U.S. patent application number 11/838677 was filed with the patent office on 2008-01-24 for endotheliase-2 ligands.
This patent application is currently assigned to Dyax Corp., a Massachusetts corporation. Invention is credited to Edwin L. Madison, Andrew Nixon.
Application Number | 20080019962 11/838677 |
Document ID | / |
Family ID | 34221374 |
Filed Date | 2008-01-24 |
United States Patent
Application |
20080019962 |
Kind Code |
A1 |
Nixon; Andrew ; et
al. |
January 24, 2008 |
ENDOTHELIASE-2 LIGANDS
Abstract
Proteins that bind to ET2, such as immunoglobulins that inhibit
ET2 with high affinity and selectivity, are provided. The ET2
binding proteins can be used to treat a variety of disorders,
including angiogenesis-associated disorders.
Inventors: |
Nixon; Andrew; (Hanover,
MA) ; Madison; Edwin L.; (San Francisco, CA) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Dyax Corp., a Massachusetts
corporation
|
Family ID: |
34221374 |
Appl. No.: |
11/838677 |
Filed: |
August 14, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10916758 |
Aug 12, 2004 |
7273610 |
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11838677 |
Aug 14, 2007 |
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60495005 |
Aug 14, 2003 |
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60520164 |
Nov 14, 2003 |
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Current U.S.
Class: |
424/130.1 ;
435/375 |
Current CPC
Class: |
A61P 17/06 20180101;
A61P 29/00 20180101; C07K 16/40 20130101; A61P 3/10 20180101; C07K
2317/76 20130101; A61P 35/00 20180101; C07K 2317/55 20130101; A61P
9/00 20180101; A61K 2039/505 20130101; A61P 27/06 20180101; C07K
2317/56 20130101; A61P 43/00 20180101; A61P 27/02 20180101 |
Class at
Publication: |
424/130.1 ;
435/375 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 43/00 20060101 A61P043/00; C12N 5/00 20060101
C12N005/00 |
Claims
1. A method of modulating an activity of an endotheliase 2
(ET2)-expressing cell, the method comprising: contacting an
ET2-expressing cell with a protein, wherein the protein comprises a
heavy chain (HC) immunoglobulin variable domain sequence and a
light chain (LC) immunoglobulin variable domain sequence, wherein
(1) the first and second immunoglobulin variable domain sequences
form an antigen binding site that specifically binds to human ET2;
and (2) the protein has one or more of the following
characteristics: (a) the protein inhibits ET2 with an inhibition
constant (Ki) of less than 300 nM; (b) the HC immunoglobulin
variable domain sequence comprises one or more CDRs that are at
least 85% identical to a CDR of a HC variable domain of A10, G3,
A6, A7, C8, H9, G10-R2, F3-R2, C6-R2, A4-R3, C1-R3, A2, B5, D2, D5,
F8, H10, or C9; (c) the LC immunoglobulin variable domain sequence
comprises one or more CDRs that are at least 85% identical to a CDR
of a LC variable domain of A10, G3, A6, A7, C8, H9, G10-R2, F3-R2,
C6-R2, A4-R3, C1-R3, A2, B5, D2, D5, F8, H10, or C9; (d) the LC
immunoglobulin variable domain sequence is at least 85% identical
to a LC variable domain of A10, G3, A6, A7, C8, H9, G10-R2, F3-R2,
C6-R2, A4-R3, C1-R3, A2, B5, D2, D5, F8, H10, or C9; (e) the HC
immunoglobulin variable domain sequence is at least 85% identical
to a HC variable domain of A10, G3, A6, A7, C8, H9, G10-R2, F3-R2,
C6-R2, A4-R3, C1-R3, A2, B5, D2, D5, F8, H10, or C9; and (f) the
protein binds an epitope that overlaps with an epitope bound by
A10, G3, A6, A7, C8, H9, G10-R2, F3-R2, C6-R2, A4-R3, C1-R3, A2,
B5, D2, D5, F8, H10, or C9; thereby modulating the activity of the
ET2-expressing cell.
2. The method of claim 1, wherein the ET2-expressing cell is in a
human subject.
3. The method of claim 1, wherein the protein prevents binding of
the ET2-expressing cell to a substrate.
4. The method of claim 1, wherein the cell is a cancer cell.
5. The method of claim 1, wherein the protein binds to the ET2
active site.
6. The method of claim 1, wherein the protein inhibits ET2
enzymatic activity.
7. The method of claim 1, wherein the protein accumulates at sites
of angiogenesis in vivo.
8. The method of claim 1, wherein the protein inhibits proteolysis
of vessel basement membrane.
9. The method of claim 1, wherein the protein inhibits angiogenesis
in vitro or in vivo.
10. The method of claim 1, wherein the HC and LC variable domain
sequences are components of the same polypeptide chain.
11. The method of claim 1, wherein the HC and LC variable domain
sequences are components of different polypeptide chains.
12. The method of claim 1, wherein the protein is a full-length
antibody.
13. The method of claim 1, wherein the antibody is a human or
humanized antibody.
14. The method of claim 1, wherein the protein comprises a human
antibody framework region.
15. The method of claim 1, wherein the protein comprises an Fc
domain.
16. The method of claim 1, wherein the HC variable domain sequence
comprises SEQ ID NO:89 and the LC variable domain sequence
comprises SEQ ID NO:90.
17. The method of claim 1, wherein the protein reduces tumor growth
in a SCID mouse model.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/916,758, filed Aug. 12, 2004, which claims priority to U.S.
Application Ser. No. 60/495,005, filed on Aug. 14, 2003, and U.S.
Application Ser. No. 60/520,164, filed on Nov. 14, 2003, the
contents of all of which are hereby incorporated by reference in
their entireties.
BACKGROUND
[0002] Angiogenesis is the biological process of producing new
blood vessels by sprouting a new branch from an existing blood
vessel. While angiogenesis is essential for normal development and
growth, it rarely occurs in adulthood except under strictly
regulated circumstances (e.g., wound healing; see, for example,
Moses et al., Science, 248:1408-1410, 1990). Angiogenesis also
occurs in a number of diseases, such as cancer, in which new
vessels are formed to support the growth and proliferation of both
primary and metastatic tumors.
[0003] Blood vessels contain endothelial cells surrounded by a
basement membrane. One of the first steps in angiogenesis is the
degradation of the basement membrane by proteolytic enzymes
produced by endothelial cells to form a breach in the membrane
through which endothelial cells can migrate and proliferate to form
a new vessel sprout. This step can be initiated as follows. First,
components of the plasminogen activator (PA)-plasmin system
stimulate a protease cascade that results in high concentrations of
plasmin and active matrix metalloproteinases (MMPs) at the site of
angiogenesis. This increased proteolytic activity leads to
degradation of the extracellular matrix (ECM) and invasion of the
vessel basal lamina. The release of ECM degradation products leads
to chemotaxis of endothelial cells.
[0004] Numerous pathological conditions are associated with
unwanted angiogenesis. For example, tumors can induce angiogenesis
in order to grow beyond minimal size and to metastasize (Hanahan
and Folkman Cell 1996, 86:353-64). Tumor development is associated
with increased release of angiogenesis factors, most prominently of
vascular endothelial growth factor (VEGF) (Brown L F et al., Exs
1997, 79:233-69). Other disorders characterized by unwanted
angiogenesis include, for example, tissue inflammation, arthritis,
diabetic retinopathy, and macular degeneration by
neovascularization of retina (see, e.g., Folkman et al., Science,
235:442-447, 1987).
[0005] The endotheliases are a class of membrane proteases that are
expressed on cells, particularly endothelial cells.
SUMMARY
[0006] In one aspect, the invention features a protein ligand that
binds to Endotheliase-2 (ET2) (also referred to herein as an ET2
ligand or ET2-binding ligand). Typically, the ligand is not
naturally occurring. In one embodiment, the protein ligand includes
a heavy chain variable domain sequence and a light chain variable
domain sequence. For example, the ligand is an antibody or an
antigen-binding fragment of a full length antibody (also referred
to herein as an anti-ET2 antibody).
