U.S. patent application number 12/403897 was filed with the patent office on 2009-11-19 for immunointeractive molecules.
This patent application is currently assigned to ZENYTH OPERATIONS PTY LTD. Invention is credited to Giuseppe MACCARONE, Andrew NASH, Pierre David SCOTNEY.
Application Number | 20090285831 12/403897 |
Document ID | / |
Family ID | 29584306 |
Filed Date | 2009-11-19 |
United States Patent
Application |
20090285831 |
Kind Code |
A1 |
NASH; Andrew ; et
al. |
November 19, 2009 |
IMMUNOINTERACTIVE MOLECULES
Abstract
The present invention relates generally to immunointeractive
molecules and more particularly antibodies which bind to vascular
endothielial growth factor-B (VEGF-B) or its functional or
structural equivalent and inhibit the biological activity of
VEGF-B. In particular the present invention relates to deimmunized
such as humanized or human antibodies that bind to VEGF-B and
inhibit the biological activity of VEGF-B. These antibodies have
uses in the treatment or prevention of diseases associated with
perturbations in normal vasculogenesis or angiogenesis or vascular
remodelling. The present invention further contemplates a method of
modulating diseases associated with perturbations in normal
vasculogenesis or angiogenesis or vascular remodelling by the
administration of the subject antibodies. The present invention
further provides an assay system useful for identifying antibodies
which bind to VEGF-B and block the biological activity of VEGF-B.
Accordingly, a method of screening for inhibitors of the biological
activity of VEGF-B is also provided.
Inventors: |
NASH; Andrew; (Victoria,
AU) ; MACCARONE; Giuseppe; (Pascoe Vale, AU) ;
SCOTNEY; Pierre David; (Greensborough, AU) |
Correspondence
Address: |
SCULLY SCOTT MURPHY & PRESSER, PC
400 GARDEN CITY PLAZA, SUITE 300
GARDEN CITY
NY
11530
US
|
Assignee: |
ZENYTH OPERATIONS PTY LTD
Richmond
AU
|
Family ID: |
29584306 |
Appl. No.: |
12/403897 |
Filed: |
March 13, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11652711 |
Jan 12, 2007 |
7517524 |
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12403897 |
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10440295 |
May 16, 2003 |
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11652711 |
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60381285 |
May 17, 2002 |
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Current U.S.
Class: |
424/158.1 ;
435/346; 530/387.1; 530/387.3; 530/388.24 |
Current CPC
Class: |
C07K 14/52 20130101;
C07K 14/71 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/158.1 ;
435/346; 530/387.1; 530/387.3; 530/388.24 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 5/00 20060101 C12N005/00; C07K 16/22 20060101
C07K016/22 |
Claims
1. An isolated antibody or an antigen-binding fragment thereof
which binds to human VEGF-B, wherein the binding of the antibody or
the fragment thereof to VEGF-B antagonizes binding between VEGF-B
and VEGF-R1.
2. The antibody or antigen-binding fragment of claim 1, wherein
said antibody competes with monoclonal antibody 2H10 in binding to
human VEGF-B.
3. The antibody or antigen-binding fragment of claim 1, wherein
said antibody binds to the same epitope in human VEGF-B as
monoclonal antibody 2H10.
4. The antibody or antigen-binding fragment of claim 1, wherein the
antibody is a monoclonal antibody.
5. The antibody or antigen-binding fragment of claim 1, wherein
said antibody is a chimeric antibody comprising a constant region
of a human antibody.
6. The antibody or antigen-binding fragment of claim 1, wherein the
antibody is a human antibody.
7. The antibody or antigen-binding fragment of claim 1, wherein the
antibody is a humanized antibody.
8. The antibody or antigen-binding fragment of claim 1, wherein the
antibody fragment is an Fv, Fab, Fab' or F(ab').sub.2 fragment.
9. The antibody or antigen-binding fragment of claim 1, wherein
said antibody is in a single chain form.
10. A composition comprising the antibody or antigen-binding
fragment of claim 1, and a pharmaceutically acceptable carrier.
11. A method of treating a disease condition in a mammal comprising
administering to said mammal an effective amount of the antibody or
antigen-binding fragment of claim 1.
12. The method of claim 1 wherein the mammal is a human.
13. The method of claim 12 wherein the disease condition is
pulmonary hypertension, the growth of angiogenic tumors and the
spread or metastases of cancer cells, chronic inflammatory diseases
such as rheumatoid arthritis and any other VEGF-B-mediated diseases
or conditions where there is known to be a significant angiogenic
component.
14. A method for the treatment or prophylaxis of a condition
mediated by VEGF-B, said method comprising administering to a
subject an effective amount of the antibody or antigen-binding
fragment according to any one of claims 5-7, for a time and under
conditions sufficient to inhibit or otherwise reduce VEGF-B
signaling via VEGF-R1.
15. The method of claim 14 wherein the mammal is a human.
16. A method for producing an antibody having specificity for human
VEGF-B, said method comprising immunizing a non-human animal with a
human VEGF-B polypeptide or an immunogenic part thereof, for a time
and under conditions sufficient for antibodies directed against the
VEGF-B polypeptide to be generated in said animal and then
subjecting said antibody to deimmunized or humanization means.
17. A method for producing a hybridoma cell line comprising
immunizing a non-human animal with human VEGF-B or an immunogenic
part thereof harvesting spleen cells from the immunized animal,
fusing the harvested spleen cells to a myeloma cell line to
generate hybridoma cells and identifying a hybridoma cell line that
produces a monoclonal antibody that binds said VEGF-B or a fragment
thereof.
18. The method of claim 16 or 17 wherein the non-human animal is a
mouse.
19. The method of claim 16 or 17 wherein the non-human animal is a
transgenic animal which produces human antibodies.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/652,711, filed Jan. 12, 2007, which is a is a continuation
of U.S. application Ser. No. 10/440,295, filed May 16, 2003, now
abandoned, which claims priority from U.S. Provisional Patent
Application Ser. No. 60/381,285, filed on May 17, 2002.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to immunointeractive
molecules and more particularly antibodies which bind to vascular
endothelial growth factor-B (VEGF-B) or its functional or
structural equivalent and inhibit the biological activity of
VEGF-B. In particular, the present invention relates to deimmunized
molecules such as humanized or human antibodies that bind to VEGF-B
and inhibit the biological activity of VEGF-B. These antibodies
have uses in the treatment or prevention of diseases associated
with perturbations in normal vasculogenesis or angiogenesis or
vascular remodelling. The present invention further contemplates a
method of modulating diseases associated with perturbations in
normal vasculogenesis or angiogenesis or vascular remodelling by
the administration of the subject antibodies. The present invention
further provides an assay system useful for identifying antibodies
which bind to VEGF-B and block the biological activity of VEGF-B.
Accordingly, a method of screening for inhibitors of the biological
activity of VEGF-B is also provided.
[0004] 2. Description of the Prior Art
[0005] The reference to any prior art in this specification is not
and should not be taken as an acknowledgment or any form of
suggestion that the prior art forms part of the common general
knowledge in any country.
[0006] Bibliographic details of the publications referred to in
this specification are also collected at the end of the
description.
[0007] The normal growth of new blood vessels, or physiological
angiogenesis, is an essential step in vertebrate growth and
development as well as in the repair of wounds and bone fractures.
This process of blood vessel formation and remodelling is kept in
close control by pro- and anti-angiogenic molecules, but
perturbations in the process can occur. Abnormal or pathological
angiogenesis occurs when the balance of blood vessel growth is
disturbed and is a contributory factor in the development of a wide
range of diseases, such as rheumatoid arthritis (Kasama et al.,
Arthritis Rheum. 44(11): 2512-2524, 2001) and malignant angiogenic
tumours and cancer-cell metastases (Liu et al., J. Surg. Res.
102(1): 31-34, 2002).
[0008] Growth and remodelling of the vascular system are mediated
by a diverse collection of polypeptide growth factors. One such
group is the peptide family known as vascular endothelial growth
factors (VEGFs) (Tuder et al., J. Pathol. 195(3): 367-374, 2001).
The VEGFs constitute a group of structurally and functionally
related growth factors that modulate many important physiological
functions of endothelial cells. The mammalian members of the VEGF
family identified to date include VEGF-A, VEGF-B, VEGF-C, VEGF-D
and placental growth factor.
[0009] The various homologues of VEGF differ slightly in the roles
they play during the various developmental stages and also in
response to vascular trauma. This is indicated by the variations in
temporal and spatial release of the various VEGFs during
physiological events such as embryonic development, regulation of
capillary growth in normal and pathological conditions in adults,
and in the maintenance of the normal vasculature. For example,
VEGF-.alpha. is a potent mitogen that plays a vital role in
vasculogenesis and angiogenesis during development (Brown et al.,
Am. J. Physiol. Lung Cell Mol. Physiol 281(4): L1001-1010, 2001).
It is also vital for revascularization during repair of dermal
wounds (Mat this et al., Am J Pathol 160(1): 289-296, 2002) and
regrowth of vasculature following bone fractures (Street et al., J.
Orthop. Res. 19(6): 1057-1066, 2001).
[0010] Gene knockout experiments have found that VEGF-B is not
essential for the growth and development of the peripheral vascular
system, although it is involved in the normal development of the
coronary vasculature (Bellomo et al., Circ. Res. 86(2): E29-35,
2000). It also plays a part in physiological responses to ischemia
and vascular occlusion (Bellomo et al. [2000; supra]). VEGF-B is
also implicated in a number of pathological angiogenic conditions
such as pulmonary hypertension (Rich et al., J. Heart Lung
Transplant 21(1): 159, 2002), the growth of angiogenic tumors (Li
et al., Growth Factors 19(1): 49-59, 2001) and the spread or
metastases of cancer cells, possibly through its effects on
plasminogen activation (Gunningham et al., J. Pathol. 193(3):
325-332, 2001).
[0011] The actions of VEGF-B are mediated through the receptor
tyrosine kinase VEGF receptor-1 (VEGF-R1). VEGF-R1 is also referred
to as Flt-1 and its extracellular domain is characterized by seven
immunoglobulin-like regions (Ma et al., Biotechnol. Appl. Biochem.
34 (Pt 3): 199-204, 2001), referred to as Ig domains 1-7.
[0012] The suspected role of VEGF-B in pathological angiogenesis
has made this growth factor a desirable control point in the
treatment of a number of diseases. Biological profiling of VEGF-B
has, however, been limited by a lack of simple in vitro assay
systems.
[0013] When further characterizing the biological effects of
VEGF-B, the inventors faced difficulties with sub-optimal
cell-based assays. Reports of activity of VEGF-B on endothelial
cells, including stimulation of proliferation and induction of mRNA
for uPA and PAI-1 have subsequently been attributed to
contaminating heterodimer and lipopolysaccharide, respectively. The
present inventors have now devised a novel, cellular based assay
for VEGF-B activity which is based on the development of a chimeric
fusion molecule encoding the extracellular portion of the VEGF-B
receptor. The assay is also useful for identifying modulators of
VEGF-B-Flt-1-mediated signalling.
[0014] Antibodies to VEGF-B may potentially act as antagonists of
VEGF-B biological activity. In accordance with the present
invention, antibodies are identified which bind to VEGF-B and block
VEGF-B binding to VEGF-R1, thereby inhibiting the biological
activity of VEGF-B.
SUMMARY OF THE INVENTION
[0015] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising", will be understood to imply the inclusion of a
stated element or integer or group of elements or integers but not
the exclusion of any other element or integer or group of elements
or integers.
[0016] Nucleotide and amino acid sequences are referred to by a
sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond
numerically to the sequence identifiers <400>1 (SEQ ID NO:1),
<400>2 (SEQ ID NO:2), etc. A summary of the sequence
identifiers is provided in Table 1. A sequence listing is provided
after the claims.
[0017] The present invention provides immunointeractive molecules
such as in the form of antibodies which function as VEGF-B
antagonists and may be used for treating certain conditions
associated with VEGF-B activity, such as pathological angiogenesis,
or other biological processes mediated by VEGF-B. The present
invention also provides methods for treating these conditions
comprising administering a VEGF-B antagonist to a patient afflicted
with such a condition. Also provided are compositions for use in
such methods which comprise one or more VEGF-B antagonists.
