SEQ
ID NO 64 <211> LENGTH: 642 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleotide sequence <400>
SEQUENCE: 64 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
cagagtcacc 60 atcacttgca aagcaagtca gagtattaac aggtacttac
actggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctataat
gcaaacggtt tgcaaacggg ggtcccatca 180 aggttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg
caacttacta ctgtttgcag cataatacgt ggccggacac gtttggcgga 300
gggaccaagg tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca
360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa
taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac
agcacctaca gcctcagcag caccctgacg 540 ctgagctgcg cagactacga
gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc
ccgtcacaaa gagcttcaac aggggagagt gt 642 <210> SEQ ID NO 65
<211> LENGTH: 446 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic peptide sequence <400> SEQUENCE:
65
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp
Tyr 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Asn Tyr Ile Tyr
Tyr Ala Glu Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Pro
Phe Val Leu Asp Ala Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260
265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380
Gly Gln Pro Glu Asn Asn Tyr Cys Thr Thr Pro Pro Val Leu Asp Ser 385
390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser
Cys Ser Pro Gly 435 440 445 <210> SEQ ID NO 66 <211>
LENGTH: 1338 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleotide sequence <400> SEQUENCE: 66 gaggtgcagc
tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cactttcagg gactatggaa tgacctgggt ccgccaggct
120 ccagggaagg ggctggagtg ggtggcctat attagtagtg gtagcaatta
catctattat 180 gcagaagcgg tgaagggccg attcaccatc tccagagaca
acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagacgaggc 300 ccgtttgttt tggatgcctg
gggccaggga accctggtca ccgtctcctc agcgtcgacc 360 aagggcccat
cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420
gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca
480 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc
aggactctac 540 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg
gcacccagac ctacatctgc 600 aacgtgaatc acaagcccag caacaccaag
gtggacaaga aagttgagcc caaatcttgt 660 gacaaaactc acacatgccc
accgtgccca gcacctgaac tcctgggggg accgtcagtc 720 ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac
840 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa
cagcacgtac 900 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc
tgaatggcaa ggagtacaag 960 tgcaaggtct ccaacaaagc cctcccagcc
cccatcgaga aaaccatctc caaagccaaa 1020 gggcagcccc gagaaccaca
ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1080 aaccaggtca
gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140
tgggagagca atgggcagcc ggagaacaac tactgcacca cgcctcccgt gctggactcc
1200 gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg
gcagcagggg 1260 aacgtcttct catgctccgt gatgcatgag gctctgcaca
accactacac gcagaagagc 1320 ctctcctgct ccccgggt 1338 <210> SEQ
ID NO 67 <211> LENGTH: 113 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic peptide sequence <400> SEQUENCE:
67 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys
Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser
<210> SEQ ID NO 68 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide sequence
<400> SEQUENCE: 68 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85
90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
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