[0007] In one embodiment, the ET2-ligand binds to human ET2 with
high affinity and specificity, and thus can be used as diagnostic,
prophylactic, or therapeutic agents in vivo and in vitro. For
example, the ligand specifically binds to ET2. As used herein,
"specific binding" refers to ability (1) to bind to ET2, e.g.,
human ET2, with an affinity (K.sub.d) of better than (i.e.,
numerically smaller than) 1.times.10.sup.-7 M, and (2) to
preferentially bind to ET2, e.g., human ET2, with an affinity that
is at least two-fold, 10-fold, 50-fold, 100-fold, or better
(smaller K.sub.d) than its affinity for binding to a non-specific
antigen (e.g., BSA, casein) other than ET2.
[0008] In one embodiment, the ligand modulates an activity of ET2,
e.g., the proteolytic activity of ET2. In one embodiment, the
ligand inhibits ET2. For example, the ligand can have a K.sub.i of
better than (i.e., numerically less than) 5 nM, 500 pM, 200 pM, 150
pM, 100 pM, 92 pM, or 75 pM, e.g., between 50 nM and 1 pM, or 200
pM and 5 pM. In one embodiment, the ligand specifically inhibits
ET2, e.g., relative to another protease (e.g., a protease whose
protease domain is between 30-90% identical to the ET2 protease
domain, or between 30-60% identical to the ET2 protease domain).
For example, the ligand does not inhibit other proteases, e.g.,
non-ET2 proteases such as trypsinogen-IV, membrane-type serine
proteases-1, -6, -7, or Endotheliase-1 (ET1), e.g., the ligand
inhibits another protease (e.g., such other proteases) with an
inhibition constant at least 2-, 5-, 10-, 50-, or 100-fold worse
(e.g., numerically greater) than its inhibition constant for ET2
(i.e., the ligand does not inhibit the other proteases as well as
they inhibit ET2).
[0009] In one embodiment, the ligand inhibits angiogenesis, e.g.,
inhibit proteolysis of one or more ECM components or vessel
basement membrane components, in vitro or in vivo. In one
embodiment, the ligands have a statistically significant effect
(e.g., on an angiogenic process) in one or more of the following
assays: a cornea neovascularization assay; a chick embryo
chorioallantoic membrane model assay; an assay using SCID mice
injected with tumors (e.g., tumors arising from injection of DU145
or LnCaP cell lines, as described in Jankun et al., Canc. Res., 57:
559-563 (1997)); or an assay in which mice are injected with bFGF,
to stimulate angiogenesis (e.g., as described by Min et al., Canc.
Res., 56: 2428-2433 (1996). Exemplary effects in these assays
include an at least 1.5, 2, 5, 10, or 20-fold improvement relative
to a negative control (e.g., no antibody).
[0010] In one embodiment, the ligand agonizes ET2 (e.g., activates
or increases an activity of ET2, e.g., a proteolytic activity),
e.g., increases activity at least 0.5, 1.5, 2, 5, 10, or 20
fold.
[0011] In one embodiment, the ligand contacts the active site of
ET2, e.g., the active site cleft of ET2 or to an amino acid
residues that is within 30, 20, or 10 Angstroms of a residue in the
catalytic triad of ET2, e.g., histidine 361 of SEQ ID NO:94 or to
serine 506 of SEQ ID NO:94, or to an amino acid residue within the
sequence LTAAHC (amino acids 357-362 of SEQ ID NO:94) or to an
amino acid within the sequence DSCQGDSGGPLV (amino acids 500-511 of
SEQ ID NO:94).
[0012] The protein ligand typically interacts with, e.g., bind to
ET2, preferably human ET2, with high affinity and specificity. For
example, the protein ligand binds to human ET2 with an affinity
constant (K.sub.d) of better than (i.e., numerically smaller than)
10.sup.-7 M, preferably, better than 10.sup.-8 M, 10.sup.-9 M, or
10.sup.-10 M. Preferably, the protein ligand interacts with, e.g.,
binds to, the extracellular domain of ET2, and most preferably, the
extracellular domain of human ET2 (e.g., amino acids 161-562 of
ET2-S or 161-688 of ET2-L). In one embodiment, the ET2-ligand binds
all or part of the epitope of an antibody described herein, e.g.,
A10, G3, A6, A7, C8, H9, G1'-R2, F3-R2, C6-R2, A4-R3, C1-R3, A2,
B5, D2, D5, F8, H10, or C9. The ET2-ligand can inhibit, e.g.,
competitively inhibit, the binding of an antibody described herein,
e.g., A10, G3, A6, A7, C8, H9, G10-R2, F3-R2, C6-R2, A4-R3, C1-R3,
A2, B5, D2, D5, F8, H10, or C9, to human ET2. An ET2-ligand may
bind to an epitope, e.g., a conformational or a linear epitope,
which epitope when bound prevents binding of an antibody described
herein, A10, G3, A6, A7, C8, H9, G10-R2, F3-R2, C6-R2, A4-R3,
C1-R3, A2, B5, D2, D5, F8, H10, or C9. The epitope can be in close
proximity spatially (e.g., within 3, 5, or 10 Angstroms of) or
functionally-associated, e.g., an overlapping or adjacent epitope
in linear sequence or conformationally similar to the one
recognized by the A10, G3, A6, A7, C8, H9, G10-R2, F3-R2, C6-R2,
A4-R3, C1-R3, A2, B5, D2, D5, F8, H10, or C9 antibody. In one
embodiment, the ET2-ligand binds to an epitope located wholly or
partially within the region of the serine protease domain of ET2,
e.g., between amino acids 321-562 for ET2-S and 321-688 for
ET2-L.
[0013] Accordingly, the invention provides anti-ET2 antibodies,
antibody fragments, and pharmaceutical compositions thereof, as
well as nucleic acids, recombinant expression vectors and host
cells for making such antibodies and fragments. An exemplary
pharmaceutical composition includes the ligand and a
pharmaceutically acceptable carrier. Methods of using the
antibodies of the invention to detect ET2, to kill, or to inhibit
growth of an ET2-expressing cell, e.g., a ET2-expressing cell,
either in vitro or in vivo, are also encompassed by the
invention.
[0014] Human ET2 is expressed at least on endothelial cells. In one
embodiment, an ET2 ligand binds to the cell surface of these cells,
and in particular, to the cell surface of living cells, e.g.,
living endothelial cells. In some cases, the protein ligand can be
internalized within the cell, e.g., to permit the intracellular
delivery of an agent conjugated to the antibody, e.g., a cytotoxic
or a labeling agent. In some embodiments, the protein ligands of
the invention can be used to target living normal, benign
hyperplastic, and cancerous cells that express ET2.
[0015] In one embodiment, an ET ligand binds to ET and alters its
conformation and/or catalytic activity, e.g., it enhances catalytic
activity or interaction with a substrate.
[0016] As used herein, the term "antibody" refers to a protein that
includes at least one immunoglobulin variable domain or
immunoglobulin variable domain sequence. For example, an antibody
can include a heavy (H) chain variable region (abbreviated herein
as VH), and a light (L) chain variable region (abbreviated herein
as VL). In another example, an antibody includes two heavy (H)
chain variable regions and two light (L) chain variable regions.
The term "antibody" encompasses antigen-binding fragments of
antibodies (e.g., single chain antibodies, Fab fragments,
F(ab').sub.2, a Fd fragment, a Fv fragments, and dAb fragments) as
well as complete antibodies.
[0017] The VH and VL regions can be further subdivided into regions
of hypervariability, termed "complementarity determining regions"
("CDR"), interspersed with regions that are more conserved, termed
"framework regions" (FR). The extent of the framework region and
CDR's has been precisely defined (see, Kabat, E. A., et al (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).
Kabat definitions are used herein. Each VH and VL is typically
composed of three CDR's and four FR's, arranged from amino-terminus
to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4.
[0018] An "immunoglobulin domain" refers to a domain from the
variable or constant domain of immunoglobulin molecules.
Immunoglobulin domains typically contain two .beta.-sheets formed
of about seven .beta.-strands, and a conserved disulphide bond
(see, e.g., A. F. Williams and A. N. Barclay 1988 Ann. Rev Immunol.