Reference to "VEGF-B" includes polypeptides and proteins having
VEGF-B-like activity. Furthermore, a VEGF-B molecule may be
naturally occurring or may be a mutant, derivative, homolog or
analog of VEGF-B.
[0018] The antibodies of the present invention bind, interact or
otherwise associate with VEGF-B or a fragment comprising an epitope
from VEGF-B. In a preferred embodiment, the antibodies bind to
VEGF-B and inhibit or at least reduce the binding of VEGF-B to
VEGF-R1, thereby blocking some or all the biological activity of
VEGF-B.
[0019] The antibodies may be specific for VEGF-B from a particular
species, such as human VEGF-B, or, in view of the level of sequence
similarity between VEGF-B from different species, the antibodies
may show some cross-reactivity with VEGF-B from two or more
species. In the case of antibodies directed towards human VEGF-B,
some level of cross-reactivity with other mammalian forms of VEGF-B
may be desirable in certain circumstances, such as for example, for
the purpose of testing antibodies in animal models of a particular
disease and for conducting toxicology studies in a manner where
VEGF-B signaling in the test animal is affected by the test
antibody. Species where cross-reactivity of an antibody to human
VEGF-B may be desirable include a non-human primate such as monkey,
gorilla, orangatang or marmoset, sheep, cow, goat, pig, donkey,
horse, dog, cat, rat, mouse and guinea pig. Accordingly, one
preferred group of antibodies are those which exhibit some level of
species cross-reactivity. A particularly preferred group of such
antibodies are those to human VEGF-B which exhibit some level of
species cross-reactivity.
[0020] Antibodies of the present invention include, but are not
limited to antibodies which bind VEGF-B) and inhibit VEGF-B induced
signaling through VEGF-R1.
[0021] Preferably, the antibodies are monoclonal antibodies or
antigen-binding fragments thereof. Most preferably, the antibodies
are deimmunized, humanized or human antibodies suitable for
administration to humans. These include humanized antibodies
prepared, for example, from murine monoclonal antibodies and human
monoclonal antibodies which may be prepared, for example, using
transgenic mice or by phage display.
[0022] Antibodies in accordance with the present invention include
the murine monoclonal antibodies 2H10, B33/02-1C6-6, B33/02-2F5-2
and 36/01-4E12-11-12 and humanized forms thereof.
[0023] The present invention contemplates methods of modulating
VEGF-B-mediated diseases or conditions by the administration of
antibodies of the present invention. Conditions to be treated in
accordance with the present invention include pulmonary
hypertension, the growth of angiogenic tumors and the spread or
metastases of cancer cells, chronic inflammatory diseases such as
rheumatoid arthritis and any other VEGF-B-mediated diseases or
conditions where there is known to be a significant angiogenic
component.
[0024] The present invention also provides an assay system useful
for identifying antibodies that inhibit the biological activity of
VEGF-B. Accordingly, a method of screening for inhibitors of VEGF-B
biological activity, which method involves the assay system, is
provided.
[0025] A summary of sequence identifiers used throughout the
subject specification is provided in Table 1.
TABLE-US-00001 TABLE 1 Summary of sequence identifiers SEQUENCE ID
NO: DESCRIPTION 1 Nucleotide sequence encoding
VEGF-R1-(Hflt1-4)gp130 fusion 2 Corresponding amino acid sequence
of VEGF-R1-(hflt1-4)gp130 fusion 3 Nucleotide sequence encoding
VEGF-R1-(hflt1-3)gp130 fusion 4 Corresponding amino acid sequence
of VEGF-R1(hflt1-4)-gp130 fusion 5-11 oligonucleotides
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1 shows the biochemical analysis of recombinant
VEGF-.beta. isoforms. (A) VEGF-.beta. isoforms expressed in E. coli
were purified and refolded then analyzed by SDS-PAGE (10-20%
gradient) under reducing and non-reducing conditions. The gel was
stained with Coomassie blue. (B) Representative example of
Biosensor dose-response analysis of VEGF-B.sub.186 binding to
VEGF-R1.sub.D2. Concentration range 0.1 nM to 500 nM. C. Scatchard
analysis of VEGF-B.sub.167 (.quadrature.), VEGF-B.sub.186 (.DELTA.)
and VEGF-B.sub.10-108 (.smallcircle.) binding to VEGF-R1.sub.D2.
KDs were determined as 1.5 nM for VEGF-B.sub.167, 2.0 nM for
VEGF-B.sub.186 and 0.8 nM for VEGF-B.sub.10-108.
[0027] FIG. 2 shows the aspects of the novel biological assay for
VEGF-R1 ligands. (A) Schematic representation of VEGF-R1 and
chimeric receptors incorporating VEGE-R1.sub.D1-4 or R1.sub.D1-3
and the transmembrane and intracellular domains of gp130. (B)
Chimeric receptors cloned into pEFBOS-S-Flag for expression as
N-terminal Flag-tagged proteins were transiently expressed in 293T
cells. Cell lysates were subjected to SDS-PAGE, transferred to a
nylon membrane and probed using an anti-Flag antibody (lane 1,
chimeric R1.sub.D1-4; lane 2, chimeric R1.sub.D1-3; lane 3, control
plasmid), (C) Clone 2.1.19.25 was derived following stable
transfection of 293A12 cells with the chimeric receptor construct
incorporating VEGF-R1.sub.D1-4. (D) VEGF-A antagonist
(VEGF-R1.sub.D1-4-IgGFc chimeric protein, R&D Systems) inhibits
the 2.1.19.25 luciferase response to VEGF-A but not to LIF (VEGF-A
[.box-solid.]; VEGF-A plus antagonist [.quadrature.]; LIF [ ]; LIF
plus antagonist [.smallcircle.]).
[0028] FIG. 3 shows an assay of VEGF-B biological activity and
characterization of VEGF-B specific mAbs. (A). Clone 2.1.19.25
response to VEGF-B.sub.167 ( ) and VEGF-B.sub.10-108 (.box-solid.).
(B). Monoclonal Ab 2H10 inhibits the 2.1.19.25 response to
VEGF-B.sub.167. 2.19.25E cells were incubated with titrating
VEGF-B.sub.167 alone (.box-solid.) or supplemented with VEGF-B
specific mAb 2H10 ( ) or 7C3 (.tangle-solidup.) or control () at a
final concentration of 50 .mu.g/ml.
[0029] FIG. 4 is a representation of the complete nucleotide and
amino acid sequences (SEQ ID NOS: 3-4) of human FLT1-3gp130
(VEGF-R1), including the sequences representing IL-3 signal, FLAG
tag, hFLT1 (domains 1-3), gp130 including gp130 transmembrane
domain and gp130 intracellular domain.
[0030] FIG. 5 is a representation of the complete nucleotide and
amino acid sequences (SEQ ID NOS: 1-2) of human Flt1-4gp130
hgp130.TM. (VEGF-R1), including the sequences representing IL-3
signal, FLAG tag, hFLT1 (domains 1-4), gp130 including gp130
transmembrane domain and gp 130 intracellular domain.
[0031] FIG. 6 shows that the VEGF-B specific mAbs 36/01-4E12-11-12,
B33/02-2F5-2, B33/02-1C6-6 and 2H10 inhibit the cellular response
to VEGF-.beta. isoforms 167 and 10-108 but not VEGF-A. Ba/F3 cells
transfected with chimeric VEGF-R1/EpoR were stimulated with
VEGF-B.sub.167 (50 .mu.M), VEGF-B.sub.10-108 (10 nM) or VEGF-A (1
nM) in the presence of test or control mAb (8H7) at a constant
concentration of 31.3 nM. Cell viability was assessed at 72 hours.
The VEGF-B specific mAbs neutralized the biological activity of
VEGF-B.sub.167 and VEGF-B.sub.10-108 but as expected had no effect
on the biological activity of VEGF-A. The control mAb 8H7 had no
neutralising effect on the biological activity of VEGF-B.sub.167,
VEGF-B.sub.10-108 and VEGF-A.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Before describing the present invention detail, it is to be
understood that unless otherwise indicated, the subject invention
is not limited to specific formulation components, manufacturing
methods, dosage regimens, or the like, as such may vary. It is also
to be understood that the terminology used herein is for the
purpose of describing particular embodiments only and is not
intended to be limiting.
[0033] It must be noted that, as used in the subject specification,
the singular forms "a", "an" and "the" include plural aspects
unless the context clearly dictates otherwise. Thus, for example,
reference to a "an antibody" includes a single compound, as well as
two or more antibodies; reference to "VEGF-B" includes a single
VEGF-B, as well as two or more VEGF-B molecules, and so forth.
[0034] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set forth below.
[0035] The terms "antibody", "immunointeractive molecule", "active
agent", "pharmacologically active agent", "medicament", "active"
and "drug" are used interchangeably herein to refer to a chemical
compound that induces a desired pharmacological, physiological
effect such as antagonizing VEGF-R1-mediated signaling. The terms
also encompass pharmaceutically acceptable and pharmacologically
active ingredients of those active agents specifically mentioned
herein including but not limited to salts, esters, amides,
prodrugs, active metabolites, analogs and the like. When the terms
"antibody", "immunointeractive molecule", "active agent",
"pharmacologically active agent", "medicament", "active" and "drug"
are used, then it is to be understood that this includes the active
agent per se as well as pharmaceutically acceptable,
pharmacologically active salts, esters, amides, prodrugs,
metabolites, analogs, etc.
[0036] By the terms "effective amount" or "therapeutically
effective amount" of an antibody, agent and the like as used herein
are meant a sufficient amount of the antibody to provide the
desired therapeutic effect including antagonism between VEGF-B and
VEGF-R1. Of course, undesirable effects, e.g. side effects, are
sometimes manifested along with the desired therapeutic effect;
hence, a practitioner balances the potential benefits against the
potential risks in determining what is an appropriate "effective
amount". The exact amount required will vary from subject to
subject, depending on the species, age and general condition of the
subject, mode of administration, the condition to be treated and
the like. Thus, it may not be possible to specify an exact
"effective amount". However, an appropriate "effective amount" in
any individual case may be determined by one of ordinary skill in
the art using only routine experimentation.
[0037] By "pharmaceutically acceptable" carrier excipient or
diluent is meant a pharmaceutical vehicle comprised of a material
that is not biologically or otherwise undesirable, i.e. the
material may be administered to a subject along with the selected
antibody without causing any or a substantial adverse reaction.
Carriers may include excipients and other additives such as
diluents, detergents, coloring agents, wetting or emulsifying
agents, pH buffering agents, preservatives, and the like.
[0038] Similarly, a "pharmacologically acceptable" salt, ester,
emide, prodrug or derivative of a compound as provided herein is a
salt, ester, amide, prodrug or derivative that this not
biologically or otherwise undesirable.
[0039] The terms "treating" and "treatment" as used herein refer to
reduction in severity and/or frequency of symptoms, elimination of
symptoms and/or underlying cause, prevention of the occurrence of
symptoms and/or their underlying cause, and improvement or
remediation of a condition or disorder. Thus, for example,
"treating" a patient involves prevention of a particular disorder
or adverse physiological event in a susceptible individual as well
as treatment of a clinically symptomatic individual by inhibiting
or causing regression of a disorder or disease. Thus, for example,
the present method of "treating" a patient in need of therapy of
the vascular system encompasses both prevention of a condition,
disease or disorder as well as treating the condition, disease or
disorder. In any event, the present invention contemplates the
treatment or prophylaxis of vascular-type disease conditions and
disorders. Such diseases, disorders and defects include pulmonary
hypertension, the growth of angiogenic tumors and the spread or
metastases of cancer cells, chronic inflammatory diseases such as
rheumatoid arthritis and any other VEGF-B-mediated diseases or
conditions where there is known to be a significant angiogenic
component.
[0040] "Patient" as used herein refers to a mammalian, preferably
human, individual who can benefit from the pharmaceutical
formulations and methods of the present invention. There is no
limitation on the type of mammal that could benefit from the
presently described pharmaceutical formulations and methods. A
patient regardless of whether a human or non-human mammal may be
referred to as an individual, subject, mammal, host or
recipient.
[0041] The preferred animals to be treated are humans or other
primates, livestock animals, laboratory test animals, companion
animals or captured wild animals.
