U.S. patent application number 13/441753 was filed with the patent office on 2013-04-11 for methods of treating liver conditions using notch2 antagonists.
This patent application is currently assigned to GENENTHECH, INC.. The applicant listed for this patent is Dorothy French, Erik Huntzicker, Christian W. Siebel. Invention is credited to Dorothy French, Erik Huntzicker, Christian W. Siebel.
Application Number | 20130089562 13/441753 |
Document ID | / |
Family ID | 45992860 |
Filed Date | 2013-04-11 |
United States Patent
Application |
20130089562 |
Kind Code |
A1 |
French; Dorothy ; et
al. |
April 11, 2013 |
METHODS OF TREATING LIVER CONDITIONS USING NOTCH2 ANTAGONISTS
Abstract
Methods and compositions for the treatment of liver conditions
are provided, such methods and compositions comprising Notch2
antagonists, e.g., anti-Notch2 antibodies. Liver conditions
include, but are not limited to, chronic liver disease.
Inventors: |
French; Dorothy; (San
Carlos, CA) ; Siebel; Christian W.; (Berkeley,
CA) ; Huntzicker; Erik; (Los Altos, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
French; Dorothy
Siebel; Christian W.
Huntzicker; Erik |
San Carlos
Berkeley
Los Altos |
CA
CA
CA |
US
US
US |
|
|
Assignee: |
GENENTHECH, INC.
|
Family ID: |
45992860 |
Appl. No.: |
13/441753 |
Filed: |
April 6, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61543483 |
Oct 5, 2011 |
|
|
|
Current U.S.
Class: |
424/172.1 ;
435/377 |
Current CPC
Class: |
C07K 2317/34 20130101;
Y02A 50/463 20180101; A61K 39/395 20130101; A61K 38/179 20130101;
C07K 14/475 20130101; C07K 14/705 20130101; C07K 16/2866 20130101;
Y02A 50/30 20180101; A61K 39/3955 20130101; C07K 14/52 20130101;
C07K 16/2863 20130101; C07K 14/71 20130101; C07K 2317/31 20130101;
C07K 2317/76 20130101; A61K 2039/505 20130101; C07K 14/715
20130101; C07K 2319/30 20130101; C07K 16/28 20130101; C07K 2317/33
20130101; C07K 2319/32 20130101; A61K 38/18 20130101; A61K 38/177
20130101; A61P 1/16 20180101 |
Class at
Publication: |
424/172.1 ;
435/377 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 5/071 20100101 C12N005/071; A61P 1/16 20060101
A61P001/16 |
Claims
1. A method of treating a liver condition characterized by liver
damage, the method comprising administering to a patient having
such condition an effective amount of a Notch2-specific
antagonist.
2. The method of claim 1, wherein the liver condition is chronic
liver disease.
3. The method of claim 2, wherein the chronic liver disease is
selected from the group consisting of liver fibrosis, cirrhosis,
viral hepatitis, autoimmune liver diseases, genetic liver diseases,
alcoholic hepatitis and nonalcoholic fatty liver disease.
4. The method of claim 3, wherein the autoimmune liver disease is
selected from the group consisting of autoimmune hepatitis, primary
biliary cirrhosis, and primary sclerosing cholangitis.
5. The method of claim 1, wherein the liver condition is an acute
liver condition.
6. The method of claim 5, wherein the acute liver condition is
acetaminophen toxicity.
7. The method of claim 1, wherein the Notch2-specific antagonist is
selected from the group consisting of a soluble Notch receptor,
soluble Notch ligand variant, aptamer, oligopeptide, anti-Notch2
antagonist antibody, and anti-Notch2 ligand antagonist
antibody.
8. The method of claim 7, wherein the anti-Notch2 antagonist
antibody is an anti-Notch2 negative regulatory region (NRR)
antibody.
9. The method of claim 8, wherein the anti-Notch2 NRR antibody
binds to the Lin 12/Notch Repeat-A and heterodimerization domain-C
domains of Notch2 NRR.
10. The method of claim 8, wherein the anti-Notch2 NRR antibody
comprises: (a) a heavy chain hypervariable region (HVR-H) 1
comprising an amino acid sequence of SEQ ID NO:3; (b) an HVR-H2
comprising the amino acid sequence of SEQ ID NO:4; (c) an HVR-H3
comprising the amino acid sequence of SEQ ID NO:5; (d) a light
chain hypervariable region (HVR-L) 1 comprising an amino acid
sequence of SEQ ID NO:10; (e) an HVR-L2 comprising an amino acid
sequence of SEQ ID NO:14; and (f) an HVR-L3 comprising an amino
acid sequence of SEQ ID NO:19.
11. The method of claim 8, wherein the anti-Notch2 NRR antibody
comprises an HVR-H1 comprising an amino acid sequence selected from
SEQ ID NOs:1-2; an HVR-H2 comprising the amino acid sequence of SEQ
ID NO:4; an HVR-H3 comprising the amino acid sequence of SEQ ID
NO:5; an HVR-L1 comprising an amino acid sequence selected from SEQ
ID NOs:6-9; an HVR-L2 comprising an amino acid sequence selected
from SEQ ID NOs:11-13; and an HVR-L3 comprising an amino acid
sequence selected from SEQ ID NOs:15-18.
12. The method of claim 7, wherein the anti-Notch2 antagonist
antibody is an anti-Notch2 antibody that binds to one or more
EGF-like repeats of Notch2.
13. The method of claim 1, wherein the Notch2-specific antagonist
is administered by intramuscular, intraperitoneal,
intracerobrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation route.
14. A method of inducing hepatic differentiation, the method
comprising the step of contacting an oval cell with an effective
amount of a Notch2-specific antagonist, thereby inducing hepatic
differentiation of the oval cell.
15. The method of claim 14, wherein the oval cell is contacted with
the Notch2-specific antagonist in vitro.
16. The method of claim 14, wherein the oval cell is contacted with
the Notch2-specific antagonist in vivo.
17. A method of reducing aberrant bile duct proliferation in a
patient in need thereof, the method comprising the step of
administering to the patient an effective amount of a
Notch2-specific antagonist, thereby reducing aberrant bile duct
proliferation.
18. The method of claim 3, wherein the genetic liver diseases is
selected from the group consisting of alpha-1 antitrypsin
deficiency, Crigler-Najjar syndrome, familial amyloidosis,
Gilbert's syndrome, Dubin-Johnson syndrome, hereditary
hemchromatosis, primary oxalosis, and Wilson's disease.
19. The method of claim 5, wherein the acute liver condition is
selected from the group consisting of acute liver failure, acute
liver injury, and acute liver toxicity.
20. The method of claim 1, 8 or 9, wherein administering to the
patient the Notch2-specific antagonist results in accelerated
differentiation of hepatocyte progenitor cells into hepatocytes
compared to hepatocyte progenitor cell differentiation in the
absence of the Notch2-specific antagonist.
21. The method of claim 1, 8 or 9, wherein administering to the
patient the Notch2-specific antagonist results in decreased
aberrant bile duct proliferation compared to aberrant bile duct
proliferation without administering the Notch2-specific
antagonist.
22. The method of claim 1, 8 or 9 wherein administering to the
patient the Notch2-specific antagonist results in improved liver
histological appearance compared to liver histological appearance
without administering the Notch2-specific antagonist.
23. The method of claim 22, wherein the improved liver histologic
appearance is selected from the group consisting of increased cell
size, decreased nuclear-to-cytoplasmic ratio and increased number
of liver cells having two nuclei.
24. The method of claim 1, 8 or 9, wherein administering to the
patient the Notch2-specific antagonist results in decreased
expression of Keratin-19 in liver cells relative to expression of
Keratin-19 in cultured adult oval cells.
25. The method of claim 1, 8 or 9, wherein administering to the
patient the Notch2-specific antagonist results in increased
expression of albumin in liver cells relative to expression of
albumin in cultured adult oval cells.
26. The method of claim 1, 8 or 9, wherein administering to the
patient the Notch2-specific antagonist results in increased
expression of .alpha.-fetoprotein in liver cells relative to
expression of .alpha.-fetoprotein in cultured adult oval cells.
27. The method of claim 1, 8 or 9, wherein administering to the
patient the Notch2-specific antagonist results in a reduced number
of Hes1-positive intrahepatic bile duct cells.
28. The method of claim 1, 8 or 9, wherein administering to the
patient the Notch2-specific antagonist results in reduced serum
bile acids, serum bilirubin, or serum alkaline phosphatase.
29. The method of claim 8, wherein the anti-Notch2 NRR antibody
further comprises at least one framework selected from a human
variable heavy acceptor 2 framework and a human variable light
kappa subgroup I consensus framework.
30. The method of claim 8, wherein the anti-Notch2 NRR antibody
comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ
ID NO:1; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID
NO:5; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID
NO:6; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID
NO:11; and (f) an HVR-L3 comprising the amino acid sequence of SEQ
ID NO:15.
31. The method of claim 8, wherein the anti-Notch2 NRR antibody
comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ
ID NO:2; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID
NO:5; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID
NO:7; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID
NO:11; and (f) an HVR-L3 comprising the amino acid sequence of SEQ
ID NO:16.
32. The method of claim 8, wherein the anti-Notch2 NRR antibody
comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ
ID NO:2; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID
NO:5; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID
NO:8; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID
NO:12; and (f) an HVR-L3 comprising the amino acid sequence of SEQ
ID NO:17.
33. The method of claim 8, wherein the anti-Notch2 NRR antibody
comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ
ID NO:2; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID
NO:5; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID
NO:9; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID
NO:13; and (f) an HVR-L3 comprising the amino acid sequence of SEQ
ID NO:18.
34. The method of claim 8, wherein the anti-Notch2 NRR antibody
comprises a heavy chain variable domain having at least 90%
sequence identity to an amino acid sequence selected from SEQ ID
NO:20-21 and a light chain variable domain having at least 90%
sequence identity to an amino acid sequence selected from SEQ ID
NO:22-25.
35. The method of claim 34, wherein the heavy chain variable domain
comprises the amino acid sequence of SEQ ID NO:21, and the light
chain variable domain comprises an amino acid sequence selected
from SEQ ID NOs:23-25.
36. The method of claim 35, wherein the heavy chain variable domain
comprises the amino acid sequence of SEQ ID NO:21, and the light
chain variable domain comprises an amino acid sequence of SEQ ID
NOs:25.
37. The method of claim 8, wherein the anti-Notch2 NRR antibody
competes for binding with an antibody comprising a heavy chain
variable domain comprising the amino acid sequence of SEQ ID NO:21,
and a light chain variable domain comprising an amino acid sequence
selected from SEQ ID NOs:23-25.
38. The method of claim 14, wherein the Notch2-specific antagonist
is an anti-Notch2 antagonist antibody.
39. The method of claim 38, wherein the anti-Notch2 antagonist
antibody is an anti-Notch2 NRR antibody.
40. The method of claim 39, wherein the anti-Notch2 NRR antibody
binds to the Lin 12/Notch Repeat-A and heterodimerization domain-C
domains of Notch2 NRR.
41. The method of claim 17, wherein the Notch2-specific antagonist
is selected from the group consisting of a soluble Notch receptor,
soluble Notch ligand variant, aptamer, oligopeptide, anti-Notch2
antagonist antibody, and anti-Notch2 ligand antagonist
antibody.
42. The method of claim 41, wherein the anti-Notch2 antagonist
antibody is an anti-Notch2 NRR antibody.
43. The method of claim 42, wherein the anti-Notch2 NRR antibody
binds to the Lin 12/Notch Repeat-A and heterodimerization domain-C
domains of Notch2 NRR.
44. A method of reducing liver progenitor cell proliferation in a
patient in need thereof comprising the step of administering to the
patient having such condition an effective amount of a
Notch2-specific antagonist.
45. The method of claim 44, wherein the Notch2-specific antagonist
is selected from the group consisting of a soluble Notch receptor,
soluble Notch ligand variant, aptamer, oligopeptide, anti-Notch2
antagonist antibody, and anti-Notch2 ligand antagonist
antibody.
46. The method of claim 45, wherein the Notch2-specific antagonist
is an anti-Notch2 antagonist antibody.
47. The method of claim 46, wherein the anti-Notch2 antagonist
antibody is an anti-Notch2 NRR antibody.
48. The method of claim 47, wherein the anti-Notch2 NRR antibody
binds to the Lin 12/Notch Repeat-A and heterodimerization domain-C
domains of Notch2 NRR.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/543,483, filed Oct. 5, 2011, the disclosure of
which is incorporated herein by reference as if set forth in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to methods of treating liver
conditions using Notch2 antagonists. Compositions for the treatment
of such conditions are also provided.
BACKGROUND
[0003] The Notch receptor family is a class of evolutionarily
conserved transmembrane receptors that transmit signals affecting
development in organisms as diverse as sea urchins and humans.
Notch receptors and their ligands Delta and Serrate (known as
Jagged in mammals) are transmembrane proteins with large
extracellular domains that contain epidermal growth factor
(EGF)-like repeats. The number of Notch paralogues differs between
species. For example, there are four Notch receptors in mammals
(Notch1-Notch4), two in Caenorhabditis elegans (LIN-12 and GLP-1)
and one in Drosophila melanogaster (Notch). Notch receptors are
proteolytically processed during transport to the cell surface by a
furin-like protease at a site 51, which is N-terminal to the
transmembrane domain, producing an extracellular Notch (ECN)
subunit and a Notch transmembrane subunit (NTM). These two subunits
remain non-covalently associated and constitute the mature
heterodimeric cell-surface receptor.
[0004] Notch2 ECN subunits contain 36 N-terminal EGF-like repeats
followed by three tandemly repeated Lin 12/Notch Repeat (LNR)
modules that precede the 51 site. Each LNR module contains three
disulfide bonds and a group of conserved acidic and polar residues
predicted to coordinate a calcium ion. Within the EGF repeat region
lie binding sites for the activating ligands.
[0005] The Notch2 NTM comprises an extracellular region (which
harbors the S2 cleavage site), a transmembrane segment (which
harbors the S3 cleavage site), and a large intracellular portion
that includes a RAM23 domain, six ankyrin repeats, a
transactivation domain and a carboxy-terminal PEST sequence. Stable
association of the ECN and NTM subunits is dependent on a
heterodimerization domain (HD) comprising the carboxy-terminal end
of the ECN (termed HD-N) and the extracellular amino-terminal end
of NTM (termed HD-C). Before ligand-induced activation, Notch is
maintained in a resting conformation by a negative regulatory
region (NRR), which comprises the three LNRs and the HD domain. The
crystal structure of the Notch2 NRR is reported in Gordon et al.,
(2007) Nature Structural & Molecular Biology 14:295-300,
2007.
[0006] Binding of a Notch ligand to the ECN subunit initiates two
successive proteolytic cleavages that occur through regulated
intramembrane proteolysis. The first cleavage by a metalloprotease
(ADAM17) at site S2 renders the Notch transmembrane subunit
susceptible to a second cleavage at site S3 close to the inner
leaflet of the plasma membrane. Site S3 cleavage, which is
catalyzed by a multiprotein complex containing presenilin and
nicastrin and promoting .gamma.-secretase activity, liberates the
intracellular portion of the Notch transmembrane subunit, allowing
it to translocate to the nucleus and activate transcription of
target genes. (For review of the proteolytic cleavage of Notch,
see, e.g., Sisodia et al., Nat. Rev. Neurosci. 3:281-290,
2002.)
[0007] Five Notch ligands of the Jagged and Delta-like classes have
been identified in humans (Jagged1 (also termed Serrate1), Jagged2
(also termed Serrate2), Delta-like1 (also termed DLL1), Delta-like3
(also termed DLL3), and Delta-like4 (also termed DLL4)). Each of
the ligands is a single-pass transmembrane protein with a conserved
N-terminal Delta, Serrate, LAG-2 (DSL) motif essential for binding
Notch. A series of EGF-like modules C-terminal to the DSL motif
precede the membrane-spanning segment. Unlike the Notch receptors,
the ligands have short cytoplasmic tails of 70-215 amino acids at
the C-terminus. In addition, other types of ligands have been
reported (e.g., DNER, NB3, and F3/Contactin). (For review of Notch
ligands and ligand-mediated Notch activation, see, e.g., D' Souza
et al., Oncogene 27:5148-5167, 2008.)
[0008] The Notch pathway functions during diverse developmental and
physiological processes including those affecting neurogenesis in
flies and vertebrates. In general, Notch signaling is involved in
lateral inhibition, lineage decisions, and the establishment of
boundaries between groups of cells. (See, e.g., Bray, Mol. Cell.
Biol. 7:678-679, 2006.) A variety of human diseases, including
cancers and neurodegenerative disorders have been shown to result
from mutations in genes encoding Notch receptors or their ligands.
(See, e.g., Nam et al., Curr. Opin. Chem. Biol. 6:501-509,
2002.)
[0009] Certain anti-Notch2 antagonist antibodies having therapeutic
efficacy have been described. (See U.S. Patent Application
Publication No. US 2009/0081238 A1, expressly incorporated by
reference in its entirety herein.) For example, such antibodies
bind to the negative regulatory region (NRR) of Notch2, block
Notch2 signaling, and inhibit the growth of melanoma cell lines,
diffuse large B-cell lymphoma (DLBCL) cell lines, and marginal zone
B cells. Certain anti-Notch2 antibodies described therein bind to
the LNR-A domain (the first of the three LIN12/Notch Repeats) and
the HD-C domain of Notch2 NRR.
[0010] Adult liver has the capacity to regenerate after injury. It
has been speculated that biliary-hepatocyte progenitor cells (oval
cells) in or near intrahepatic bile ducts can differentiate into
adult hepatocytes (Brues and Marble, J. Exp. Med., 65(1):15 (1937);
Zajicek et al., Liver, 5(6):293 (1985)), which subsequently mature
as they move toward the central vein and eventually undergo
apoptosis and elimination (Benedetti et al., J. Hepatol., 7(3):319
(1988)). Recent lineage-tracing studies have supported a role of
progenitor cells in liver homeostasis and repair, but the signals
that govern precursor differentiation into hepatocytes are poorly
understood. While Notch signaling is known to be critical for the
proper formation of the intrahepatic biliary system during
development (Lozier et al., PLoS One 3(3):e1851 (2008); McCright et
al., Development 129(4):1075 (2002)), it was not known what role,
if any, Notch signaling plays in adult hepatocyte formation and in
adult hepatobiliary disease.
[0011] Chronic liver disease is marked by gradual destruction of
liver tissue, especially of hepatocytes and the functional lobular
unit, leading to fibrosis (replacement of liver tissue with scar
tissue) and cirrhosis (fibrosis with ineffective nodular
regeneration and associated loss of liver function). Moreover,
chronic liver disease often includes pathological biliary
hyperplasia and may increase the risk of liver cancer.
[0012] There is a need in the art for further therapeutic methods
of treating liver conditions. The invention described herein meets
the above-described needs and provides other benefits.
SUMMARY
[0013] The present invention relates to the treatment of liver
conditions using Notch2 antagonists. The present invention is
based, in part, on the observation that anti-Notch2 NRR antibodies
(a) improve liver histologic appearance and hepatocyte function in
an acute liver damage model in vivo and (b) reduce biliary damage
and improve hepatocyte function in a chronic liver damage model in
vivo.
[0014] In one aspect, a method of treating a liver condition
characterized by liver damage is provided, the method comprising
administering to a patient having such condition an effective
amount of a Notch2-specific antagonist. In certain embodiments, the
liver condition is chronic liver disease. In certain embodiments,
the liver condition is liver fibrosis.
[0015] In any of the above embodiments, the Notch2-specific
antagonist may be an anti-Notch2 antagonist antibody. In certain
embodiments, the anti-Notch2 antagonist antibody is an anti-Notch2
NRR antibody. In one such embodiment, the anti-Notch2 NRR antibody
binds to the LNR-A and HD-C domains of Notch2 NRR. In another such
embodiment, the anti-Notch2 NRR antibody is Antibody D, Antibody
D-1, Antibody D-2, or Antibody D-3. In another such embodiment, the
anti-Notch2 NRR antibody comprises the heavy and light chain
variable region CDRs of Antibody D, Antibody D-1, Antibody D-2, or
Antibody D-3. In certain embodiments, the anti-Notch2 antagonist
antibody is an anti-Notch2 antibody that binds to one or more
EGF-like repeats of Notch2.
[0016] The above and further aspects and embodiments of the
invention are provided herein.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIGS. 1A-G show transcriptional profiling of hepatic
progenitor cells and Identification of active Notch signaling in
hepatic progenitors in vivo.
[0018] FIGS. 2A-O show that Notch signaling inhibition promotes
hepatocyte differentiation of hepatic progenitors in vitro.
[0019] FIGS. 3A-J show that inhibition of Notch2 signaling in vivo
promotes hepatocyte differentiation and improved liver function in
chronic and acute liver damage models.
[0020] FIGS. 4A-E illustrate a liver progenitor cell isolation
strategy.
[0021] FIGS. 5A-H show an analysis of oval cell-specific gene
expression signature.
[0022] FIGS. 6A-E show a validation of oval cell gene signature and
expression pattern of putative hepatic stem cell markers.
[0023] FIGS. 7A-B show a strategy for in vitro differentiation.
[0024] FIGS. 8A-F show the efficacy of anti-Notch2 antibody
treatment in vivo and its effect on liver growth and proliferation
following partial hepatectomy.
[0025] FIGS. 9A-H show the effect of anti-Notch2 antibody treatment
on serum hepatobiliary function markers following partial
hepatectomy.
[0026] FIGS. 10A-F show the effect of anti-Notch2 antibody
treatment on hepatobiliary and Notch signaling gene expression
following partial hepatectomy.
[0027] FIGS. 11A-I show serum hepatobiliary function markers
following 4 weeks of antibody administration in normal and DDC-fed
mice.
[0028] FIG. 12 shows the H1, H2, and H3 heavy chain hypervariable
region (HVR) sequences of anti-Notch2 NRR monoclonal antibodies
designated Antibody D, Antibody D-1, Antibody D-2, and Antibody
D-3. Amino acid positions are numbered according to the Kabat
numbering system as described below.
[0029] FIG. 13 shows the L1, L2, and L3 light chain HVR sequences
of anti-Notch2 NRR monoclonal antibodies designated Antibody D,
Antibody D-1, Antibody D-2, and Antibody D-3. Amino acid positions
are numbered according to the Kabat numbering system as described
below.
[0030] FIG. 14 shows an alignment of the heavy chain variable
region sequences of Antibody D, Antibody D-1, Antibody D-2, and
Antibody D-3. HVRs are enclosed in boxes.
[0031] FIG. 15 shows an alignment of the light chain variable
region sequences of Antibody D, Antibody D-1, Antibody D-2, and
Antibody D-3. HVRs are enclosed in boxes.
[0032] FIGS. 16A-B show exemplary acceptor human variable heavy
(VH) consensus framework sequences for use in practicing the
instant invention. Sequence identifiers are as follows: [0033]
human VH subgroup I consensus framework "A" minus Kabat CDRs (SEQ
ID NOs:32, 33, 34, 35). [0034] human VH subgroup I consensus
frameworks "B," "C," and "D" minus extended hypervariable regions
(SEQ ID NOs:36, 37, 34, 35; SEQ ID NOs:36, 37, 38, 35; and SEQ ID
NOs:36, 37, 39, 35). [0035] human VH subgroup II consensus
framework "A" minus Kabat CDRs (SEQ ID NOs:40, 41, 42, 35). [0036]
human VH subgroup II consensus frameworks "B," "C," and "D" minus
extended hypervariable regions (SEQ ID NOs:43, 44, 42, 35; SEQ ID
NOs:43, 44, 45, 35; and SEQ ID NOs:43, 44, 46, and 35). [0037]
human VH subgroup III consensus framework "A" minus Kabat CDRs (SEQ
ID NOs:47, 48, 49, 35). [0038] human VH subgroup III consensus
frameworks "B," "C," and "D" minus extended hypervariable regions
(SEQ ID NOs:50, 51, 49, 35; SEQ ID NOs:50, 51, 52, 35; and SEQ ID
NOs:50, 51, 53, 35). [0039] human VH acceptor framework "A" minus
Kabat CDRs (SEQ ID NOs:54, 48, 55, 35). [0040] human VH acceptor
frameworks "B" and "C" minus extended hypervariable regions (SEQ ID
NOs:50, 51, 55, 35; and SEQ ID NOs:50, 51, 56, 35). [0041] human VH
acceptor 2 framework "A" minus Kabat CDRs (SEQ ID NOs:54, 48, 57,
35). [0042] human VH acceptor 2 framework "B," "C," and "D" minus
extended hypervariable regions (SEQ ID NOs:50, 51, 57, 35; SEQ ID
NOs:50, 51, 58, 35; and SEQ ID NOs:50, 51, 59, 35).