6:381-405). The canonical structures of hypervariable loops of an
immunoglobulin variable can be inferred from its sequence, as
described in Chothia et al. (1992) J. Mol. Biol. 227:799-817;
Tomlinson et al. (1992) J. Mol. Biol. 227:776-798); and Tomlinson
et al. (1995) EMBO J. 14(18):4628-38.
[0019] As used herein, an "immunoglobulin variable domain sequence"
refers to an amino acid sequence which can form the structure of an
immunoglobulin variable domain. For example, the sequence may
include all or part of the amino acid sequence of a
naturally-occurring variable domain. For example, the sequence may
omit one, two or more N- or C-terminal amino acids, internal amino
acids, may include one or more insertions or additional terminal
amino acids, or may include other alterations. In one embodiment, a
polypeptide that includes immunoglobulin variable domain sequence
can associate with another immunoglobulin variable domain sequence
to form a target binding structure (or "antigen binding site"),
e.g., a structure that interacts with ET2, e.g., binds to or
inhibits ET2.
[0020] The VH or VL chain of the antibody can further include all
or part of a heavy or light chain constant region, to thereby form
a heavy or light immunoglobulin chain, respectively. In one
embodiment, the antibody is a tetramer of two heavy immunoglobulin
chains and two light immunoglobulin chains, wherein the heavy and
light immunoglobulin chains are inter-connected by, e.g., disulfide
bonds. The heavy chain constant region includes three domains, CH1,
CH2 and CH3. The light chain constant region includes a CL domain.
The variable region of the heavy and light chains contains a
binding domain that interacts with an antigen. The constant regions
of the antibodies typically mediate the binding of the antibody to
host tissues or factors, including various cells of the immune
system (e.g., effector cells) and the first component (Clq) of the
classical complement system. The term "antibody" includes intact
immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as
subtypes thereof). The light chains of the immunoglobulin may be of
types kappa or lambda. In one embodiment, the antibody is
glycosylated. An antibody can be functional for antibody-dependent
cytotoxicity and/or complement-mediated cytotoxicity.
[0021] In one embodiment, the HC or LC of an antibody includes
sequences that correspond (e.g., are identical to or have a
threshold degree of similarity) to an amino acid sequence encoded
by a human germline sequence, e.g., the framework regions and/or in
the CDRs. For example, the antibody can include sequences from the
human DP47 antibody. In one embodiment, one or more codons for the
antibody are altered relative to the germline nucleic acid
sequence, but are chosen to encode the same amino acid sequence.
Codons can be selected, e.g., to optimize expression in a
particular system, create restriction enzyme sites, create a silent
fingerprint, etc. CDR sequences can also be substantially human,
e.g., are at least 70, 80, 85, 87, 90, 91, 92, 93, 94, or 95%
identical to a completely human CDR (e.g., a CDR in a human
germline sequence or in a mature human antibody). Accordingly,
synthetic nucleic acid sequences can be used to encode completely
human or substantially human CDRs.
[0022] In one embodiment, CDR2 of the antibody HC includes at least
11, 12, 13, 14, or 15 amino acid positions that are identical to
the amino acids found in CDR2 of DP47.
[0023] As used herein, the term "immunoglobulin" refers to a
protein consisting of one or more polypeptides or regions thereof
substantially encoded by immunoglobulin genes (e.g., natural or
synthetic). Exemplary natural human immunoglobulin genes include
the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3,
IgG4), delta, epsilon and mu constant region genes, as well as the
myriad immunoglobulin variable region genes. Full-length
immunoglobulin "light chains" (about 25 Kd or 214 amino acids) can
be encoded by a variable region gene at the NH.sub.2-terminus
(about 110 amino acids) and a kappa or lambda constant region gene
at the COOH-terminus. Full-length immunoglobulin "heavy chains"
(about 50 Kd or 446 amino acids), can be similarly encoded by a
variable region gene (about 116 amino acids) and one of the other
aforementioned constant region genes, e.g., gamma (encoding about
330 amino acids).
[0024] The term "antigen-binding fragment" of an antibody (or
simply "antibody portion," or "fragment"), as used herein, refers
to one or more fragments of a full-length antibody that retain the
ability to specifically bind to ET2 (e.g., human ET2). Examples of
binding fragments encompassed within the term "antigen-binding
fragment" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL and CH1 domains; (ii) a
F(ab').sub.2 fragment, a bivalent fragment including two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the VH and CH1 domains; (iv) a Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which consists of a VH domain; and (vi) an isolated
complementarity determining region (CDR). Furthermore, although the
two domains of the Fv fragment, VL and VH, are coded for by
separate genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the VL and VH regions pair to form monovalent
molecules (known as single chain Fv (scFv); see e.g., Bird et al.
(1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883). Monomers and dimers of such single
chain antibodies are also intended to be encompassed within the
term "antigen-binding fragment" of an antibody. These antibody
fragments are obtained using conventional techniques known to those
with skill in the art, and the fragments are screened for activity
in the same manner as are intact antibodies.
[0025] The antibody is preferably monospecific, e.g., a monoclonal
antibody, or antigen-binding fragment thereof. The term
"monospecific antibody" refers to an antibody that displays a
single binding specificity and affinity for a particular target,
e.g., epitope. This term includes a "monoclonal antibody" or
"monoclonal antibody composition," which as used herein refer to a
preparation of antibodies or fragments thereof of a single
molecular composition.
[0026] The anti-ET2 antibodies can be full-length (e.g., an IgG
(e.g., an IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2),
IgD, and IgE, but preferably an IgG) or can include only an
antigen-binding fragment (e.g., a Fab, F(ab').sub.2 or scFv
fragment). The antibody, or antigen-binding fragment thereof, can
include two heavy chain immunoglobulins and two light chain
immunoglobulins, or can be a single chain antibody. The antibodies
can, optionally, include a constant region chosen from a kappa,
lambda, alpha, gamma, delta, epsilon or a mu constant region gene.
A preferred anti-ET2 antibody includes a heavy and light chain
constant region substantially from a human antibody, e.g., a human
IgG1 constant region or a portion thereof. As used herein,
"isotype" refers to the antibody class (e.g., IgM or IgG1) that is
encoded by heavy chain constant region genes.
[0027] In one embodiment, the antibody (or fragment thereof) is a
recombinant or modified anti-ET2 antibody, e.g., a chimeric, a
humanized, a deimmunized, or an in vitro generated antibody. The
term "recombinant" or "modified" human antibody, as used herein, is
intended to include all antibodies that are prepared, expressed,
created or isolated by recombinant means, such as antibodies
expressed using a recombinant expression vector transfected into a
host cell, antibodies isolated from a recombinant, combinatorial
antibody library, antibodies isolated from an animal (e.g., a
mouse) that is transgenic for human immunoglobulin genes or
antibodies prepared, expressed, created or isolated by any other
means that involves splicing of human immunoglobulin gene sequences
to other DNA sequences. Such recombinant antibodies include
humanized, CDR grafted, chimeric, deimmunized, in vitro generated
antibodies, and may optionally include constant regions derived
from human germline immunoglobulin sequences. In one embodiment,
the antibody does not elicit an anti-globulin response in a
human.
[0028] In other embodiments, the anti-ET2 antibody is a human
antibody.
[0029] Also within the scope of the invention are antibodies, or
antigen-binding fragments thereof, which bind overlapping epitopes
of, or competitively inhibit, the binding of the anti-ET2
antibodies disclosed herein to ET2, e.g., antibodies which bind
overlapping epitopes of, or competitively inhibit, the binding of
monospecific antibodies A10, G3, A6, A7, C8, H9, G10-R2, F3-R2,
C6-R2, A4-R3, C1-R3, A2, B5, D2, D5, F8, H10, or C9 to ET2. Any
combination of anti-ET2 antibodies is within the scope of the
invention, e.g., two or more antibodies that bind to different
regions of ET2, e.g., antibodies that bind to two different
epitopes on the serine protease domain of ET2, e.g., a bispecific
antibody.
[0030] In one embodiment, the anti-ET2 antibody, or antigen-binding
fragment thereof, includes at least one light or heavy chain
variable domain sequence (e.g., at least one light chain
immunoglobulin and at least one heavy chain immunoglobulin).