[0042] The present invention relates generally to immunointeractive
molecules which bind, interact or otherwise associated to or with
VEGF-B or a fragment, portion or part thereof and inhibit or
otherwise reduce the biological activity of VEGF-B and which may be
employed in the methods of the present invention. An
immunointeractive molecule includes antibodies and derivatives,
fragments and recombinant or modified forms thereof including Fv,
Fab, Fab', F(ab').sub.2, single chain antibodies and Fc fragments.
The preferred antibodies are monoclonal antibodies or
antigen-binding fragments thereof. Preferably, the antibodies are
in isolated, homogenous or fully or partially purified form.
[0043] An antibody may be a chimeric antibody including a fusion of
antibody portions or molecules.
[0044] Most preferably, the antibodies are deimmunized, humanized
or human antibodies suitable for administration to humans. These
include deimmunized or humanized antibodies prepared, for example,
from murine monoclonal antibodies, and human monoclonal antibodies
which may be prepared, for example, using transgenic mice as
described below, or by phage display.
[0045] Reference to "VEGF-B" is reference to the protein and its
encoding nucleotide sequence described in the literature as
VEGF-related factor or VEGF-B (Grimmond et al., Genome Res. 6(2):
122-129, 1996; Townson et al., Biochem. Biophys. Res. Commun.
220(3): 922-928, 1996), and in International Patent Publication
Nos. WO 96/26736 and WO 96/27007.
[0046] Reference to "binding" of an antibody means binding,
interacting or associating with or to a target antigen such as
VEGF-B. Reference to "VEGF-B" includes fragments or portions which
comprise the epitopes to which an antibody binds. Consequently,
reference to an antibody binding to VEGF-B includes the binding,
interaction or association of the antibody or an antigen-binding
portion thereof to VEGF-B or a part, fragment or epitope-containing
region thereof. A "VEGF-B" protein includes a polypeptide or
protein having VEGF-B-like properties including an ability to
interact with VEGF-R1.
[0047] Generally, "binding", "interaction" or "association" means
or includes the specific binding, interaction or association of the
antibody to VEGF-B or a portion thereof.
[0048] The biological effects of VEGF-B are mediated by
VEGF-R1.
[0049] The present invention is hereinafter described with
reference to antibodies and VEGF-B. This is done, however, with the
understanding that other immunointeractive molecules may be used
and antibodies may be directed to polypeptides having at least one
biological property in common with VEGF-B. Furthermore, in terms of
animal studies, rather than humanized antibodies, mammalianized or
other deimmunized antibodies may be employed for use in non-human
primates or laboratory test mammals.
[0050] Examples of antibodies contemplated by the present invention
include those that bind to VEGF-B and inhibit or otherwise reduce
the biological activity of VEGF-B. Such antibodies, referred to
herein as blocking or neutralising antibodies, may be raised with
VEGF-B or immunogenic parts thereof and screened in assays for the
ability to block the signaling of VEGF-B through VEGF-R1. Suitable
assays are assays that test the antibodies for the ability to
inhibit the binding of VEGF-B to cells expressing VEGF-R1, or that
test antibodies for the ability to reduce a biological or cellular
response that results from the signaling of VEGF-B through
VEGF-R1.
[0051] In one embodiment, the present invention provides antibodies
that bind to VEGF-B and inhibit or otherwise reduce the biological
activity of VEGF-3.
[0052] Preferably the antibodies are monoclonal antibodies or
antigen-binding fragments thereof.
[0053] Most preferably, the antibodies are deimmunized, humanized
or human monoclonal antibodies suitable for use in human
therapeutics.
[0054] As such, in a preferred embodiment, the present invention
provides antibodies that are deimmunized, humanized or human
monoclonal antibodies which bind to VEGF-B and inhibit or otherwise
reduce VEGF-B signaling through VEGF-R1 or a hybrid-type
receptor.
[0055] In an especially preferred embodiment, the present invention
provides antibodies that are deimmunized, humanized or human
monoclonal antibodies which bind to VEGF-B and inhibit the
biological activity of VEGF-B.
[0056] Reference to an "antibody" or "antibodies" includes
reference to all the various forms of antibodies, including but not
limited to whole antibodies, antibody fragments, including, for
example, Fv, Fab, Fab' and F(ab').sub.2 fragments, humanized
antibodies, human antibodies (e.g., produced in transgenic animals
or through phage display) and immunoglobulin-derived polypeptides
produced through genetic engineering techniques. An Fc portion from
these antibodies is also contemplated even if this does not have
any binding specificity.
[0057] Unless stated otherwise, specificity in respect of an
antibody of the present invention is intended to mean that the
antibody does not exhibit any meaningful cross-reactivity with
non-VEGF-B proteins. However, it is not intended to indicate that
there is no cross-reactivity with other forms of VEGF-B which may
exist, (for example, splice variants or fragments of VEGF-B), nor
is it intended to indicate that no cross-reactivity with VEGF-1
from other species may exist. The amino acid sequence of VEGF-B is
a well conserved across species, with other mammalian forms of the
receptor showing high levels of amino acid homology with the human
VEGF-B chain. For example, the human and mouse VEGF-B.sub.10-108
protein has 91.9% identity over the 99 amino acids, the human and
mouse VEGF-B.sub.167 protein has 88.0% identity over the 167 amino
acids and the human and mouse VEGF-B.sub.186 protein has 87.1%
identity over the 186 amino acids. Reference to "identity"
generally means after optimal alignment.
[0058] The antibodies may be specific for VEGF-B from a particular
species, such as human VEGF-B, or, because of the level sequence
similarity between VEGF-B from certain mammalian species, may show
some cross-reactivity with VEGF-B from other mammalian species. In
the case of antibodies directed towards human VEGF-B, some level of
cross reactivity with other mammalian forms of VEGF-B may be
desirable in certain circumstances. For example, such antibodies
are useful for the purpose of testing antibodies in animal models
of a particular disease, and for conducting toxicology studies in a
manner where VEGF-B signaling in the test animal is affected by the
test antibody. Species where cross reactivity of an antibody to
human VEGF-B may be desirable include primates such as monkeys,
orangatangs, marmosets and gorillas, livestock animals such as
sheep, cattle, horses, goats, donkeys, pigs, laboratory test
animals such as mice, rats, guinea pigs, hamsters and companion
animals such as dog and rat. Accordingly, one preferred group of
antibodies are those which exhibit some level of species cross
reactivity. A particularly preferred group of antibodies are those
antibodies to human VEGF-B which exhibit some level of species
cross-reactivity.
[0059] In a preferred embodiment, the present invention provides
antibodies that bind to human VEGF-B and to cynamolgus monkey
VEGF-B and inhibit the biological activity of VEGF-B.
[0060] In a further preferred embodiment, the present invention
provides antibodies that bind to human VEGF-B and to ovine VEGF-B
and inhibit the biological activity of VEGF-B.
[0061] In still a further preferred embodiment, the present
invention provides antibodies that bind to human VEGF-B and to
canine VEGF-B and inhibit the biological activity of VEGF-B.
[0062] In yet a further preferred embodiment, the present invention
provides antibodies that bind to human VEGF-B and to rat VEGF-B and
inhibit the biological activity of VEGF-B.
[0063] In yet a further preferred embodiment, the present invention
provides antibodies that bind to human VEGF-B and to murine VEGF-B
and inhibit the biological activity of VEGF-B.
[0064] The antibodies of the present invention may be prepared by
well known procedures. See, for example, Monoclonal Antibodies,
Hybridomas: A New Dimension in Biological Analyses, Kennet et al.
(eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory
Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., (1988).
[0065] One method for producing an antibody of the present
invention comprises immunizing a non-human animal, such as a mouse
or a transgenic mouse, with a VEGF-B polypeptide, or immunogenic
parts thereof whereby antibodies directed against the VEGF-B
polypeptide are generated in said animal.
[0066] Both polyclonal and monoclonal antibodies can be produced by
this method. The methods for obtaining both types of sera are well
known in the art. Polyclonal sera are less preferred but are
relatively easily prepared by injection of a suitable laboratory
animal with an effective amount of an VEGF-B polypeptide, or
immunogenic parts thereof, collecting serum from the animal and
isolating VEGF-B specific sera by any of the known immunoabsorbent
techniques. Antibodies produced by this technique are generally
less favoured, because of the potential for heterogeneity of the
product.
[0067] The use of monoclonal antibodies is particularly preferred
because of the ability to produce them in large quantities and the
homogeneity of the product. Monoclonal antibodies may be produced
by conventional procedures.
[0068] The present invention contemplates a method for producing a
hybridoma cell line comprises immunizing a non-human animal, such
as a mouse or a transgenic mouse, with an VEGF-B polypeptide, or
immunogene parts thereof; harvesting spleen cells from the
immunized animal; fusing the harvested spleen cells to a myeloma
cell line to generate hybridoma cells; and identifying a hybridoma
cell line that produces a monoclonal antibody that binds a VEGF-B
polypeptide.
[0069] Such hybridoma cell lines and the anti-VEGF-B monoclonal
antibodies produced by them are encompassed by the present
invention. Monoclonal antibodies secreted by the hybridoma cell
lines are purified by conventional techniques. Hybridomas or the
monoclonal antibodies produced by them may be screened further to
identify monoclonal antibodies with particularly desirable
properties, such as the ability to inhibit the biological activity
of VEGF-B.
[0070] The VEGF-B polypeptide or immunogenic part thereof that may
be used to immunize animals in the initial stages of the production
of the antibodies of the present invention may be from any
mammalian source. Preferably, the VEGF-B polypeptide or immunogenic
part thereof is human VEGF-B.
[0071] Antigen-binding fragments of antibodies of the present
invention may be produced by conventional techniques. Examples of
such fragments include, but are not limited to, Fab, Fab', F(ab') 2
and Fv fragments, including single chain Fv fragments (termed sFv
or scFv). Antibody fragments and derivatives produced by genetic
engineering techniques, such as disulphide stabilized Fv fragments
(dsFv), single chain variable region domain (Abs) molecules and
minibodies are also contemplated for use. Unless otherwise
specified, the terms "antibody" and "monoclonal antibody" as used
herein encompass both whole antibodies and antigen-binding
fragments thereof.
[0072] Such derivatives of monoclonal antibodies directed against
VEGF-B may be prepared and screened for desired properties, by
known techniques, including the assays described herein. The assays
described herein provide the means to identify derivatives of the
antibodies of the present invention that bind to VEGF-B and inhibit
the biological activity of VEGF-B. Certain of the techniques
involve isolating DNA encoding a polypeptide chain (or a portion
thereof) of a mAb of interest, and manipulating the DNA through
recombinant DNA technology. The DNA may be fused to another DNA of
interest, or altered (e.g. by mutagenesis or other conventional
techniques) to add, delete, or substitute one or more amino acid
residues, for example.
[0073] DNA encoding antibody polypeptides (e.g. heavy or light
chain, variable region only or full length) may be isolated from
B-cells of mice that have been immunized with VEGF-B. The DNA may
be isolated by conventional procedures such as polymerase chain
reaction (PCR). Phage display is another example of a known
technique whereby derivatives of antibodies may be prepared, U one
approach, polypeptides that are components of all antibody of
interest are expressed in any suitable recombinant expression
system, and the expressed polypeptides are allowed to assemble to
form antibody molecules.
[0074] Single chain antibodies may be formed by linking heavy and
light chain variable region (Fv region) fragments via an amino acid
bridge (short peptide linker), resulting in a single polypeptide
chain. Such single-chain Fvs (scFvs) have been prepared by fusing
DNA encoding a peptide linker between DNAs encoding the two
variable region polypeptides (VL and VH). The resulting antibody
fragments can form dimers or trimers, depending on the length of a
flexible linker between the two variable domains (Kortt et al.,
Protein Engineering 10: 423, 1997). Techniques developed for the
production of single chain antibodies include those described in
U.S. Pat. No. 4,946,778; Bird (Science 242: 423, 1988), Huston et
al. (Proc. Natl. Acad. Sci. USA 85: 5879, 1988) and Ward et al.,
(Nature 334: 544, 1989). Single chain antibodies derived from
antibodies provided herein are encompassed by the present
invention.
[0075] In one embodiment, the present invention provides
derivatives of the antibodies of the present invention that bind to
VEGF-B and inhibit the biological activity of VEGF-B.