[0043] FIG. 17 shows exemplary acceptor human variable light (VL)
consensus framework sequences for use in practicing the instant
invention. Sequence identifiers are as follows: [0044] human VL
kappa subgroup I consensus framework (.kappa.v1): SEQ ID NOs:60,
61, 62, 63 [0045] human VL kappa subgroup II consensus framework
(.kappa.v2): SEQ ID NOs:64, 65, 66, 63 [0046] human VL kappa
subgroup III consensus framework (.kappa.v3): SEQ ID NOs:67, 68,
69, 63 [0047] human VL kappa subgroup IV consensus framework
(.kappa.v4): SEQ ID NOs:70, 71, 72, 63
[0048] FIG. 18 shows framework sequences of huMAb4D5-8 light and
heavy chains. Numbers in superscript/bold indicate amino acid
positions according to Kabat.
[0049] FIG. 19 shows framework sequences of huMAb4D5-8 light and
heavy chains with the indicated modifications. Numbers in
superscript/bold indicate amino acid positions according to
Kabat.
DETAILED DESCRIPTION OF EMBODIMENTS
I. Definitions
[0050] For purposes of interpreting this specification, the
following definitions will apply and whenever appropriate, terms
used in the singular will also include the plural and vice versa.
In the event that any definition set forth below conflicts with any
document incorporated herein by reference, the definition set forth
below shall control.
[0051] The term "Notch," as used herein, refers, unless
specifically or contextually indicated otherwise, to any native or
variant (whether native or synthetic) Notch polypeptide (Notch1-4).
The term "native sequence" specifically encompasses naturally
occurring truncated forms (e.g., an extracellular domain sequence
or a transmembrane subunit sequence), naturally occurring variant
forms (e.g., alternatively spliced forms) and naturally-occurring
allelic variants. The term "wild-type Notch" generally refers to a
polypeptide comprising an amino acid sequence of a naturally
occurring, non-mutated Notch protein. The term "wild-type Notch
sequence" generally refers to an amino acid sequence found in a
naturally occurring, non-mutated Notch.
[0052] The term "Notch2," as used herein, refers, unless
specifically or contextually indicated otherwise, to any native or
variant (whether native or synthetic) Notch2 polypeptide. The term
"native sequence" specifically encompasses naturally occurring
truncated forms (e.g., an extracellular domain sequence or a
transmembrane subunit sequence), naturally occurring variant forms
(e.g., alternatively spliced forms) and naturally occurring allelic
variants. The term "wild-type Notch2" generally refers to a
polypeptide comprising an amino acid sequence of a naturally
occurring, non-mutated Notch2 protein. The term "wild type Notch2
sequence" generally refers to an amino acid sequence found in a
naturally occurring, non-mutated Notch2.
[0053] The term "Notch2 ligand," as used herein, refers, unless
specifically or contextually indicated otherwise, to any native or
variant (whether native or synthetic) Notch2 ligand (for example,
Jagged1, Jagged2, Delta-like1, Delta-like3, and/or Delta-like4)
polypeptide. The term "native sequence" specifically encompasses
naturally occurring truncated forms (e.g., an extracellular domain
sequence or a transmembrane subunit sequence), naturally occurring
variant forms (e.g., alternatively spliced forms) and naturally
occurring allelic variants. The term "wild-type Notch2 ligand"
generally refers to a polypeptide comprising an amino acid sequence
of a naturally occurring, non-mutated Notch2 ligand. The term "wild
type Notch2 ligand sequence" generally refers to an amino acid
sequence found in a naturally occurring, non-mutated Notch2
ligand.
[0054] The term "Notch2 NRR," as used herein, refers, unless
specifically or contextually indicated otherwise, to any native or
variant (whether native or synthetic) polypeptide region of Notch2
consisting of the 3 LNR modules and the amino acid sequences
extending from the carboxy-terminus of the LNR modules to the
transmembrane domain, such sequences including the HD domain (HD-N
and HD-C). An exemplary Notch2 NRR consists of the region from
about amino acid 1422-1677 of human Notch2 (SEQ ID NO:73). An
exemplary human Notch2 NRR is also shown in SEQ ID NO:74. The term
"native sequence Notch2 NRR" specifically encompasses naturally
occurring truncated forms, naturally occurring variant forms (e.g.,
alternatively spliced forms) and naturally-occurring allelic
variants of a Notch2 NRR. The term "wild-type Notch2 NRR" generally
refers to a naturally occurring, non-mutated Notch2 NRR. In some
embodiments, a Notch2 NRR is contained in a Notch2, such as, for
example, a Notch2 processed at the 51, S2 and/or S3 site(s), or an
unprocessed Notch2. In some embodiments, a Notch2 NRR contains two
or more non-covalently linked fragments of a Notch2 NRR amino acid
sequence, e.g., a fragment containing amino acids 1422 to 1608 of
SEQ ID NO:73 non-covalently linked to a fragment containing amino
acids 1609 to 1677 of SEQ ID NO:73.
[0055] The term "increased Notch2 signaling," as used herein,
refers to an increase in Notch2 signaling that is significantly
above the level of Notch2 signaling observed in a control under
substantially identical conditions. In certain embodiments, the
increase in Notch2 signaling is at least two fold, three fold, four
fold, five fold, or ten fold above the level observed in the
control. The term "decreased Notch1 signaling," as used herein,
refers to a decrease in Notch2 signaling that is significantly
below the level of Notch2 signaling observed in a control under
substantially identical conditions. In certain embodiments, the
decrease in Notch2 signaling is at least two fold, three fold, four
fold, five fold, or ten fold below the level observed in the
control.
[0056] In certain embodiments, Notch2 signaling (i.e., increased or
decreased Notch2 signaling) is assessed using a suitable reporter
assay, e.g, as described in U.S. Patent Application Publication No.
US 2010/0080808 A1.
[0057] The term "anti-Notch2 antibody" or "an antibody that binds
to Notch2" refers to an antibody that is capable of binding Notch2
with sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting Notch2.
Preferably, the extent of binding of an anti-Notch2 antibody to an
unrelated, non-Notch protein is less than about 10% of the binding
of the antibody to Notch2 as measured, e.g., by a radioimmunoassay
(RIA). In certain embodiments, an antibody that binds to Notch2 has
a dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.0.5 .mu.M,
.ltoreq.100 nM, .ltoreq.50 nM, .ltoreq.10 nM, .ltoreq.5 nM,
.ltoreq.1 nM, .ltoreq.0.5 nM, or .ltoreq.0.1 nM. In certain
embodiments, an anti-Notch2 antibody binds to an epitope of Notch2
that is conserved among Notch2 from different species, e.g.,
rodents (mice, rats) and primates.
[0058] The term "anti-Notch2 NRR antibody" or "an antibody that
binds to Notch2 NRR" refers to an antibody that is capable of
binding Notch2 NRR with sufficient affinity such that the antibody
is useful as a diagnostic and/or therapeutic agent in targeting
Notch2. Preferably, the extent of binding of an anti-Notch2 NRR
antibody to an unrelated, non-Notch protein is less than about 10%
of the binding of the antibody to Notch2 NRR as measured, e.g., by
a radioimmunoassay (RIA). In certain embodiments, an antibody that
binds to Notch2 NRR has a dissociation constant (Kd) of .ltoreq.1
.mu.M, .ltoreq.0.5 .mu.M, .ltoreq.100 nM, .ltoreq.50 nM, .ltoreq.10
nM, .ltoreq.5 nM, .ltoreq.1 nM, .ltoreq.0.5 nM, or .ltoreq.0.1 nM.
In certain embodiments, an anti-Notch2 NRR antibody binds to an
epitope of Notch that is conserved among Notch from different
species, e.g., rodents (mice, rats) and primates.
[0059] The term "Notch2-specific antagonist" refers to an agent
that effects decreased Notch2 signaling, as defined above, and does
not significantly affect signaling by another Notch receptor
(Notch1, 3, or 4 in mammals).
[0060] An "anti-Notch2 antagonist antibody" is an anti-Notch2
antibody (including an anti-Notch2 NRR antibody) that effects
decreased Notch2 signaling, as defined above.
[0061] The term "antagonist" refers to an agent that significantly
inhibits (either partially or completely) the biological activity
of a target molecule.
[0062] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies, polyclonal antibodies,
multispecific antibodies (e.g. bispecific antibodies) formed from
at least two intact antibodies, and antibody fragments so long as
they exhibit the desired biological activity.
[0063] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with research, diagnostic or
therapeutic uses for the antibody, and may include enzymes,
hormones, and other proteinaceous or nonproteinaceous solutes. In
some embodiments, an antibody is purified (1) to greater than 95%
by weight of antibody as determined by, for example, the Lowry
method, and in some embodiments, to greater than 99% by weight; (2)
to a degree sufficient to obtain at least 15 residues of N-terminal
or internal amino acid sequence by use of, for example, a spinning
cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or
nonreducing conditions using, for example, Coomassie blue or silver
stain. Isolated antibody includes the antibody in situ within
recombinant cells since at least one component of the antibody's
natural environment will not be present. Ordinarily, however,
isolated antibody will be prepared by at least one purification
step.
[0064] "Native antibodies" are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies among the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (V.sub.H) followed by
a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) and a constant domain at its other end;
the constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light chain and heavy chain variable domains.
[0065] The "variable region" or "variable domain" of an antibody
refers to the amino-terminal domains of the heavy or light chain of
the antibody. The variable domain of the heavy chain may be
referred to as "VH." The variable domain of the light chain may be
referred to as "VL." These domains are generally the most variable
parts of an antibody and contain the antigen-binding sites.
[0066] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions (HVRs) both in the light-chain and the
heavy-chain variable domains. The more highly conserved portions of
variable domains are called the framework regions (FR). The
variable domains of native heavy and light chains each comprise
four FR regions, largely adopting a beta-sheet configuration,
connected by three HVRs, which form loops connecting, and in some
cases forming part of, the beta-sheet structure. The HVRs in each
chain are held together in close proximity by the FR regions and,
with the HVRs from the other chain, contribute to the formation of
the antigen-binding site of antibodies (see Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, National
Institute of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in the binding of an antibody to an
antigen, but exhibit various effector functions, such as
participation of the antibody in antibody-dependent cellular
toxicity.
[0067] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains.
[0068] Depending on the amino acid sequences of the constant
domains of their heavy chains, antibodies (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., IgG.sub.1,
IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and IgA.sub.2. The
heavy chain constant domains 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 and described generally in, for
example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B.
Saunders, Co., 2000). An antibody may be part of a larger fusion
molecule, formed by covalent or non-covalent association of the
antibody with one or more other proteins or peptides.
[0069] The terms "full length antibody," "intact antibody" and
"whole antibody" are used herein interchangeably to refer to an
antibody in its substantially intact form, not antibody fragments
as defined below. The terms particularly refer to an antibody with
heavy chains that contain an Fc region.
[0070] A "naked antibody" for the purposes herein is an antibody
that is not conjugated to a cytotoxic moiety or radiolabel.
[0071] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen binding region thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments.
[0072] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen.
[0073] "Fv" is the minimum antibody fragment which contains a
complete antigen-binding site. In one embodiment, a two-chain Fv
species consists of a dimer of one heavy- and one light-chain
variable domain in tight, non-covalent association. In a
single-chain Fv (scFv) species, one heavy- and one light-chain
variable domain can be covalently linked by a flexible peptide
linker such that the light and heavy chains can associate in a
"dimeric" structure analogous to that in a two-chain Fv species. It
is in this configuration that the three HVRs of each variable
domain interact to define an antigen-binding site on the surface of
the VH-VL dimer. Collectively, the six HVRs confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three HVRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0074] The Fab fragment contains the heavy- and light-chain
variable domains and also contains the constant domain of the light
chain and the first constant domain (CH1) of the heavy chain. Fab'
fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0075] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Generally, the scFv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv, see, e.g., Pluckthun, in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York, 1994), pp. 269-315.
[0076] The term "diabodies" refers to antibody fragments with two
antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (V.sub.H-V.sub.L). By using a
linker that is too short to allow pairing between the two domains
on the same chain, the domains are forced to pair with the
complementary domains of another chain and create two
antigen-binding sites. Diabodies may be bivalent or bispecific.
Diabodies are described more fully in, for example, EP 404,097; WO
1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and
Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).
Triabodies and tetrabodies are also described in Hudson et al.,
Nat. Med. 9:129-134 (2003).
[0077] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible mutations, e.g.,
naturally occurring mutations, that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies. In
certain embodiments, such a monoclonal antibody typically includes
an antibody comprising a polypeptide sequence that binds a target,
wherein the target-binding polypeptide sequence was obtained by a
process that includes the selection of a single target binding
polypeptide sequence from a plurality of polypeptide sequences. For
example, the selection process can be the selection of a unique
clone from a plurality of clones, such as a pool of hybridoma
clones, phage clones, or recombinant DNA clones. It should be
understood that a selected target binding sequence can be further
altered, for example, to improve affinity for the target, to
humanize the target binding sequence, to improve its production in
cell culture, to reduce its immunogenicity in vivo, to create a
multispecific antibody, etc., and that an antibody comprising the
altered target binding sequence is also a monoclonal antibody of
this invention. In contrast to polyclonal antibody preparations,
which typically include different antibodies directed against
different determinants (epitopes), each monoclonal antibody of a
monoclonal antibody preparation is directed against a single
determinant on an antigen. In addition to their specificity,
monoclonal antibody preparations are advantageous in that they are
typically uncontaminated by other immunoglobulins.
[0078] The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including, for
example, the hybridoma method (e.g., Kohler and Milstein, Nature,
256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995),
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal
Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)),
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),
phage-display technologies (see, e.g., Clackson et al., Nature,
352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597
(1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et
al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl.
Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J.
Immunol. Methods 284(1-2): 119-132 (2004), and technologies for
producing human or human-like antibodies in animals that have parts
or all of the human immunoglobulin loci or genes encoding human
immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096;
WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad.
Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258
(1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg
et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813
(1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996);
Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and
Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
[0079] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc.
Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies
include PRIMATIZED.RTM. antibodies wherein the antigen-binding
region of the antibody is derived from an antibody produced by,
e.g., immunizing macaque monkeys with the antigen of interest.
[0080] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. In one embodiment, a humanized antibody
is a human immunoglobulin (recipient antibody) in which residues
from a HVR of the recipient are replaced by residues from a HVR of
a non-human species (donor antibody) such as mouse, rat, rabbit, or
nonhuman primate having the desired specificity, affinity, and/or
capacity. In some instances, FR residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues that are
not found in the recipient antibody or in the donor antibody. These
modifications may be made to further refine antibody performance.
In general, a humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin, and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally will also comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see, e.g., Jones et al.,
Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329
(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See
also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma &
Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions
23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433
(1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.
[0081] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues. Human antibodies can be
produced using various techniques known in the art, including
phage-display libraries. Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also
available for the preparation of human monoclonal antibodies are
methods described in Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,
147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr.
Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be
prepared by administering the antigen to a transgenic animal that
has been modified to produce such antibodies in response to
antigenic challenge, but whose endogenous loci have been disabled,
e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and
6,150,584 regarding XENOMOUSE.TM. technology). See also, for
example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562
(2006) regarding human antibodies generated via a human B-cell
hybridoma technology.
[0082] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six HVRs; three in the VH
(H1, H2, H3), and three in the VL (L1, L2, L3). In native
antibodies, H3 and L3 display the most diversity of the six HVRs,
and H3 in particular is believed to play a unique role in
conferring fine specificity to antibodies. See, e.g., Xu et al.,
Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular
Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).
Indeed, naturally occurring camelid antibodies consisting of a
heavy chain only are functional and stable in the absence of light
chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448
(1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).
[0083] A number of HVR delineations are in use and are encompassed
herein. The Kabat Complementarity Determining Regions (CDRs) are
based on sequence variability and are the most commonly used (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Chothia refers instead to the location of the structural
loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM
HVRs represent a compromise between the Kabat HVRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software. The "contact" HVRs are based on an analysis of
the available complex crystal structures. The residues from each of
these HVRs are noted below.
TABLE-US-00001 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0084] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34
(L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and
26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3)
in the VH. The variable domain residues are numbered according to
Kabat et al., supra, for each of these definitions.
[0085] "Framework" or "FR" residues are those variable domain
residues other than the HVR residues as herein defined.
[0086] The term "variable domain residue numbering as in Kabat" or
"amino acid position numbering as in Kabat," and variations
thereof, refers to the numbering system used for heavy chain
variable domains or light chain variable domains of the compilation
of antibodies in Kabat et al., supra. Using this numbering system,
the actual linear amino acid sequence may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FR or HVR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0087] The Kabat numbering system is generally used when referring
to a residue in the variable domain (approximately residues 1-107
of the light chain and residues 1-113 of the heavy chain) (e.g.,
Kabat et al., supra). The "EU numbering system" or "EU index" is
generally used when referring to a residue in an immunoglobulin
heavy chain constant region (e.g., the EU index reported in Kabat
et al., supra). The "EU index as in Kabat" refers to the residue
numbering of the human IgG1 EU antibody. Unless stated otherwise
herein, references to residue numbers in the variable domain of
antibodies means residue numbering by the Kabat numbering system.
Unless stated otherwise herein, references to residue numbers in
the constant domain of antibodies means residue numbering by the EU
numbering system (e.g., see United States Patent Application
Publication US 2008/0181888 A1, Figures for EU numbering).
[0088] An "affinity matured" antibody is one with one or more
alterations in one or more HVRs thereof which result in an
improvement in the affinity of the antibody for antigen, compared
to a parent antibody which does not possess those alteration(s). In
one embodiment, an affinity matured antibody has nanomolar or even
picomolar affinities for the target antigen. Affinity matured
antibodies may be produced using certain procedures known in the
art. For example, Marks et al. Bio/Technology 10:779-783 (1992)
describe affinity maturation by VH and VL domain shuffling. Random
mutagenesis of HVR and/or framework residues is described by, for
example, in Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813
(1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J.
Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol.
154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896
(1992).
[0089] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: C1q binding and complement dependent
cytotoxicity (CDC); Fc receptor binding; antibody-dependent
cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of
cell surface receptors (e.g. B cell receptor); and B cell
activation.
[0090] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Low-affinity antibodies generally
bind antigen slowly and tend to dissociate readily, whereas
high-affinity antibodies generally bind antigen faster and tend to
remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for
purposes of the present invention. Specific illustrative and
exemplary embodiments for measuring binding affinity are described
in the following.
[0091] In one embodiment, the "Kd" or "Kd value" according to this
invention is measured by a radiolabeled antigen binding assay (RIA)
performed with the Fab version of an antibody of interest and its
antigen as described by the following assay. Solution binding
affinity of Fabs for antigen is measured by equilibrating Fab with
a minimal concentration of (.sup.125I)-labeled antigen in the
presence of a titration series of unlabeled antigen, then capturing
bound antigen with an anti-Fab antibody-coated plate (see, e.g.,
Chen, et al., J. Mol. Biol. 293:865-881 (1999)). To establish
conditions for the assay, MICROTITER.RTM. multi-well plates (Thermo
Scientific) are coated overnight with 5 .mu.g/ml of a capturing
anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6),
and subsequently blocked with 2% (w/v) bovine serum albumin in PBS
for two to five hours at room temperature (approximately 23.degree.
C.). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM
[.sup.125I]-antigen are mixed with serial dilutions of a Fab of
interest (e.g., consistent with assessment of the anti-VEGF
antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599
(1997)). The Fab of interest is then incubated overnight; however,
the incubation may continue for a longer period (e.g., about 65
hours) to ensure that equilibrium is reached. Thereafter, the
mixtures are transferred to the capture plate for incubation at
room temperature (e.g., for one hour). The solution is then removed
and the plate washed eight times with 0.1% TWEEN-20.TM. in PBS.
When the plates have dried, 150 .mu.l/well of scintillant
(MICROSCINT-20.TM.; Packard) is added, and the plates are counted
on a TOPCOUNT.TM. gamma counter (Packard) for ten minutes.
Concentrations of each Fab that give less than or equal to 20% of
maximal binding are chosen for use in competitive binding assays.
According to another embodiment, the Kd or Kd value is measured by
using surface plasmon resonance assays using a BIACORE.RTM.-2000 or
a BIACORE.RTM.-3000 (BIAcore, Inc., Piscataway, N.J.) at 25.degree.
C. with immobilized antigen CM5 chips at .about.10 response units
(RU). Briefly, carboxymethylated dextran biosensor chips (CM5,
BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% TWEEN-20.TM. surfactant (PBST) at
25.degree. C. at a flow rate of approximately 25 .mu.l/min.
Association rates (k.sub.on) and dissociation rates (k.sub.off) are
calculated using a simple one-to-one Langmuir binding model
(BIACORE.RTM. Evaluation Software version 3.2) by simultaneously
fitting the association and dissociation sensorgrams. The
equilibrium dissociation constant (Kd) is calculated as the ratio
k.sub.off/k.sub.on. See, e.g., Chen et al., J. Mol. Biol.
293:865-881 (1999). If the on-rate exceeds 10.sup.6 M.sup.-1
s.sup.-1 by the surface plasmon resonance assay above, then the
on-rate can be determined by using a fluorescent quenching
technique that measures the increase or decrease in fluorescence
emission intensity (excitation=295 nm; emission=340 nm, 16 nm
band-pass) at 25.degree. C. of a 20 nM anti-antigen antibody (Fab
form) in PBS, pH 7.2, in the presence of increasing concentrations
of antigen as measured in a spectrometer, such as a stop-flow
equipped spectrophometer (Aviv Instruments) or a 8000-series
SLM-AMINCO.TM. spectrophotometer (ThermoSpectronic) with a stirred
cuvette.
[0092] An "on-rate," "rate of association," "association rate," or
"k.sub.on" according to this invention can also be determined as
described above using a BIACORE.RTM.-2000 or a BIACORE.RTM.-3000
system (BIAcore, Inc., Piscataway, N.J.).
[0093] The term "substantially similar" or "substantially the
same," as used herein, denotes a sufficiently high degree of
similarity between two numeric values (for example, one associated
with an antibody of the invention and the other associated with a
reference/comparator antibody), such that one of skill in the art
would consider the difference between the two values to be of
little or no biological and/or statistical significance within the
context of the biological characteristic measured by said values
(e.g., Kd values). The difference between said two values is, for
example, less than about 50%, less than about 40%, less than about
30%, less than about 20%, and/or less than about 10% as a function
of the reference/comparator value.
[0094] The phrase "substantially reduced," or "substantially
different," as used herein, denotes a sufficiently high degree of
difference between two numeric values (generally one associated
with a molecule and the other associated with a
reference/comparator molecule) such that one of skill in the art
would consider the difference between the two values to be of
statistical significance within the context of the biological
characteristic measured by said values (e.g., Kd values). The
difference between said two values is, for example, greater than
about 10%, greater than about 20%, greater than about 30%, greater
than about 40%, and/or greater than about 50% as a function of the
value for the reference/comparator molecule.
[0095] An "acceptor human framework" or a "human acceptor
framework" for the purposes herein is a framework comprising the
amino acid sequence of a VL or VH framework derived from a human
immunoglobulin framework or a human consensus framework. An
acceptor human framework "derived from" a human immunoglobulin
framework or a human consensus framework may comprise the same
amino acid sequence thereof, or it may contain pre-existing amino
acid sequence changes. In some embodiments, the number of
pre-existing amino acid changes are 10 or less, 9 or less, 8 or
less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2
or less. Where pre-existing amino acid changes are present in a VH,
preferably those changes occur at only three, two, or one of
positions 71H, 73H and 78H; for instance, the amino acid residues
at those positions may be 71A, 73T and/or 78A. In one embodiment,
the VL acceptor human framework is identical in sequence to the VL
human immunoglobulin framework sequence or human consensus
framework sequence.
[0096] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., supra. In
one embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
[0097] A "VH subgroup III consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable heavy subgroup III of Kabat et al., supra. In one
embodiment, a human acceptor framework is derived from the VH
subgroup III consensus framework and comprises an amino acid
sequence comprising at least a portion or all of each of the
following sequences: (SEQ ID NO:50)-H1-(SEQ ID NO:51)-H2-(SEQ ID
NO:57 or 59)-H3-(SEQ ID NO: 35). In some embodiments, the last
residue (S11) of SEQ ID NO:35 is substituted with an alanine
[0098] A "VL subgroup I consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable light kappa subgroup I of Kabat et al., supra. In one
embodiment, the VH subgroup I consensus framework amino acid
sequence comprises at least a portion or all of each of the
following sequences: (SEQ ID NO:60)-L1-(SEQ ID NO:61)-L2-(SEQ ID
NO:62)-L3-(SEQ ID NO:63).
[0099] A "disorder" is any condition or disease that would benefit
from treatment with a composition or method of the invention. This
includes chronic and acute disorders including those pathological
conditions which predispose the mammal to the disorder in question.
Non-limiting examples of disorders to be treated herein include
conditions such as cancer.
[0100] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer.
[0101] "Tumor," as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. The terms "cancer,"
"cancerous," "cell proliferative disorder," "proliferative
disorder," and "tumor" are not mutually exclusive as referred to
herein.