Preferably, each immunoglobulin includes a light or a heavy chain
variable domain sequence having at least one, two and, preferably,
three complementarity determining regions (CDR's) substantially
identical to a CDR from a light or heavy chain variable domain
sequence of an antibody that interacts with ET2, e.g., an antibody
described herein, e.g., A10, G3, A6, A7, C8, H9, G10-R2, F3-R2,
C6-R2, A4-R3, C1-R3, A2, B5, D2, D5, F8, H10, or C9. The amino acid
and nucleic acid sequences of exemplary light chain and heavy chain
variable regions are shown in Table 1. In some embodiments, the
residue listed as a "q" in SEQ ID NO:10 and SEQ ID NO:89 of Table 1
and 2 is a lysine. TABLE-US-00001 TABLE 1 Exemplary Sequences
Antibody Sequence Identifier C9 VLC
CAGAGCGTCTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTC SEQ ID NO:3
Nucleic GATCACCATCTCCTGCACTGGAACCAGTAGTGACGTTGGTCATTATAATT Acid
ATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAAGTCATGATT Sequence
TATGATGTCAGTAGTCGGCCCTCCGGGGTTTCTGATCGCTTCTCTGGGTC
CAAGTCTGGCAACACGGCCTCCCTGGCCATCTCTGGGCTCCAGGCTGAGG
ACGAGGCTGATTATTACTGCAGTTCGTATACAAGCGGTGACACTCTTTAT
GTCTTCGGAACTGGGACCAAGGTCACCGTCCTAGGTCAGCCCAAGGCCAA
CCCCACTGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTCCAAGCCAACA
AGGCCACACTAGTGTGTCTGATCAGTGACTTCTACCCGGGAGCTGTGACA
GTGGCCTGGAAGGCAGATGGCAGCCCCGTCAAGGCGGGAGTGGAGACCAC
CAAACCCTCCAAACAGAGCAACAACAAGTACGCGGCCAGCAGCTACCTGA
GCCTGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTC
ACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTGCAGAATGCTC TTAATAA C9 VLC
QSVLTQPASVSGSPGQSITISCTGTSSDVGHYNYVSWYQQHPGKAPKVMI SEQ ID NO:4
Amino Acid YDVSSRPSGVSDRFSGSKSGNTASLAISGLQAEDEADYYCSSYTSGDTLY
Sequence VFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVT
VAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV
THEGSTVEKTVAPAECS C9 VHC
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC SEQ ID NO:5
Nucleic TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCCTA Acid
TGTTTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTAT Sequence
ATCTCTTCTTCTGGTGGCTTTACTGGTTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGGGGGA
CCGCGGGGTAACAAGTACTACTTTGACTACTGGGGCCAGGGAACCCTGGT
CACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGCTAGC C9 VHC
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYPMFWVRQAPGKGLEWVSY SEQ ID NO:6
Amino Acid ISSSGGFTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGG
Sequence PRGNKYYFDYWGQGTLVTVSSASTKGPSVFPL B5 VLC
AGCTACGAATTGACTCAGCCACCCTCAGTGTCCGTGTCCCTAGGACAGGC SEQ ID NO:7
Nucleic AGCCAACATCTCCTGCTCTGGAGATAGATTGGGGGATAAATATACTTCCT Acid
GGTATCAACAACAGTCAGGACAGTCCCCTGTCCTGGTCATCTATCAAGAT Sequence
AAGAAGCGACCCTCAGGGATCCCCGAGCGATTCTCTGGCTCCTCCTCTGG
GAACACAGCCACTCTGACCATCAGCGGGGCCCAGGCCATAGATGAGGCTG
CCTATTACTGTCAGGCGTGGGCCACCAATGTGGTTTTCGGCGCTGGGACC
AAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTT
CCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTC
TCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGAT
AGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAG
CAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGT
GGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACC
GTGGAGAAGACAGTGGCCCCTACAGGATGTTCATAATAA B5 VLC
SYELTQPPSVSVSLGQAANISCSGDRLGDKYTSWYQQQSGQSPVLVIYQD SEQ ID NO:8
Amino Acid KKRPSGIPERFSGSSSGNTATLTISGAQAIDEAAYYCQAWATNVVFGAGT
Sequence KLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD
SSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGST VEKTVAPTGCS
B5-H10-A2- GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC SEQ
ID NO:9 D2 VHC TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTA
Nucleic TGTATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCT Acid
ATCTCTCCTTCTGGTGGCGATACTCGTTATGCTGACTCCGTTAAAGGTCG Sequence
CTTCACTATCTCTAGAGACAACTCTTAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGGGGGA
CCGCGGGGTAACAAGTACTACTTTGACTACTGGGGCCAGGGAACCCTGGT
CACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGCTAGC B5-H10-A2-
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYRMYWVRQAPGKGLEWVSS SEQ ID NO:10 D2
VHC ISPSGGDTRYADSVKGRFTISRDNSqNTLYLQMNSLRAEDTAVYYCARGG Amino Acid
PRGNKYYFDYWGQGTLVTVSSASTKGPSVFPL Sequence F8 VLC
GACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA SEQ ID NO:11
Nucleic AAGAGTCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAGCAGCGACT Acid
TAGCCTGGTACCAGCAGAAACCTGGTCAGGCTCCCAGGCTCCTCATTTCT Sequence
GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG
GTCTGGGACAGACTTCACCCTCACCATCAGCAGACTGGAACCTGAAGATT
TTGCAGTGTATTACTGTCAGCAGTATGGTAACTCACCTGGGACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTT
CATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTG
TGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAG
GTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCA
GGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCA
AAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG
GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAATA A F8 VLC
DIQMTQSPGTLSLSPGERVTLSCRASQSVTSSDLAWYQQKPGQAPRLLIS SEQ ID NO:12
Amino Acid GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGNSPGTFG
Sequence QGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC
F8 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC SEQ ID
NO:13 Nucleic TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCATA
Acid TGTGGTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGT Sequence
ATCTCTTCTTCTCGTGGCATTACTAAGTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGGGGGA
CCGCGGGGTAACAAGTACTACTTTGACTACTGGGGCCAGGGAACCCTGGT
CACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGCTAGC F8 VHC
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYHMWWVRQAPGKGLEWVSG SEQ ID NO:14
Amino Acid ISSSRGITKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGG
Sequence PRGNKYYFDYWGQGTLVTVSSASTKGPSVFPL H10 VLC
GACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA SEQ ID NO:15
Nucleic AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACT Acid
TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT Aequence
GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG
GTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT
TTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCAACGTGGACGTTCGGC
CAAGGGACCAAAGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTT
CATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTG
TGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAG
GTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCA
GGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCA
AAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG
GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAATA A H10 VLC
DIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY SEQ ID NO:16
Amino Acid GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSTWTFG
Sequence QGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYFKHKVYACEVTHQ GLSSPVTKSFNRGEC
A2 VLC GACATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATTTGTAGGAGA SEQ ID
NO:17 Nucleic CAGGGTCACCATCACTTGCCGGGCCAGTCAGGACATTAGAAGTGATTTAG
Acid CCTGGTATCAGCAAACACCAGGGAAAGCCCCAAAGCTCCTGATCTATGCT Sequence
GCATCCACTTTGAAAGATGGGGCCCCATCAAGATTCAGCGGCAGTGGATC
TGGGACAGAATTTACTCTCACAATCAGCAGCCTGCACCCTGAAGATCTTG
CGACTTATTACTGTCAACACCTTAATGGTCACCCTGCTTTCGGCCCTGGG
ACCAAAGTGAATATCCAAAGAACTGTGGCTGCACCATCTGTCTTCATCTT
CCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCC
TGCTGAATAACTTCTATCCCAGAGAAGCCAAAGTACAGTGGAAGGTGGAT
AACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAAGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAG
ACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG
AGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAATAA A2 VLC
DIQMTQSPSFLSAFVGDRVTITCRASQDIRSDLAWYQQTPGKAPKLLIYA SEQ ID NO:18
Amino Acid ASTLKDGAPSRFSGSGSGTEFTLTISSLHPEDLATYYCQHLNGHPAFGPG
Sequence TKVNIQRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC D2
VLC GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCTTCTGTTGGAGA SEQ ID NO:19
Nucleic CAGAGTCACCATCACTTGCCGGGCAAGCCAGACCATTGACAATTATTTGA Acid
ATTGGTATCAGCAGAAACCAGGGAAAGCCCCCAAACTCGTGGTCTATGCT Sequence
GCATCCACTTTGCAAACTAGGGTCCCATCAAGGTTCAGTGGCAGTGGGTC
TGGGACAGACTTCACTCTCACCATCGACAGTCTGAAACCTGAAGATTTTG
CAACTTACTTCTGTCAACAGGGTTTCAGTAATCCTTGGACGTTCGGCCAA
GGGACCACGGTGGCAATGATACGAACTGTGGCTGCACCATCTGTCTTCAT
CTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGT
GCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTG
GATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGA
CAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG
CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC
CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAATAA D2 VLC
DIQMTQSPSSLSASVGDRVTITCRASQTIDNYLNWYQQKPGKAPKLVVYA SEQ ID NO:20
Amino Acid ASTLQTRVPSRFSGSGSGTDFTLTIDSLKPEDFATYFCQQGFSNPWTFGQ
Sequence GTTVAMIRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC
D5 VLC GACATCCAGATGACCCAGTCTCCAGGCACCCTGTCATTGTCTCCAGGGGA SEQ ID
NO:21 Nucleic AAGAGGCACCCTCTCCTGCAGGGCCAGTCAGTTTGTTAGTTACAGCTACT
Acid TAGCCTGGTACCAGCAGAAGCCTGGCCAGGCTCCCCGGCTCCTCATCTAT Sequence
GGCGCATCCAGCAGGGCCAAAGGCATCCCAGACAGGTTCAGTGGCAGTGG
GTCTGGGACAGACTTCACTCTCACCATCACCAGACTGGAGCCTGAAGACT
TTGCAGTTTATTACTGTCAGCAGTATGTTCCCTCAGTTCCGTGGACGTTC
GGCCAAGGGACCAAGGTGGAAGTCAAACGAACTGTGGCTGCACCATCTGT
CTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTG
TTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGG
AAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGA
GCAGGACGGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGA
GCAAAGCAGACTACGAGGAACACAAAGTCTACGCCTGCGAAGTCACCCAT
CAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA ATAA D5 VLC
DIQMTQSPGTLSLSPGERGTLSCRASQFVSYSYLAWYQQKPGQAPRLLIY SEQ ID NO:22
Amino Acid GASSRAKGIPDRFSGSGSGTDFTLTITRLEPEDFAVYYCQQYVPSVPWTF
Sequence GQGTKVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDGKDSTYSLSSTLTLSKADYEEHKVYACEVTH QGLSSPVTKSFNRGEC
D5 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC SEQ ID
NO:23 Nucleic TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGATA
Acid TGCATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCT Sequence
ATCTCTTCTTCTGGTGGCTATACTGCTTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGGCGCC
CGAGGTACCAGCCAAGGCTACTGGGGCCAGGGAACCCTGGTCACCGTCTC
AAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGCTAGC D5 VHC
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSS SEQ ID NO:24
Amino Acid ISSSGGYTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGA
Sequence RGTSQGYWGQGTLVTVSSASTKGPSVFPL
[0031] In one embodiment, the antibody (or fragment thereof)
includes at least one, two and preferably three CDR's from the
light or heavy chain variable region of an antibody disclosed
herein, e.g., A10, G3, A6, A7, C8, H9, G10-R2, F3-R2, C6-R2, A4-R3,
C1-R3, A2, B5, D2, D5, F8, H10, or C9, or a sequence substantially
identical thereto, e.g., 80%, 85%, 90%, 95%, 99%, or more,
identical. In other embodiments, the antibody (or fragment thereof)
can have at least one, two, and preferably three CDR's from the
light or heavy chain variable region of an antibody disclosed
herein, e.g., A10, G3, A6, A7, C8, H9, G10-R2, F3-R2, C6-R2, A4-R3,
C1-R3, A2, B5, D2, D5, F8, H10, or C9. In one preferred embodiment,
the antibody, or antigen-binding fragment thereof, includes all six
CDR's from the human anti-ET2 antibody, e.g., A10, G3, A6, A7, C8,
H9, G10-R2, F3-R2, C6-R2, A4-R3, C1-R3, A2, B5, D2, D5, F8, H10, or
C9.
[0032] The CDR and framework sequences of some exemplary antibodies
are shown in Table 2 and Table 3. TABLE-US-00002 TABLE 2 HC CDRs
Name H-CDR1 H-CDR2 H-CDR3 A10 RYRMW (residues 31 YISSSGGFTNYADSVKG
(residues NARRALPSMDV (residues 99 to 109 of to 35 of 50 to 66 of
SEQ ID NO:25) SEQ ID NO:25) SEQ ID NO:25) G3 RYGMS (residues 31
VIYSSGGITRYADSVKG (residues RAPRGEVAFDI (residues 99 to 109 of to
35 of 50 to 66 of SEQ ID NO:29) SEQ ID NO:29) SEQ ID NO:29) A6
RYKMW (residues 31 YISPSGGYTGYADSVKG (residues NARRAFPSMDV
(residues 99 to 109 of to 35 of SEQ ID 50 to 66 of SEQ ID NO:33)
SEQ ID NO:33) SEQ ID NO:33) A7 RYRMS (residues 31 SISSSGGITTYADSVKG
(residues NARRAFPSMDV (residues 99 to 109 of to 35 of 50 to 66 of
SEQ ID NO:37) SEQ ID NO:37) SEQ ID NO:37) C8 RYTMS (residues 31
YIVPSGGMTKYADSVKG (residues RAPRGEVAFDI (residues 99 to 109 of to
35 of 50 to 66 of SEQ ID NO:41) SEQ ID NO:41) SEQ ID NO:41) H9
RYSMH (residues 31 SIGPSGGKTKYADSVKG (residues PFRGSYYYFDY
(residues 99 to 109 of to 35 of 50 to 66 of SEQ ID NO:45) SEQ ID
NO:45) SEQ ID NO:45) G10-R2 RYKMW (residues 31 YISPSGGYTGYADSVKG
(residues NARRAFPSMDV (residues 99 to 109 of to 35 of 50 to 66 of
SEQ ID NO:49) SEQ ID NO:49) SEQ ID NO:49) F3-R2 RYRMH (residues 31
GISSSGGDTNYADSVKG (residues NARRAFPSMDV (residues 99 to 109 of to
35 of 50 to 66 of SEQ ID NO:53) SEQ ID NO:53) SEQ ID NO:53) C6-R2