[0076] Techniques are known for deriving an antibody of a different
subclass or isotype from an antibody of interest, i.e., subclass
switching. Thus, IgG1 or IgG4 monoclonal antibodies may be derived
from an IgM monoclonal antibody, for example, and vice versa. Such
techniques allow the preparation of new antibodies that possess the
antigen-binding properties of a given antibody (the parent
antibody), but also exhibit biological properties associated with
an antibody isotype or subclass different from that of the parent
antibody. Recombinant DNA techniques may be employed, Cloned DNA
encoding particular antibody polypeptides may be employed in such
procedures, e.g. DNA encoding the constant region of an antibody of
the desired isotype.
[0077] The monoclonal production process described above may be
used in animals, for example mice, to produce monoclonal
antibodies. Conventional antibodies derived from such animals, for
example murine antibodies, are known to be generally unsuitable for
administration to humans as they may cause an immune response.
Therefore, such antibodies may need to be subjected to a
humanization process in order to provide antibodies suitable for
administration to humans. Such humanization processes are well
known in the art and are described in further detail below.
[0078] Additional embodiments include chimeric antibodies and
humanized versions of immune monoclonal antibodies. Such chimeric
or humanized antibodies may be prepared by known techniques, for
example, CDR grafting, and offer the advantage of reduced
immunogenicity when the antibodies are administered to humans. In
one embodiment, a chimeric monoclonal antibody comprises the
variable region of a murine antibody (or just the antigen binding
site thereof) and a constant region derived from a human
antibody.
[0079] Alternatively, a humanized antibody fragment may comprise
the antigen binding sites (complementarity determining regions
CDRs) of a murine monoclonal antibody and a variable region
fragment (lacking the antigen-binding site) derived from a human
antibody. Procedures for the production of chimeric and humanized
monoclonal antibodies include those described in Riechmann et al.
(Nature 332: 323, 1988) Liu et al. (Proc. Natl. Acad. Sci. USA 84:
3439, 1987), Larrick et al. (Bio/Technology 7: 934, 1989) and
Winter and Harris (TIPS 14: 139, 1993).
[0080] The complementarity determining regions (CDRs) of a given
antibody may be identified using the system described by Kabat et
al. in Sequences of Proteins of Immunological Interest, 5th Ed.,
U.S. Department of Health and Human Services, PHS, NIH, NIH
Publication No. 91-3242, 1991).
[0081] For example, the murine monoclonal antibody 2H10 may be
subjected to humanization to reduce the immunogenicity of the
antibody in a target host. Murine monoclonal antibody 2H10 has a
specific and potent antagonistic effect against VEGF-B and inhibits
the biological activity of VEGF-B. However, the potential
immunogenicity of mAb 2H10 in other hosts, and in particular
humans, makes the use of mAb 2H10 unsuitable as a therapeutic agent
in these hosts. The murine monoclonal antibodies B33/02-1C6-6,
B33/02-2F5-2 and 36/01-4E12-11-12 may also be subjected to
humanization. The present invention, however, extends to any
deimmunized, humanized or human monoclonal antibodies directed to
VEGF-B.
[0082] In a particular embodiment contemplated by the present
invention, the antibodies of the present invention comprise within
the variable region of their light chain, at least one of the CDRs
found in the light chain of mAb 2H10. Thus, among the antibodies
contemplated by the present invention are those that comprise from
one to all three of the CDR sequences from the light chain variable
region of mAb 2H10. Further, among the antibodies contemplated by
the present invention are those that comprise from one to all three
of the CDR sequences from the heavy chain variable region of mAb
2H10. In a preferred embodiment, among the antibodies contemplated
by the present invention are those that comprise from one to all
six CDR sequences from the heavy and light chain variable regions
of mAb 2H10. In further embodiments contemplated by the present
invention, the antibodies of the present invention comprise within
the variable region of their light chain one or more CDRs found in
the light chain of monoclonal antibodies B33/02-1C6-6 or
B33/02-2F5-2 or 36/01-4E12-11-12.
[0083] Procedures for generating human antibodies in non-human
animals have also been developed and are well known to those
skilled in the art. The antibodies may be partially human; or
preferably completely human. For example, transgenic mice into
which genetic material encoding one or more human immunoglobulin
chains has been introduced may be used to produce the antibodies of
the present invention. Such mice may be genetically altered in a
variety of ways. The genetic manipulation may result in human
immunoglobulin polypeptide chains replacing endogenous
immunoglobulin chains in at least some (preferably virtually all)
antibodies produced by the animal upon immunization.
[0084] Mice in which one or more endogenous immunoglobulin genes
have been inactivated by various means have been prepared. Human
immunoglobulin genes have been introduced into the mice to replace
the inactivated mouse genes. Antibodies produced in the animals
incorporate 22 human immunoglobulin polypeptide chains encoded by
the human genetic material introduced into the animal. Examples of
techniques for production and use of such transgenic animals are
described in U.S. Pat. Nos. 5,814,318, 5,569,825, and 5,545,806,
which are incorporated by reference herein.
[0085] As such, antibodies of the present invention may include,
but are not limited to, partially human (preferably fully human)
monoclonal antibodies that inhibit the biological activity of
VEGF-B.
[0086] Another method for generating human antibodies is phage
display. Phage display techniques for generating human antibodies
are well known to those skilled in the art, and include the methods
used by companies such as Cambridge Antibody Technology and
MorphoSys and which are described in International Patent
Publication Nos. WO 92/01047, WO 92/20791, WO 93/06213 and WO
93/11236.
[0087] Antibodies of the present invention may be employed in vitro
or in vivo. Among the uses for antibodies of the present invention
are assays (either in vitro or in vivo) to detect the presence of
VEGF-B1 polypeptides and immunoaffinity chromatography to purify
VEGF-B polypeptides. Further; those antibodies of the present
invention that can inhibit the biological activity of VEGF-B may be
used to inhibit a biological activity that results from VEGF-B
signalling through the VEGF-R1 receptor.
[0088] Therefore, in one embodiment, such antibodies may be used in
therapeutic applications to treat disorders caused or exacerbated
(directly or indirectly) by the signaling of VEGF-B through the
VEGF-R1 receptor. A therapeutic application involves in vivo
administration of a blocking antibody to a mammal in an amount
effective to inhibit signaling by VEGF-B3 through the VEGF-R1
receptor. Preferably, the antibodies are human or humanized
monoclonal antibodies of the present invention.
[0089] The antibodies may be used to treat diseases or conditions
induced by VEGF-B, including but not limited to pulmonary
hypertension, the growth of angiogenic tumors and the spread or
metastases of cancer cells, chronic inflamatory diseases such as
rheumatoid arthritis and any other VEGF-B-mediated diseases or
conditions where there is known to be a significant angiogenic
component.
[0090] Antibodies in accordance with the present invention include
the murine monoclonal antibodies 2H10, B33/02-1C6-6, B33/02-2F5-2
and 36/01-4E12-11-12 and humanized forms thereof.
[0091] Particular monoclonal antibodies of the invention are
selected from the group consisting of mAb 2H10; a mAb that is
cross-reactive with in mAb 2H10; a nab that binds to the same
epitope as mAb 2H10; a mAb that competes with mAb 2H10 for binding
to VEGF-B; a mAb that possesses a biological activity of mAb 2H10;
and an antigen-binding fragment of any of the foregoing
antibodies.
[0092] In one embodiment, the antibody has a binding affinity for
human VEGF-B that is substantially equivalent to the binding
affinity of mAb 2H10 for human VEGF-B. mAb 2H10 is an IgG2a
antibody. mAbs of other isotypes (including but not limited to
IgG4), derived from mAb 2H10 are also encompassed by the present
invention. Hybridoma cell lines that produce any such monoclonal
antibodies also are provided by the present invention.
[0093] Procedures for switching (altering) the subclass or isotype
of an antibody are also well known to those skilled in the alt.
Such procedures may involve, for examples recombinant DNA
technology, whereby DNA encoding antibody polypeptide chains that
confer the desired subclass is substituted for DNA encoding the
corresponding polypeptide chain of the parent antibody. This
procedure is useful, for example, in certain antibody therapeutic
applications where are particular antibody isotope is preferred,
such as in the treatment of asthma where IgG4 may be the preferred
antibody isotype.
[0094] One example of a biological activity of in mAb 2H10 is the
ability to bind to VEGF-B and inhibit the biological activity of
VEGF-B. In one embodiment, a mAb of the invention possesses VEGF-B
biological activity blocking activity substantially equivalent to
that of mAb 2H10.
[0095] The ability of the antibodies of the present invention to
inhibit the biological activity of VEGF-B can be confirmed in a
number of assays.
[0096] One assay that may be used for identifying antibodies which
function as VEGF-B antagonists and inhibit the biological activity
of VEGF-B is described below and in the Examples.
[0097] In this assay, 293A12-cells are engineered to express
chimeric polypeptides comprising the extracellular domain of either
VEGF-R1 operably connected to the transmembrane and cytoplasmic
domains of the protein, gp130. When the engineered 293A12-cells are
in the presence of VEGF-B the chimeric polypeptides form a
homodimeric receptor complex which permits signal transduction to
occur. The VEGF-B-mediated signal transduction is observable via an
identifiable signal, such as the activation of a gene encoding a
reporter molecule (Example 5).
[0098] Anti-VEGF-B antibodies that antagonize VEGF-B signaling
through the VEGF-R1 receptor will inhibit VEGF-B-mediated
activation of the reporter molecule.
[0099] The level of signal transduction is conveniently determined
by selecting cells wherein signal transduction activates a pathway
regulating the expression of a gene encoding a reporter molecule
that provides an identifiable signal. Preferred reporter molecules
are enzymes such as luciferase.
[0100] 293A12 cells are particularly preferred in this assay as
they are 293T cells which stably express genetic material encoding
a luciferase reporter molecule (Example 1). The expression of the
luciferase reporter molecule is regulated by a STAT-3 signaling
pathway which is activated by gp130 signaling.
[0101] The signal transduction portion from gp130 is particularly
preferred, as it induces STAT-3 phosphorylation which leads to the
expression of the STAT-3 activated luciferase reporter gene.
However, the signal transduction portion from other molecules may
also be employed. The choice of the signal transduction portion of
the polypeptides must be matched to the activation or promoter
portion of the gene encoding the reporter molecule.
[0102] Those skilled in the art appreciate that the cell based
assays of the invention, for example described above and in Example
4, may be utilised as a basis for screening for modulators of
VEGF-B/VEGF-R1 interaction. While such methods are well known to
those skilled in the art, a brief description of the method is
provided herein. The method involves subjecting appropriately
engineered cells to a signal producing amount of VEGF-B under
conditions where, in the absence of any antagonism of ligand
receptor binding, a signal, for example luciferase expression, may
be detected. The exposure is then conducted in the presence of test
compounds and the level of signal detected compared with that
detected in the absence of a test compound. Test compounds may
include compound libraries, for example libraries of natural
product extracts or libraries of synthetic compounds.
Alternatively, phage display libraries of antibody variable domains
and the like, or panels of monoclonal antibodies against VEGF-B may
be screened across the assay.
[0103] Chimeric polypeptides that may be used in the assay of the
present invention are described in Example 1 and comprise the amino
acid sequences set forth in SEQ ID NO:2 and SEQ ID NO:4.
[0104] cDNA encoding the chimeric polypeptides contemplated for use
in this assay comprise a nucleotide sequence selected from SEQ ID
NO: 1 and SEQ ID NO: 3. The sequence defined by SEQ ID NO: 1
comprises a sequence which encodes the extracellular immunoglobulin
(Ig) domains (D) 1 to 4 of human VEGF-R1 fused to the transmembrane
and cytoplasmic domains of gp130. SEQ ID NO: 3 comprises a sequence
which encodes the extracellular immunoglobulin (Ig) domains (D) 1
to 3 of human VEGF-R1 fused to the transmembrane and cytoplasmic
domains of gp130.
[0105] Although 293A12 cells are described in the assay of the
present invention, other cells may be used. Generally a eukaryotic
cell is employed, and more particularly, a mammalian cell. The
mammalian cells may be derived from humans, livestock animals,
laboratory test animals and companion animals, Non-mammalian cells
contemplated herein include cells from avian species, reptilian
species, amphibian species and insect species.
[0106] The term "operably connected" is used in its broadest
context to include molecules which have associated together such
that they are in functional interaction with each other Generally,
the association is by a chemical linkage or bond. Preferably, the
chemical linkage or bond is a peptide bond. The terms include,
therefore, a polypeptide comprising a contiguous series of amino
acids each linked via a peptide bond wherein one contiguous series
of amino acids has ligand-binding properties and another contiguous
series of amino acids has signal transduction properties.