[0102] A cancer that "responds" to a therapeutic agent is one that
shows a significant decrease in cancer or tumor progression,
including but not limited to, (1) inhibition, to some extent, of
tumor growth, including slowing down and complete growth arrest;
(2) reduction in the number of cancer or tumor cells; (3) reduction
in tumor size; (4) inhibition (i.e., reduction, slowing down or
complete stopping) of cancer cell infiltration into adjacent
peripheral organs and/or tissues; and/or (5) inhibition (i.e.
reduction, slowing down or complete stopping) of metastasis.
[0103] As used herein, "treatment" (and variations such as "treat"
or "treating") refers to clinical intervention in an attempt to
alter the natural course of the individual or cell being treated,
and can be performed either for prophylaxis or during the course of
clinical pathology. Desirable effects of treatment include
preventing occurrence or recurrence of disease, alleviation of
symptoms, diminishment of any direct or indirect pathological
consequences of the disease, preventing metastasis, decreasing the
rate of disease progression, amelioration or palliation of the
disease state, and remission or improved prognosis. In some
embodiments, Notch2 antagonists of the invention are used to delay
development of a disease or disorder or to slow the progression of
a disease or disorder.
[0104] An "individual," "subject," or "patient" is a vertebrate. In
certain embodiments, the vertebrate is a mammal. Mammals include,
but are not limited to, farm animals (such as cows), sport animals,
pets (such as cats, dogs, and horses), primates, mice and rats. In
certain embodiments, a mammal is a human.
[0105] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of the active ingredient to be effective, and which
contains no additional components which are unacceptably toxic to a
subject to which the formulation would be administered. Such
formulations may be sterile.
[0106] An "effective amount" refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result.
[0107] The term "progenitor," "hepatic progenitor," "liver
progenitor" or "oval cell" refers to small epithelial cells that
can differentiate into both hepatocytes and intra-hepatic bile duct
cells.
II. Embodiments of the Invention
[0108] The present invention relates to the treatment of liver
conditions using Notch2 antagonists. The present invention is
based, in part, on the observation that anti-Notch2 NRR antibodies
(a) improve liver appearance and hepatocyte function in an acute
liver damage model in vivo and (b) reduce biliary damage and
improve hepatocyte function in a chronic liver damage model in
vivo. Without being bound by any particular theory or operation,
the Notch2 antagonist might improve liver conditions by promoting
hepatocyte differentiation and/or by decreasing aberrant bile duct
proliferation.
[0109] In various aspects of the invention, a method of treating a
liver condition characterized by liver damage is provided, the
method comprising administering to a patient having such condition
an effective amount of a Notch2-specific antagonist. In certain
embodiments, the liver condition is chronic liver disease,
including but not limited to fibrosis, cirrhosis, viral hepatitis
(e.g., hepatitis A, B, C, D, E, or G), autoimmune liver diseases
(e.g., autoimmune hepatitis, primary biliary cirrhosis, or primary
sclerosing cholangitis), genetic liver diseases (e.g., alpha-1
antitrypsin deficiency, Crigler-Najjar syndrome, familial
amyloidosis, Gilbert's syndrome, Dubin-Johnson syndrome, hereditary
hemchromatosis, primary oxalosis, or Wilson's disease), alcoholic
hepatitis or nonalcoholic fatty liver disease. In certain
embodiments, the liver condition is an acute liver condition, such
as acute liver failure, acute liver injury, or acute liver
toxicity, e.g., acetaminophen toxicity. In certain embodiments, the
liver condition is liver cancer, e.g., hepatocellular carcinoma
(HCC), intrahepatic cholangiocarcinoma (bile duct cancer), or
hepatoblastoma.
[0110] In some embodiments, treatment results in improved liver
histological appearance, including but not limited to, e.g., larger
cell size, lower nuclear-to-cytoplasmic ratio, two nuclei, as
compared to cell size, nuclear-to-cytoplasmic ration and nuclei
number in cultured adult oval cells. In some embodiments, treatment
results in a more differentiated morphology, e.g., as compared to
morphology of cultured adult oval cells.
[0111] In some embodiments, treatment results in decreased
expression of Keratin-19 biomarker in liver cells, e.g., decreased
expression relative to expression of Keratin-19 biomarker in
cultured adult oval cells. Methods for detecting keratin-19
biomarker (e.g., Keratin-19 gene expression, e.g., mRNA expression)
are well known in the art and are also exemplified herein.
[0112] In some embodiments, treatment results in increased
expression of albumin and AFP e.g., increased expression relative
to expression of albumin and/or AFP biomarkers in cultured adult
oval cells. Methods for detecting albumin and/or AFP biomarkers
(e.g., gene expression, e.g., mRNA expression) are well known in
the art and are also exemplified herein.
[0113] In some embodiments, treatment results in a reduced number
of Hes1 positive intrahepatic bile duct cells.
[0114] In some embodiment, treatment results in reduced liver
progenitor cells (e.g., adult liver oval cell) proliferation within
the bile ducts. Reduced proliferation may be determined, e.g., by
determining average cross-sectional area of K19-positive tissue as
compared to the total liver cross sectional area.
[0115] In some embodiments, treatment results in improved
hepatocyte function. Hepatocyte function may be measured by methods
known in the art, including but not limited to: no significant
elevation of biomarkers associated with biliary dysfunction, such
as those biomarkers described in FIG. 11. In some embodiments, a
biomarker associated with biliary dysfunction is total and/or
direct serum bilirubin level. In some embodiments, a biomarker
associated with biliary dysfunction is the differentiation
quotient, as further described and exemplified herein.
[0116] In some embodiments, improved hepatocyte function is
determined, e.g., by assessment of heptobiliary function
biomarkers, including but not limited to the serum heptobiliary
function biomarker described in FIGS. 2 and 5. In some embodiments,
serum heptobiliary function biomarker is serum albumin level.
[0117] In some embodiments, improved hepatocyte function is
increased rate of recovery of liver function.
[0118] The invention also provides methods for promoting hepatocyte
differentiation and/or by decreasing aberrant bile duct
proliferation, the method comprising administering to a patient in
need of such treatment an effective amount of a Notch2-specific
antagonist. In some embodiments, the patient has a liver condition
characterized by liver damage. In certain embodiments, the liver
condition is chronic liver disease, including but not limited to
fibrosis, cirrhosis, viral hepatitis (e.g., hepatitis A, B, C, D,
E, or G), autoimmune liver diseases (e.g., autoimmune hepatitis,
primary biliary cirrhosis, or primary sclerosing cholangitis),
genetic liver diseases (e.g., alpha-1 antitrypsin deficiency,
Crigler-Najjar syndrome, familial amyloidosis, Gilbert's syndrome,
Dubin-Johnson syndrome, hereditary hemchromatosis, primary
oxalosis, or Wilson's disease), alcoholic hepatitis or nonalcoholic
fatty liver disease. In certain embodiments, the liver condition is
an acute liver condition, such as acute liver failure, acute liver
injury, or acute liver toxicity, e.g., acetaminophen toxicity. In
certain embodiments, the liver condition is liver cancer, e.g.,
hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma
(bile duct cancer), or hepatoblastoma. In some embodiment,
treatment results in reduced liver progenitor cell (e.g., adult
liver oval cell) proliferation. Reduced proliferation may be
determined, e.g., by determining average cross-sectional area of
K19 positive tissue as compared to the total liver cross sectional
area.
[0119] The invention also provides methods for improving liver
histological appearance, the method comprising administering to a
patient in need of such treatment an effective amount of a
Notch2-specific antagonist. In some embodiments, treatment results
in improved liver histological appearance, including but not
limited to: larger cell size, lower nuclear-to-cytoplasmic ratio,
two nucleic, e.g., as compared to cell size, nuclear-to-cytoplasmic
ratio and nuclei number in cultured adult oval cells. In some
embodiments, treatment results in a more differentiated morphology,
e.g., as compared to morphology of cultured adult oval cells.
[0120] In some embodiments, treatment results in decreased
expression of Keratin-19 biomarker in liver cells, e.g., decreased
expression relative to expression of Keratin-19 biomarker in
cultured adult oval cells. Methods for detecting keratin-19
biomarker (e.g., Keratin-19 gene expression, e.g., mRNA expression)
are well known in the art and are also exemplified herein.
[0121] In some embodiments, treatment results in increased
expression of albumin and AFP e.g., increased expression relative
to expression of albumin and/or AFP biomarkers in cultured adult
oval cells. Methods for detecting albumin and/or AFP biomarkers
(e.g., gene expression, e.g., mRNA expression) are well known in
the art and are also exemplified herein.
[0122] In some embodiments, treatment results in reduced number of
Hes1 positive intrahepatic bile duct cells.
[0123] The invention also provides methods for reducing serum bile
acids, serum bilirubin, serum alkaline phosphatase, serum ALT,
and/or serum AST following hepatic injury, the method comprising
administering to a patient in need thereof an effective amount of a
Notch2-specific antagonist.
[0124] The invention also provides methods for reducing the number
of CK19-positive cells in cell population that comprises an oval
cell, the method comprising the step of contacting the oval cell
with a Notch2-specific antagonist.
[0125] The invention also provides methods for reducing the
expression or secretion of bile acids, bilirubin, alkaline
phosphatase, ALT, and/or AST, the method comprising contacting an
oval cell with an effective amount of a Notch2-specific
antagonist.
[0126] The invention provides methods for identifying a patient
eligible for receiving treatment of a liver condition characterized
by liver damage by administering to a patient having such condition
an effective amount of a Notch2-specific antagonist, the method
comprising determining expression of one or more of the genes
listed in Table 2 in a sample obtained from the patient. In some
embodiments, the genes belong to the Notch pathway, e.g., JAG1. In
some embodiments, a sample or biopsy from the patient is analyzed
for mRNA expression of one of the genes listed in Table 1 using
methods well known in the art, such as, e.g., quantitative PCR
analysis, and compared to expression of the same gene or genes in a
biopsy obtained from a control individual or compared to a
reference value. In some embodiments, elevated expression of one or
more genes listed in Table 1 in the biopsy obtained from the
patient, relative to the control, identifies the patient as
suitable for receiving treatment with a Notch2-specific antagonist,
as described herein. In some embodiments, additional parameters,
such as, e.g., examination by a physician, histologic evaluation of
a biopsy, determination of serum levels indicative of liver damage,
etc. are employed to identify the patient for receiving the
treatment. Also, elevated hepatic expression by a patient of one or
more of the genes identified in Table 2 is specifically
contemplated as one possible embodiment of any of the methods
provided herein.
[0127] In some embodiments patients are selected for treatment with
a Notch2-specific antagonist as described herein by measuring other
known markers of oval cells or aggressive HCC (see, e.g., Woo et al
2011 Mol. Carcinog. 2011 April; 50(4):235-43). In some embodiments,
patients are selected for treatment by analyzing hepatic Notch2
activation, for example by detection of the activated form of
Notch2 as described herein.
[0128] Examples of Notch2-specific antagonists include, but are not
limited to, soluble Notch receptors, soluble Notch ligand variants,
e.g., dominant negative ligand variants, aptamers or oligopeptides
that bind Notch2 or Notch2 ligands, organic or inorganic molecules
that interfere specifically with Notch2 signaling, anti-Notch2
antagonist antibodies and anti-Notch2 ligand antagonist antibodies.
Examples of Notch2-specific antagonists include those described in
U.S. Patent Application Publication No. US 2010/0111958.
[0129] In certain embodiments, the Notch2-specific antagonist is an
anti-Notch2 antagonist antibody. In one such embodiment, the
anti-Notch2 antagonist antibody is an antibody that binds to the
extracellular domain of Notch2 and effects decreased Notch2
signaling. In one such embodiment, the anti-Notch2 antagonist
antibody is an anti-Notch2 NRR antibody. Anti-Notch2 NRR antibodies
include, but are not limited to, any of the anti-Notch2 NRR
antibodies disclosed in U.S. Patent Application Publication No. US
2010/0080808 A1, which is expressly incorporated by reference
herein in its entirety. Such antibodies include, but are not
limited to anti-Notch2 NRR antibodies that bind to the LNR-A and
HD-C domains of Notch2 NRR. Exemplary anti-Notch2 NRR antibodies
are monoclonal antibodies designated Antibody D, Antibody D-1,
Antibody D-2, or Antibody D-3 that were derived from a phage
library, as disclosed in US 2010/0080808. Antibody D that binds to
Notch2 NRR was isolated. That antibody was affinity matured to
generate Antibody D-1, Antibody D-2, and Antibody D-3. The
sequences of the heavy chain and light chain hypervariable regions
(HVRs) of Antibody D, Antibody D-1, Antibody D-2, and Antibody D-3
are shown in FIGS. 12 and 13. The sequences of the heavy and light
chain variable domains of Antibody D, Antibody D-1, Antibody D-2,
and Antibody D-3 are shown in FIGS. 14 and 15. Further embodiments
of anti-Notch2 NRR antibodies are provided as follows.
[0130] In one aspect, an antagonist antibody that specifically
binds to Notch2 NRR is provided, wherein the antibody comprises at
least one, two, three, four, five, or six HVRs selected from:
[0131] (a) an HVR-H1 comprising an amino acid sequence that
conforms to the consensus sequence of SEQ ID NO:3; [0132] (b) an
HVR-H2 comprising the amino acid sequence of SEQ ID NO:4; [0133]
(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5;
[0134] (d) an HVR-L1 comprising an amino acid sequence that
conforms to the consensus sequence of SEQ ID NO:10; [0135] (e) an
HVR-L2 comprising an amino acid sequence that conforms to the
consensus sequence of SEQ ID NO:14; and [0136] (f) an HVR-L3
comprising an amino acid sequence that conforms to the consensus
sequence of SEQ ID NO:19.
[0137] In a further aspect, the antibody comprises an HVR-H3
comprising the amino acid sequence of SEQ ID NO:5 and at least one,
two, three, four, or five HVRs selected from (a), (b), (d), (e),
and (f) above. In a further aspect, the antibody comprises (a),
(b), (c), (d), (e), and (f) above. With respect to (a), (d), (e),
and (f), any one or more of the following embodiments are
contemplated: HVR-H1 comprises an amino acid sequence selected from
SEQ ID NOs:1-2; HVR-L1 comprises an amino acid sequence selected
from SEQ ID NOs:6-9; HVR-L2 comprises an amino acid sequence
selected from SEQ ID NOs:11-13; and HVR-L3 comprises an amino acid
sequence selected from SEQ ID NOs:15-18.
[0138] In another aspect, an antibody that specifically binds to
Notch2 NRR is provided, wherein the antibody comprises an HVR-H1
comprising an amino acid sequence that conforms to the consensus
sequence of SEQ ID NO:3, an HVR-H2 comprising the amino acid
sequence of SEQ ID NO:4, and an HVR-H3 comprising the amino acid
sequence of SEQ ID NO:5. In one embodiment, HVR-H1 comprises an
amino acid sequence selected from SEQ ID NOs:1-2.
[0139] In another aspect, an antibody that specifically binds to
Notch2 NRR is provided, wherein the antibody comprises an HVR-L1
comprising an amino acid sequence that conforms to the consensus
sequence of SEQ ID NO:10, an HVR-L2 comprising an amino acid
sequence that conforms to the consensus sequence of SEQ ID NO:14,
and an HVR-L3 comprising an amino acid sequence that conforms to
the consensus sequence of SEQ ID NO:19. The following embodiments
are contemplated in any combination: HVR-L1 comprises an amino acid
sequence selected from SEQ ID NOs:6-9; HVR-L2 comprises an amino
acid sequence selected from SEQ ID NOs:11-13; and HVR-L3 comprises
an amino acid sequence selected from SEQ ID NOs:15-18. In one
embodiment, an antibody that binds to Notch2 NRR comprises an
HVR-L1 comprising the amino acid sequence of SEQ ID NO:6; an HVR-L2
comprising the amino acid sequence of SEQ ID NO:11; and an HVR-L3
comprising the amino acid sequence of SEQ ID NO:15. In another
embodiment, an antibody that binds to Notch2 NRR comprises an
HVR-L1 comprising the amino acid sequence of SEQ ID NO:7; an HVR-L2
comprising the amino acid sequence of SEQ ID NO:11; and an HVR-L3
comprising the amino acid sequence of SEQ ID NO:16. In another
embodiment, an antibody that binds to Notch2 NRR comprises an
HVR-L1 comprising the amino acid sequence of SEQ ID NO:8; an HVR-L2
comprising the amino acid sequence of SEQ ID NO:12; and an HVR-L3
comprising the amino acid sequence of SEQ ID NO:17. In another
embodiment, an antibody that binds to Notch2 NRR comprises an
HVR-L1 comprising the amino acid sequence of SEQ ID NO:9; an HVR-L2
comprising the amino acid sequence of SEQ ID NO:13; and an HVR-L3
comprising the amino acid sequence of SEQ ID NO:18.
[0140] In one embodiment, an antibody that specifically binds to
Notch2 NRR is provided, wherein the antibody comprises: [0141] (a)
an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; [0142]
(b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;
[0143] (c) an HVR-H3 comprising the amino acid sequence of SEQ ID
NO:5; [0144] (d) an HVR-L1 comprising the amino acid sequence of
SEQ ID NO:6; [0145] (e) an HVR-L2 comprising the amino acid
sequence of SEQ ID NO:11; and [0146] (f) an HVR-L3 comprising the
amino acid sequence of SEQ ID NO:15.
[0147] In another embodiment, an antibody that specifically binds
to Notch2 NRR is provided, wherein the antibody comprises: [0148]
(a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2;
[0149] (b) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4; [0150] (c) an HVR-H3 comprising the amino acid sequence of
SEQ ID NO:5; [0151] (d) an HVR-L1 comprising the amino acid
sequence of SEQ ID NO:7; [0152] (e) an HVR-L2 comprising the amino
acid sequence of SEQ ID NO:11; and [0153] (f) an HVR-L3 comprising
the amino acid sequence of SEQ ID NO:16.
[0154] In another embodiment, an antibody that specifically binds
to Notch2 NRR is provided, wherein the antibody comprises: [0155]
(a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2;
[0156] (b) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4; [0157] (c) an HVR-H3 comprising the amino acid sequence of
SEQ ID NO:5; [0158] (d) an HVR-L1 comprising the amino acid
sequence of SEQ ID NO:8; [0159] (e) an HVR-L2 comprising the amino
acid sequence of SEQ ID NO:12; and [0160] (f) an HVR-L3 comprising
the amino acid sequence of SEQ ID NO:17.
[0161] In another embodiment, an antibody that specifically binds
to Notch2 NRR is provided, wherein the antibody comprises: [0162]
(a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2;
[0163] (b) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4; [0164] (c) an HVR-H3 comprising the amino acid sequence of
SEQ ID NO:5; [0165] (d) an HVR-L1 comprising the amino acid
sequence of SEQ ID NO:9; [0166] (e) an HVR-L2 comprising the amino
acid sequence of SEQ ID NO:13; and [0167] (f) an HVR-L3 comprising
the amino acid sequence of SEQ ID NO:18.
[0168] In certain embodiments, any of the above antibodies further
comprises at least one framework selected from a VH subgroup III
consensus framework and a VL subgroup I consensus framework.
[0169] In certain embodiments, an anti-Notch2 NRR antibody is
affinity matured. For example, any one or more of the following
substitutions in the indicated HVR positions (Kabat numbered) may
be made in any combination: [0170] in HVR-H1 (SEQ ID NO:1): S28T;
T30S; [0171] in HVR-L1 (SEQ ID NO:6): S28N; 129N or V; S30R or K;
S31R; Y32F [0172] in HVR-L2 (SEQ ID NO:11): G50R; S53I or T; A55E
[0173] in HVR-L3 (SEQ ID NO:15): S93I or R; L96W or H The specific
antibodies disclosed herein, i.e., Antibody D as well as affinity
matured forms of Antibody D (D-1, D-2, and D-3), may undergo
further affinity maturation. Accordingly, affinity matured forms of
any of the antibodies described herein are provided.
[0174] In certain embodiments, an anti-Notch2 NRR antibody having
any of the above HVR sequences can further comprise any suitable
framework variable domain sequence, provided binding activity to
Notch2 NRR is substantially retained. In certain embodiments, an
anti-Notch2 NRR antibody comprises a human variable heavy (VH)
consensus framework sequence, as in any of the VH consensus
framework sequences shown in FIGS. 16A and 16B. In one embodiment,
the VH consensus framework sequence comprises a human subgroup III
heavy chain framework consensus sequence, e.g., as shown in FIGS.
16A and 16B. In another embodiment, the VH consensus framework
sequence comprises an "Acceptor 2" framework sequence, e.g., as
shown in FIGS. 16A and 16B. In a particular embodiment, the VH
framework consensus sequence comprises FR1-FR4 of Acceptor 2B or
Acceptor 2D, wherein the FR4 comprises SEQ ID NO:35 (FIGS. 16A and
16B), with the last residue of SEQ ID NO:35 (S11) optionally being
substituted with alanine 1n a further particular embodiment, the VH
framework consensus sequence comprises the sequences of SEQ ID
NOs:50; 51; 57 or 59; and 35, wherein S11 of SEQ ID NO:35 is
optionally substituted with alanine
[0175] In certain embodiments, an anti-Notch2 NRR antibody having
any of the above HVR sequences can further comprise a human
variable light (VL) consensus framework sequence as shown in FIG.
17. In one embodiment, the VL consensus framework sequence
comprises a human VL kappa subgroup I consensus framework
(.kappa.v1) sequence, e.g., as shown in FIG. 17.
[0176] In another embodiment, the VL framework consensus sequence
comprises FR1-FR4 of huMAb4D5-8 as shown in FIG. 18 or 19. In a
particular embodiment, the VL framework consensus sequence
comprises the sequences of SEQ ID NOs:60, 61, 62, and 63.
[0177] In another aspect, an anti-Notch2 NRR antibody comprises a
heavy chain variable domain (VH) sequence having at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to an amino acid sequence selected from SEQ ID NOs:20-21. In
certain embodiments, a VH sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-Notch2
NRR antibody comprising that sequence retains the ability to bind
to Notch2 NRR. In certain embodiments, a total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in an amino
acid sequence selected from SEQ ID NOs:20-21. In certain
embodiments, substitutions, insertions, or deletions occur in
regions outside the HVRs (i.e., in the FRs). In a particular
embodiment, the VH comprises one, two or three HVRs selected from:
(a) an HVR-H1 comprising an amino acid sequence that conforms to
the consensus sequence of SEQ ID NO:3, (b) an HVR-H2 comprising the
amino acid sequence of SEQ ID NO:4, and (c) an HVR-H3 comprising
the amino acid sequence of SEQ ID NO:5. In one such embodiment,
HVR-H1 comprises an amino acid sequence selected from SEQ ID
NOs:1-2.
[0178] In another aspect, an antibody that specifically binds to
Notch2 NRR is provided, wherein the antibody comprises a light
chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid
sequence selected from SEQ ID NOs:22-25. In certain embodiments, a
VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity contains substitutions (e.g., conservative
substitutions), insertions, or deletions relative to the reference
sequence, but an anti-Notch2 NRR antibody comprising that sequence
retains the ability to bind to Notch2 NRR. In certain embodiments,
a total of 1 to 10 amino acids have been substituted, inserted
and/or deleted in an amino acid sequence selected from SEQ ID
NOs:22-25. In certain embodiments, the substitutions, insertions,
or deletions occur in regions outside the HVRs (i.e., in the FRs).
In a particular embodiment, the VL comprises one, two or three HVRs
selected from (a) an HVR-L1 comprising an amino acid sequence that
conforms to the consensus sequence of SEQ ID NO:10; (b) an HVR-L2
comprising an amino acid sequence that conforms to the consensus
sequence of SEQ ID NO:14; and (c) an HVR-L3 comprising an amino
acid sequence that conforms to the consensus sequence of SEQ ID
NO:19. In one such embodiment, the VL comprises one, two or three
HVRs selected from (a) an HVR-L1 comprising an amino acid sequence
selected from SEQ ID NOs:6-9; (b) an HVR-L2 comprising an amino
acid sequence selected from SEQ ID NOs:11-13; and (c) an HVR-L3
comprising an amino acid sequence selected from SEQ ID NOs:15-18.
In one such embodiment, the VL comprises one, two or three HVRs
selected from (a) an HVR-L1 comprising the amino acid sequence of
SEQ ID NO:6; (b) an HVR-L2 comprising the amino acid sequence of
SEQ ID NO:11; and (c) an HVR-L3 comprising the amino acid sequence
of SEQ ID NO:15. In another such embodiment, the VL comprises one,
two or three HVRs selected from (a) an HVR-L1 comprising the amino
acid sequence of SEQ ID NO:7; (b) an HVR-L2 comprising the amino
acid sequence of SEQ ID NO:11; and (c) an HVR-L3 comprising the
amino acid sequence of SEQ ID NO:16. In another such embodiment,
the VL comprises one, two or three HVRs selected from (a) an HVR-L1
comprising the amino acid sequence of SEQ ID NO:8; (b) an HVR-L2
comprising the amino acid sequence of SEQ ID NO:12; and (c) an
HVR-L3 comprising the amino acid sequence of SEQ ID NO:17. In
another such embodiment, the VL comprises one, two or three HVRs
selected from (a) an HVR-L1 comprising the amino acid sequence of
SEQ ID NO:9; (b) an HVR-L2 comprising the amino acid sequence of
SEQ ID NO:13; and (c) an HVR-L3 comprising the amino acid sequence
of SEQ ID NO:18.