RYSMH (residues 31 RIVPSGGTTFYADSVKG (residues NARRAFPSMDV
(residues 99 to 109 of to 35 of 50 to 66 of SEQ ID NO:57) SEQ ID
NO:57) SEQ ID NO:57) A4-R3 RYNMY (residues 31 GIRPSGGSTQYADSVKG
(residues NARRAFPSMDV (residues 99 to 109 of to 35 of 50 to 66 of
SEQ ID NO:61) SEQ ID NO:61) SEQ ID NO:61) C1-R3 RYSMH (residues 31
GIRPSGGSTKYADSVKG (residues NARRAFPSMDV (residues 99 to 109 of to
35 of 50 to 66 of SEQ ID NO:65) SEQ ID NO:65) SEQ ID NO:65) A2
RYRMY (residues 31 SISPSGGDTRYADSVKG (residues GGPRGNKYYFDY
(residues 98 to 109 of to 35 of 50 to 66 of SEQ ID NO:69) SEQ ID
NO:69) SEQ ID NO:69) B5 RYRMY (residues 31 SISPSGGDTRYADSVKG
(residues GGPRGNKYYFDY (residues 98 to 109 of to 35 of 50 to 66 of
SEQ ID NO:73) SEQ ID NO:73) SEQ ID NO:73) D2 RYRMY (residues 31
SISPSGGDTRYADSVKG (residues GGPRGNKYYFDY (residues 98 to 109 of to
35 of 50 to 66 of SEQ ID NO:77) SEQ ID NO:77) SEQ ID NO:77) D5
RYDMH (residues 31 SISSSGGYTAYADSVKG (residues GARGTSQGY (residues
99 to 107 of to 35 of 50 to 66 of SEQ ID NO:81) SEQ ID NO:81) SEQ
ID NO:81) F8 RYHMW (residues 31 GISSSRGITKYADSVKG (residues
GGPRGNKYYFDY (residues 99 to 110 of to 35 of 50 to 66 of SEQ ID
NO:85) SEQ ID NO:85) SEQ ID NO:85) H10 RYRMY (residues 31
SISPSGGDTRYADSVKG (residues GGPRGNKYYFDY (residues 99 to 110 of to
35 of 50 to 66 of SEQ ID NO:89) SEQ ID NO:89) SEQ ID NO:89) C9
RYPMF (residues 31 YISSSGGFTGYADSVKG (residues GGPRGNKYYFDY
(residues 99 to 110 of to 35 of 50 to 66 of SEQ ID NO:6) SEQ ID
NO:6) SEQ ID NO:6)
[0033] TABLE-US-00003 TABLE 3 LC CDRs Name L-CDR1 L-CDR2 L-CDR3 A10
SGSSSNIGSNYVY (residues SNNQRPS (residues 51 AAWDDSLSGPV (residues
90 to 100 of 23 to 35 of SEQ ID NO:26) to 57 of SEQ ID NO:26) SEQ
ID NO:26) G3 WASQGISNYLA (residues 25 SASTLQS (residues 51
QQANSFPWT (residues 90 to 98 of to 35 of SEQ ID NO:30) to 57 of SEQ
ID NO:30) SEQ ID NO:30) A6 RGDRLRSYYSS (residues 23 GRNNRPS
(residues 49 SSRDGSGNFL (residues 88 to 97 of to 33 of SEQ ID
NO:34) to 55x of SEQ ID NO:34) SEQ ID NO:34) A7 RASQSISSYLN
(residues 25 AASSLQS (residues 51 QQLTGYPSIT (residues 90 to 99 of
to 35 of SEQ ID NO:38) to 57 of SEQ ID NO:38) SEQ ID NO:38) C8
TGTSSDVGGYNYVS (residues DVSKRPS (residues 52 TSYTSSSTWV (residues
91 to 100 of 23 to 36 of SEQ ID NO:42) to 58 of SEQ ID NO:42) SEQ
ID NO:42) H9 QASQDTYNRLH (residues 25 DAVNLKR (residues 51
QHSDDLSLA (residues 90 to 98 of to 35 of SEQ ID NO:46) to 57 of SEQ
ID NO:46) SEQ ID NO:46) G10-R2 RSSQSLLYSNGYNYLD LGSNRAS (residues
56 MQALQIPRT (residues 95 to 103 of (residues 25 to 40 of to 62 of
SEQ ID NO:50) SEQ ID NO:50) SEQ ID NO:50) F3-R2 RASLPVNTWLA
(residues 25 AASRLQS (residues 51 QQANTFPYT (residues 90 to 98 of
to 35 of SEQ ID NO:54) to 57 of SEQ ID NO:54) SEQ ID NO:54) C6-R2
QGDSLRSYYAS (residues 23 SKSNRPS (residues 49 NSRDSSGNHLV (residues
88 to 98 of to 33 of SEQ ID NO:58) to 55 of SEQ ID NO:58) SEQ ID
NO:58) A4-R3 RGDRLRSYYSS (residues 23 GRKNRPS (residues 49
SSRDGSGNFL (residues 88 to 97 of to 33 of SEQ ID NO:62) to 55 of
SEQ ID NO:62) SEQ ID NO:62) C1-R3 RASQSISTYLN (residues 25 GASSLVS
(residues 51 HQSYITSWT (residues 90 to 98 of to 35 of SEQ ID NO:66)
to 57 of SEQ ID NO:66) SEQ ID NO:66) A2 RASQDIRSDLA (residues 25
AASTLKD (residues 51 QHLNGHPA (residues 90 to 97 of to 35 of SEQ ID
NO:70) to 57 of SEQ ID NO:70) SEQ ID NO:70) B5 SGDRLGDKYTS
(residues 23 QDKKRPS (residues 49 QAWATNVV (residues 88 to 95 of to
33 of SEQ ID NO:74) to 55 of SEQ ID NO:74) SEQ ID NO:74) D2
RASQTIDNYLN (residues 25 AASTLQT (residues 51 QQGFSNPWT (residues
90 to 98 of to 35 of SEQ ID NO:78) to 57 of SEQ ID NO:78) SEQ ID
NO:78) D5 RASQFVSYSYLA (residues 25 GASSRAK (residues 52 QQYVPSVPWT
(residues 91 to 100 of to 35 of SEQ ID NO:82) to 58 of SEQ ID
NO:82) SEQ ID NO:82) F8 RASQSVTSSDLA (residues 25 GASSRAT (residues
52 QQYGNSPGT (residues 91 to 99 of to 36 of SEQ ID NO:86) to 58 of
SEQ ID NO:86) SEQ ID NO:86) H10 RASQSVSSSYLA (residues 25 GASSRAT
(residues 52 QQYGSSTWT (residues 91 to 99 of to 36 of SEQ ID NO:90)
to 58 of SEQ ID NO:90) SEQ ID NO:90) C9 TGTSSDVGHYNYVS (residues
DVSSRPS (residues 52 SSYTSGDTLYV (residues 91 to 101 of 23 to 36 of
SEQ ID NO:4) to 58 of SEQ ID NO:4) SEQ ID NO:4)
[0034] In another preferred embodiment, the antibody (or fragment
thereof) includes at least one, two and preferably three CDR's from
the light and/or heavy chain variable region of an antibody
disclosed herein, e.g., A10, G3, A6, A7, C8, H9, G10-R2, F3-R2,
C6-R2, A4-R3, C1-R3, A2, B5, D2, D5, F8, H10, or C9, having an
amino acid sequence that differs by no more than 3, 2.5, 2, 1.5, or
1, 0.5 substitutions, insertions or deletions for every 10 amino
acids (e.g., the number of differences being proportional to the
CDR length) relative to the corresponding CDR's of the disclosed
antibody. Further, the antibody, or antigen-binding fragment
thereof, can include six CDR's, each of which differs by no more
than 3, 2.5, 2, 1.5, or 1, 0.5 substitutions, insertions or
deletions for every 10 amino acids relative to the corresponding
CDRs of the human anti-ET2 antibody, e.g., A10, G3, A6, A7, C8, H9,
G10-R2, F3-R2, C6-R2, A4-R3, C1-R3, A2, B5, D2, D5, F8, H10, or
C9.
[0035] In one embodiment, the heavy chain variable region includes
a CDR1 including the following amino acid sequence: Y-X-M-X-W (SEQ
ID NO:95) or R-Y-X-M-X (SEQ ID NO: 96) or R-Y-(SRK)-M-(SYWH) (SEQ
ID NO:97), wherein X is any amino acid.
[0036] In one embodiment, the heavy chain variable region includes
a CDR2 including the following sequence:
I/S-I/S-S-X-X-G-X-X-X-X*-Y-A-D-S (SEQ ID NO:98), wherein X is any
amino acid and wherein X* may be absent, or
(GSVYR)-I-(GSVYR)-(SP)-S-(GR)-G-(STIMYFKD)-T-(AGTFRKNQ)-Y-A-D-S-V-K-G
(SEQ ID NO: 112) or
(GSY)-I-(SVR)-(SP)-S-G-G-(SIYD)-T-(GRKN)-Y-A-D-S-V-K-G (SEQ ID
NO:113).
[0037] In one embodiment, the heavy chain variable region includes
a CDR3 that includes
(GN)-(AG)-(RP)-R-(AG)-(FN)-(KP)-(SY)-(MY)-(FD)-(VD)-Y (SEQ ID
NO:99) or
(GRN)-(AG)-(RP)-(GR)-(AG)-(FNE)-(VKP)-(ASY)-(MYF)-(FD)-(IVD)-Y (SEQ
ID NO:100) or one of the following sequences: GPRGNKYY (SEQ ID
NO:101) or ARGTSQ (SEQ ID NO:102).
[0038] In one embodiment, the light chain variable region includes
a CDR1 including the following sequence:
R-A-S-Q-S-(IV)-S-(ST)-(SY)-(LY)-(ALN)-A (SEQ ID NO:103) or
R-A-S-(LQ)-(STFDP)-(IV)-(STRDN)-(STYN)-(SYWD)-(LYD)-(ALN)-A (SEQ ID
NO:104).