[0107] Pharmaceutically acceptable carriers and/or diluents include
any and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, agents used for adjusting tonicity, buffers;
chelating agents, and absorption delaying agents and the like. The
use of such media and agents for pharmaceutical active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredient, use thereof in
the therapeutic compositions is contemplated. Supplementary active
ingredients can also be incorporated into the compositions.
[0108] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions (where water soluble) and sterile powders
for the extemporaneous preparation of sterile injectable solutions.
It must be stable under the conditions of manufacture and storage
and must be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dilution medium comprising, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol and liquid
polyethylene glycol, and the like), suitable mixtures thereof and
vegetable oils. The proper fluidity can be maintained, for example,
by the use of superfactants. The preventions of the action of
microorganisms can be brought about by various anti-bacterial and
anti-fungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thirmerosal and the like. In many cases, it will be
preferable to include agents to adjust tonicity, for example,
sugars or sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminium monostearate and
gelatin. The compositions may also include buffers and chelating
agents.
[0109] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with the active ingredient and optionally other active
ingredients as required, followed by filtered sterilization or
other appropriate means of sterilization. In the case of sterile
powders for the preparation of sterile injectable solutions,
suitable methods of preparation include vacuum drying and the
freeze-drying technique which yield a powder of active ingredient
plus any additionally desired ingredient.
[0110] The amount of active compound in such therapeutically useful
compositions is such that a suitable dosage will be obtained.
[0111] The compositions of the present invention are useful in
modifying a VEGF-B-mediated condition including but not limited to
pulmonary hypertension, the growth of angiogenic tumors and the
spread or metastases of cancer cells, chronic inflammatory diseases
such as rheumatoid arthritis and any other VEGF-B-mediated diseases
or conditions where there is known to be a significant angiogenic
component.
[0112] The human and humanized antibodies of the present invention
are useful in the treatment of such conditions. Any adverse
condition resulting from VEGF-B interaction with VEGF-R1 may be
treated or prevented by the administration of the human and
humanised monoclonal antibodies of the present invention.
[0113] Accordingly, another aspect of the present invention
contemplates a method for the treatment or prophylaxis of a
condition mediated by VEGF-B such as but not limited to a chronic
inflammatory condition, said method comprising administering to a
subject an effective amount of a deimmunized, humanized or human
monoclonal antibody of the present invention for a time and under
conditions sufficient to inhibit the biological activity of
VEGF-B.
[0114] An "effective amount" in this context is an amount of an
antibody sufficient to reduce VEGF-B signaling through the VEGF-R1
receptor by at least 40%, preferably at least 50%, more preferably
by at least 60%, still more preferably by at least 70-80% or
greater than 90%. For example, the reduction in signal may be by at
least 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. Reduction in
signaling may be measured in any number of ways including
inhibition or antagonism of binding between VEGF-D and VEGF-41 or
reduction in activity of VEGF-R1 fused to a receptor molecule.
[0115] The method may also be measured at the level of amelioration
of symptoms. Hence, an effective amount would be that amount
required to at least partially alleviate symptoms of, for example,
inflammation.
[0116] Preferably, the subject is a human. However, veterinary
applications are also contemplated for livestock animals as well as
companion animals. In such cases it would be necessary to prepare
an appropriate antibody designed to avoid an immunogenic response
to the antibody by the mammal.
[0117] In a specific embodiment, the present invention contemplates
a method for ameliorating the effects of VEGF-B mediated conditions
in a human subject, said method comprising administering to said
subject an effective amount of a humanized monoclonal antibody of
the present invention or its equivalent for a time and under
conditions sufficient to ameliorate the effects of
inflammation.
[0118] The present invention further contemplates the use of a
humanized monoclonal antibody of the present invention or its
equivalent in the manufacture of a medicament in the treatment or
prophylaxis of an inflammatory condition in a subject.
[0119] The present invention is further described by the following
non-limiting Examples.
Example 1
Development of Assays for Analysis of VEGF-B-Receptor
Interaction
[0120] Endothelial cells do not proliferate in response to VEGF-R1
ligands and no simple, biological assay system for the measurement
of VEGF-B activity has been described.
[0121] The present inventors reasoned that an assay system that
would provide a more reliable and quantifiable response to
ligand-induced receptor activation would facilitate the analysis of
the biological activities of VEGF-B.
Mammalian Cell Culture and Transfection
[0122] Human 293T cells were transfected using Lipofectamine 2000
according to the manufacturer's instructions. Cells co-transfected
with plasmids encoding either puromycin resistance or hygromycin
resistance were subsequently selected and maintained in media
supplemented with puromycin (25 .mu.g/ml) or hygromycin (60
.mu.g/ml) respectively. 293A12 cells were derived from 293T cells
following stable transfection with a luciferase reporter under the
control of a STAT-3 promoter (Nicholson et al., Proc. Natl. Acad.
Sci. USA 97; 6493-6498, 2000). When stimulated with cytokines that
activate STAT-3 such as leukaemia inhibitory factor (LIF) and
interleukin-6 (IL-6), luciferase expression 10-15 fold in excess of
background is induced within 24 hours.
[0123] Clone 2.1.19.25 was derived from 293A12 cells following
stable transfection with a chimeric receptor construct (see below).
For assay of VEGF-R1 ligand activity 2.1.19.25 cells were plated
into 96 well ViewPlates (Packard Bioscience, Australia) at
5.times.10.sup.4/well and ligands added to the indicated
concentration to give a final assay volume of 100 .mu.l. Luciferase
was assessed at 18-24 hours (LucLite Kit, Packard Bioscience,
Australia).
Expression, Purification and Refolding of VEGF-B Isoforms
[0124] The VEGF-.beta. isoforms, VEGF-B.sub.167 and VEGF-B.sub.186,
and a truncated form, VEGF-B.sub.10-108, are expressed in E. coli
as N-terminal His.sub.6-tagged proteins.
[0125] Recombinant VEGF-B.sub.167 is expressed in E. coli using the
pET15b vector with downstream purification and refolding as
previously described (Scrofani et al., Protein Science 9:
2018-2025, 2000).
[0126] The coding region of mature human VEGF-B.sub.10-108 protein
is amplified using PCR [95.degree. C. for 2 minutes, 1 cycle;
94.degree. C. for 1 minute, 60.degree. C. for 1 minute, 72.degree.
C. for 1 minute--30 cycles; 72.degree. C. for 1 minute--1 cycle;
1.5U Expand High Fidelity PCT System enzyme mix to introduce in
frame BamHI HindIII restriction enzyme sites, at the 5' and 3' ends
respectively, using the oligonucleotides:
TABLE-US-00002 [SEQ ID NO: 5] 5'Oligo:
5'-CACGGATCCGCAGCACACTATCACCAGAGGAAAG-3' [SEQ ID NO: 6] 3'Oligo:
3'-GCATAAGCTTTCACTTTTTTTTAGGTCTGCATTC-3'
[0127] The resulting PCR-derived DNA fragment is digested with
BamHI and HindIII and ligated into BamHI and HindIII digested pQE30
(QIAGEN, Germany). The VEGF-B.sub.10-108-pQE30 is transformed into
M15[pREP4] E. coli (QIAGEN) using an electroporator according to
the manufacturer's instructions. The VEGF-B.sub.10-108 protein
displays an additional 16 amino acids at the N-terminus which
incorporate a His.sub.6 tag and a Genenase I cleavage site. The
VEGF-B.sub.10-108 protein is isolated from E. coli inclusion bodies
and purified and refolded as previously described (Scrofani et al.,
2000, supra).
[0128] The coding region of mature human VEGF-B186 is amplified by
PCR and cloned into pET15b. In contrast to the other isoforms,
VEGF-B.sub.186 is purified directly from whole E. coli cell lysate
rather than inclusion bodies. Pelleted cells are suspended in a
buffer of 6M guanidine hydrochloride (GdCl), 0.1 M
NaH.sub.2PO.sub.4, 10 mM Tric-HCl, 10 mM 2-mercaptoethanol, 0.02%
w/v Tween-20, pH 8.0 at 10 mL per gram of cells and incubated
overnight at 37.degree. C. The solution is centrifuged and the
supernatant is decanted and filtered. Nickel affinity
chromatography and further downstream purification and refolding
are performed as previously described (Scrofani et al., 2000,
supra).
[0129] Metal affinity chromatography under reducing and denaturing
conditions was used to purify monomeric VEGF-B proteins and,
following dialysis refolding, dimeric protein is separated from
monomeric and high molecular weight multimeric forms using a
combination of reverse-phase HPLC and hydrophilic chromatography.
SDS-PAGE analysis of the three, refolded VEGF-B proteins is shown
(FIG. 1).
Development of a Cell-Based Assay
[0130] The present inventors used splice-overlap extension PCR to
generate a series of chimeric receptors and developed an
assay-based on a chimeric receptor strategy. The strategy involves
joining the extracellular immunoglobulin (Ig) domains of VEGF-R1
(preferably D1 to D4 or D1 to D3) to the cytoplasmic domains of
gp130 (gp130 transmembrane domain--amino acids 574 to 595 and gp130
cytoplasmic domain--amino acids 595 to 918) (FIG. 2).
[0131] Using VEGF-R1 and gp130 cDNAs as templates, a human
VEGF-R-gp130 chimeric receptor cDNA is generated by
splice-overlap-extension PCR. Briefly, the coding region of
extracellular immunoglobulin (Ig) domains (D) 1 to 4 and 1 to 3 of
human VEGF-R1 are amplified by PCR [96.degree. C. for 2 mins, 1
cycle; 94.degree. C. for 30 seconds, 55.degree. C. for 30 seconds,
68.degree. C. for 1.5 minutes--35 cycles; 1.5U Expand High Fidelity
PCT System enzyme mix (Roche Diagnostics, Mannheim, Germany) using
the oligonucleotides:
TABLE-US-00003 5'Oligo [SEQ ID NO: 7]
5'-ATATGGCGCGCCTAGTCAGCTACTGGGACACCGGGGTC-3' 3'Oligo (domains 1 to
4): [SEQ ID NO: 8] 5'-CAGGCACGACTATGGCTTCAATTTCTCCGGCCTTTTCGTAAATC
TGGGTTTTCAC-3' 3'Oligo (domains 1 to 3): [SEQ ID NO: 9]
5'-CACGACTATGGCTTCAATTTCTCCTATATGCACTGAGGTGTTAA CAGATTTG-3'
[0132] Similar PCR conditions are used to amplify the human gp130
transmembrane and cytoplasmic domains using the following
oligonucleotides:
TABLE-US-00004 5'Oligo: [SEQ ID NO: 10]
5'-ACGTACGCGTTCACTGAGGCATGTAGCCGCCTTGCCG-3 3'Oligo: [SEQ ID NO: 11]
5'-GGAGAAATTGAAGCCATAGTCGTGCCTGTTTGCTTAGC-3'
[0133] To generate chimeric cDNA the PCR products are mixed and a
further PCR using the same conditions with the 5' sense VEGF-R1
oligonucleotide and the 3'antisense gp130 oligonucleotide are
performed. This PCR product is designed to incorporate 5' Asc1 site
and 3' Mlu1 restriction enzyme sites and after digestion of the PCR
product with these enzymes, the chimeric cDNA is ligated into an
Mlu1 digested mammalian expression vector, pEFBOS-S-FLAG (Nicholson
et al., 2000, supra) for expression as an N-terminal FLAG-tagged
protein.
[0134] Details of both chimeric receptors are provided in schematic
form in FIG. 2. Transient expression in 293T cells, followed by
Western blot analysis with anti-FLAG antibodies confirmed that the
constructs encode a protein of the expected molecular weight (FIG.
2B).
[0135] For assay development, the chimeric receptor construct
incorporating VEGF-R1 D1 to D4 and a vector incorporating a
hygromycin resistance gene were co-transfected into 293A12 cells.
Following hygromycin selection, isolated resistant colonies were
picked and expanded, then assayed for luciferase after incubation
in the presence of VEGF-A. Eleven of the 63 colonies assayed
expressed luciferase in response to VEGF-A and colony 2.1.19 was
subsequently cloned by limit dilution. Dose-response analysis of
clone 2.1.19.25 to VEGF-A is shown in FIG. 2C. In further analysis,
this response was shown to be completely inhibited by soluble
VEGF-R1-IgG-Fc chimeric receptor protein (R&D Systems, UK; FIG.