[0179] In certain embodiments of the variant VH and VL sequences
provided above, substitutions, insertions, or deletions may occur
within the HVRs. In such embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations that do not
substantially reduce binding affinity may be made in HVRs. In
certain instances, alterations in HVRs may actually improve
antibody affinity. Such alterations may be made in HVR "hotspots"
(i.e., residues encoded by codons that undergo mutation at high
frequency during the somatic maturation process) in order to
increase antibody affinity. (See, e.g., Chowdhury, Methods Mol.
Biol. 207:179-196, 2008.) In certain embodiments of the variant VH
and VL sequences provided above, each HVR either is conserved
(unaltered), or contains no more than a single amino acid
substitution, insertion or deletion.
[0180] In another aspect, an antibody that specifically binds
Notch2 NRR is provided, wherein the antibody comprises a VH as in
any of the embodiments provided above, and a VL as in any of the
embodiments provided above. In one embodiment, the antibody
comprises a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% sequence identity to the amino acid sequence
of SEQ ID NO:20, and a VL having at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino
acid sequence of SEQ ID NO:22. In one such embodiment, the VH
comprises one, two or three HVRs selected from: (a) an HVR-H1
comprising the amino acid sequence of SEQ ID NO:1, (b) an HVR-H2
comprising the amino acid sequence of SEQ ID NO:4, and (c) an
HVR-H3 comprising the amino acid sequence of SEQ ID NO:5, and the
VL comprises one, two or three HVRs selected from (a) an HVR-L1
comprising the amino acid sequence of SEQ ID NO:6; (b) an HVR-L2
comprising the amino acid sequence of SEQ ID NO:11; and (c) an
HVR-L3 comprising the amino acid sequence of SEQ ID NO:15. In a
particular embodiment, the antibody comprises a VH comprising the
amino acid sequence of SEQ ID NO:20, and a VL comprising the amino
acid sequence of SEQ ID NO:22.
[0181] In another embodiment, an anti-Notch2 NRR antibody that
specifically binds Notch2 NRR comprises a VH having at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the amino acid sequence of SEQ ID NO:21, and a VL
having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% sequence identity to an amino acid sequence selected from
SEQ ID NOs:23-25. In one such embodiment, the VH comprises one, two
or three HVRs selected from: (a) an HVR-H1 comprising the amino
acid sequence of SEQ ID NO:2, (b) an HVR-H2 comprising the amino
acid sequence of SEQ ID NO:4, and (c) an HVR-H3 comprising the
amino acid sequence of SEQ ID NO:5, and the VL comprises one, two
or three HVRs selected from (a) an HVR-L1 comprising an amino acid
sequence selected from SEQ ID NOs:7-9; (b) an HVR-L2 comprising an
amino acid sequence selected from SEQ ID NOs:11-13; and (c) an
HVR-L3 comprising an amino acid sequence selected from SEQ ID
NOs:16-18. In particular embodiments, the antibody comprises a VH
comprising the amino acid sequence of SEQ ID NO:21 and a VL
comprising an amino acid sequence selected from SEQ ID
NOs:23-25.
[0182] In certain embodiments, an affinity-matured form of any of
the above antibodies is provided. In further embodiments, a
recombinant protein that specifically binds Notch2 NRR is provided,
wherein the recombinant protein comprises an antigen binding
site(s) of any of the above antibodies. In one such embodiment, a
recombinant protein comprises any one or more of the HVRs provided
above.
[0183] In certain embodiments, a polynucleotide encoding any of the
above antibodies is provided. In one embodiment, a vector
comprising the polynucleotide is provided. In one embodiment, a
host cell comprising the vector is provided. In one embodiment, the
host cell is eukaryotic. In one embodiment, the host cell is a CHO
cell. In one embodiment, a method of making an anti-Notch2 NRR
antibody is provided, wherein the method comprises culturing the
host cell under conditions suitable for expression of the
polynucleotide encoding the antibody, and isolating the
antibody.
[0184] In another embodiment, an isolated antibody is provided that
binds to the same epitope as an antibody provided herein. In one
embodiment, an isolated anti-Notch2 NRR antibody is provided that
binds to the same epitope as an antibody selected from Antibody D,
Antibody D-1, Antibody D-2, and Antibody D-3. In another
embodiment, the invention provides an anti-Notch2 NRR antibody that
competes for binding with an antibody selected from Antibody D,
Antibody D-1, Antibody D-2, and Antibody D-3. In another
embodiment, an isolated antibody is provided that binds to at least
one domain selected from the LNR-A domain and the HD-C domain of
Notch2. In one such embodiment, the antibody binds to both the
LNR-A domain and the HD-C domain. In another such embodiment, the
antibody further binds to the LNR-B and/or HD-N domains.
[0185] Any of the Notch2-specific antagonists provided herein may
be used in therapeutic methods. In one aspect, a Notch2-specific
antagonist for use as a medicament is provided. In further aspects,
a Notch2-specific antagonist for use in treating a liver condition
characterized by liver damage is provided. In certain embodiments,
a Notch2-specific antagonist for use in a method of treatment is
provided. In certain embodiments, the invention provides a
Notch2-specific antagonist for use in a method of treating an
individual having a liver condition characterized by liver damage
comprising administering to the individual an effective amount of
the Notch2-specific antagonist. In one such embodiment, the method
further comprises administering to the individual an effective
amount of at least one additional therapeutic agent, e.g., as
described below. An "individual" according to any of the above
embodiments is preferably a human.
[0186] In a further aspect, the invention provides for the use of a
Notch2-specific antagonist in the manufacture or preparation of a
medicament. In one embodiment, the medicament is for treatment of a
liver condition characterized by liver damage. In a further
embodiment, the medicament is for use in a method of treating a
liver condition characterized by liver damage comprising
administering to an individual having a liver condition
characterized by liver damage an effective amount of the
medicament. In one such embodiment, the method further comprises
administering to the individual an effective amount of at least one
additional therapeutic agent, e.g., as described below. An
"individual" according to any of the above embodiments may be a
human.
[0187] In a further aspect, the invention provides pharmaceutical
formulations comprising any of the Notch2-specific antagonists
provided herein, e.g., for use in any of the above therapeutic
methods. In one embodiment, a pharmaceutical formulation comprises
any of the Notch2-specific antagonists provided herein and a
pharmaceutically acceptable carrier. In another embodiment, a
pharmaceutical formulation comprises any of the Notch2-specific
antagonists provided herein and at least one additional therapeutic
agent, e.g., as described below.
[0188] Antibodies of the invention can be used either alone or in
combination with other agents in a therapy. For instance, an
antibody of the invention may be co-administered with at least one
additional therapeutic agent.
[0189] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of the antagonist of the invention
can occur prior to, simultaneously, and/or following,
administration of the additional therapeutic agent and/or adjuvant.
Antagonists of the invention can also be used in combination with
radiation therapy.
[0190] The antagonist can be administered to a human patient by any
known method, such as intravenous administration, e.g., as a bolus
or by continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, topical, or inhalation routes.
The Notch2-specific antagonist might be administered as a protein
or as a nucleic acid encoding a protein (see, for example,
WO96/07321). Other therapeutic regimens may be combined with the
administration of the Notch2-specific antagonist. The combined
administration includes co-administration, using separate
formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities. In some
embodiments, such combined therapy results in a synergistic
therapeutic effect.
[0191] The dosage and mode of administration will be chosen by the
physician according to known criteria. The appropriate dosage of
antibody, oligopeptide or organic molecule will depend on the type
of disease to be treated, the severity and course of the disease,
whether the antibody, oligopeptide or organic molecule is
administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the
Notch2-specific antagonist, and the discretion of the attending
physician. The Notch2-specific antagonist can be administered to
the patient at one time or over a series of treatments.
[0192] Success of treatment of liver disease can be monitored by
assessing parameters of liver function and recovery. Such
parameters include, but are not limited to, improved liver function
tests, (e.g., assessing serum albumin, bilirubin, bile acids, total
protein, clotting times), liver enzymes (e.g., alanine
transaminase, aspartate transaminase, alkaline phosphatase, gamma
glutamyl transpeptidase), histologic appearance (e.g., needle
biopsy showing improved hepatic architecture), and imaging
modalities (e.g., ultrasound, magnetic resonance imaging for
fibrosis and liver size).
[0193] In a further aspect, an anti-Notch2 antibody used in any of
the above embodiments may incorporate any of the features, singly
or in combination, as described in Sections 1-7 below.
[0194] 1. Antibody Affinity
[0195] In certain embodiments, an antibody provided herein has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g., 10.sup.-8M or less, e.g. from 10.sup.-8M to
10.sup.-13M, e.g., from 10.sup.-9M to 10.sup.-13 M). For example,
the exemplary phage Antibody D-3 binds to Notch2 with a Kd of 5
nM.
[0196] In one embodiment, Kd is measured by a radiolabeled antigen
binding assay (RIA) performed with the Fab version of an antibody
of interest and its antigen as described by the following assay.
Solution binding affinity of Fabs for antigen is measured by
equilibrating Fab with a minimal concentration of
(.sup.125I)-labeled antigen in the presence of a titration series
of unlabeled antigen, then capturing bound antigen with an anti-Fab
antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.
293:865-881 (1999)). To establish conditions for the assay,
MICROTITER.RTM. multi-well plates (Thermo Scientific) are coated
overnight with 5 .mu.g/ml of a capturing anti-Fab antibody (Cappel
Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked
with 2% (w/v) bovine serum albumin in PBS for two to five hours at
room temperature (approximately 23.degree. C.). In a non-adsorbent
plate (Nunc #269620), 100 .mu.M or 26 .mu.M [.sup.125I]-antigen are
mixed with serial dilutions of a Fab of interest (e.g., consistent
with assessment of the anti-VEGF antibody, Fab-12, in Presta et
al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then
incubated overnight; however, the incubation may continue for a
longer period (e.g., about 65 hours) to ensure that equilibrium is
reached. Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
polysorbate 20 (TWEEN-20.RTM.) in PBS. When the plates have dried,
150 .mu.l/well of scintillant (MICROSCINT-20.TM.; Packard) is
added, and the plates are counted on a TOPCOUNT.TM. gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays.
[0197] According to another embodiment, Kd is measured using
surface plasmon resonance assays using a BIACORE.RTM.-2000 or a
BIACORE.RTM.-3000 (BIAcore, Inc., Piscataway, N.J.) at 25.degree.
C. with immobilized antigen CM5 chips at .about.10 response units
(RU). Briefly, carboxymethylated dextran biosensor chips (CM5,
BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% polysorbate 20 (TWEEN-20.TM.)
surfactant (PBST) at 25.degree. C. at a flow rate of approximately
25 .mu.l/min. Association rates (k.sub.on) and dissociation rates
(k.sub.off) are calculated using a simple one-to-one Langmuir
binding model (BIACORE.RTM. Evaluation Software version 3.2) by
simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant (Kd) is
calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen et al.,
J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10.sup.6
M.sup.-1 s.sup.-1 by the surface plasmon resonance assay above,
then the on-rate can be determined by using a fluorescent quenching
technique that measures the increase or decrease in fluorescence
emission intensity (excitation=295 nm; emission=340 nm, 16 nm
band-pass) at 25.degree. C. of a 20 nM anti-antigen antibody (Fab
form) in PBS, pH 7.2, in the presence of increasing concentrations
of antigen as measured in a spectrometer, such as a stop-flow
equipped spectrophometer (Aviv Instruments) or a 8000-series
SLM-AMINCO.TM. spectrophotometer (ThermoSpectronic) with a stirred
cuvette.
[0198] 2. Antibody Fragments
[0199] In certain embodiments, an antibody provided herein is an
antibody fragment. Antibody fragments include, but are not limited
to, Fab, Fab', Fab'-SH, F(ab').sub.2, Fv, and scFv fragments, and
other fragments described below. For a review of certain antibody
fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a
review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology
of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York), pp. 269-315 (1994); see also WO
93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab').sub.2 fragments comprising salvage
receptor binding epitope residues and having increased in vivo
half-life, see U.S. Pat. No. 5,869,046.
[0200] Diabodies are antibody fragments with two antigen-binding
sites that may be bivalent or bispecific. See, for example, EP
404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003);
and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448
(1993). Triabodies and tetrabodies are also described in Hudson et
al., Nat. Med. 9:129-134 (2003). Single-domain antibodies are
antibody fragments comprising all or a portion of the heavy chain
variable domain or all or a portion of the light chain variable
domain of an antibody. In certain embodiments, a single-domain
antibody is a human single-domain antibody (Domantis, Inc.,
Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).
[0201] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g. E.
coli or phage).
[0202] 3. Chimeric and Humanized Antibodies
[0203] In certain embodiments, an antibody provided herein is a
chimeric antibody. Certain chimeric antibodies are described, e.g.,
in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody
comprises a non-human variable region (e.g., a variable region
derived from a mouse, rat, hamster, rabbit, or non-human primate,
such as a monkey) and a human constant region. In a further
example, a chimeric antibody is a "class switched" antibody in
which the class or subclass has been changed from that of the
parent antibody. Chimeric antibodies include antigen-binding
fragments thereof.
[0204] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0205] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods
36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol.
Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua
et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and
Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J.
Cancer, 83:252-260 (2000) (describing the "guided selection"
approach to FR shuffling).
[0206] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296
(1993)); framework regions derived from the consensus sequence of
human antibodies of a particular subgroup of light or heavy chain
variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci.
USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623
(1993)); human mature (somatically mutated) framework regions or
human germline framework regions (see, e.g., Almagro and Fransson,
Front. Biosci. 13:1619-1633 (2008)); and framework regions derived
from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem.
272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.
271:22611-22618 (1996)).
[0207] 4. Human Antibodies
[0208] In certain embodiments, an antibody provided herein is a
human antibody. Human antibodies can be produced using various
techniques known in the art. Human antibodies are described
generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:
368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459
(2008).
[0209] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology; U.S.
Pat. No. 5,770,429 describing HUMAB.RTM. technology; U.S. Pat. No.
7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VELOCIMOUSE.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0210] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.
Immunol., 147: 86 (1991).) Human antibodies generated via human
B-cell hybridoma technology are also described in Li et al., Proc.
Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods
include those described, for example, in U.S. Pat. No. 7,189,826
(describing production of monoclonal human IgM antibodies from
hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268
(2006) (describing human-human hybridomas). Human hybridoma
technology (Trioma technology) is also described in Vollmers and
Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and
Vollmers and Brandlein, Methods and Findings in Experimental and
Clinical Pharmacology, 27(3):185-91 (2005).
[0211] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain.
[0212] 5. Library-Derived Antibodies
[0213] Antibodies of the invention may be isolated by screening
combinatorial libraries for antibodies with the desired activity or
activities. For example, a variety of methods are known in the art
for generating phage display libraries and screening such libraries
for antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom et al. in Methods in
Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press,
Totowa, N.J., 2001) and further described, e.g., in the McCafferty
et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628
(1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and
Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed.,
Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.
338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093
(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472
(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132
(2004).
[0214] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self antigens without any
immunization as described by Griffiths et al., EMBO J, 12: 725-734
(1993). Finally, naive libraries can also be made synthetically by
cloning unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable
CDR3 regions and to accomplish rearrangement in vitro, as described
by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Patent publications describing human antibody phage libraries
include, for example: U.S. Pat. No. 5,750,373, and US Patent
Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,
2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0215] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0216] 6. Multispecific Antibodies
[0217] In certain embodiments, an antibody provided herein is a
multispecific antibody, e.g. a bispecific antibody. Multispecific
antibodies are monoclonal antibodies that have binding
specificities for at least two different sites. In certain
embodiments, one of the binding specificities is for Notch2 and the
other is for any other antigen. In certain embodiments, bispecific
antibodies may bind to two different epitopes of Notch2. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express Notch2. Bispecific antibodies can be prepared as full
length antibodies or antibody fragments.
[0218] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and
Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole"
engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific
antibodies may also be made by engineering electrostatic steering
effects for making antibody Fc-heterodimeric molecules (WO
2009/089004A1); cross-linking two or more antibodies or fragments
(see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science,
229: 81 (1985)); using leucine zippers to produce bi-specific
antibodies (see, e.g., Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992)); using "diabody" technology for making
bispecific antibody fragments (see, e.g., Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain
Fv (sFv) dimers (see, e.g. Gruber et al., J. Immunol., 152:5368
(1994)); and preparing trispecific antibodies as described, e.g.,
in Tutt et al. J. Immunol. 147: 60 (1991).
[0219] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies," are also included
herein (see, e.g. US 2006/0025576A1).
[0220] The antibody or fragment herein also includes a "Dual Acting
FAb" or "DAF" comprising an antigen binding site that binds to
Notch2 as well as another, different antigen (see, US 2008/0069820,
for example).
[0221] 7. Antibody Variants
[0222] In certain embodiments, amino acid sequence variants of the
antibodies provided herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody. Amino acid sequence variants of an
antibody may be prepared by introducing appropriate modifications
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0223] a) Substitution, Insertion, and Deletion Variants
[0224] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in Table 1 under the heading of
"conservative substitutions." More substantial changes are provided
in Table 1 under the heading of "exemplary substitutions," and as
further described below in reference to amino acid side chain
classes. Amino acid substitutions may be introduced into an
antibody of interest and the products screened for a desired
activity, e.g., retained/improved antigen binding, decreased
immunogenicity, or improved ADCC or CDC.
[0225] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0226] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g. binding
affinity).
TABLE-US-00002 TABLE 1 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
Amino acids may be grouped according to common side-chain
properties: [0227] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu,
Ile; [0228] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0229] (3) acidic: Asp, Glu; [0230] (4) basic: H is, Lys, Arg;
[0231] (5) residues that influence chain orientation: Gly, Pro;
[0232] (6) aromatic: Trp, Tyr, Phe.
[0233] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs
(a-CDRs), with the resulting variant VH or VL being tested for
binding affinity. Affinity maturation by constructing and
reselecting from secondary libraries has been described, e.g., in
Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien
et al., ed., Human Press, Totowa, N.J., (2001).) In some
embodiments of affinity maturation, diversity is introduced into
the variable genes chosen for maturation by any of a variety of
methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves HVR-directed approaches, in which several HVR
residues (e.g., 4-6 residues at a time) are randomized. HVR
residues involved in antigen binding may be specifically
identified, e.g., using alanine scanning mutagenesis or modeling.
CDR-H3 and CDR-L3 in particular are often targeted.
[0234] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in HVRs. Such
alterations may be outside of HVR "hotspots" or SDRs. In certain
embodiments of the variant VH and VL sequences provided above, each
HVR either is unaltered, or contains no more than one, two or three
amino acid substitutions.
[0235] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham and Wells (1989)
Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as arg, asp, his, lys,
and glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0236] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0237] b) Glycosylation Variants
[0238] In certain embodiments, an antibody provided herein is
altered to increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
[0239] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.
TIBTECH 15:26-32 (1997). The oligosaccharide may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc),
galactose, and sialic acid, as well as a fucose attached to a
GlcNAc in the "stem" of the biantennary oligosaccharide structure.
In some embodiments, modifications of the oligosaccharide in an
antibody of the invention may be made in order to create antibody
variants with certain improved properties.
[0240] In one embodiment, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. The amount of fucose is determined by
calculating the average amount of fucose within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn
297 (e.g. complex, hybrid and high mannose structures) as measured
by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc region (Eu numbering of Fc region residues);
however, Asn297 may also be located about +3 amino acids upstream
or downstream of position 297, i.e., between positions 294 and 300,
due to minor sequence variations in antibodies. Such fucosylation
variants may have improved ADCC function. See, e.g., US Patent
Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621
(Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to
"defucosylated" or "fucose-deficient" antibody variants include: US
2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US
2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US
2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO
2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki
et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al.
Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of
producing defucosylated antibodies include Lec13 CHO cells
deficient in protein fucosylation (Ripka et al. Arch. Biochem.
Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 A1,
Presta, L; and WO 2004/056312 A1, Adams et al., especially at
Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,
e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda,
Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and
WO2003/085107).
[0241] Antibodies variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.
No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
Antibody variants with at least one galactose residue in the
oligosaccharide attached to the Fc region are also provided. Such
antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in WO 1997/30087 (Patel et al.); WO
1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
[0242] c) Fc Region Variants
[0243] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc region of an antibody provided
herein, thereby generating an Fc region variant. The Fc region
variant may comprise a human Fc region sequence (e.g., a human
IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g. a substitution) at one or more amino acid
positions.
[0244] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the antibody lacks
Fc.gamma.R binding (hence likely lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC,
NK cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting
examples of in vitro assays to assess ADCC activity of a molecule
of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.
Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063
(1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA
82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp.
Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays
methods may be employed (see, for example, ACTI.TM. non-radioactive
cytotoxicity assay for flow cytometry (CellTechnology, Inc.
Mountain View, Calif.; and CytoTox 96.RTM. non-radioactive
cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells
for such assays include peripheral blood mononuclear cells (PBMC)
and Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. Proc.
Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also
be carried out to confirm that the antibody is unable to bind C1q
and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA
in WO 2006/029879 and WO 2005/100402. To assess complement
activation, a CDC assay may be performed (see, for example,
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg,
M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M.
J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo
clearance/half life determinations can also be performed using
methods known in the art (see, e.g., Petkova, S. B. et al., Intl
Immunol. 18(12):1759-1769 (2006)).
[0245] Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0246] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604
(2001).)
[0247] In certain embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0248] In some embodiments, alterations are made in the Fc region
that result in altered (i.e., either improved or diminished) C1q
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et
al. J. Immunol. 164: 4178-4184 (2000).
[0249] Antibodies with increased half lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are
described in US2005/0014934A1 (Hinton et al.). Those antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238,
256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc
region residue 434 (U.S. Pat. No. 7,371,826).
[0250] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351
concerning other examples of Fc region variants.
[0251] d) Cysteine Engineered Antibody Variants
[0252] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," in which one or
more residues of an antibody are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
S400 (EU numbering) of the heavy chain Fc region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
[0253] e) Antibody Derivatives
[0254] In certain embodiments, an antibody provided herein may be
further modified to contain additional nonproteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer are attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0255] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA
102: 11600-11605 (2005)). The radiation may be of any wavelength,
and includes, but is not limited to, wavelengths that do not harm
ordinary cells, but which heat the nonproteinaceous moiety to a
temperature at which cells proximal to the
antibody-nonproteinaceous moiety are killed.
[0256] B. Recombinant Methods and Compositions
[0257] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, isolated nucleic acid encoding an anti-Notch2 antibody
described herein is provided. Such nucleic acid may encode an amino
acid sequence comprising the VL and/or an amino acid sequence
comprising the VH of the antibody (e.g., the light and/or heavy
chains of the antibody). In a further embodiment, one or more
vectors (e.g., expression vectors) comprising such nucleic acid are
provided. In a further embodiment, a host cell comprising such
nucleic acid is provided. In one such embodiment, a host cell
comprises (e.g., has been transformed with): (1) a vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VL of the antibody and an amino acid sequence
comprising the VH of the antibody, or (2) a first vector comprising
a nucleic acid that encodes an amino acid sequence comprising the
VL of the antibody and a second vector comprising a nucleic acid
that encodes an amino acid sequence comprising the VH of the
antibody. In one embodiment, the host cell is eukaryotic, e.g. a
Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,
Sp20 cell). In one embodiment, a method of making an anti-Notch2
antibody is provided, wherein the method comprises culturing a host
cell comprising a nucleic acid encoding the antibody, as provided
above, under conditions suitable for expression of the antibody,
and optionally recovering the antibody from the host cell (or host
cell culture medium).
[0258] For recombinant production of an anti-Notch2 antibody,
nucleic acid encoding an antibody, e.g., as described above, is
isolated and inserted into one or more vectors for further cloning
and/or expression in a host cell. Such nucleic acid may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0259] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular
Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.,
2003), pp. 245-254, describing expression of antibody fragments in
E. coli.) After expression, the antibody may be isolated from the
bacterial cell paste in a soluble fraction and can be further
purified.
[0260] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414
(2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
[0261] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0262] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0263] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham et al.,
J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse
sertoli cells (TM4 cells as described, e.g., in Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African
green monkey kidney cells (VERO-76); human cervical carcinoma cells
(HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL
3A); human lung cells (W138); human liver cells (Hep G2); mouse
mammary tumor (MMT 060562); TR1 cells, as described, e.g., in
Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5
cells; and FS4 cells. Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR.sup.- CHO
cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980));
and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of
certain mammalian host cell lines suitable for antibody production,
see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268
(2003).