[0039] In one embodiment, the light chain variable region includes
a CDR2 including the following sequence: X-A-S-S-L-X-X (SEQ ID
NO:105) or (AG)-A-S-(STR)-(LR)-(AVKQ)-(STKD) (SEQ ID NO:106),
wherein X is any amino acid.
[0040] In one embodiment, the light chain variable region includes
a CDR3 including the following sequence: Q-Q-X-X-X-X-P-X-T-X (SEQ
ID NO:107) or
Q-Q-(AGSLY)-(GTVYFN)-(GSTINP)-(STYFN)-(STVP)-(AGSYWP)-(TIW)-T (SEQ
ID NO:108).
[0041] In one embodiment, the light chain variable region includes
a CDR1 including the following sequence: S-X-D-X-X-X-X-X-Y-X-S-W
(SEQ ID NO:109) or R-A-S-Q-X-V/I-X-X-X-(X)-L-A/N-W (SEQ ID NO:110),
wherein X is any amino acid and wherein (X) may be absent;
[0042] In one embodiment, the light chain variable region includes
a CDR2 including the following sequence: A-S-S/T-R/L-X-X-G-R (SEQ
ID NO:111), wherein X is any amino acid.
[0043] In one embodiment, two or three of the CDRs of the HC
variable domain sequence match motifs described herein such that
the motifs also match a HC variable domain of an antibody described
herein. Similarly, in one embodiment, two or three of the CDRs of
the LC variable domain sequence match motifs described herein such
that the motifs also match a LC variable domain of an antibody
described herein. In still another embodiment, the matched motifs
for the CDRs are based on a HC and a LC that are paired in an
antibody described herein.
[0044] In one embodiment, the H1 and H2 hypervariable loops have
the same canonical structure as an antibody described herein. In
one embodiment, the L1 and L2 hypervariable loops have the same
canonical structure as an antibody described herein.
[0045] In another embodiment, the light or heavy chain
immunoglobulin of the anti-ET2 antibody, or antigen-binding
fragment thereof, can further include a light or a heavy chain
variable framework that has no more than 3, 2.5, 2, 1.5, or 1, 0.5
substitutions, insertions or deletions for every 10 amino acids in
FR1, FR2, FR3, or FR4 relative to the corresponding frameworks of
an antibody disclosed herein, e.g., A10, G3, A6, A7, C8, H9,
G10-R2, F3-R2, C6-R2, A4-R3, C1-R3, A2, B5, D2, D5, F8, H10, or C9.
In one embodiment, the light or heavy chain immunoglobulin of the
anti-ET2 antibody, or antigen-binding fragment thereof, further
includes a light or a heavy chain variable framework, e.g., FR1,
FR2, FR3, or FR4, that is identical to a framework of an antibody
disclosed herein, e.g., A10, G3, A6, A7, C8, H9, G10-R2, F3-R2,
C6-R2, A4-R3, C1-R3, A2, B5, D2, D5, F8, H10, or C9.
[0046] In one embodiment, the light or the heavy chain variable
framework can be chosen from: (a) a light or heavy chain variable
framework including at least 80%, 90%, 95%, or preferably 100% of
the amino acid residues from a human light or heavy chain variable
framework, e.g., a light or heavy chain variable framework residue
from a human mature antibody, a human germline sequence, a
consensus sequence, or an antibody described herein; (b) a light or
heavy chain variable framework including from 20% to 80%, 40% to
80%, or 60% to 90% of the amino acid residues from a human light or
heavy chain variable framework, e.g., a light or heavy chain
variable framework residue from a human mature antibody, a human
germline sequence, or a consensus sequence; (c) a non-human
framework (e.g., a rodent framework); or (d) a non-human framework
that has been modified, e.g., to remove antigenic or cytotoxic
determinants, e.g., deimmunized, or partially humanized. In one
embodiment, the ET2-ligand is not antigenic in humans.
[0047] In one embodiment, the heavy or light chain framework
includes an amino acid sequence, which is at least 80%, 85%, 90%,
95%, 97%, 98%, 99% or higher identical to the heavy chain framework
of an antibody disclosed herein, e.g., A10, G3, A6, A7, C8, H9,
G10-R2, F3-R2, C6-R2, A4-R3, C1-R3, A2, B5, D2, D5, F8, H10, or C9;
or which differs at least 1 or 5 but at less than 40, 30, 20, or 10
residues from, the amino acid sequence of a variable domain of an
antibody disclosed herein, e.g., A10, G3, A6, A7, C8, H9, G10-R2,
F3-R2, C6-R2, A4-R3, C1-R3, A2, B5, D2, D5, F8, H10, or C9.
[0048] In one embodiment, the heavy or light chain variable domain
sequence of the ET2 antibody includes an amino acid sequence, which
is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher identical
to a variable domain sequence of an antibody described herein,
e.g., A10, G3, A6, A7, C8, H9, G10-R2, F3-R2, C6-R2, A4-R3, C1-R3,
A2, B5, D2, D5, F8, H10, or C9; or which differs at least 1 or 5
but at less than 40, 30, 20, or 10 residues from a variable domain
sequence of an antibody described herein, e.g., A10, G3, A6, A7,
C8, H9, G10-R2, F3-R2, C6-R2, A4-R3, C1-R3, A2, B5, D2, D5, F8,
H10, or C9.
[0049] In one embodiment, an anti-ET2 antibody includes at least
one, preferably two, light chain variable regions that include a
light chain variable domain sequence of an antibody described
herein, e.g., A10, G3, A6, A7, C8, H9, G10-R2, F3-R2, C6-R2, A4-R3,
C1-R3, A2, B5, D2, D5, F8, H10, or C9, and at least one, preferably
two, heavy chain variable regions that include a heavy chain
variable domain sequence of an antibody described herein, e.g.,
A10, G3, A6, A7, C8, H9, G10-R2, F3-R2, C6-R2, A4-R3, C1-R3, A2,
B5, D2, D5, F8, H10, or C9.
[0050] In one embodiment, the light or heavy chain variable
framework of the anti-ET2 antibody or antigen-binding fragment
thereof includes at least one, two, three, four, five, six, seven,
eight, nine, ten, fifteen, sixteen, or seventeen amino acid
residues from a human light or heavy chain variable framework,
e.g., a light or heavy chain variable framework residue from a
human mature antibody, a human germline sequence, a consensus
sequence, or an antibody described herein. In one embodiment, the
amino acid residue from the human light or heavy chain variable
framework is the same as the residue found at the same position in
a human germline. Preferably, the amino acid residue from the human
light or heavy chain variable framework is the most common residue
in the human germline at the same position.
[0051] An ET2-ligand described herein can be used alone, e.g., can
be administered to a subject or used in vitro in non-derivatized or
unconjugated forms. In other embodiments, the ET2-ligand can be
derivatized, modified or linked to another functional molecule,
e.g., another compound, peptide, protein, isotope, cell, or
insoluble support. For example, the ET2-ligand can be functionally
linked (e.g., by chemical coupling, genetic fusion, non-covalent
association or otherwise) to one or more other molecular entities,
such as an antibody (e.g., if the ligand is an antibody to form a
bi-specific or a multi-specific antibody), a toxin, a radioisotope,
a therapeutic (e.g., a cytotoxic or cytostatic) agent or moiety,
among others. For example, the ET2-ligand can be coupled to a
radioactive ion (e.g., an .alpha.-, .gamma.-, or .beta.-emitter),
e.g., iodine (.sup.131I or .sup.125I), yttrium (.sup.90Y), lutetium
(.sup.177Lu), actinium (.sup.225Ac), rhenium (.sup.186Re), or
bismuth (.sup.212 or .sup.213Bi).
[0052] In another aspect, the invention provides, compositions,
e.g., pharmaceutical compositions, which include a pharmaceutically
acceptable carrier, excipient or stabilizer, and at least one of
the ET2-ligands (e.g., antibodies or fragments thereof) described
herein. In one embodiment, the compositions, e.g., the
pharmaceutical compositions, include a combination of two or more
of the aforesaid ET2-ligands.