2D). As expected the VEGF-R1-IgG-Fc chimeric protein did not
inhibit 2.1.19.25 luciferase production in response to LIF. Over a
large number of assays the VEGF-A signal to background ratio have
varied between 2.5 to 3.
[0136] The refolded VEGF-.beta. isoforms were assessed for
biological activity in the 2.1.19.25 cell-based assay. Both
VEGF-B.sub.167 and VEGF-B.sub.10-108 were shown to be active (FIG.
3A). The ED.sub.50 for both isoforms of VEGF-B is routinely in the
order of 150-300 ng/ml. VEGF-B.sub.186 preparations have not shown
activity despite display of an interaction with VEGF-R1 in
Biosensor-based analysis.
[0137] The VEGF family members retain a complex secondary structure
making the refolding of these proteins difficult. However, recently
there has been considerable success in refolding these proteins
from insoluble inclusion bodies (reviewed in Scrofani and Nash, J.
Microbiol. Biotechnol. 11(4): 543-511, 2001). The inventors have
previously described a protocol for production of dimeric
VEGF-B.sub.167 based on E. coli fermentation, inclusion body
isolation and dialysis refolding. In the present invention, they
have applied a similar strategy to express, purify and refold the
other naturally occurring isoform and a truncated form of the
protein that retains the core cystine-knot motif. All three
proteins were purified as homodimers and demonstrated to interact
with the minimal ligand binding domain of VEGF-R1.
[0138] Although the Biosensor analysis indicates appropriate
folding within each monomeric subunit, it does not confirm correct
inter-chain disulphide bond formation to yield a biologically
active dimer. To date, the biological assay of VEGF-R1 ligand
activity has been based on relatively complex readouts such as
monocyte migration, smooth muscle cell MMP production and
osteoclast function (Clauss et al., J. Biol. Chem. 271:
17269-17634, 1996; Wang and Keiser, Circ. Res. 83: 832-840, 1998;
Niida et al., J. Exp. Med. 190: 293-298, 1999). The simple chimeric
receptor-based assay of the present invention utilizes a reporter
gene readout and is used to demonstrate the activity of
VEGF-B.sub.167 and VEGF-B.sub.10-108. Surprisingly, the inventors
did not detect activity of refolded VEGF-B.sub.186. One explanation
may be inappropriate dimerization as noted above, however, Makinen
et al. (J. Biol. Chem. 274: 21217-21222, 1999), have reported that
VEGF-B.sub.186 expressed in mammalian cells is processed at the
C-terminus and it is only alter this processing occurs that it is
able to interact with neuropilin-1. It is possible that the
full-length C-terminal domain retained in the E. coli expressed
protein may interfere with receptor dimerization and
signalling.
[0139] In addition to allowing demonstration of recombinant protein
biological activity, the new cell-based assay has facilitated the
identification of VEGF-B antagonists, such as specific neutralizing
mAbs that inhibit VEGF-1 signalling mediated though VEGF-R1.
Development of a Molecular Assay
[0140] A molecular assay based oil the interaction of VEGF-R1 with
VEGF-B represents the best primary screen for both monoclonal
antibodies and, potentially, small molecule antagonists.
[0141] The coding region of 1 g domain 2 (D2) of the human VEGF-R1
protein (residues 129-229) was amplified by PCR and ligated into
pQE30 vector (QIAGEN). VEGF-R1.sub.D2 protein was isolated from E.
coli inclusion bodies using a previously described protocol
(Weismann et al., Cell 91: 695-704, 1997) and further purified
under denaturing conditions by RP-HPLC (QIAGEN).
[0142] For initial Biosensor analysis of the binding of the
purified and refolded VEGF-.beta. isoforms, surface plasmon
resonance (Biosensor 2000; Biacore, Sweden) and recombinant
VEGF-R1.sub.D2 are used. D2 has previously been demonstrated to
represent the minimal ligand binding domain of VEGF-R1 (Weismann et
al., 1997, supra). The three forms of VEGF-B were immobilized on
separate channels of a CM5 sensor chip, while murine LIF was
immobilized to a fourth channel to serve as a negative control.
Interaction with VEGF-R1.sub.D2 was monitored on all channels
simultaneously.
[0143] The target molecule is immobilized to a CM5 dextran chip
using amine-coupling chemistry according to the manufacturer's
instructions. Briefly, 35 .mu.L NHS/EDC (1:1) was injected onto the
sensor chip at a flow rate of 5 .mu.L/min to activate the sensor
surface. Test and negative control (LIF) proteins were resuspended
in 20 mM sodium acetate, pH 4.5 (final concentration 7-20 .mu.g/mL)
and injected directly onto the sensor surface. Post coupling, 50 mM
diaminoethane, pH 9.0 was used to quench residual activated sites
on the biosensor surface. Two cycles of 0.1 M phosphoric acid (30
.mu.L; 50 .mu.L/min) were performed at the end of each run to
regenerate the sensor chip surface.
[0144] Purified VEGF-R1.sub.D2 is diluted to varying concentrations
in 0.1% w/v BSA, 20 mM HEPES, 0.15 M NaCl, 0.005% w/v Tween 20, 3.4
mM EDTA, pH 7.4. Receptor binding is simultaneously monitored on
VEGF-B.sub.10-108, VEGF-B.sub.167, VEGF-B.sub.186 and mLIF control
channel at a flow rate of 5 .mu.L/min. Scatchard analysis is used
to determine binding kinetics at steady state equilibrium.
[0145] A dose-response analysis of human VEGF-R1.sub.D2 binding is
completed. The molecules VEGF.sub.10-108, VEGF-B.sub.167 and
VEGF-B.sub.186 clearly associate with VEGE-R1.sub.D2 with similar
kinetics (FIGS. 2B and 2C). The truncated VEGF-B10-108 appears to
have a slightly higher affinity for VEGE-R1.sub.D2 (KD=0.8 nM) than
exhibited by either VEGF-B.sub.176 (KD 1.5 nN) or VEGF-B.sub.186
(KD 2.0 nM).
Example 2
Analysis of VEGF-B-Specific Neutralizing in mAbs Using New
Assays
Analysis Using Biochemical Assays--Biosensor and ELISA
[0146] Monoclonal antibodies which bind to and inhibit the
biological activity of VEGF-B (neutralizing antibodies) would
represent valuable tools for characterization of VEGF-B function
and may be used to generate valuable therapeutic agents though the
process of mouse antibody humanisation.
[0147] A panel of mAbs is raised against the VEGF-B.sub.167 isoform
and are screened for VEGF-B antagonist activity in the cell-based
assay described above. The results are presented in FIG. 3. The mAb
2H10 but not the VEGF-B specific mAb 7C3 or control unrelated in
mAb 6A9 was able to inhibit VEGF-B binding activity in the entire
test range. mAb 2H10 is unable to block the cellular responses to
VEGF-A.
[0148] ELISA-based analysis is performed and reveals that mAb 2H10
binds to both of the naturally occurring isoforms of VEGF-B,
VEGF-B.sub.167 and VEGE-B.sub.186 as well as the short 10-108 form
used for structural studies. Western blot analysis shows that this
monoclonal antibody reacts only with these proteins under
non-denaturing conditions, suggesting that 2H10 targets the core
receptor-binding domain of VEGF-B and, as a consequence, the mAb is
anticipated to inhibit the activity of all VEGF-.beta.
isoforms.
[0149] In addition to 2H10, the anti-VEGF-B mAbs B33/02-1C6-6,
B33/02-2F5-2 and 36/01-4E12-11-12 have been identified as
antagonists of VEGF-B biological activity (see FIG. 6). Antagonist
activity was characterised using a cell viability assay similar to
the cell-based assay described in Example 1 above. The cell
viability assay used a murine IL-3-dependant pro-B cell line,
Ba/F3, transfected to stably express chimeric VEGFR-1 extracellular
domains with the cytoplasmic domain of the erythropoietin receptor
(VEGFR-1/EpoR). These cells, in addition to proliferating in
response to IL-3, will also proliferate in response to cytokines
that signal through VEGF-R1 such as VEGF-A and VEGF-B. Cell
viability was estimated colourmetrically by the enzymatic reduction
of a tetrazolium dye
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium (MTS) and phenylmetha-sulfazone (PMS).
[0150] The hybridoma that produces monoclonal antibody 2H10 was
deposited on Jul. 27, 2005 with American Type Culture Collection
(ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, U.S.A.
under Accession No. PTA-6889.
[0151] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more said steps or features.