[0264] C. Assays
[0265] Anti-Notch2 antibodies provided herein may be identified,
screened for, or characterized for their physical/chemical
properties and/or biological activities by various assays known in
the art.
[0266] 1. Binding Assays and Other Assays
[0267] In one aspect, an antibody of the invention is tested for
its antigen binding activity, e.g., by known methods such as ELISA,
Western blot, etc.
[0268] In another aspect, competition assays may be used to
identify an antibody that competes with Antibody D, Antibody D-1,
Antibody D-2, or Antibody D-3 for binding to Notch2. In certain
embodiments, such a competing antibody binds to the same epitope
(e.g., a linear or a conformational epitope) that is bound by
Antibody D, Antibody D-1, Antibody D-2, or Antibody D-3. Detailed
exemplary methods for mapping an epitope to which an antibody binds
are provided in Morris (1996) "Epitope Mapping Protocols," in
Methods in Molecular Biology vol. 66 (Humana Press, Totowa,
N.J.).
[0269] In an exemplary competition assay, immobilized Notch2 is
incubated in a solution comprising a first labeled antibody that
binds to Notch2 (e.g., Antibody D, Antibody D-1, Antibody D-2, or
Antibody D-3) and a second unlabeled antibody that is being tested
for its ability to compete with the first antibody for binding to
Notch2. The second antibody may be present in a hybridoma
supernatant. As a control, immobilized Notch2 is incubated in a
solution comprising the first labeled antibody but not the second
unlabeled antibody. After incubation under conditions permissive
for binding of the first antibody to Notch2, excess unbound
antibody is removed, and the amount of label associated with
immobilized Notch2 is measured. If the amount of label associated
with immobilized Notch2 is substantially reduced in the test sample
relative to the control sample, then that indicates that the second
antibody is competing with the first antibody for binding to
Notch2. See Harlow and Lane (1988) Antibodies: A Laboratory Manual
ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.).
[0270] 2. Activity Assays
[0271] In one aspect, assays are provided for identifying
anti-Notch2 antibodies thereof having biological activity.
Biological activity may include, e.g., inhibition or reduction of
Notch2 activity, e.g., Notch2 signaling. Antibodies having such
biological activity in vivo and/or in vitro are also provided.
[0272] In certain embodiments, an anti-Notch2 NRR antibody of the
invention is tested for its ability to inhibit generation of
marginal zone B cells. An exemplary assay is provided in the
Examples. In certain other embodiments, an antibody of the
invention is tested for its ability to inhibit expression of a
reporter gene that is responsive to Notch2 signaling.
[0273] D. Immunoconjugates
[0274] The invention also provides immunoconjugates comprising an
anti-Notch2 antibody herein conjugated to one or more cytotoxic
agents, such as chemotherapeutic agents or drugs, growth inhibitory
agents, toxins (e.g., protein toxins, enzymatically active toxins
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or radioactive isotopes.
[0275] In one embodiment, an immunoconjugate is an antibody-drug
conjugate (ADC) in which an antibody is conjugated to one or more
drugs, including but not limited to a maytansinoid (see U.S. Pat.
Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an
auristatin such as monomethylauristatin drug moieties DE and DF
(MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and
7,498,298); a dolastatin; a calicheamicin or derivative thereof
(see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,
5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al.,
Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res.
58:2925-2928 (1998)); an anthracycline such as daunomycin or
doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523
(2006); Jeffrey et al., Bioorganic & Med. Chem. Letters
16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005);
Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000);
Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532
(2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S.
Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as
docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a
trichothecene; and CC1065.
[0276] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to an enzymatically active
toxin or fragment thereof, including but not limited to diphtheria
A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A
chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,
dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and
PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes.
[0277] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to a radioactive atom to
form a radioconjugate. A variety of radioactive isotopes are
available for the production of radioconjugates. Examples include
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive
isotopes of Lu. When the radioconjugate is used for detection, it
may comprise a radioactive atom for scintigraphic studies, for
example tc99m or I123, or a spin label for nuclear magnetic
resonance (NMR) imaging (also known as magnetic resonance imaging,
mri), such as iodine-123 again, iodine-131, indium-111,
fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,
manganese or iron.
[0278] Conjugates of an antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of a cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No.
5,208,020) may be used.
[0279] The immunuoconjugates or ADCs herein expressly contemplate,
but are not limited to such conjugates prepared with cross-linker
reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS,
LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A).
III. Examples
Example 1
Isolation and Transcriptional Profiling of Liver Progenitor
Cells
[0280] To identify the signals that regulate hepatocyte
differentiation from liver progenitor cells, transcriptional
profiling was performed on adult liver progenitors (oval cells)
from mice fed a choline deficient, ethionine supplemented (CDE)
diet (FIG. 1A; FIG. 5A). This CDE model is known in the art as a
model of chronic liver disease. CDE is also a steatohepatitis model
(NASH). Chronic CDE can also lead to hepatocellular carcinoma
(HCC), thereby also serving as a model for HCC. To induce an oval
cell response, 8-12 week-old female C57BL/6N mice (Charles River)
were fed a choline deficient diet (20% Lard; Teklad TD.04523)
supplemented with 0.15% (w/v) Ethionine in the drinking water
(Akhurst et al., Hepatology 34(3):519 (2001)). Liver
non-parenchymal cells were isolated according to the protocol of
del Castillo (del Castillo et al., Am. J. Pathol., 172(5):1238
(2008)) with the addition of 0.04% Hyaluronidase (Sigma) to the in
vitro dissociation step.
[0281] Epithelial Cell Adhesion Molecule (EpCAM)-expressing
progenitor cells and normal bile duct cells from livers of CDE-fed
and control mice were isolated by fluorescence activated cell
sorting (FACS; FIG. 4A-B). C57BL/6 mouse livers were perfused with
a Collagenase/Pronase solution and the liver was dissociated and
further incubated in the presence of DNase and Hyaluronidase (FIG.
4B). The resulting cell suspension was passed over a 30%/70%
Percoll density gradient by centrifugation at 2500 RPM for 30
minutes. Cells from the 30%/70% Percoll interface, consisting
mostly of non-parenchymal cells, were stained with fluorescently
labeled antibodies to EpCAM (BioLegend) and CD45 (BD Pharmingen).
Flow cytometry was used to isolate EpCAM.sup.+/CD45.sup.- cells
from mice fed CDE or standard rodent diet. The majority of
EpCAM.sup.+/CD45.sup.- cells from CDE-fed mice were oval cells,
while the majority of EpCAM.sup.+/CD45.sup.- cells from standard
diet-fed mice were bile duct cells. QRT-PCR analysis on RNA from
FACS sorted cells confirmed that EpCAM.sup.+/CD45.sup.--sorted
cells were greatly enriched for EpCAM as well as CK19 (FIG. 4B),
indicating a successful positive selection of EpCAM.sup.+
progenitor and bile duct cells.
[0282] In addition to isolating progenitor cells by FACS sorting,
progenitor and normal bile duct cells were isolated by laser
capture microdissection (LCM) from hematoxylin and eosin (H&E)
stained liver sections. Livers from C57BL/6N 8-12 week-old female
mice fed normal chow or CDE diet were removed and immediately flash
frozen in liquid Nitrogen. Flash frozen liver pieces were placed in
prechilled plastic molds, embedded in TISSUE-TEK OCT Compound
(Sakura, The Netherlands) and immediately placed on a dry
ice/2-methylbutane bath until frozen. The embedded frozen liver
pieces were cut into 7-8 .mu.m sections at -14.degree. C., adhered
to metal frame membrane slides (MMI, Eching, Germany), fixed,
stained with hematoxylin and eosin, and dried. Laser
microdissection was used to isolate 1-2 mm.sup.2 of oval cell or
normal bile duct tissue per sample.
[0283] RNA from flow-sorted and laser microdissected tissue was
isolated using the RNEASY Micro Kit (Qiagen). For microarray
analysis RNA from flow-sorted and laser microdissected tissue, as
well as whole liver controls, spiked with Agilent RNA Spike-In RNA
(Agilent), was submitted to two rounds of amplification with
Message AmpII (Ambion) and hybridized to Whole Mouse Genome Oligo
44k microarrays. Log expression ratios were exported and analyzed
using Partek Genomic Suite (Partek). Quantitative real-time PCR
(QRTPCR) was performed using the TAQMAN One-Step RT-PCR Kit for one
step reactions using the 7900 HT RT-PCR system (Applied Biosystems)
with TAQMAN probes (Applied Biosystems) or High Capacity cDNA RT
kit with TAQMAN Fast Advanced Master Mix using the Viia7 RT-PCR
system (Applied Biosystems) with custom designed low density
arrays.
[0284] Enrichment of bile duct and progenitor cell-associated
transcripts, such as EpCAM and Keratin19, confirmed effective
isolation of bile duct and progenitor cells (FIG. 1E). Using
supervised analysis (FIG. 5B), genes expressed more highly in liver
progenitor cells than in closely related normal bile duct cells
were identified (Table 2). This supervised analysis correlated
highly with progenitor cell associated transcripts identified by
unbiased principle components analysis (FIG. 5C-H). The expression
pattern of these genes was validated on independent samples by
QRT-PCR (FIG. 6) and immunofluorescence, which confirmed that
members of the Notch signaling pathway, including Jag1 and Notch2,
as well as Hes1 and Hey1, among other target genes, were
upregulated in liver progenitor cells compared to bile duct cells
(FIG. 1C-G). A selection of microarray probes for Notch
pathway-associated transcripts distinguished flow-sorted oval cells
from flow-sorted biliary cells and whole liver by supervised
clustering (FIG. 1B). Supervised hierarchical clustering of 29
Notch pathway-associated genes, including receptors, ligands,
transcription factors, and select target genes, differentiated
whole liver, bile ducts, and oval cells into separate clusters
(Rand index=1, 3 clusters; Rand index=0.7607, 3 clusters, for
randomly generated list of 29 genes) and revealed differences
between normal bile duct cells and progenitors. The data showed
significant upregulation of Jag1 (p=0.0006; FIG. 1C), Notch2
(p=0.0006; FIG. 1D), Hes1 (p=0.0035; FIG. 1E), and Hey1 (p=0.0087;
FIG. 1F) in liver progenitor cells. Immunofluorescence staining
confirmed that Jag1 was more highly expressed in EpCAM.sup.+ oval
cells radiating out from the portal vein in a CDE liver than in
adjacent normal bile duct cells. Immunhistochemistry for Hes1
confirmed that Hes1 positive cells were largely confined to the
oval cell and bile duct compartments, with some staining in other
non-parenchymal cells of the liver (FIG. 1G).
TABLE-US-00003 TABLE 2 Oval Cell Associated Genes log2 (Fold
UNQ_Short_Name ProbeID t-statistic pvalue Change) ADAMTS9
A_52_P49321 5.272 0 2.132 ANXA9 A_51_P451482 6.839 0 2.578 APP
A_52_P381311 5.045 0 2.531 BMP8B A_51_P411926 6.273 0 3.562 CHRNB1
A_51_P475342 5.32 0 1.912 CTGF A_51_P157042 7.146 0 3.336 DTNA
A_52_P108607 5.585 0 3.558 Embigin A_51_P382849 6.196 0 3.246 Epdr2
A_52_P577388 5.703 0 2.431 EPHA7 A_52_P504787 5.855 0 2.255 FADS3
A_52_P451796 5.809 0 2.163 Foxc1 A_51_P107686 5.098 0 2.77 GSPT1
A_52_P354785 5.672 0 2.726 Hig2I A_52_P321150 5.522 0 1.696 ID2
A_52_P240542 5.054 0 1.504 Ifrd1 A_51_P367060 5.888 0 1.609 JAG1
A_52_P634090 5.368 0 2.377 LTB A_51_P302358 6.15 0 2.428 MAL
A_52_P562661 5.619 0 3.02 Mex3a A_52_P706060 5.228 0 3.582 MFI2
A_51_P324351 7.735 0 3.397 MYC A_51_P102096 5.498 0 1.522 NFAM1
A_52_P686701 5.472 0 2.09 NFKB1 A_52_P32733 5.348 0 2.398 Nrarp
A_51_P504354 5.261 0 2.585 peg3 A_51_P206037 6.329 0 2.84 RASL11A
A_51_P340699 5.425 0 2.303 SLIT2 A_51_P496569 5.964 0 3.261 SPATA7
A_52_P134680 5.187 0 1.926 SPRR1A A_51_P139678 7.825 0 2.8 TNFAIP8
A_51_P435968 6.322 0 2.403 TNFRSF12A A_51_P131408 6.641 0 2.397
tp53 A_52_P957260 5.399 0 2.388 TRIM47 A_51_P437176 5.633 0 1.592
Trio A_51_P319662 6.257 0 2.81 TTYH1 A_52_P475052 5.668 0 2.442
USP47 A_52_P610967 5.021 0 2.018 VCAM1 A_51_P210956 5.696 0
1.847
Example 2
Identification of Expression Patterns of Oval Cell-Associated
Genes
[0285] The microarray data from RNA isolated from oval and bile
duct cells isolated FACS (Flow) or microdisection (LCM) and control
and CDE livers were examined for the expression of putative markers
of oval cells and markers of other select cell types. Albumin
transcript was detected at relatively high levels in all groups
(FIG. 6A-1), whereas AFP, a marker of immature hepatocytes that is
upregulated during chronic liver damage, was greatly enriched in
LCM oval cells, but virtually absent from the other cell types
examined (FIG. 6A-1). To determine why AFP was present only in LCM
oval cells, immunofluorescence was performed on 7 .mu.m frozen CDE
liver sections that had been briefly air-dried and fixed in 4%
paraformaldehyde in PBS. After blocking with normal horse serum in
PBS, sections were incubated with fluorescently labeled antibodies
to EpCAM (BioLegend), Sca1 (BD Pharmingen), CD90 (BioLegend), or
with unlabeled primary antibodies to AFP (R&D Systems) and CK19
(Santa Cruz Biotech) followed by incubation with fluorescently
labeled secondary antibodies (Invitrogen). AFP was expressed in
only a subset of hepatocytes, often in close proximity to
EpCAM.sup.+ oval cells. However, AFP expression could not be
observed in oval cells themselves.
[0286] LCM isolates expressed high levels of the myofibroblast
marker SMA and the mesenchymal cell marker CD90/Thy1 (FIG. 6A-3),
possibly as a result of inclusion of periportal myofibroblasts,
which are positive for both CD90/Thy1 and SMA. Thus, it appears
that the LCM samples contain a heterogeneous mixture of cell types
that at least include myofibroblasts, and in the case of LCM
samples from CDE livers, AFP positive hepatocytes adjacent to cords
of microdissected oval cells. Though Sca1 also marks mesenchymal
cells, it also appears to be expressed in bile duct cells and oval
cells themselves, as the transcript is found at high levels in both
the FACS-sorted and LCM samples (FIG. 6A-2). The oval cell markers
CD13, Sox9, FoxL, and FoxJ1 were also expressed in both FACS-sorted
and LCM samples. Except for Sox9, which was more highly expressed
in FACS oval cells, each of these markers was expressed at
comparable levels in CDE oval cells and in normal bile ducts.
Expression patterns of oval cell-associated genes and genes marking
other hepatic cell types were confirmed in independent samples by
QRT-PCR. For these experiments, CDE oval cells were enriched by
Magnetically Activated Cell Sorting (MACS), first by depleting
CD45.sup.+ cells from the lower band of a 30%/70% Percoll gradient
followed by positive selection for EpCAM.sup.+ cells (FIG. 6B).
Purity of the resulting cell suspensions was >95%. c, Relative
to CD45-/EpCAM- cells, CD45-/EpCAM+ cells expressed high levels of
EpCAM, CK19, Trop2, and CD133 and low levels of AFP, LGR5, CD90,
and Vimentin (FIG. 6C). Albumin was expressed at comparable levels
in CD45.sup.- /EpCAM.sup.- and CD45.sup.-/EpCAM.sup.+
fractions.
Example 3
Notch Signaling in Liver Progenitors In Vitro
[0287] To elucidate the role of Notch signaling in liver
regeneration, an in vitro culture system was developed. Cell
cultures were established by culturing primary oval cells on Sw-3T3
fibroblasts (ATCC), arrested with Mitomycin C (Sigma), in High
Glucose Dulbecco's Modified Eagle Medium (DMEM; Invitrogen)
supplemented with 15% Fetal Calf Serum (Sigma), non-essential amino
acids (Invitrogen), Glutamax (Invitrogen), and ITS
(Invitrogen).
[0288] The anti-activated Notch2 antibody (clone 40-2-7) was
generated against the peptide VIMAKRKRKHGSLW, corresponding to
amino acids 1697-1710 of the human Notch2 protein sequence (SEQ ID
NO:73), coupled to KLH (YenZym Custom Antibodies, LLC). Splenocytes
from a rabbit producing an antibody with the appropriate
specificity were used to generate hybridomas (Epitomics, Inc.).
Clone 40-2-7 was identified by screening the resulting rabbit
monoclonal antibodies by immunoblotting and immunohistochemistry.
This antibody recognizes both human (FIG. 7B, left panel) and mouse
(FIG. 7B, right panel) active Notch2 at endogenous levels.
[0289] Progenitors cultured in the culture system maintained growth
(FIG. 2A) and formed colonies consisting of small, tightly packed
cells with a high nuclear-cytoplasmic ratio and a distinct raised
edge (FIG. 2A, left panel). The cells within these colonies were
uniformly EpCAM positive (FIG. 2A, right panel). Progenitor
cultures also maintained the characteristic progenitor expression
signature in vitro (FIG. 2C). Activated Notch2 was detected by
Western blot analysis using a rabbit monoclonal antibody raised
against the S3 cleaved form of the human Notch2 protein (FIG. 7B).
Activated Notch2 signal was increased upon ligand stimulation (Jag)
or EDTA stimulation (EDTA), and the activated form was greatly
enriched in the nuclear fraction (FIG. 7B; lanes "N").
[0290] Treatment of the primary cultures with the .gamma.-secretase
and Notch pathway inhibitor
N--[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl
ester (DAPT) unexpectedly enhanced colony formation approximately
ten-fold, from approximately 1 colony formed per 100,000 plated
CD45 negative, non-parenchymal cells to approximately 1 colony per
10,000 cells plated (FIG. 2B) suggesting that inhibition of Notch
pathway activity promoted either liver progenitor cell maintenance
or proliferation. Treatment with DAPT resulted in a small increase
in progenitor cell proliferation (FIG. 2D).
[0291] To determine if Notch signaling inhibition also suppresses
the differentiation of progenitor cells, thereby allowing for
long-term maintenance, the biliary and hepatic differentiation
potential of these cells in vitro was analyzed (FIG. 7). Cultured
oval cells were maintained on a mitomycin-C treated feeder layer of
Swiss-3T3 fibroblasts by culture in the presence of 15% Fetal
Bovine Serum (FBS) and the .gamma.-secretase inhibitor DAPT.
[0292] Differentiation along the hepatocyte lineage was induced by
plating oval cells without feeder cells onto tissue-culture treated
plastic coated with diluted Rat Tail Collagen (BD) in the presence
of 10 ng/ml Oncostatin M (R&D) and 25 ng/mL HGF (Lonza). For
some experiments, DAPT or vehicle (DMSO) and/or the anti-Notch2 NRR
antibody, Antibody D-3 (also referred to herein as anti-N2,
anti-Notch2, or anti-NRR2) or an isotype control antibody were
added to the medium at the time of cell plating and replenished
every three days. Bile duct differentiation was induced by
suspending oval cells in a 1:1 mixture of MATRIGEL and oval cell
growth medium supplemented with 7.5% FBS and plating onto plastic
tissue culture dishes. Following solidification, the MATRIGEL
cultures were overlain with growth medium supplemented with 15%
FBS. In some experiments, DAPT or vehicle and/or anti-Notch2
antibody or isotype control were added to the MATRIGEL as well as
the overlying medium, which was replenished every three days.
[0293] Progenitor cells that were cultured on a collagen substrate
in the presence of Hepatocyte Growth Factor and Oncostatin M
displayed a changed cellular morphology consistent with hepatocyte
differentiation, including larger cell size, lower
nuclear-cytoplasmic ratio, and two nuclei (FIG. 2E-F).
Hepatocyte-associated transcripts Albumin and a-Fetoprotein (AFP;
FIG. 2G) were also increased in these cells. Notch2 signaling was
active in cultured oval cells, as transcriptionally active Notch2
intracellular domain (ICD) could be detected using an antibody
specific to the .gamma.-secretase cleaved form of this receptor
(FIG. 2H). The appearance of the cleaved form was dependent on
.gamma.-secretase activity, as it was absent from DAPT treated
cells (FIG. 2H). Specific binding of an anti-Notch2 inhibitory
antibody also blocked formation of this active form of Notch2 (FIG.
2H). As expected, treatment with the Notch2 inhibitory antibody
leads to a decrease in Notch target gene Hes1 (p=6E-05) (FIG. 2I).
Surprisingly, progenitor cells that were cultured on a collagen
substrate in the presence of Hepatocyte Growth Factor and
Oncostatin M in the presence of an anti-Notch2 inhibitory antibody
(Wu et al., Nature 464(7291):1052 (2010)) (FIG. 2H-I) resulted in a
more pronounced hepatocyte-like morphology (FIG. 2 K) and increased
albumin expression level (FIG. 2L), compared to cells cultured with
the isotype control (FIG. 2J, L).
[0294] In contrast, differentiation of cultured oval cells along
the biliary lineage, assessed in three dimensional culture, was
decreased by inhibition of Notch2. Cells cultured in the presence
of an anti-Notch2 inhibitory antibody displayed a less
differentiated morphology (FIG. 2N) and a 50% decreased expression
of the biliary marker Keratin 19 (CK19) (p=2E-05) (FIG. 2O)
compared to cells grown in the absence of the inhibitory antibody
(FIG. 2M, O). Together, these results are consistent with the
notion that Notch2 inhibition biases differentiation away from the
biliary lineage and toward hepatocyte formation.
Example 4
Inhibition of Notch2 Signaling In Vivo
[0295] To determine the effect of Notch2 inhibition in liver
damage, a rodent model of liver damage was employed. Mice were
partially hepatectomized by removal of left lateral and median
lobes according to Yokoyama et al. (Yokoyama et al., Cancer
Research 13(1):80-85 (1953)), which results in compensatory
proliferation of hepatocytes and recovery of liver mass within 7-10
days (Higgins and Anderson, Arch. Pathol., 12:186 (1931); Yokoyama
et al., Cancer Res. 13(1):80 (1953)). Mice were injected
intraperitoneally with an anti-Notch2 NRR antagonist antibody (Wu
et al., Nature 464(7291):1052 (2010)) or an anti-Ragweed isotype
control antibody at a dose of 5 mg/kg twice per week, including
twice prior to hepatectomy. Two hours prior to liver harvest, mice
were injected intraperitoneally with Bromodeoxyuridine at 50 mg/kg.
Immunohistochemistry was performed on 5 .mu.m liver sections using
antibodies for BrdU (DAKO), pan-Cytokeratin (WSS; DAKO), and Hes1
(MBL International). Antibody binding was detected using standard
streptavidin-HRP/DAB for BrdU and pan-Cytokeratin and tyramide
signal amplification (TSA)/DAB for Hes1.
[0296] Treatment with anti-Notch2 antibody resulted in effective
Notch2 inhibition as determined by significant splenic Marginal
Zone B-cell (MZB) depletion (p<0.0001, FIG. 8A-C) (Wu et al.).
MZB represent approximately 5% of the splenic B lymphocytes in
normal C57Bl/6 mice. Treatment with Notch2 antagonist (5 mg/kg,
2.times./week) resulted in a virtual disappearance of MZB (FIG.
8A-C). Notch2 inhibition was maintained through the course of
partial hepatectomy experiments as indicated by persistent
depression in MZB population (FIG. 8C). Inhibition of Notch2 did
not significantly alter the rate of liver mass recovery immediately
following partial hepatectomy (FIG. 8D), which in early stages is
due largely to hepatocyte hypertrophy and division of pre-existing
polyploid hepatocytes (St. Aubin and Bucher, The Anatomical Record
112(4):797 (1952); Higgins and Ingle, The Anatomical Record,
73(1):95 (1939)). Notch2 inhibition caused a small decrease in
overall BrdU incorporation at 40 hours (FIG. 8E; (p=0.027)) and a
much larger and more significant decrease in BrdU incorporation in
intrahepatic bile ducts (FIG. 8F), suggesting that anti-Notch2
treatment affected liver progenitor cells within the bile ducts.
Consistent with this observation, expression of the Notch target
gene Hes1 was detected primarily in intra-hepatic bile duct cells
(FIG. 3C-1) rather than in hepatocytes. Moreover, the percent
Hes1-positive intrahepatic bile duct cells was significantly
reduced in anti-Notch2 treated mice 40 hours after surgery,
compared to control antibody treated mice, and in many portal areas
Hes1 staining was not observed (FIG. 3C-2). Despite the reduction
in Hes1-positive cells, bile duct morphology was unaffected (FIG.
3, compare panels A-1-D-1 on left (isotype) to panels A-2-D-2 on
right (anti-Notch2)) and no significant elevation of markers of
biliary dysfunction was observed, even after one month of ongoing
Notch2 inhibition (FIG. 11F-H).
Example 5
Effects of Notch2 Inhibition on Liver Regeneration In Vivo
[0297] To determine whether Notch2 inhibition affects the recovery
of hepatocyte function following partial hepatectomy, serum
hepatobiliary function markers were assessed following 2/3 partial
hepatectomy in mice treated with anti-Notch2 or control antibody.
Unexpectedly, recovery in liver function began earlier in
anti-Notch2-treated animals compared to controls. Serum albumin
levels started increasing by 40 hours post surgery (FIG. 9A) and
reached significantly (p<0.05) higher levels in anti-Notch2
antibody-treated mice at both the 40 hour and 72 hour time points
(FIG. 3E; FIG. 9A), compared to serum levels in control animals
that started to increase by 72 hours after surgery (2.8 versus 2.4
g/dL, p<0.02). This improvement in recovery of pre-operative
serum albumin levels was accompanied by reduced expression of Hes1
(p<0.02; FIG. 3F). These results suggest that Notch2 inhibition
results in enhanced recovery of hepatocyte function. Markers of
hepatocyte damage were not significantly different between
treatment and control groups (FIG. 5B-F). However, a consistent and
significant increase in the ratio of albumin to K19 transcripts,
referred to herein as Differentiation Quotient, was observed (FIG.
10E). The average Differentiation Quotient of anti-Notch2
antibody-treated livers, normalized to pre-surgical levels,
recovered more quickly, regaining 75% of pre-surgical values 3 days
after surgery and 100% of pre-surgical values between 3 and 6 days
after surgery (FIG. 10E). In contrast, Differentiation Quotient
values in isotype control antibody-treated animals did not recover
to pre-surgical levels until 14 days after surgery (FIG. 10E).
These results suggest that inhibition of Notch2 biases the
differentiation of bipotent liver progenitor cells away from the
biliary (K19-positive) towards the hepatic (albumin-positive)
lineage. Differences between anti-Notch2 and isotype control
antibody-treated livers in apparent de novo hepatocyte formation
could be detected as early as 24 hours after partial hepatectomy.
Transcript levels of in the form of the immature hepatocyte marker
alpha-fetoprotein (AFP) were significantly elevated in anti-Notch2
antibody-treated mice (FIG. 10B). Also, morphological and
functional differences between anti-Notch2 and control
antibody-treated livers persisted for up to 2 weeks after surgery
as the anti-Notch2 antibody-treated livers appeared more robust,
with a noticeably more uniform parenchymal architecture (FIG. 3A-2)
compared to control-treated livers (FIG. 3A-1).
[0298] The effects of Notch2 inhibition on liver regeneration were
also studied in a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)
model of chronic liver disease. This DDC model is known in the art
as a model of chronic liver disease. The mechanism of liver damage
in response to DDC is abnormal heme metabolism with accumulation of
protoporphyrin which is toxic to the hepatocytes. Thus, the DDC
model can also serve as model for hereditary or acquired defects in
the heme metabolic pathway.
[0299] C57BL/6N female mice 8-12 weeks of age (Charles River) were
fed a choline deficient diet (20% Lard; Teklad TD.04523)
supplemented with 0.15% (w/v) Ethionine (supplier) in the drinking
water to induce oval cells (Akhurst et al., Hepatology 34(3):519
(2001)). The prolonged hepatotoxic influence of a DDC diet led to a
proliferation of cytokeratin (CK)19-positive progenitor cells (FIG.
3D-1), referred to as oval cell response, reminiscent of the
ductular reaction common in human hepatobiliary disease (Farber,
Cancer Research, 16(2):142 (1956)). After four weeks of DDC, the
oval cell reaction had increased such that CK19-positive oval cells
occupied an average of about 15% (10-20%) of the total hepatic
cross sectional area (FIG. 3D-1; FIG. 3G), while serum markers of
hepatobiliary injury were greatly elevated (p<0.0001, FIG. 3H;
FIG. 11A-H). However, treatment with the anti-Notch2 inhibitory
antibody significantly impeded the oval cell reaction (FIG. 3D-2),
reducing the average cross-sectional area of CK19-positive tissue
from approximately 15% to only 5% of total liver cross sectional
area (p<0.0001, FIG. 3G). Despite this striking reduction in
oval cell proliferation, hepatic architecture was not adversely
affected by anti-Notch2 antibody treatment and was grossly
indistinguishable from control-treated tissue (FIG. 3D-2). The
decrease in CK19-positive oval cells associated with Notch 2
inhibition was accompanied by improved hepatobiliary function, with
significantly decreased total and direct serum bilirubin levels, a
marker of cholestasis and other forms of hepatobiliary damage
(p=0.0003, FIG. 3H). Also, the Differentiation Quotient was
significantly elevated in livers from mice treated with the
anti-Notch2 antibody (p<0.0001, FIG. 3J) suggesting improved
hepatocyte function Inhibition of Notch2 signaling by treatment
with an anti-Notch2 antibody was confirmed by a greater than 70%
reduction in Hes1 expression in the biliary and progenitor cells in
anti-Notch2 antibody-treated livers (p<0.0001, FIG. 3I),
reflecting the central role of Notch2 signaling in governing oval
cell fate choice.
[0300] Taken together, these results show that treatment with
anti-Notch2 NRR antibody facilitates the recovery of liver function
after two different types of liver damage, one by partial
hepatectomy and one by chemical damage (choline-limiting diet).
Mechanistically, anti-Notch2 NRR antibody facilitates liver
recovery by favoring hepatocyte differentiation and by preventing
aberrant (or pathologic) bile duct proliferation. Accordingly,
treatment with anti-Notch2 NRR antibody could, e.g., prevent
progression of chronic liver disease, such as liver fibrosis.
[0301] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literatures cited herein are expressly
incorporated in their entirety by reference.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 74 <210> SEQ ID NO 1 <211> LENGTH: 10 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic HVR-H1 of Antibody D peptide <400>
SEQUENCE: 1 Gly Tyr Ser Phe Thr Ser Tyr Gly Met Ser 1 5 10
<210> SEQ ID NO 2 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic HVR-H1 of Antibodies D-1, D-2, and D-3 peptide
<400> SEQUENCE: 2 Gly Tyr Thr Phe Ser Ser Tyr Gly Met Ser 1 5
10 <210> SEQ ID NO 3 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic HVR-H1 consensus sequence <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (3)..(3)
<223> OTHER INFORMATION: Ser or Thr <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5)..(5)
<223> OTHER INFORMATION: Ser or Thr <400> SEQUENCE: 3
Gly Tyr Xaa Phe Xaa Ser Tyr Gly Met Ser 1 5 10 <210> SEQ ID
NO 4 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
HVR-H2 of Antibodies D, D-1, D-2, and D-3 peptide <400>
SEQUENCE: 4 Ser Tyr Ile Tyr Pro Tyr Ser Gly Ala Thr Tyr Tyr Ala Asp
Ser Val 1 5 10 15 Lys Gly <210> SEQ ID NO 5 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic HVR-H3 of Antibodies
D, D-1, D-2, and D-3 peptide <400> SEQUENCE: 5 His Ser Gly
Tyr Tyr Arg Ile Ser Ser Ala Met Asp Val 1 5 10 <210> SEQ ID
NO 6 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
HVR-L1 of Antibody D peptide <400> SEQUENCE: 6 Arg Ala Ser
Gln Ser Ile Ser Ser Tyr Leu Ala 1 5 10 <210> SEQ ID NO 7
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic HVR-L1
of Antibody D-1 peptide <400> SEQUENCE: 7 Arg Ala Ser Gln Ser
Asn Arg Arg Phe Leu Ala 1 5 10 <210> SEQ ID NO 8 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic HVR-L1 of Antibody
D-2 peptide <400> SEQUENCE: 8 Arg Ala Ser Gln Ser Val Arg Ser
Phe Leu Ala 1 5 10 <210> SEQ ID NO 9 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic HVR-L1 of Antibody D-3 peptide
<400> SEQUENCE: 9 Arg Ala Ser Gln Asn Ile Lys Arg Phe Leu Ala
1 5 10 <210> SEQ ID NO 10 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic HVR-L1 consensus sequence <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (5)..(5)
<223> OTHER INFORMATION: Ser or Asn <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (6)..(6)
<223> OTHER INFORMATION: Ile, Asn or Val <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (7)..(7)
<223> OTHER INFORMATION: Ser, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (8)..(8)
<223> OTHER INFORMATION: Ser or Arg <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (9)..(9)
<223> OTHER INFORMATION: Tyr or Phe <400> SEQUENCE: 10
Arg Ala Ser Gln Xaa Xaa Xaa Xaa Xaa Leu Ala 1 5 10 <210> SEQ
ID NO 11 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
HVR-L2 of Antibodies D and D-1 peptide <400> SEQUENCE: 11 Gly
Ala Ser Ser Arg Ala Ser 1 5 <210> SEQ ID NO 12 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic HVR-L2 of Antibody
D-2 peptide <400> SEQUENCE: 12 Arg Ala Ser Ile Arg Ala Ser 1
5 <210> SEQ ID NO 13 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic HVR-L2 of Antibody D-3 peptide <400> SEQUENCE: 13
Gly Ala Ser Thr Arg Glu Ser 1 5 <210> SEQ ID NO 14
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic HVR-L2
consensus sequence <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: Gly or Arg <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: Ser, Ile or Thr <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (6)..(6) <223> OTHER
INFORMATION: Ala or Glu <400> SEQUENCE: 14 Xaa Ala Ser Xaa
Arg Xaa Ser 1 5 <210> SEQ ID NO 15 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic HVR-L3 of Antibody D peptide
<400> SEQUENCE: 15 Gln Gln Tyr Tyr Ser Ser Pro Leu Thr 1 5
<210> SEQ ID NO 16 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic HVR-L3 of Antibody D-1 peptide <400> SEQUENCE: 16
Gln Gln Tyr Tyr Ile Ser Pro Leu Thr 1 5 <210> SEQ ID NO 17
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic HVR-L3
of Antibody D-2 peptide <400> SEQUENCE: 17 Gln Gln Tyr Tyr
Ile Ser Pro Trp Thr 1 5 <210> SEQ ID NO 18 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic HVR-L3 of Antibody
D-3 peptide <400> SEQUENCE: 18 Gln Gln Tyr Tyr Arg Ser Pro
His Thr 1 5 <210> SEQ ID NO 19 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic HVR-L3 consensus sequence
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Ser, Ile or Arg
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (8)..(8) <223> OTHER INFORMATION: Leu, Trp or His
<400> SEQUENCE: 19 Gln Gln Tyr Tyr Xaa Ser Pro Xaa Thr 1 5
<210> SEQ ID NO 20 <211> LENGTH: 122 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic Heavy chain variable region of Antibody D polypeptide
<400> SEQUENCE: 20 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 Ser Phe Thr Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Tyr
Pro Tyr Ser Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg His Ser Gly Tyr Tyr Arg Ile Ser Ser Ala Met Asp Val
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 120
<210> SEQ ID NO 21 <211> LENGTH: 122 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic Heavy chain variable region of Antibodies D-1, D-2, and
D-3 polypeptide <400> SEQUENCE: 21 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 Thr Phe Ser Ser Tyr 20 25 30 Gly Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Tyr Ile Tyr Pro Tyr Ser Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg His Ser Gly Tyr Tyr Arg Ile Ser Ser
Ala Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser
Ala 115 120 <210> SEQ ID NO 22 <211> LENGTH: 108
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Light chain variable region of
Antibody D polypeptide <400> SEQUENCE: 22 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
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Gly Ala Ser Ser Arg Ala 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 Tyr Tyr
Ser Ser Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 <210> SEQ ID NO 23 <211> LENGTH: 108
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Light chain variable region of
Antibody D-1 polypeptide <400> SEQUENCE: 23 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 Asn Arg Arg Phe 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Gly Ala Ser Ser Arg Ala 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 Tyr
Tyr Ile Ser Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 <210> SEQ ID NO 24 <211> LENGTH:
108 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Light chain variable region of
Antibody D-2 polypeptide <400> SEQUENCE: 24 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 Val Arg Ser Phe 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Arg Ala Ser Ile Arg Ala 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 Tyr
Tyr Ile Ser Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 <210> SEQ ID NO 25 <211> LENGTH:
108 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Light chain variable region of
Antibody D-3 polypeptide <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 Arg Ala Ser Gln Asn Ile Lys Arg Phe 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Gly Ala Ser Thr Arg Glu 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 Tyr
Tyr Arg Ser Pro His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 <210> SEQ ID NO 26 <400> SEQUENCE:
26 000 <210> SEQ ID NO 27 <400> SEQUENCE: 27 000
<210> SEQ ID NO 28 <400> SEQUENCE: 28 000 <210>
SEQ ID NO 29 <400> SEQUENCE: 29 000 <210> SEQ ID NO 30
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 30 Gly Val Pro Ser Arg Phe Ser
Gly Ser Arg Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 <210>
SEQ ID NO 31 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic framework region 4 peptide <400> SEQUENCE: 31 Phe
Arg Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> SEQ ID NO 32
<211> LENGTH: 30 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 32 Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 <210> SEQ ID NO
33 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 33 Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met Gly 1 5 10 <210> SEQ ID NO 34 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 34 Arg Val Thr Ile Thr Ala Asp Thr Ser Thr
Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> SEQ ID NO 35
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 35 Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 1 5 10 <210> SEQ ID NO 36 <211> LENGTH: 25
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 36 Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 <210> SEQ ID NO
37 <211> LENGTH: 13 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 37 Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 1 5 10 <210> SEQ ID NO 38 <211>
LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 38 Arg Val Thr Ile Thr Ala Asp Thr Ser Thr
Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys Ala 20 25 30 <210> SEQ ID NO 39
<211> LENGTH: 30 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 39 Arg Val Thr Ile Thr Ala Asp
Thr Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 <210> SEQ ID NO
40 <211> LENGTH: 30 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 40 Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Gly Ser Val Ser 20 25 30 <210> SEQ ID NO
41 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 41 Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Ile Gly 1 5 10 <210> SEQ ID NO 42 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 42 Arg Val Thr Ile Ser Val Asp Thr Ser Lys
Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> SEQ ID NO 43
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 43 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val
Ser 20 25 <210> SEQ ID NO 44 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 44 Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 1 5 10 <210>
SEQ ID NO 45 <211> LENGTH: 31 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 45 Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 20 25 30
<210> SEQ ID NO 46 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 46 Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 20 25 30 <210>
SEQ ID NO 47 <211> LENGTH: 30 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 47 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 20 25 30 <210>
SEQ ID NO 48 <211> LENGTH: 14 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 48 Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> SEQ ID NO 49
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 49 Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210>
SEQ ID NO 50 <211> LENGTH: 25 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 50 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 20 25 <210> SEQ ID NO 51 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <400>
SEQUENCE: 51 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 1
5 10 <210> SEQ ID NO 52 <211> LENGTH: 31 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 52 Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 20 25
30 <210> SEQ ID NO 53 <211> LENGTH: 30 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 53 Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30
<210> SEQ ID NO 54 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 54 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 Asn Ile Lys 20 25 30 <210>
SEQ ID NO 55 <211> LENGTH: 32 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 55 Arg Phe Thr Ile Ser
Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg 20 25 30
<210> SEQ ID NO 56 <211> LENGTH: 31 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 56 Arg Phe Thr Ile Ser
Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser 20 25 30
<210> SEQ ID NO 57 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 57 Arg Phe Thr Ile Ser
Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30
<210> SEQ ID NO 58 <211> LENGTH: 31 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 58 Arg Phe Thr Ile Ser
Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 20 25 30
<210> SEQ ID NO 59 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 59 Arg Phe Thr Ile Ser
Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 <210>
SEQ ID NO 60 <211> LENGTH: 23 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 60 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 20 <210> SEQ ID NO 61 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 61 Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15
<210> SEQ ID NO 62 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 62 Gly Val Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30
<210> SEQ ID NO 63 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 63 Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 1 5 10 <210> SEQ ID NO 64 <211> LENGTH:
23 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 64 Asp
Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys 20 <210> SEQ ID NO 65
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 65 Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro
Gln Leu Leu Ile Tyr 1 5 10 15 <210> SEQ ID NO 66 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 66 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 <210> SEQ ID NO 67
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 67 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20
<210> SEQ ID NO 68 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 68 Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile Tyr 1 5 10 15 <210> SEQ ID NO
69 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 69 Gly Ile Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Arg
Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30 <210>
SEQ ID NO 70 <211> LENGTH: 23 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 70 Asp Ile Val Met Thr Gln
Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr
Ile Asn Cys 20 <210> SEQ ID NO 71 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 71 Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 1 5 10 15
<210> SEQ ID NO 72 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 72 Gly Val Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile
Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys 20 25 30
<210> SEQ ID NO 73 <211> LENGTH: 2471 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 73 Met
Pro Ala Leu Arg Pro Ala Leu Leu Trp Ala Leu Leu Ala Leu Trp 1 5 10
15 Leu Cys Cys Ala Ala Pro Ala His Ala Leu Gln Cys Arg Asp Gly Tyr
20 25 30 Glu Pro Cys Val Asn Glu Gly Met Cys Val Thr Tyr His Asn