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Sequence CWU 1
1
1112277DNAhumanCDS(1)..(2277) 1atg gtt ctt gcc agc tct acc acc agc
atc cac acc atg ctg ctc ctg 48Met Val Leu Ala Ser Ser Thr Thr Ser
Ile His Thr Met Leu Leu Leu1 5 10 15ctc ctg atg ctc ttc cac ctg gga
ctc caa gct tca atc tcg gcg cgc 96Leu Leu Met Leu Phe His Leu Gly
Leu Gln Ala Ser Ile Ser Ala Arg 20 25 30cag gac tac aag gac gac gat
gac aag acg cgc cag tct agt tca ggt 144Gln Asp Tyr Lys Asp Asp Asp
Asp Lys Thr Arg Gln Ser Ser Ser Gly 35 40 45tca aaa tta aaa gat cct
gaa ctg agt tta aaa ggc acc cag cac atc 192Ser Lys Leu Lys Asp Pro
Glu Leu Ser Leu Lys Gly Thr Gln His Ile 50 55 60atg caa gca ggc cag
aca ctg cat ctc caa tgc agg ggg gaa gca gcc 240Met Gln Ala Gly Gln
Thr Leu His Leu Gln Cys Arg Gly Glu Ala Ala65 70 75 80cat aaa tgg
tct ttg cct gaa atg gtg agt aag gaa agc gaa agg ctg 288His Lys Trp
Ser Leu Pro Glu Met Val Ser Lys Glu Ser Glu Arg Leu 85 90 95agc ata
act aaa tct gcc tgt gga aga aat ggc aaa caa ttc tgc agt 336Ser Ile
Thr Lys Ser Ala Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser 100 105
110act tta acc ttg aac aca gct caa gca aac cac act ggc ttc tac agc
384Thr Leu Thr Leu Asn Thr Ala Gln Ala Asn His Thr Gly Phe Tyr Ser
115 120 125tgc aaa tat cta gct gta cct act tca aag aag aag gaa aca
gaa tct 432Cys Lys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr
Glu Ser 130 135 140gca atc tat ata ttt att agt gat aca ggt aga cct
ttc gta gag atg 480Ala Ile Tyr Ile Phe Ile Ser Asp Thr Gly Arg Pro
Phe Val Glu Met145 150 155 160tac agt gaa atc ccc gaa att ata cac
atg act gaa gga agg gag ctc 528Tyr Ser Glu Ile Pro Glu Ile Ile His
Met Thr Glu Gly Arg Glu Leu 165 170 175gtc att ccc tgc cgg gtt acg
tca cct aac atc act gtt act tta aaa 576Val Ile Pro Cys Arg Val Thr
Ser Pro Asn Ile Thr Val Thr Leu Lys 180 185 190aag ttt cca ctt gac
act ttg atc cct gat gga aaa cgc ata atc tgg 624Lys Phe Pro Leu Asp
Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp 195 200 205gac agt aga
aag ggc ttc atc ata tca aat gca acg tac aaa gaa ata 672Asp Ser Arg
Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile 210 215 220ggg
ctt ctg acc tgt gaa gca aca gtc aat ggg cat ttg tat aag aca 720Gly
Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr225 230
235 240aac tat ctc aca cat cga caa acc aat aca atc ata gat gtc caa
ata 768Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Gln
Ile 245 250 255agc aca cca cgc cca gtc aaa tta ctt aga ggc cat act
ctt gtc ctc 816Ser Thr Pro Arg Pro Val Lys Leu Leu Arg Gly His Thr
Leu Val Leu 260 265 270aat tgt act gct acc act ccc ttg aac acg aga
gtt caa atg acc tgg 864Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr Arg
Val Gln Met Thr Trp 275 280 285agt tac cct gat gaa aaa aat aag aga
gct tcc gta agg cga cga att 912Ser Tyr Pro Asp Glu Lys Asn Lys Arg
Ala Ser Val Arg Arg Arg Ile 290 295 300gac caa agc aat tcc cat gcc
aac ata ttc tac agt gtt ctt act att 960Asp Gln Ser Asn Ser His Ala
Asn Ile Phe Tyr Ser Val Leu Thr Ile305 310 315 320gac aaa atg cag
aac aaa gac aaa gga ctt tat act tgt cgt gta agg 1008Asp Lys Met Gln
Asn Lys Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg 325 330 335agt gga
cca tca ttc aaa tct gtt aac acc tca gtg cat ata tat gat 1056Ser Gly
Pro Ser Phe Lys Ser Val Asn Thr Ser Val His Ile Tyr Asp 340 345
350aaa gca ttc atc act gtg aaa cat cga aaa cag cag gtg ctt gaa acc
1104Lys Ala Phe Ile Thr Val Lys His Arg Lys Gln Gln Val Leu Glu Thr
355 360 365gta gct ggc aag cgg tct tac cgg ctc tct atg aaa gtg aag
gca ttt 1152Val Ala Gly Lys Arg Ser Tyr Arg Leu Ser Met Lys Val Lys
Ala Phe 370 375 380ccc tcg ccg gaa gtt gta tgg tta aaa gat ggg tta
cct gcg act gag 1200Pro Ser Pro Glu Val Val Trp Leu Lys Asp Gly Leu
Pro Ala Thr Glu385 390 395 400aaa tct gct cgc tat ttg act cgt ggc
tac tcg tta att atc aag gac 1248Lys Ser Ala Arg Tyr Leu Thr Arg Gly
Tyr Ser Leu Ile Ile Lys Asp 405 410 415gta act gaa gag gat gca ggg
aat tat aca atc ttg ctg agc ata aaa 1296Val Thr Glu Glu Asp Ala Gly
Asn Tyr Thr Ile Leu Leu Ser Ile Lys 420 425 430cag tca aat gtg ttt
aaa aac ctc act gcc act cta att gtc aat gtg 1344Gln Ser Asn Val Phe
Lys Asn Leu Thr Ala Thr Leu Ile Val Asn Val 435 440 445aaa ccc cag
att tac gaa aag gga gaa att gaa gcc ata gtc gtg cct 1392Lys Pro Gln
Ile Tyr Glu Lys Gly Glu Ile Glu Ala Ile Val Val Pro 450 455 460gtt
tgc tta gca ttc cta ttg aca act ctt ctg gga gtg ctg ttc tgc 1440Val
Cys Leu Ala Phe Leu Leu Thr Thr Leu Leu Gly Val Leu Phe Cys465 470
475 480ttt aat aag cga gac cta att aaa aaa cac atc tgg cct aat gtt
cca 1488Phe Asn Lys Arg Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val
Pro 485 490 495gat cct tca aag agt cat att gcc cag tgg tca cct cac
act cct cca 1536Asp Pro Ser Lys Ser His Ile Ala Gln Trp Ser Pro His
Thr Pro Pro 500 505 510agg cac aat ttt aat tca aaa gat caa atg tat
tca gat ggc aat ttc 1584Arg His Asn Phe Asn Ser Lys Asp Gln Met Tyr
Ser Asp Gly Asn Phe 515 520 525act gat gta agt gtt gtg gaa ata gaa
gca aat gac aaa aag cct ttt 1632Thr Asp Val Ser Val Val Glu Ile Glu
Ala Asn Asp Lys Lys Pro Phe 530 535 540cca gaa gat ctg aaa tta ttg
gac ctg ttc aaa aag gaa aaa att aat 1680Pro Glu Asp Leu Lys Leu Leu
Asp Leu Phe Lys Lys Glu Lys Ile Asn545 550 555 560act gaa gga cac
agc agt ggt att ggg ggg tct tca tgc atg tca tct 1728Thr Glu Gly His
Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser 565 570 575tct agg
cca agc att tct agc agt gat gaa aat gaa tct tca caa aac 1776Ser Arg
Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser Gln Asn 580 585
590act tcg agc act gtc cag tat tct acc gtg gta cac agt ggc tac aga
1824Thr Ser Ser Thr Val Gln Tyr Ser Thr Val Val His Ser Gly Tyr Arg
595 600 605cac caa gtt ccg tca gtc caa gtc ttc tca aga tcc gag tct
acc cag 1872His Gln Val Pro Ser Val Gln Val Phe Ser Arg Ser Glu Ser
Thr Gln 610 615 620ccc ttg tta gat tca gag gag cgg cca gaa gat cta
caa tta gta gat 1920Pro Leu Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu
Gln Leu Val Asp625 630 635 640cat gta gat ggc ggt gat ggt att ttg
ccc agg caa cag tac ttc aaa 1968His Val Asp Gly Gly Asp Gly Ile Leu
Pro Arg Gln Gln Tyr Phe Lys 645 650 655cag aac tgc agt cag cat gaa
tcc agt cca gat att tca cat ttt gaa 2016Gln Asn Cys Ser Gln His Glu
Ser Ser Pro Asp Ile Ser His Phe Glu 660 665 670agg tca aag caa gtt
tca tca gtc aat gag gaa gat ttt gtt aga ctt 2064Arg Ser Lys Gln Val
Ser Ser Val Asn Glu Glu Asp Phe Val Arg Leu 675 680 685aaa cag cag
att tca gat cat att tca caa tcc tgt gga tct ggg caa 2112Lys Gln Gln
Ile Ser Asp His Ile Ser Gln Ser Cys Gly Ser Gly Gln 690 695 700atg
aaa atg ttt cag gaa gtt tct gca gca gat gct ttt ggt cca ggt 2160Met
Lys Met Phe Gln Glu Val Ser Ala Ala Asp Ala Phe Gly Pro Gly705 710
715 720act gag gga caa gta gaa aga ttt gaa aca gtt ggc atg gag gct
gcg 2208Thr Glu Gly Gln Val Glu Arg Phe Glu Thr Val Gly Met Glu Ala
Ala 725 730 735act gat gaa ggc atg cct aaa agt tac tta cca cag act
gta cgg caa 2256Thr Asp Glu Gly Met Pro Lys Ser Tyr Leu Pro Gln Thr
Val Arg Gln 740 745 750ggc ggc tac atg cct cag tga 2277Gly Gly Tyr
Met Pro Gln 7552758PRThuman 2Met Val Leu Ala Ser Ser Thr Thr Ser
Ile His Thr Met Leu Leu Leu1 5 10 15Leu Leu Met Leu Phe His Leu Gly
Leu Gln Ala Ser Ile Ser Ala Arg 20 25 30Gln Asp Tyr Lys Asp Asp Asp
Asp Lys Thr Arg Gln Ser Ser Ser Gly 35 40 45Ser Lys Leu Lys Asp Pro
Glu Leu Ser Leu Lys Gly Thr Gln His Ile 50 55 60Met Gln Ala Gly Gln
Thr Leu His Leu Gln Cys Arg Gly Glu Ala Ala65 70 75 80His Lys Trp
Ser Leu Pro Glu Met Val Ser Lys Glu Ser Glu Arg Leu 85 90 95Ser Ile
Thr Lys Ser Ala Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser 100 105
110Thr Leu Thr Leu Asn Thr Ala Gln Ala Asn His Thr Gly Phe Tyr Ser
115 120 125Cys Lys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr
Glu Ser 130 135 140Ala Ile Tyr Ile Phe Ile Ser Asp Thr Gly Arg Pro
Phe Val Glu Met145 150 155 160Tyr Ser Glu Ile Pro Glu Ile Ile His
Met Thr Glu Gly Arg Glu Leu 165 170 175Val Ile Pro Cys Arg Val Thr
Ser Pro Asn Ile Thr Val Thr Leu Lys 180 185 190Lys Phe Pro Leu Asp
Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp 195 200 205Asp Ser Arg
Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile 210 215 220Gly
Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr225 230
235 240Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Gln
Ile 245 250 255Ser Thr Pro Arg Pro Val Lys Leu Leu Arg Gly His Thr
Leu Val Leu 260 265 270Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr Arg
Val Gln Met Thr Trp 275 280 285Ser Tyr Pro Asp Glu Lys Asn Lys Arg
Ala Ser Val Arg Arg Arg Ile 290 295 300Asp Gln Ser Asn Ser His Ala
Asn Ile Phe Tyr Ser Val Leu Thr Ile305 310 315 320Asp Lys Met Gln
Asn Lys Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg 325 330 335Ser Gly
Pro Ser Phe Lys Ser Val Asn Thr Ser Val His Ile Tyr Asp 340 345
350Lys Ala Phe Ile Thr Val Lys His Arg Lys Gln Gln Val Leu Glu Thr
355 360 365Val Ala Gly Lys Arg Ser Tyr Arg Leu Ser Met Lys Val Lys
Ala Phe 370 375 380Pro Ser Pro Glu Val Val Trp Leu Lys Asp Gly Leu
Pro Ala Thr Glu385 390 395 400Lys Ser Ala Arg Tyr Leu Thr Arg Gly
Tyr Ser Leu Ile Ile Lys Asp 405 410 415Val Thr Glu Glu Asp Ala Gly
Asn Tyr Thr Ile Leu Leu Ser Ile Lys 420 425 430Gln Ser Asn Val Phe
Lys Asn Leu Thr Ala Thr Leu Ile Val Asn Val 435 440 445Lys Pro Gln
Ile Tyr Glu Lys Gly Glu Ile Glu Ala Ile Val Val Pro 450 455 460Val
Cys Leu Ala Phe Leu Leu Thr Thr Leu Leu Gly Val Leu Phe Cys465 470
475 480Phe Asn Lys Arg Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val
Pro 485 490 495Asp Pro Ser Lys Ser His Ile Ala Gln Trp Ser Pro His
Thr Pro Pro 500 505 510Arg His Asn Phe Asn Ser Lys Asp Gln Met Tyr
Ser Asp Gly Asn Phe 515 520 525Thr Asp Val Ser Val Val Glu Ile Glu
Ala Asn Asp Lys Lys Pro Phe 530 535 540Pro Glu Asp Leu Lys Leu Leu
Asp Leu Phe Lys Lys Glu Lys Ile Asn545 550 555 560Thr Glu Gly His
Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser 565 570 575Ser Arg
Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser Gln Asn 580 585
590Thr Ser Ser Thr Val Gln Tyr Ser Thr Val Val His Ser Gly Tyr Arg