Gly Thr 35 40 45 Gly Tyr Cys Lys Cys Pro Glu Gly Phe Leu Gly Glu
Tyr Cys Gln His 50 55 60 Arg Asp Pro Cys Glu Lys Asn Arg Cys Gln
Asn Gly Gly Thr Cys Val 65 70 75 80 Ala Gln Ala Met Leu Gly Lys Ala
Thr Cys Arg Cys Ala Ser Gly Phe 85 90 95 Thr Gly Glu Asp Cys Gln
Tyr Ser Thr Ser His Pro Cys Phe Val Ser 100 105 110 Arg Pro Cys Leu
Asn Gly Gly Thr Cys His Met Leu Ser Arg Asp Thr 115 120 125 Tyr Glu
Cys Thr Cys Gln Val Gly Phe Thr Gly Lys Glu Cys Gln Trp 130 135 140
Thr Asp Ala Cys Leu Ser His Pro Cys Ala Asn Gly Ser Thr Cys Thr 145
150 155 160 Thr Val Ala Asn Gln Phe Ser Cys Lys Cys Leu Thr Gly Phe
Thr Gly 165 170 175 Gln Lys Cys Glu Thr Asp Val Asn Glu Cys Asp Ile
Pro Gly His Cys 180 185 190 Gln His Gly Gly Thr Cys Leu Asn Leu Pro
Gly Ser Tyr Gln Cys Gln 195 200 205 Cys Pro Gln Gly Phe Thr Gly Gln
Tyr Cys Asp Ser Leu Tyr Val Pro 210 215 220 Cys Ala Pro Ser Pro Cys
Val Asn Gly Gly Thr Cys Arg Gln Thr Gly 225 230 235 240 Asp Phe Thr
Phe Glu Cys Asn Cys Leu Pro Gly Phe Glu Gly Ser Thr 245 250 255 Cys
Glu Arg Asn Ile Asp Asp Cys Pro Asn His Arg Cys Gln Asn Gly 260 265
270 Gly Val Cys Val Asp Gly Val Asn Thr Tyr Asn Cys Arg Cys Pro Pro
275 280 285 Gln Trp Thr Gly Gln Phe Cys Thr Glu Asp Val Asp Glu Cys
Leu Leu 290 295 300 Gln Pro Asn Ala Cys Gln Asn Gly Gly Thr Cys Ala
Asn Arg Asn Gly 305 310 315 320 Gly Tyr Gly Cys Val Cys Val Asn Gly
Trp Ser Gly Asp Asp Cys Ser 325 330 335 Glu Asn Ile Asp Asp Cys Ala
Phe Ala Ser Cys Thr Pro Gly Ser Thr 340 345 350 Cys Ile Asp Arg Val
Ala Ser Phe Ser Cys Met Cys Pro Glu Gly Lys 355 360 365 Ala Gly Leu
Leu Cys His Leu Asp Asp Ala Cys Ile Ser Asn Pro Cys 370 375 380 His
Lys Gly Ala Leu Cys Asp Thr Asn Pro Leu Asn Gly Gln Tyr Ile 385 390
395 400 Cys Thr Cys Pro Gln Gly Tyr Lys Gly Ala Asp Cys Thr Glu Asp
Val 405 410 415 Asp Glu Cys Ala Met Ala Asn Ser Asn Pro Cys Glu His
Ala Gly Lys 420 425 430 Cys Val Asn Thr Asp Gly Ala Phe His Cys Glu
Cys Leu Lys Gly Tyr 435 440 445 Ala Gly Pro Arg Cys Glu Met Asp Ile
Asn Glu Cys His Ser Asp Pro 450 455 460 Cys Gln Asn Asp Ala Thr Cys
Leu Asp Lys Ile Gly Gly Phe Thr Cys 465 470 475 480 Leu Cys Met Pro
Gly Phe Lys Gly Val His Cys Glu Leu Glu Ile Asn 485 490 495 Glu Cys
Gln Ser Asn Pro Cys Val Asn Asn Gly Gln Cys Val Asp Lys 500 505 510
Val Asn Arg Phe Gln Cys Leu Cys Pro Pro Gly Phe Thr Gly Pro Val 515
520 525 Cys Gln Ile Asp Ile Asp Asp Cys Ser Ser Thr Pro Cys Leu Asn
Gly 530 535 540 Ala Lys Cys Ile Asp His Pro Asn Gly Tyr Glu Cys Gln
Cys Ala Thr 545 550 555 560 Gly Phe Thr Gly Val Leu Cys Glu Glu Asn
Ile Asp Asn Cys Asp Pro 565 570 575 Asp Pro Cys His His Gly Gln Cys
Gln Asp Gly Ile Asp Ser Tyr Thr 580 585 590 Cys Ile Cys Asn Pro Gly
Tyr Met Gly Ala Ile Cys Ser Asp Gln Ile 595 600 605 Asp Glu Cys Tyr
Ser Ser Pro Cys Leu Asn Asp Gly Arg Cys Ile Asp 610 615 620 Leu Val
Asn Gly Tyr Gln Cys Asn Cys Gln Pro Gly Thr Ser Gly Val 625 630 635
640 Asn Cys Glu Ile Asn Phe Asp Asp Cys Ala Ser Asn Pro Cys Ile His
645 650 655 Gly Ile Cys Met Asp Gly Ile Asn Arg Tyr Ser Cys Val Cys
Ser Pro 660 665 670 Gly Phe Thr Gly Gln Arg Cys Asn Ile Asp Ile Asp
Glu Cys Ala Ser 675 680 685 Asn Pro Cys Arg Lys Gly Ala Thr Cys Ile
Asn Gly Val Asn Gly Phe 690 695 700 Arg Cys Ile Cys Pro Glu Gly Pro
His His Pro Ser Cys Tyr Ser Gln 705 710 715 720 Val Asn Glu Cys Leu
Ser Asn Pro Cys Ile His Gly Asn Cys Thr Gly 725 730 735 Gly Leu Ser
Gly Tyr Lys Cys Leu Cys Asp Ala Gly Trp Val Gly Ile 740 745 750 Asn
Cys Glu Val Asp Lys Asn Glu Cys Leu Ser Asn Pro Cys Gln Asn 755 760
765 Gly Gly Thr Cys Asp Asn Leu Val Asn Gly Tyr Arg Cys Thr Cys Lys
770 775 780 Lys Gly Phe Lys Gly Tyr Asn Cys Gln Val Asn Ile Asp Glu
Cys Ala 785 790 795 800 Ser Asn Pro Cys Leu Asn Gln Gly Thr Cys Phe
Asp Asp Ile Ser Gly 805 810 815 Tyr Thr Cys His Cys Val Leu Pro Tyr
Thr Gly Lys Asn Cys Gln Thr 820 825 830 Val Leu Ala Pro Cys Ser Pro
Asn Pro Cys Glu Asn Ala Ala Val Cys 835 840 845 Lys Glu Ser Pro Asn
Phe Glu Ser Tyr Thr Cys Leu Cys Ala Pro Gly 850 855 860 Trp Gln Gly
Gln Arg Cys Thr Ile Asp Ile Asp Glu Cys Ile Ser Lys 865 870 875 880
Pro Cys Met Asn His Gly Leu Cys His Asn Thr Gln Gly Ser Tyr Met 885
890 895 Cys Glu Cys Pro Pro Gly Phe Ser Gly Met Asp Cys Glu Glu Asp
Ile 900 905 910 Asp Asp Cys Leu Ala Asn Pro Cys Gln Asn Gly Gly Ser
Cys Met Asp 915 920 925 Gly Val Asn Thr Phe Ser Cys Leu Cys Leu Pro
Gly Phe Thr Gly Asp 930 935 940 Lys Cys Gln Thr Asp Met Asn Glu Cys
Leu Ser Glu Pro Cys Lys Asn 945 950 955 960 Gly Gly Thr Cys Ser Asp
Tyr Val Asn Ser Tyr Thr Cys Lys Cys Gln 965 970 975 Ala Gly Phe Asp
Gly Val His Cys Glu Asn Asn Ile Asn Glu Cys Thr 980 985 990 Glu Ser
Ser Cys Phe Asn Gly Gly Thr Cys Val Asp Gly Ile Asn Ser 995 1000
1005 Phe Ser Cys Leu Cys Pro Val Gly Phe Thr Gly Ser Phe Cys Leu
1010 1015 1020 His Glu Ile Asn Glu Cys Ser Ser His Pro Cys Leu Asn
Glu Gly 1025 1030 1035 Thr Cys Val Asp Gly Leu Gly Thr Tyr Arg Cys
Ser Cys Pro Leu 1040 1045 1050 Gly Tyr Thr Gly Lys Asn Cys Gln Thr
Leu Val Asn Leu Cys Ser 1055 1060 1065 Arg Ser Pro Cys Lys Asn Lys
Gly Thr Cys Val Gln Lys Lys Ala 1070 1075 1080 Glu Ser Gln Cys Leu
Cys Pro Ser Gly Trp Ala Gly Ala Tyr Cys 1085 1090 1095 Asp Val Pro
Asn Val Ser Cys Asp Ile Ala Ala Ser Arg Arg Gly 1100 1105 1110 Val
Leu Val Glu His Leu Cys Gln His Ser Gly Val Cys Ile Asn 1115 1120
1125 Ala Gly Asn Thr His Tyr Cys Gln Cys Pro Leu Gly Tyr Thr Gly
1130 1135 1140 Ser Tyr Cys Glu Glu Gln Leu Asp Glu Cys Ala Ser Asn
Pro Cys 1145 1150 1155 Gln His Gly Ala Thr Cys Ser Asp Phe Ile Gly
Gly Tyr Arg Cys 1160 1165 1170 Glu Cys Val Pro Gly Tyr Gln Gly Val
Asn Cys Glu Tyr Glu Val 1175 1180 1185 Asp Glu Cys Gln Asn Gln Pro
Cys Gln Asn Gly Gly Thr Cys Ile 1190 1195 1200 Asp Leu Val Asn His
Phe Lys Cys Ser Cys Pro Pro Gly Thr Arg 1205 1210 1215 Gly Leu Leu
Cys Glu Glu Asn Ile Asp Asp Cys Ala Arg Gly Pro 1220 1225 1230 His
Cys Leu Asn Gly Gly Gln Cys Met Asp Arg Ile Gly Gly Tyr 1235 1240
1245 Ser Cys Arg Cys Leu Pro Gly Phe Ala Gly Glu Arg Cys Glu Gly
1250 1255 1260 Asp Ile Asn Glu Cys Leu Ser Asn Pro Cys Ser Ser Glu
Gly Ser 1265 1270 1275 Leu Asp Cys Ile Gln Leu Thr Asn Asp Tyr Leu
Cys Val Cys Arg 1280 1285 1290 Ser Ala Phe Thr Gly Arg His Cys Glu
Thr Phe Val Asp Val Cys 1295 1300 1305 Pro Gln Met Pro Cys Leu Asn
Gly Gly Thr Cys Ala Val Ala Ser 1310 1315 1320 Asn Met Pro Asp Gly
Phe Ile Cys Arg Cys Pro Pro Gly Phe Ser 1325 1330 1335 Gly Ala Arg
Cys Gln Ser Ser Cys Gly Gln Val Lys Cys Arg Lys 1340 1345 1350 Gly
Glu Gln Cys Val His Thr Ala Ser Gly Pro Arg Cys Phe Cys 1355 1360
1365 Pro Ser Pro Arg Asp Cys Glu Ser Gly Cys Ala Ser Ser Pro Cys
1370 1375 1380 Gln His Gly Gly Ser Cys His Pro Gln Arg Gln Pro Pro
Tyr Tyr 1385 1390 1395 Ser Cys Gln Cys Ala Pro Pro Phe Ser Gly Ser
Arg Cys Glu Leu 1400 1405 1410 Tyr Thr Ala Pro Pro Ser Thr Pro Pro
Ala Thr Cys Leu Ser Gln 1415 1420 1425 Tyr Cys Ala Asp Lys Ala Arg
Asp Gly Val Cys Asp Glu Ala Cys 1430 1435 1440 Asn Ser His Ala Cys
Gln Trp Asp Gly Gly Asp Cys Ser Leu Thr 1445 1450 1455 Met Glu Asn
Pro Trp Ala Asn Cys Ser Ser Pro Leu Pro Cys Trp 1460 1465 1470 Asp
Tyr Ile Asn Asn Gln Cys Asp Glu Leu Cys Asn Thr Val Glu 1475 1480
1485 Cys Leu Phe Asp Asn Phe Glu Cys Gln Gly Asn Ser Lys Thr Cys
1490 1495 1500 Lys Tyr Asp Lys Tyr Cys Ala Asp His Phe Lys Asp Asn
His Cys 1505 1510 1515 Asp Gln Gly Cys Asn Ser Glu Glu Cys Gly Trp
Asp Gly Leu Asp 1520 1525 1530 Cys Ala Ala Asp Gln Pro Glu Asn Leu
Ala Glu Gly Thr Leu Val 1535 1540 1545 Ile Val Val Leu Met Pro Pro
Glu Gln Leu Leu Gln Asp Ala Arg 1550 1555 1560 Ser Phe Leu Arg Ala
Leu Gly Thr Leu Leu His Thr Asn Leu Arg 1565 1570 1575 Ile Lys Arg
Asp Ser Gln Gly Glu Leu Met Val Tyr Pro Tyr Tyr 1580 1585 1590 Gly
Glu Lys Ser Ala Ala Met Lys Lys Gln Arg Met Thr Arg Arg 1595 1600
1605 Ser Leu Pro Gly Glu Gln Glu Gln Glu Val Ala Gly Ser Lys Val
1610 1615 1620 Phe Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Asp Ser
Asp His 1625 1630 1635 Cys Phe Lys Asn Thr Asp Ala Ala Ala Ala Leu
Leu Ala Ser His 1640 1645 1650 Ala Ile Gln Gly Thr Leu Ser Tyr Pro
Leu Val Ser Val Val Ser 1655 1660 1665 Glu Ser Leu Thr Pro Glu Arg
Thr Gln Leu Leu Tyr Leu Leu Ala 1670 1675 1680 Val Ala Val Val Ile
Ile Leu Phe Ile Ile Leu Leu Gly Val Ile 1685 1690 1695 Met Ala Lys
Arg Lys Arg Lys His Gly Ser Leu Trp Leu Pro Glu 1700 1705 1710 Gly
Phe Thr Leu Arg Arg Asp Ala Ser Asn His Lys Arg Arg Glu 1715 1720
1725 Pro Val Gly Gln Asp Ala Val Gly Leu Lys Asn Leu Ser Val Gln
1730 1735 1740 Val Ser Glu Ala Asn Leu Ile Gly Thr Gly Thr Ser Glu
His Trp 1745 1750 1755 Val Asp Asp Glu Gly Pro Gln Pro Lys Lys Val
Lys Ala Glu Asp 1760 1765 1770 Glu Ala Leu Leu Ser Glu Glu Asp Asp
Pro Ile Asp Arg Arg Pro 1775 1780 1785 Trp Thr Gln Gln His Leu Glu
Ala Ala Asp Ile Arg Arg Thr Pro 1790 1795 1800 Ser Leu Ala Leu Thr
Pro Pro Gln Ala Glu Gln Glu Val Asp Val 1805 1810 1815 Leu Asp Val
Asn Val Arg Gly Pro Asp Gly Cys Thr Pro Leu Met 1820 1825 1830 Leu
Ala Ser Leu Arg Gly Gly Ser Ser Asp Leu Ser Asp Glu Asp 1835 1840
1845 Glu Asp Ala Glu Asp Ser Ser Ala Asn Ile Ile Thr Asp Leu Val
1850 1855 1860 Tyr Gln Gly Ala Ser Leu Gln Ala Gln Thr Asp Arg Thr
Gly Glu 1865 1870 1875 Met Ala Leu His Leu Ala Ala Arg Tyr Ser Arg
Ala Asp Ala Ala 1880 1885 1890 Lys Arg Leu Leu Asp Ala Gly Ala Asp
Ala Asn Ala Gln Asp Asn 1895 1900 1905 Met Gly Arg Cys Pro Leu His
Ala Ala Val Ala Ala Asp Ala Gln 1910 1915 1920 Gly Val Phe Gln Ile
Leu Ile Arg Asn Arg Val Thr Asp Leu Asp 1925 1930 1935 Ala Arg Met
Asn Asp Gly Thr Thr Pro Leu Ile Leu Ala Ala Arg 1940 1945 1950 Leu
Ala Val Glu Gly Met Val Ala Glu Leu Ile Asn Cys Gln Ala 1955 1960
1965 Asp Val Asn Ala Val Asp Asp His Gly Lys Ser Ala Leu His Trp
1970 1975 1980 Ala Ala Ala Val Asn Asn Val Glu Ala Thr Leu Leu Leu
Leu Lys 1985 1990 1995 Asn Gly Ala Asn Arg Asp Met Gln Asp Asn Lys
Glu Glu Thr Pro 2000 2005 2010 Leu Phe Leu Ala Ala Arg Glu Gly Ser
Tyr Glu Ala Ala Lys Ile 2015 2020 2025 Leu Leu Asp His Phe Ala Asn
Arg Asp Ile Thr Asp His Met Asp 2030 2035 2040 Arg Leu Pro Arg Asp
Val Ala Arg Asp Arg Met His His Asp Ile 2045 2050 2055 Val Arg Leu
Leu Asp Glu Tyr Asn Val Thr Pro Ser Pro Pro Gly 2060 2065 2070 Thr
Val Leu Thr Ser Ala Leu Ser Pro Val Ile Cys Gly Pro Asn 2075 2080
2085 Arg Ser Phe Leu Ser Leu Lys His Thr Pro Met Gly Lys Lys Ser
2090 2095 2100 Arg Arg Pro Ser Ala Lys Ser Thr Met Pro Thr Ser Leu
Pro Asn 2105 2110 2115 Leu Ala Lys Glu Ala Lys Asp Ala Lys Gly Ser
Arg Arg Lys Lys 2120 2125 2130 Ser Leu Ser Glu Lys Val Gln Leu Ser
Glu Ser Ser Val Thr Leu 2135 2140 2145 Ser Pro Val Asp Ser Leu Glu
Ser Pro His Thr Tyr Val Ser Asp 2150 2155 2160 Thr Thr Ser Ser Pro
Met Ile Thr Ser Pro Gly Ile Leu Gln Ala 2165 2170 2175 Ser Pro Asn
Pro Met Leu Ala Thr Ala Ala Pro Pro Ala Pro Val 2180 2185 2190 His
Ala Gln His Ala Leu Ser Phe Ser Asn Leu His Glu Met Gln 2195 2200
2205 Pro Leu Ala His Gly Ala Ser Thr Val Leu Pro Ser Val Ser Gln
2210 2215 2220 Leu Leu Ser His His His Ile Val Ser Pro Gly Ser Gly
Ser Ala 2225 2230 2235 Gly Ser Leu Ser Arg Leu His Pro Val Pro Val
Pro Ala Asp Trp 2240 2245 2250 Met Asn Arg Met Glu Val Asn Glu Thr
Gln Tyr Asn Glu Met Phe 2255 2260 2265 Gly Met Val Leu Ala Pro Ala
Glu Gly Thr His Pro Gly Ile Ala 2270 2275 2280 Pro Gln Ser Arg Pro
Pro Glu Gly Lys His Ile Thr Thr Pro Arg 2285 2290 2295 Glu Pro Leu
Pro Pro Ile Val Thr Phe Gln Leu Ile Pro Lys Gly 2300 2305 2310 Ser
Ile Ala Gln Pro Ala Gly Ala Pro Gln Pro Gln Ser Thr Cys 2315 2320
2325 Pro Pro Ala Val Ala Gly Pro Leu Pro Thr Met Tyr Gln Ile Pro
2330 2335 2340 Glu Met Ala Arg Leu Pro Ser Val Ala Phe Pro Thr Ala
Met Met 2345 2350 2355 Pro Gln Gln Asp Gly Gln Val Ala Gln Thr Ile
Leu Pro Ala Tyr 2360 2365 2370 His Pro Phe Pro Ala Ser Val Gly Lys
Tyr Pro Thr Pro Pro Ser 2375 2380 2385 Gln His Ser Tyr Ala Ser Ser
Asn Ala Ala Glu Arg Thr Pro Ser 2390 2395 2400 His Ser Gly His Leu
Gln Gly Glu His Pro Tyr Leu Thr Pro Ser 2405 2410 2415 Pro Glu Ser
Pro Asp Gln Trp Ser Ser Ser Ser Pro His Ser Ala 2420 2425 2430 Ser
Asp Trp Ser Asp Val Thr Thr Ser Pro Thr Pro Gly Gly Ala 2435 2440
2445 Gly Gly Gly Gln Arg Gly Pro Gly Thr His Met Ser Glu Pro Pro
2450 2455 2460 His Asn Asn Met Gln Val Tyr Ala 2465 2470
<210> SEQ ID NO 74 <211> LENGTH: 256 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 74 Pro
Ala Thr Cys Leu Ser Gln Tyr Cys Ala Asp Lys Ala Arg Asp Gly 1 5 10
15 Val Cys Asp Glu Ala Cys Asn Ser His Ala Cys Gln Trp Asp Gly Gly
20 25 30 Asp Cys Ser Leu Thr Met Glu Asn Pro Trp Ala Asn Cys Ser
Ser Pro 35 40 45 Leu Pro Cys Trp Asp Tyr Ile Asn Asn Gln Cys Asp
Glu Leu Cys Asn 50 55 60 Thr Val Glu Cys Leu Phe Asp Asn Phe Glu
Cys Gln Gly Asn Ser Lys 65 70 75 80 Thr Cys Lys Tyr Asp Lys Tyr Cys
Ala Asp His Phe Lys Asp Asn His 85 90 95 Cys Asp Gln Gly Cys Asn
Ser Glu Glu Cys Gly Trp Asp Gly Leu Asp 100 105 110 Cys Ala Ala Asp
Gln Pro Glu Asn Leu Ala Glu Gly Thr Leu Val Ile 115 120 125 Val Val
Leu Met Pro Pro Glu Gln Leu Leu Gln Asp Ala Arg Ser Phe 130 135 140
Leu Arg Ala Leu Gly Thr Leu Leu His Thr Asn Leu Arg Ile Lys Arg 145
150 155 160 Asp Ser Gln Gly Glu Leu Met Val Tyr Pro Tyr Tyr Gly Glu
Lys Ser 165 170 175 Ala Ala Met Lys Lys Gln Arg Met Thr Arg Arg Ser
Leu Pro Gly Glu 180 185 190 Gln Glu Gln Glu Val Ala Gly Ser Lys Val
Phe Leu Glu Ile Asp Asn 195 200 205 Arg Gln Cys Val Gln Asp Ser Asp
His Cys Phe Lys Asn Thr Asp Ala 210 215 220 Ala Ala Ala Leu Leu Ala
Ser His Ala Ile Gln Gly Thr Leu Ser Tyr 225 230 235 240 Pro Leu Val
Ser Val Val Ser Glu Ser Leu Thr Pro Glu Arg Thr Gln 245 250 255
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 74 <210>
SEQ ID NO 1 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic HVR-H1 of Antibody D peptide <400> SEQUENCE: 1 Gly
Tyr Ser Phe Thr Ser Tyr Gly Met Ser 1 5 10 <210> SEQ ID NO 2
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic HVR-H1
of Antibodies D-1, D-2, and D-3 peptide <400> SEQUENCE: 2 Gly
Tyr Thr Phe Ser Ser Tyr Gly Met Ser 1 5 10 <210> SEQ ID NO 3
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic HVR-H1
consensus sequence <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Ser or Thr <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (5)..(5) <223> OTHER
INFORMATION: Ser or Thr <400> SEQUENCE: 3 Gly Tyr Xaa Phe Xaa
Ser Tyr Gly Met Ser 1 5 10 <210> SEQ ID NO 4 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic HVR-H2 of Antibodies
D, D-1, D-2, and D-3 peptide <400> SEQUENCE: 4 Ser Tyr Ile
Tyr Pro Tyr Ser Gly Ala Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys
Gly <210> SEQ ID NO 5 <211> LENGTH: 13 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic HVR-H3 of Antibodies D, D-1, D-2, and D-3
peptide <400> SEQUENCE: 5 His Ser Gly Tyr Tyr Arg Ile Ser Ser
Ala Met Asp Val 1 5 10 <210> SEQ ID NO 6 <211> LENGTH:
11 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic HVR-L1 of Antibody D peptide
<400> SEQUENCE: 6 Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ala
1 5 10 <210> SEQ ID NO 7 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic HVR-L1 of Antibody D-1 peptide <400>
SEQUENCE: 7 Arg Ala Ser Gln Ser Asn Arg Arg Phe Leu Ala 1 5 10
<210> SEQ ID NO 8 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic HVR-L1 of Antibody D-2 peptide <400> SEQUENCE: 8
Arg Ala Ser Gln Ser Val Arg Ser Phe Leu Ala 1 5 10 <210> SEQ
ID NO 9 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
HVR-L1 of Antibody D-3 peptide <400> SEQUENCE: 9 Arg Ala Ser
Gln Asn Ile Lys Arg Phe Leu Ala 1 5 10 <210> SEQ ID NO 10
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic HVR-L1
consensus sequence <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (5)..(5) <223> OTHER
INFORMATION: Ser or Asn <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (6)..(6) <223> OTHER
INFORMATION: Ile, Asn or Val <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (7)..(7) <223> OTHER
INFORMATION: Ser, Arg or Lys <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (8)..(8) <223> OTHER
INFORMATION: Ser or Arg <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (9)..(9) <223> OTHER
INFORMATION: Tyr or Phe <400> SEQUENCE: 10 Arg Ala Ser Gln
Xaa Xaa Xaa Xaa Xaa Leu Ala 1 5 10 <210> SEQ ID NO 11
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic HVR-L2
of Antibodies D and D-1 peptide <400> SEQUENCE: 11 Gly Ala
Ser Ser Arg Ala Ser 1 5 <210> SEQ ID NO 12 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic HVR-L2 of Antibody
D-2 peptide <400> SEQUENCE: 12 Arg Ala Ser Ile Arg Ala Ser 1
5 <210> SEQ ID NO 13 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic HVR-L2 of Antibody D-3 peptide <400> SEQUENCE: 13
Gly Ala Ser Thr Arg Glu Ser 1 5 <210> SEQ ID NO 14
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic HVR-L2
consensus sequence <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: Gly or Arg <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: Ser, Ile or Thr <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (6)..(6)
<223> OTHER INFORMATION: Ala or Glu <400> SEQUENCE: 14
Xaa Ala Ser Xaa Arg Xaa Ser 1 5 <210> SEQ ID NO 15
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic HVR-L3
of Antibody D peptide <400> SEQUENCE: 15 Gln Gln Tyr Tyr Ser
Ser Pro Leu Thr 1 5 <210> SEQ ID NO 16 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic HVR-L3 of Antibody D-1 peptide
<400> SEQUENCE: 16 Gln Gln Tyr Tyr Ile Ser Pro Leu Thr 1 5
<210> SEQ ID NO 17 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic HVR-L3 of Antibody D-2 peptide <400> SEQUENCE: 17
Gln Gln Tyr Tyr Ile Ser Pro Trp Thr 1 5 <210> SEQ ID NO 18
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic HVR-L3
of Antibody D-3 peptide <400> SEQUENCE: 18 Gln Gln Tyr Tyr
Arg Ser Pro His Thr 1 5 <210> SEQ ID NO 19 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic HVR-L3 consensus
sequence <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Ser,
Ile or Arg <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (8)..(8) <223> OTHER INFORMATION: Leu,
Trp or His <400> SEQUENCE: 19 Gln Gln Tyr Tyr Xaa Ser Pro Xaa
Thr 1 5 <210> SEQ ID NO 20 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Heavy chain variable region of
Antibody D polypeptide <400> SEQUENCE: 20 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 Ser Phe Thr Ser Tyr 20 25 30 Gly
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Tyr Ile Tyr Pro Tyr Ser Gly Ala Thr Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg His Ser Gly Tyr Tyr Arg Ile Ser
Ser Ala Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val
Ser Ala 115 120 <210> SEQ ID NO 21 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Heavy chain variable region of
Antibodies D-1, D-2, and D-3 polypeptide <400> SEQUENCE: 21
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 Thr Phe Ser Ser
Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Tyr Ile Tyr Pro Tyr Ser Gly Ala Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Ser Gly
Tyr Tyr Arg Ile Ser Ser Ala Met Asp Val Trp 100 105 110 Gly Gln Gly
Thr Leu Val Thr Val Ser Ala 115 120 <210> SEQ ID NO 22
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic Light
chain variable region of Antibody D polypeptide <400>
SEQUENCE: 22 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 Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Arg Ala
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 Tyr Tyr Ser Ser Pro Leu 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ
ID NO 23 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Light chain variable region of Antibody D-1 polypeptide <400>
SEQUENCE: 23 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 Asn Arg Arg Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Arg Ala
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 Tyr Tyr Ile Ser Pro Leu 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ
ID NO 24 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Light chain variable region of Antibody D-2 polypeptide <400>
SEQUENCE: 24 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 Val Arg Ser Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Ile Arg Ala
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 Tyr Tyr
Ile Ser Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 <210> SEQ ID NO 25 <211> LENGTH: 108
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Light chain variable region of
Antibody D-3 polypeptide <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 Arg Ala Ser Gln Asn Ile Lys Arg Phe 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Gly Ala Ser Thr Arg Glu 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 Tyr
Tyr Arg Ser Pro His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 <210> SEQ ID NO 26 <400> SEQUENCE:
26 000 <210> SEQ ID NO 27 <400> SEQUENCE: 27 000
<210> SEQ ID NO 28 <400> SEQUENCE: 28 000 <210>
SEQ ID NO 29 <400> SEQUENCE: 29 000 <210> SEQ ID NO 30
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 30 Gly Val Pro Ser Arg Phe Ser
Gly Ser Arg Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 <210>
SEQ ID NO 31 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic framework region 4 peptide <400> SEQUENCE: 31 Phe
Arg Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> SEQ ID NO 32
<211> LENGTH: 30 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 32 Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 <210> SEQ ID NO
33 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 33 Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met Gly 1 5 10 <210> SEQ ID NO 34 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 34 Arg Val Thr Ile Thr Ala Asp Thr Ser Thr
Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> SEQ ID NO 35
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 35 Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 1 5 10 <210> SEQ ID NO 36 <211> LENGTH: 25
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 36 Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 <210> SEQ ID NO
37 <211> LENGTH: 13 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 37 Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 1 5 10 <210> SEQ ID NO 38 <211>
LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 38 Arg Val Thr Ile Thr Ala Asp Thr Ser Thr
Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys Ala 20 25 30 <210> SEQ ID NO 39
<211> LENGTH: 30 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 39 Arg Val Thr Ile Thr Ala Asp
Thr Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 <210> SEQ ID NO
40 <211> LENGTH: 30 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 40
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5
10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser 20 25
30 <210> SEQ ID NO 41 <211> LENGTH: 14 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <400> SEQUENCE: 41 Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 <210> SEQ
ID NO 42 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 42 Arg Val Thr Ile Ser Val Asp
Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr
Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210>
SEQ ID NO 43 <211> LENGTH: 25 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 43 Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu
Thr Cys Thr Val Ser 20 25 <210> SEQ ID NO 44 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <400>
SEQUENCE: 44 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 1
5 10 <210> SEQ ID NO 45 <211> LENGTH: 31 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 45 Arg Val
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 20 25
30 <210> SEQ ID NO 46 <211> LENGTH: 30 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 46 Arg Val
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 20 25 30
<210> SEQ ID NO 47 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 47 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 20 25 30 <210>
SEQ ID NO 48 <211> LENGTH: 14 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 48 Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> SEQ ID NO 49
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 49 Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210>
SEQ ID NO 50 <211> LENGTH: 25 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 50 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 20 25 <210> SEQ ID NO 51 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <400>
SEQUENCE: 51 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 1
5 10 <210> SEQ ID NO 52 <211> LENGTH: 31 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 52 Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 20 25
30 <210> SEQ ID NO 53 <211> LENGTH: 30 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 53 Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30
<210> SEQ ID NO 54 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 54 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 Asn Ile Lys 20 25 30 <210>
SEQ ID NO 55 <211> LENGTH: 32 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 55 Arg Phe Thr Ile Ser
Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg 20 25 30
<210> SEQ ID NO 56 <211> LENGTH: 31 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 56 Arg Phe Thr Ile Ser
Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser 20 25 30
<210> SEQ ID NO 57 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 57 Arg Phe Thr Ile Ser
Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30
<210> SEQ ID NO 58 <211> LENGTH: 31 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 58 Arg Phe Thr Ile Ser
Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 20 25 30
<210> SEQ ID NO 59 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 59 Arg Phe Thr Ile Ser
Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 <210>
SEQ ID NO 60 <211> LENGTH: 23 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 60 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 20 <210> SEQ ID NO 61 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 61 Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15
<210> SEQ ID NO 62 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 62 Gly Val Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30
<210> SEQ ID NO 63 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 63 Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 1 5 10 <210> SEQ ID NO 64 <211> LENGTH:
23 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 64 Asp
Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys 20 <210> SEQ ID NO 65
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 65 Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro
Gln Leu Leu Ile Tyr 1 5 10 15 <210> SEQ ID NO 66 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 66 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 <210> SEQ ID NO 67
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 67 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20
<210> SEQ ID NO 68 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 68 Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile Tyr 1 5 10 15 <210> SEQ ID NO
69 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 69 Gly Ile Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15
Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 20
25 30 <210> SEQ ID NO 70 <211> LENGTH: 23 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <400> SEQUENCE: 70 Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu
Arg Ala Thr Ile Asn Cys 20 <210> SEQ ID NO 71 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <400>
SEQUENCE: 71 Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
Ile Tyr 1 5 10 15 <210> SEQ ID NO 72 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 72
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5
10 15 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr
Cys 20 25 30 <210> SEQ ID NO 73 <211> LENGTH: 2471
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 73 Met Pro Ala Leu Arg Pro Ala Leu Leu Trp
Ala Leu Leu Ala Leu Trp 1 5 10 15 Leu Cys Cys Ala Ala Pro Ala His
Ala Leu Gln Cys Arg Asp Gly Tyr 20 25 30 Glu Pro Cys Val Asn Glu
Gly Met Cys Val Thr Tyr His Asn Gly Thr 35 40 45 Gly Tyr Cys Lys
Cys Pro Glu Gly Phe Leu Gly Glu Tyr Cys Gln His 50 55 60 Arg Asp
Pro Cys Glu Lys Asn Arg Cys Gln Asn Gly Gly Thr Cys Val 65 70 75 80
Ala Gln Ala Met Leu Gly Lys Ala Thr Cys Arg Cys Ala Ser Gly Phe 85
90 95 Thr Gly Glu Asp Cys Gln Tyr Ser Thr Ser His Pro Cys Phe Val
Ser 100 105 110 Arg Pro Cys Leu Asn Gly Gly Thr Cys His Met Leu Ser
Arg Asp Thr 115 120 125 Tyr Glu Cys Thr Cys Gln Val Gly Phe Thr Gly
Lys Glu Cys Gln Trp 130 135 140 Thr Asp Ala Cys Leu Ser His Pro Cys
Ala Asn Gly Ser Thr Cys Thr 145 150 155 160 Thr Val Ala Asn Gln Phe
Ser Cys Lys Cys Leu Thr Gly Phe Thr Gly 165 170 175 Gln Lys Cys Glu
Thr Asp Val Asn Glu Cys Asp Ile Pro Gly His Cys 180 185 190 Gln His
Gly Gly Thr Cys Leu Asn Leu Pro Gly Ser Tyr Gln Cys Gln 195 200 205
Cys Pro Gln Gly Phe Thr Gly Gln Tyr Cys Asp Ser Leu Tyr Val Pro 210
215 220 Cys Ala Pro Ser Pro Cys Val Asn Gly Gly Thr Cys Arg Gln Thr
Gly 225 230 235 240 Asp Phe Thr Phe Glu Cys Asn Cys Leu Pro Gly Phe
Glu Gly Ser Thr 245 250 255 Cys Glu Arg Asn Ile Asp Asp Cys Pro Asn
His Arg Cys Gln Asn Gly 260 265 270 Gly Val Cys Val Asp Gly Val Asn
Thr Tyr Asn Cys Arg Cys Pro Pro 275 280 285 Gln Trp Thr Gly Gln Phe
Cys Thr Glu Asp Val Asp Glu Cys Leu Leu 290 295 300 Gln Pro Asn Ala
Cys Gln Asn Gly Gly Thr Cys Ala Asn Arg Asn Gly 305 310 315 320 Gly
Tyr Gly Cys Val Cys Val Asn Gly Trp Ser Gly Asp Asp Cys Ser 325 330
335 Glu Asn Ile Asp Asp Cys Ala Phe Ala Ser Cys Thr Pro Gly Ser Thr
340 345 350 Cys Ile Asp Arg Val Ala Ser Phe Ser Cys Met Cys Pro Glu
Gly Lys 355 360 365 Ala Gly Leu Leu Cys His Leu Asp Asp Ala Cys Ile
Ser Asn Pro Cys 370 375 380 His Lys Gly Ala Leu Cys Asp Thr Asn Pro
Leu Asn Gly Gln Tyr Ile 385 390 395 400 Cys Thr Cys Pro Gln Gly Tyr
Lys Gly Ala Asp Cys Thr Glu Asp Val 405 410 415 Asp Glu Cys Ala Met
Ala Asn Ser Asn Pro Cys Glu His Ala Gly Lys 420 425 430 Cys Val Asn
Thr Asp Gly Ala Phe His Cys Glu Cys Leu Lys Gly Tyr 435 440 445 Ala
Gly Pro Arg Cys Glu Met Asp Ile Asn Glu Cys His Ser Asp Pro 450 455
460 Cys Gln Asn Asp Ala Thr Cys Leu Asp Lys Ile Gly Gly Phe Thr Cys
465 470 475 480 Leu Cys Met Pro Gly Phe Lys Gly Val His Cys Glu Leu
Glu Ile Asn 485 490 495 Glu Cys Gln Ser Asn Pro Cys Val Asn Asn Gly
Gln Cys Val Asp Lys 500 505 510 Val Asn Arg Phe Gln Cys Leu Cys Pro
Pro Gly Phe Thr Gly Pro Val 515 520 525 Cys Gln Ile Asp Ile Asp Asp
Cys Ser Ser Thr Pro Cys Leu Asn Gly 530 535 540 Ala Lys Cys Ile Asp
His Pro Asn Gly Tyr Glu Cys Gln Cys Ala Thr 545 550 555 560 Gly Phe
Thr Gly Val Leu Cys Glu Glu Asn Ile Asp Asn Cys Asp Pro 565 570 575
Asp Pro Cys His His Gly Gln Cys Gln Asp Gly Ile Asp Ser Tyr Thr 580
585 590 Cys Ile Cys Asn Pro Gly Tyr Met Gly Ala Ile Cys Ser Asp Gln
Ile 595 600 605 Asp Glu Cys Tyr Ser Ser Pro Cys Leu Asn Asp Gly Arg
Cys Ile Asp 610 615 620 Leu Val Asn Gly Tyr Gln Cys Asn Cys Gln Pro
Gly Thr Ser Gly Val 625 630 635 640 Asn Cys Glu Ile Asn Phe Asp Asp
Cys Ala Ser Asn Pro Cys Ile His 645 650 655 Gly Ile Cys Met Asp Gly
Ile Asn Arg Tyr Ser Cys Val Cys Ser Pro 660 665 670 Gly Phe Thr Gly
Gln Arg Cys Asn Ile Asp Ile Asp Glu Cys Ala Ser 675 680 685 Asn Pro
Cys Arg Lys Gly Ala Thr Cys Ile Asn Gly Val Asn Gly Phe 690 695 700
Arg Cys Ile Cys Pro Glu Gly Pro His His Pro Ser Cys Tyr Ser Gln 705
710 715 720 Val Asn Glu Cys Leu Ser Asn Pro Cys Ile His Gly Asn Cys
Thr Gly 725 730 735 Gly Leu Ser Gly Tyr Lys Cys Leu Cys Asp Ala Gly
Trp Val Gly Ile 740 745 750 Asn Cys Glu Val Asp Lys Asn Glu Cys Leu
Ser Asn Pro Cys Gln Asn 755 760 765 Gly Gly Thr Cys Asp Asn Leu Val
Asn Gly Tyr Arg Cys Thr Cys Lys 770 775 780 Lys Gly Phe Lys Gly Tyr
Asn Cys Gln Val Asn Ile Asp Glu Cys Ala 785 790 795 800 Ser Asn Pro
Cys Leu Asn Gln Gly Thr Cys Phe Asp Asp Ile Ser Gly 805 810 815 Tyr
Thr Cys His Cys Val Leu Pro Tyr Thr Gly Lys Asn Cys Gln Thr 820 825
830 Val Leu Ala Pro Cys Ser Pro Asn Pro Cys Glu Asn Ala Ala Val Cys
835 840 845 Lys Glu Ser Pro Asn Phe Glu Ser Tyr Thr Cys Leu Cys Ala
Pro Gly 850 855 860 Trp Gln Gly Gln Arg Cys Thr Ile Asp Ile Asp Glu
Cys Ile Ser Lys 865 870 875 880 Pro Cys Met Asn His Gly Leu Cys His
Asn Thr Gln Gly Ser Tyr Met 885 890 895 Cys Glu Cys Pro Pro Gly Phe
Ser Gly Met Asp Cys Glu Glu Asp Ile 900 905 910 Asp Asp Cys Leu Ala
Asn Pro Cys Gln Asn Gly Gly Ser Cys Met Asp 915 920 925 Gly Val Asn
Thr Phe Ser Cys Leu Cys Leu Pro Gly Phe Thr Gly Asp 930 935 940 Lys
Cys Gln Thr Asp Met Asn Glu Cys Leu Ser Glu Pro Cys Lys Asn 945 950
955 960 Gly Gly Thr Cys Ser Asp Tyr Val Asn Ser Tyr Thr Cys Lys Cys
Gln 965 970 975 Ala Gly Phe Asp Gly Val His Cys Glu Asn Asn Ile Asn
Glu Cys Thr 980 985 990 Glu Ser Ser Cys Phe Asn Gly Gly Thr Cys Val
Asp Gly Ile Asn Ser 995 1000 1005
Phe Ser Cys Leu Cys Pro Val Gly Phe Thr Gly Ser Phe Cys Leu 1010
1015 1020 His Glu Ile Asn Glu Cys Ser Ser His Pro Cys Leu Asn Glu
Gly 1025 1030 1035 Thr Cys Val Asp Gly Leu Gly Thr Tyr Arg Cys Ser
Cys Pro Leu 1040 1045 1050 Gly Tyr Thr Gly Lys Asn Cys Gln Thr Leu
Val Asn Leu Cys Ser 1055 1060 1065 Arg Ser Pro Cys Lys Asn Lys Gly
Thr Cys Val Gln Lys Lys Ala 1070 1075 1080 Glu Ser Gln Cys Leu Cys
Pro Ser Gly Trp Ala Gly Ala Tyr Cys 1085 1090 1095 Asp Val Pro Asn
Val Ser Cys Asp Ile Ala Ala Ser Arg Arg Gly 1100 1105 1110 Val Leu
Val Glu His Leu Cys Gln His Ser Gly Val Cys Ile Asn 1115 1120 1125
Ala Gly Asn Thr His Tyr Cys Gln Cys Pro Leu Gly Tyr Thr Gly 1130
1135 1140 Ser Tyr Cys Glu Glu Gln Leu Asp Glu Cys Ala Ser Asn Pro
Cys 1145 1150 1155 Gln His Gly Ala Thr Cys Ser Asp Phe Ile Gly Gly
Tyr Arg Cys 1160 1165 1170 Glu Cys Val Pro Gly Tyr Gln Gly Val Asn
Cys Glu Tyr Glu Val 1175 1180 1185 Asp Glu Cys Gln Asn Gln Pro Cys
Gln Asn Gly Gly Thr Cys Ile 1190 1195 1200 Asp Leu Val Asn His Phe
Lys Cys Ser Cys Pro Pro Gly Thr Arg 1205 1210 1215 Gly Leu Leu Cys
Glu Glu Asn Ile Asp Asp Cys Ala Arg Gly Pro 1220 1225 1230 His Cys
Leu Asn Gly Gly Gln Cys Met Asp Arg Ile Gly Gly Tyr 1235 1240 1245
Ser Cys Arg Cys Leu Pro Gly Phe Ala Gly Glu Arg Cys Glu Gly 1250
1255 1260 Asp Ile Asn Glu Cys Leu Ser Asn Pro Cys Ser Ser Glu Gly
Ser 1265 1270 1275 Leu Asp Cys Ile Gln Leu Thr Asn Asp Tyr Leu Cys
Val Cys Arg 1280 1285 1290 Ser Ala Phe Thr Gly Arg His Cys Glu Thr
Phe Val Asp Val Cys 1295 1300 1305 Pro Gln Met Pro Cys Leu Asn Gly
Gly Thr Cys Ala Val Ala Ser 1310 1315 1320 Asn Met Pro Asp Gly Phe
Ile Cys Arg Cys Pro Pro Gly Phe Ser 1325 1330 1335 Gly Ala Arg Cys
Gln Ser Ser Cys Gly Gln Val Lys Cys Arg Lys 1340 1345 1350 Gly Glu
Gln Cys Val His Thr Ala Ser Gly Pro Arg Cys Phe Cys 1355 1360 1365
Pro Ser Pro Arg Asp Cys Glu Ser Gly Cys Ala Ser Ser Pro Cys 1370
1375 1380 Gln His Gly Gly Ser Cys His Pro Gln Arg Gln Pro Pro Tyr
Tyr 1385 1390 1395 Ser Cys Gln Cys Ala Pro Pro Phe Ser Gly Ser Arg
Cys Glu Leu 1400 1405 1410 Tyr Thr Ala Pro Pro Ser Thr Pro Pro Ala
Thr Cys Leu Ser Gln 1415 1420 1425 Tyr Cys Ala Asp Lys Ala Arg Asp
Gly Val Cys Asp Glu Ala Cys 1430 1435 1440 Asn Ser His Ala Cys Gln
Trp Asp Gly Gly Asp Cys Ser Leu Thr 1445 1450 1455 Met Glu Asn Pro
Trp Ala Asn Cys Ser Ser Pro Leu Pro Cys Trp 1460 1465 1470 Asp Tyr
Ile Asn Asn Gln Cys Asp Glu Leu Cys Asn Thr Val Glu 1475 1480 1485
Cys Leu Phe Asp Asn Phe Glu Cys Gln Gly Asn Ser Lys Thr Cys 1490
1495 1500 Lys Tyr Asp Lys Tyr Cys Ala Asp His Phe Lys Asp Asn His
Cys 1505 1510 1515 Asp Gln Gly Cys Asn Ser Glu Glu Cys Gly Trp Asp
Gly Leu Asp 1520 1525 1530 Cys Ala Ala Asp Gln Pro Glu Asn Leu Ala
Glu Gly Thr Leu Val 1535 1540 1545 Ile Val Val Leu Met Pro Pro Glu
Gln Leu Leu Gln Asp Ala Arg 1550 1555 1560 Ser Phe Leu Arg Ala Leu
Gly Thr Leu Leu His Thr Asn Leu Arg 1565 1570 1575 Ile Lys Arg Asp
Ser Gln Gly Glu Leu Met Val Tyr Pro Tyr Tyr 1580 1585 1590 Gly Glu
Lys Ser Ala Ala Met Lys Lys Gln Arg Met Thr Arg Arg 1595 1600 1605
Ser Leu Pro Gly Glu Gln Glu Gln Glu Val Ala Gly Ser Lys Val 1610
1615 1620 Phe Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Asp Ser Asp
His 1625 1630 1635 Cys Phe Lys Asn Thr Asp Ala Ala Ala Ala Leu Leu
Ala Ser His 1640 1645 1650 Ala Ile Gln Gly Thr Leu Ser Tyr Pro Leu
Val Ser Val Val Ser 1655 1660 1665 Glu Ser Leu Thr Pro Glu Arg Thr
Gln Leu Leu Tyr Leu Leu Ala 1670 1675 1680 Val Ala Val Val Ile Ile
Leu Phe Ile Ile Leu Leu Gly Val Ile 1685 1690 1695 Met Ala Lys Arg
Lys Arg Lys His Gly Ser Leu Trp Leu Pro Glu 1700 1705 1710 Gly Phe
Thr Leu Arg Arg Asp Ala Ser Asn His Lys Arg Arg Glu 1715 1720 1725
Pro Val Gly Gln Asp Ala Val Gly Leu Lys Asn Leu Ser Val Gln 1730
1735 1740 Val Ser Glu Ala Asn Leu Ile Gly Thr Gly Thr Ser Glu His
Trp 1745 1750 1755 Val Asp Asp Glu Gly Pro Gln Pro Lys Lys Val Lys
Ala Glu Asp 1760 1765 1770 Glu Ala Leu Leu Ser Glu Glu Asp Asp Pro
Ile Asp Arg Arg Pro 1775 1780 1785 Trp Thr Gln Gln His Leu Glu Ala
Ala Asp Ile Arg Arg Thr Pro 1790 1795 1800 Ser Leu Ala Leu Thr Pro
Pro Gln Ala Glu Gln Glu Val Asp Val 1805 1810 1815 Leu Asp Val Asn
Val Arg Gly Pro Asp Gly Cys Thr Pro Leu Met 1820 1825 1830 Leu Ala
Ser Leu Arg Gly Gly Ser Ser Asp Leu Ser Asp Glu Asp 1835 1840 1845
Glu Asp Ala Glu Asp Ser Ser Ala Asn Ile Ile Thr Asp Leu Val 1850
1855 1860 Tyr Gln Gly Ala Ser Leu Gln Ala Gln Thr Asp Arg Thr Gly
Glu 1865 1870 1875 Met Ala Leu His Leu Ala Ala Arg Tyr Ser Arg Ala
Asp Ala Ala 1880 1885 1890 Lys Arg Leu Leu Asp Ala Gly Ala Asp Ala
Asn Ala Gln Asp Asn 1895 1900 1905 Met Gly Arg Cys Pro Leu His Ala
Ala Val Ala Ala Asp Ala Gln 1910 1915 1920 Gly Val Phe Gln Ile Leu
Ile Arg Asn Arg Val Thr Asp Leu Asp 1925 1930 1935 Ala Arg Met Asn
Asp Gly Thr Thr Pro Leu Ile Leu Ala Ala Arg 1940 1945 1950 Leu Ala
Val Glu Gly Met Val Ala Glu Leu Ile Asn Cys Gln Ala 1955 1960 1965
Asp Val Asn Ala Val Asp Asp His Gly Lys Ser Ala Leu His Trp 1970
1975 1980 Ala Ala Ala Val Asn Asn Val Glu Ala Thr Leu Leu Leu Leu
Lys 1985 1990 1995 Asn Gly Ala Asn Arg Asp Met Gln Asp Asn Lys Glu
Glu Thr Pro 2000 2005 2010 Leu Phe Leu Ala Ala Arg Glu Gly Ser Tyr
Glu Ala Ala Lys Ile 2015 2020 2025 Leu Leu Asp His Phe Ala Asn Arg
Asp Ile Thr Asp His Met Asp 2030 2035 2040 Arg Leu Pro Arg Asp Val
Ala Arg Asp Arg Met His His Asp Ile 2045 2050 2055 Val Arg Leu Leu
Asp Glu Tyr Asn Val Thr Pro Ser Pro Pro Gly 2060 2065 2070 Thr Val
Leu Thr Ser Ala Leu Ser Pro Val Ile Cys Gly Pro Asn 2075 2080 2085
Arg Ser Phe Leu Ser Leu Lys His Thr Pro Met Gly Lys Lys Ser 2090
2095 2100 Arg Arg Pro Ser Ala Lys Ser Thr Met Pro Thr Ser Leu Pro
Asn 2105 2110 2115 Leu Ala Lys Glu Ala Lys Asp Ala Lys Gly Ser Arg
Arg Lys Lys 2120 2125 2130 Ser Leu Ser Glu Lys Val Gln Leu Ser Glu
Ser Ser Val Thr Leu 2135 2140 2145 Ser Pro Val Asp Ser Leu Glu Ser
Pro His Thr Tyr Val Ser Asp 2150 2155 2160 Thr Thr Ser Ser Pro Met
Ile Thr Ser Pro Gly Ile Leu Gln Ala 2165 2170 2175 Ser Pro Asn Pro
Met Leu Ala Thr Ala Ala Pro Pro Ala Pro Val 2180 2185 2190 His Ala
Gln His Ala Leu Ser Phe Ser Asn Leu His Glu Met Gln 2195 2200 2205
Pro Leu Ala His Gly Ala Ser Thr Val Leu Pro Ser Val Ser Gln 2210
2215 2220 Leu Leu Ser His His His Ile Val Ser Pro Gly Ser Gly Ser
Ala 2225 2230 2235 Gly Ser Leu Ser Arg Leu His Pro Val Pro Val Pro
Ala Asp Trp 2240 2245 2250 Met Asn Arg Met Glu Val Asn Glu Thr Gln
Tyr Asn Glu Met Phe 2255 2260 2265
Gly Met Val Leu Ala Pro Ala Glu Gly Thr His Pro Gly Ile Ala 2270
2275 2280 Pro Gln Ser Arg Pro Pro Glu Gly Lys His Ile Thr Thr Pro
Arg 2285 2290 2295 Glu Pro Leu Pro Pro Ile Val Thr Phe Gln Leu Ile
Pro Lys Gly 2300 2305 2310 Ser Ile Ala Gln Pro Ala Gly Ala Pro Gln
Pro Gln Ser Thr Cys 2315 2320 2325 Pro Pro Ala Val Ala Gly Pro Leu
Pro Thr Met Tyr Gln Ile Pro 2330 2335 2340 Glu Met Ala Arg Leu Pro
Ser Val Ala Phe Pro Thr Ala Met Met 2345 2350 2355 Pro Gln Gln Asp
Gly Gln Val Ala Gln Thr Ile Leu Pro Ala Tyr 2360 2365 2370 His Pro
Phe Pro Ala Ser Val Gly Lys Tyr Pro Thr Pro Pro Ser 2375 2380 2385
Gln His Ser Tyr Ala Ser Ser Asn Ala Ala Glu Arg Thr Pro Ser 2390
2395 2400 His Ser Gly His Leu Gln Gly Glu His Pro Tyr Leu Thr Pro
Ser 2405 2410 2415 Pro Glu Ser Pro Asp Gln Trp Ser Ser Ser Ser Pro
His Ser Ala 2420 2425 2430 Ser Asp Trp Ser Asp Val Thr Thr Ser Pro
Thr Pro Gly Gly Ala 2435 2440 2445 Gly Gly Gly Gln Arg Gly Pro Gly
Thr His Met Ser Glu Pro Pro 2450 2455 2460 His Asn Asn Met Gln Val
Tyr Ala 2465 2470 <210> SEQ ID NO 74 <211> LENGTH: 256
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 74 Pro Ala Thr Cys Leu Ser Gln Tyr Cys Ala
Asp Lys Ala Arg Asp Gly 1 5 10 15 Val Cys Asp Glu Ala Cys Asn Ser
His Ala Cys Gln Trp Asp Gly Gly 20 25 30 Asp Cys Ser Leu Thr Met
Glu Asn Pro Trp Ala Asn Cys Ser Ser Pro 35 40 45 Leu Pro Cys Trp
Asp Tyr Ile Asn Asn Gln Cys Asp Glu Leu Cys Asn 50 55 60 Thr Val
Glu Cys Leu Phe Asp Asn Phe Glu Cys Gln Gly Asn Ser Lys 65 70 75 80
Thr Cys Lys Tyr Asp Lys Tyr Cys Ala Asp His Phe Lys Asp Asn His 85
90 95 Cys Asp Gln Gly Cys Asn Ser Glu Glu Cys Gly Trp Asp Gly Leu
Asp 100 105 110 Cys Ala Ala Asp Gln Pro Glu Asn Leu Ala Glu Gly Thr
Leu Val Ile 115 120 125 Val Val Leu Met Pro Pro Glu Gln Leu Leu Gln
Asp Ala Arg Ser Phe 130 135 140 Leu Arg Ala Leu Gly Thr Leu Leu His
Thr Asn Leu Arg Ile Lys Arg 145 150 155 160 Asp Ser Gln Gly Glu Leu
Met Val Tyr Pro Tyr Tyr Gly Glu Lys Ser 165 170 175 Ala Ala Met Lys
Lys Gln Arg Met Thr Arg Arg Ser Leu Pro Gly Glu 180 185 190 Gln Glu
Gln Glu Val Ala Gly Ser Lys Val Phe Leu Glu Ile Asp Asn 195 200 205
Arg Gln Cys Val Gln Asp Ser Asp His Cys Phe Lys Asn Thr Asp Ala 210
215 220 Ala Ala Ala Leu Leu Ala Ser His Ala Ile Gln Gly Thr Leu Ser
Tyr 225 230 235 240 Pro Leu Val Ser Val Val Ser Glu Ser Leu Thr Pro
Glu Arg Thr Gln 245 250 255
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