595 600 605His Gln Val Pro Ser Val Gln Val Phe Ser Arg Ser Glu Ser
Thr Gln 610 615 620Pro Leu Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu
Gln Leu Val Asp625 630 635 640His Val Asp Gly Gly Asp Gly Ile Leu
Pro Arg Gln Gln Tyr Phe Lys 645 650 655Gln Asn Cys Ser Gln His Glu
Ser Ser Pro Asp Ile Ser His Phe Glu 660 665 670Arg Ser Lys Gln Val
Ser Ser Val Asn Glu Glu Asp Phe Val Arg Leu 675 680 685Lys Gln Gln
Ile Ser Asp His Ile Ser Gln Ser Cys Gly Ser Gly Gln 690 695 700Met
Lys Met Phe Gln Glu Val Ser Ala Ala Asp Ala Phe Gly Pro Gly705 710
715 720Thr Glu Gly Gln Val Glu Arg Phe Glu Thr Val Gly Met Glu Ala
Ala 725 730 735Thr Asp Glu Gly Met Pro Lys Ser Tyr Leu Pro Gln Thr
Val Arg Gln 740 745 750Gly Gly Tyr Met Pro Gln
75531842DNAhumanCDS(1)..(1842) 3atg gtt ctt gcc agc tct acc acc agc
atc cac acc atg ctg ctc ctg 48Met Val Leu Ala Ser Ser Thr Thr Ser
Ile His Thr Met Leu Leu Leu1 5 10 15ctc ctg atg ctc ttc cac ctg gga
ctc caa gct tca atc tcg gcg cgc 96Leu Leu Met Leu Phe His Leu Gly
Leu Gln Ala Ser Ile Ser Ala Arg 20 25 30cag gac tac aag gac gac gat
gac aag acg cgc cag tct agt tca ggt 144Gln Asp Tyr Lys Asp Asp Asp
Asp Lys Thr Arg Gln Ser Ser Ser Gly 35 40 45tca aaa tta aaa gat cct
gaa ctg agt tta aaa ggc acc cag cac atc 192Ser Lys Leu Lys Asp Pro
Glu Leu Ser Leu Lys Gly Thr Gln His Ile 50 55 60atg caa gca ggc cag
aca ctg cat ctc caa tgc agg ggg gaa gca gcc 240Met Gln Ala Gly Gln
Thr Leu His Leu Gln Cys Arg Gly Glu Ala Ala65 70 75 80cat aaa tgg
tct ttg cct gaa atg gtg agt aag gaa agc gaa agg ctg 288His Lys Trp
Ser Leu Pro Glu Met Val Ser Lys Glu Ser Glu Arg Leu 85 90 95agc ata
act aaa tct gcc tgt gga aga aat ggc aaa caa ttc tgc agt 336Ser Ile
Thr Lys Ser Ala Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser 100 105
110act tta acc ttg aac aca gct caa gca aac cac act ggc ttc tac agc
384Thr Leu Thr Leu Asn Thr Ala Gln Ala Asn His Thr Gly Phe Tyr Ser
115 120 125tgc aaa tat cta gct gta cct act tca aag aag aag gaa aca
gaa tct 432Cys Lys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr
Glu Ser 130 135 140gca atc tat ata ttt att agt gat aca ggt aga cct
ttc gta gag atg 480Ala Ile Tyr Ile Phe Ile Ser Asp Thr Gly Arg Pro
Phe Val Glu Met145 150 155 160tac agt gaa atc ccc gaa att ata cac
atg act gaa gga agg gag ctc 528Tyr Ser Glu Ile Pro Glu Ile Ile His
Met Thr Glu Gly Arg Glu Leu 165 170 175gtc att ccc tgc cgg gtt acg
tca cct aac atc act gtt act tta aaa 576Val Ile Pro Cys Arg Val Thr
Ser Pro Asn Ile Thr Val Thr Leu Lys 180 185 190aag ttt cca ctt gac
act ttg atc cct gat gga aaa cgc ata atc tgg 624Lys Phe Pro Leu Asp
Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp 195 200 205gac agt aga
aag ggc ttc atc ata tca aat gca acg tac aaa gaa ata 672Asp Ser Arg
Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile 210 215 220ggg
ctt ctg acc tgt gaa gca aca gtc aat ggg cat ttg tat aag aca 720Gly
Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr225 230
235 240aac acg aga gtt caa atg acc tgg agt tac cct gat gaa aaa aat
aag 768Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys Asn
Lys 245 250 255aga gct tcc gta agg cga cga att gac caa agc aat tcc
cat gcc aac 816Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser
His Ala Asn 260 265 270ata ttc tac agt gtt ctt act att gac aaa atg
cag aac aaa gac aaa 864Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met
Gln Asn Lys Asp Lys 275 280 285gga ctt tat act tgt cgt gta agg agt
gga cca tca ttc aaa tct gtt 912Gly Leu Tyr Thr Cys Arg Val Arg Ser
Gly Pro Ser Phe Lys Ser Val 290 295 300aac acc tca gtg cat
ata gga gaa att gaa gcc ata gtc gtg cct gtt 960Asn Thr Ser Val His
Ile Gly Glu Ile Glu Ala Ile Val Val Pro Val305 310 315 320tgc tta
gca ttc cta ttg aca act ctt ctg gga gtg ctg ttc tgc ttt 1008Cys Leu
Ala Phe Leu Leu Thr Thr Leu Leu Gly Val Leu Phe Cys Phe 325 330
335aat aag cga gac cta att aaa aaa cac atc tgg cct aat gtt cca gat
1056Asn Lys Arg Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val Pro Asp
340 345 350cct tca aag agt cat att gcc cag tgg tca cct cac act cct
cca agg 1104Pro Ser Lys Ser His Ile Ala Gln Trp Ser Pro His Thr Pro
Pro Arg 355 360 365cac aat ttt aat tca aaa gat caa atg tat tca gat
ggc aat ttc act 1152His Asn Phe Asn Ser Lys Asp Gln Met Tyr Ser Asp
Gly Asn Phe Thr 370 375 380gat gta agt gtt gtg gaa ata gaa gca aat
gac aaa aag cct ttt cca 1200Asp Val Ser Val Val Glu Ile Glu Ala Asn
Asp Lys Lys Pro Phe Pro385 390 395 400gaa gat ctg aaa tta ttg gac
ctg ttc aaa aag gaa aaa att aat act 1248Glu Asp Leu Lys Leu Leu Asp
Leu Phe Lys Lys Glu Lys Ile Asn Thr 405 410 415gaa gga cac agc agt
ggt att ggg ggg tct tca tgc atg tca tct tct 1296Glu Gly His Ser Ser
Gly Ile Gly Gly Ser Ser Cys Met Ser Ser Ser 420 425 430agg cca agc
att tct agc agt gat gaa aat gaa tct tca caa aac act 1344Arg Pro Ser
Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser Gln Asn Thr 435 440 445tcg
agc act gtc cag tat tct acc gtg gta cac agt ggc tac aga cac 1392Ser
Ser Thr Val Gln Tyr Ser Thr Val Val His Ser Gly Tyr Arg His 450 455
460caa gtt ccg tca gtc caa gtc ttc tca aga tcc gag tct acc cag ccc
1440Gln Val Pro Ser Val Gln Val Phe Ser Arg Ser Glu Ser Thr Gln
Pro465 470 475 480ttg tta gat tca gag gag cgg cca gaa gat cta caa
tta gta gat cat 1488Leu Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu Gln
Leu Val Asp His 485 490 495gta gat ggc ggt gat ggt att ttg ccc agg
caa cag tac ttc aaa cag 1536Val Asp Gly Gly Asp Gly Ile Leu Pro Arg
Gln Gln Tyr Phe Lys Gln 500 505 510aac tgc agt cag cat gaa tcc agt
cca gat att tca cat ttt gaa agg 1584Asn Cys Ser Gln His Glu Ser Ser
Pro Asp Ile Ser His Phe Glu Arg 515 520 525tca aag caa gtt tca tca
gtc aat gag gaa gat ttt gtt aga ctt aaa 1632Ser Lys Gln Val Ser Ser
Val Asn Glu Glu Asp Phe Val Arg Leu Lys 530 535 540cag cag att tca
gat cat att tca caa tcc tgt gga tct ggg caa atg 1680Gln Gln Ile Ser
Asp His Ile Ser Gln Ser Cys Gly Ser Gly Gln Met545 550 555 560aaa
atg ttt cag gaa gtt tct gca gca gat gct ttt ggt cca ggt act 1728Lys
Met Phe Gln Glu Val Ser Ala Ala Asp Ala Phe Gly Pro Gly Thr 565 570
575gag gga caa gta gaa aga ttt gaa aca gtt ggc atg gag gct gcg act
1776Glu Gly Gln Val Glu Arg Phe Glu Thr Val Gly Met Glu Ala Ala Thr
580 585 590gat gaa ggc atg cct aaa agt tac tta cca cag act gta cgg
caa ggc 1824Asp Glu Gly Met Pro Lys Ser Tyr Leu Pro Gln Thr Val Arg
Gln Gly 595 600 605ggc tac atg cct cag tga 1842Gly Tyr Met Pro Gln
6104613PRThuman 4Met Val Leu Ala Ser Ser Thr Thr Ser Ile His Thr
Met Leu Leu Leu1 5 10 15Leu Leu Met Leu Phe His Leu Gly Leu Gln Ala
Ser Ile Ser Ala Arg 20 25 30Gln Asp Tyr Lys Asp Asp Asp Asp Lys Thr
Arg Gln Ser Ser Ser Gly 35 40 45Ser Lys Leu Lys Asp Pro Glu Leu Ser
Leu Lys Gly Thr Gln His Ile 50 55 60Met Gln Ala Gly Gln Thr Leu His
Leu Gln Cys Arg Gly Glu Ala Ala65 70 75 80His Lys Trp Ser Leu Pro
Glu Met Val Ser Lys Glu Ser Glu Arg Leu 85 90 95Ser Ile Thr Lys Ser
Ala Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser 100 105 110Thr Leu Thr
Leu Asn Thr Ala Gln Ala Asn His Thr Gly Phe Tyr Ser 115 120 125Cys
Lys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser 130 135
140Ala Ile Tyr Ile Phe Ile Ser Asp Thr Gly Arg Pro Phe Val Glu
Met145 150 155 160Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu
Gly Arg Glu Leu 165 170 175Val Ile Pro Cys Arg Val Thr Ser Pro Asn
Ile Thr Val Thr Leu Lys 180 185 190Lys Phe Pro Leu Asp Thr Leu Ile
Pro Asp Gly Lys Arg Ile Ile Trp 195 200 205Asp Ser Arg Lys Gly Phe
Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile 210 215 220Gly Leu Leu Thr
Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr225 230 235 240Asn
Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys Asn Lys 245 250
255Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His Ala Asn
260 265 270Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys
Asp Lys 275 280 285Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser
Phe Lys Ser Val 290 295 300Asn Thr Ser Val His Ile Gly Glu Ile Glu
Ala Ile Val Val Pro Val305 310 315 320Cys Leu Ala Phe Leu Leu Thr
Thr Leu Leu Gly Val Leu Phe Cys Phe 325 330 335Asn Lys Arg Asp Leu
Ile Lys Lys His Ile Trp Pro Asn Val Pro Asp 340 345 350Pro Ser Lys
Ser His Ile Ala Gln Trp Ser Pro His Thr Pro Pro Arg 355 360 365His
Asn Phe Asn Ser Lys Asp Gln Met Tyr Ser Asp Gly Asn Phe Thr 370 375
380Asp Val Ser Val Val Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe
Pro385 390 395 400Glu Asp Leu Lys Leu Leu Asp Leu Phe Lys Lys Glu
Lys Ile Asn Thr 405 410 415Glu Gly His Ser Ser Gly Ile Gly Gly Ser
Ser Cys Met Ser Ser Ser 420 425 430Arg Pro Ser Ile Ser Ser Ser Asp
Glu Asn Glu Ser Ser Gln Asn Thr 435 440 445Ser Ser Thr Val Gln Tyr
Ser Thr Val Val His Ser Gly Tyr Arg His 450 455 460Gln Val Pro Ser
Val Gln Val Phe Ser Arg Ser Glu Ser Thr Gln Pro465 470 475 480Leu
Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu Gln Leu Val Asp His 485 490
495Val Asp Gly Gly Asp Gly Ile Leu Pro Arg Gln Gln Tyr Phe Lys Gln
500 505 510Asn Cys Ser Gln His Glu Ser Ser Pro Asp Ile Ser His Phe
Glu Arg 515 520 525Ser Lys Gln Val Ser Ser Val Asn Glu Glu Asp Phe
Val Arg Leu Lys 530 535 540Gln Gln Ile Ser Asp His Ile Ser Gln Ser
Cys Gly Ser Gly Gln Met545 550 555 560Lys Met Phe Gln Glu Val Ser
Ala Ala Asp Ala Phe Gly Pro Gly Thr 565 570 575Glu Gly Gln Val Glu
Arg Phe Glu Thr Val Gly Met Glu Ala Ala Thr 580 585 590Asp Glu Gly
Met Pro Lys Ser Tyr Leu Pro Gln Thr Val Arg Gln Gly 595 600 605Gly
Tyr Met Pro Gln 610534DNAartificial sequence5' oligo 5cacggatccg
cagcacacta tcaccagagg aaag 34634DNAartificial sequence3' oligo
6gcataagctt tcactttttt ttaggtctgc attc 34738DNAartificial
sequence5' oligo 7atatggcgcg cctagtcagc tactgggaca ccggggtc
38855DNAartificial sequence3' oligo (domains 1 to 4) 8caggcacgac
tatggcttca atttctccgg ccttttcgta aatctgggtt ttcac
55952DNAartificial sequence3' oligo (domains 1 to 3) 9cacgactatg
gcttcaattt ctcctatatg cactgaggtg ttaacagatt tg 521037DNAartificial
sequence5' oligo 10acgtacgcgt tcactgaggc atgtagccgc cttgccg
371138DNAartificial sequence3' oligo 11ggagaaattg aagccatagt
cgtgcctgtt tgcttagc 38
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