[0053] In another aspect, the invention features a kit that
includes an anti-ET2 antibody (or fragment thereof), e.g., an
anti-ET2 antibody (or fragment thereof) as described herein, for
use alone or in combination with other therapeutic modalities,
e.g., a cytotoxic or labeling agent, e.g., a cytotoxic or labeling
agent as described herein, along with instructions on how to use
the ET2 antibody or the combination of such agents, e.g., to treat,
prevent or detect cancerous lesions.
[0054] The invention also features nucleic acid sequences that
encode a heavy and light chain immunoglobulin or immunoglobulin
fragment described herein. For example, the invention features, a
first and second nucleic acid encoding a heavy and light chain
variable region, respectively, of an anti-ET2 antibody molecule as
described herein. In another aspect, the invention features host
cells and vectors containing the nucleic acids of the
invention.
[0055] In another aspect, the invention features, a method of
producing an anti-ET2 antibody, or antigen-binding fragment
thereof. The method includes: providing a first nucleic acid
encoding a heavy chain variable region, e.g., a heavy chain
variable region as described herein; providing a second nucleic
acid encoding a light chain variable region, e.g., a light chain
variable region as described herein; and expressing said first and
second nucleic acids in a host cell under conditions that allow
assembly of said light and heavy chain variable regions to form an
antigen binding protein. The first and second nucleic acids can be
linked or unlinked, e.g., expressed on the same or different
vector, respectively. The first and second nucleic acids can
further encode constant regions of heavy and light chains.
[0056] The host cell can be a eukaryotic cell, e.g., a mammalian
cell, an insect cell, a yeast cell, or a prokaryotic cell, e.g., E.
coli. For example, the mammalian cell can be a cultured cell or a
cell line. Exemplary mammalian cells include lymphocytic cell lines
(e.g., NSO), Chinese hamster ovary cells (CHO), COS cells, oocyte
cells, and cells from a transgenic animal, e.g., mammary epithelial
cells. For example, nucleic acids encoding the antibodies described
herein can be expressed in a transgenic animal. In one embodiment,
the nucleic acids are placed under the control of a tissue-specific
promoter (e.g., a mammary specific promoter) and the antibody is
produced in the transgenic animal. For example, the antibody
molecule is secreted into the milk of the transgenic animal, such
as a transgenic cow, pig, horse, sheep, goat or rodent.
[0057] The invention also features a method of treating, e.g.,
inhibiting a cellular activity (e.g., cell growth, cell
differentiation, cell migration, or cell organization), a
physiological activity (e.g., blood vessel growth, organization,
etc.) and/or cell or ablating, or killing, a cell, e.g., a normal,
benign or hyperplastic cell (e.g., a cell found in pulmonary,
breast, renal, urothelial, colonic, prostatic, or hepatic cancer
and/or metastasis). The treating may have direct and/or indirect
effects on the growth of a cancer, e.g., by targeting a tumor cell
directly, or by inhibiting tumor angiogenesis, thereby inhibiting
growth of tumor cell indirectly. Methods of the invention include
contacting the cell with an ET2-ligand, in an amount sufficient to
treat, e.g., inhibit cell growth, or ablate or kill, the cell. The
ligand can include a cytotoxic entity. Methods of the invention can
be used, for example, to treat or prevent a disorder, e.g., a
cancerous (e.g., a malignant or metastatic disorder), or
non-cancerous disorder (e.g., a benign or hyperplastic disorder) by
administering to a subject an ET2-ligand in an amount effective to
treat or prevent such disorder.
[0058] A ET2-ligand that increases ET2 activity can be used, for
example, to treat or prevent disorders, e.g., a disorder in which
increased proteolysis and/or increased angiogenesis is desirable.
For example, the ligand can be used to treat a wound (e.g., to
assist wound healing). For example, the wound can be a laceration,
a burn, or a surgical incision.
[0059] The subject method can be used on cells in culture, e.g. in
vitro or ex vivo. For example, cancerous or metastatic cells (e.g.,
pulmonary, breast, renal, urothelial, colonic, prostatic, or
hepatic cancer or metastatic cells) can be cultured in vitro in
culture medium and the contacting step can be effected by adding
the ET2-ligand to the culture medium. The method can be performed
on cells (e.g., cancerous or metastatic cells) present in a
subject, as part of an in vivo (e.g., therapeutic or prophylactic)
protocol. For in vivo embodiments, the contacting step is effected
in a subject and includes administering the ET2-ligand to the
subject under conditions effective to permit both binding of the
ligand to the cell, and the treating, e.g., the inhibiting of cell
growth and/or cell division, or the killing or ablating of the
cell.
[0060] The method of the invention can be used to treat or prevent
disorders characterized by unwanted angiogenesis, such as cancerous
disorders, e.g., including but are not limited to, solid tumors,
soft tissue tumors, and metastatic lesions. Examples of solid
tumors include malignancies, e.g., sarcomas, adenocarcinomas, and
carcinomas, of the various organ systems, such as those affecting
lung, breast, lymphoid, gastrointestinal (e.g., colon), and
genitourinary tract (e.g., renal, urothelial cells), pharynx, as
well as adenocarcinomas which include malignancies such as most
colon cancers, rectal cancer, renal-cell carcinoma, liver cancer,
non-small cell carcinoma of the lung, cancer of the small intestine
and cancer of the esophagus. Metastatic lesions of the
aforementioned cancers can also be treated or prevented using the
methods and compositions of the invention.
[0061] The method of the invention can be used to treat or prevent
disorders in which increased angiogenesis is desirable, e.g., using
an ET2-ligand that increases ET2 activity.
[0062] The subject can be a mammal, e.g., a primate, preferably a
higher primate, e.g., a human (e.g., a patient having, or at risk
of, a disorder described herein, e.g., cancer).
[0063] The anti-ET2 antibody or fragment thereof, e.g., an anti-ET2
antibody or fragment thereof as described herein, can be
administered to the subject systemically (e.g., orally,
parenterally, subcutaneously, intravenously, intramuscularly,
intraperitoneally, intranasally, transdermally, or by inhalation),
topically, or by application to mucous membranes, such as the nose,
throat and bronchial tubes. In one embodiment, the protein
accumulates at sites of angiogenesis and/or tumor growth in
vivo.
[0064] The methods of the invention can further include the step of
monitoring the subject, e.g., for a reduction in one or more of: a
reduction in tumor size; reduction in cancer markers; reduction in
the appearance of new lesions, e.g., in a bone scan; a reduction in
the appearance of new disease-related symptoms; or decreased or
stabilization of size of soft tissue mass; or any parameter related
to improvement in clinical outcome. The subject can be monitored in
one or more of the following periods: prior to beginning of
treatment; during the treatment; or after one or more elements of
the treatment have been administered. Monitoring can be used to
evaluate the need for further treatment with the same ET2-ligand or
for additional treatment with additional agents. Generally, a
decrease in one or more of the parameters described above is
indicative of the improved condition of the subject.
[0065] The ET2-ligand can be used alone in unconjugated form to
thereby ablate, kill, or inhibit growth of the ET2-expressing
cells. For example, if the ligand is an antibody, the ablation,
killing, or growth inhibition can be mediated by an
antibody-dependent cell killing mechanisms such as
complement-mediated cell lysis and/or effector cell-mediated cell
killing. In other embodiments, the ET2-ligand can be bound to a
substance, e.g., a cytotoxic agent or moiety, effective to kill or
ablate the ET2-expressing cells. For example, the ET2-ligand can be
coupled to a radioactive ion (e.g., an .alpha.-, .gamma.-, or
.beta.-emitter), e.g., iodine (.sup.131I or .sup.125I), yttrium
(.sup.90Y), lutetium (.sup.177Lu), actinium (.sup.225Ac), or
bismuth (.sup.213Bi). The methods and compositions of the invention
can be used in combination with other therapeutic modalities, e.g.,
other anti-cancer and/or anti-angiogenic treatments. In one
embodiment, the methods of the invention include administering to
the subject an ET2-ligand, e.g., an anti-ET2 antibody or fragment
thereof, in combination with a cytotoxic agent, in an amount
effective to treat or prevent said disorder. The ligand and the
cytotoxic agent can be administered simultaneously or sequentially.
In other embodiments, the methods and compositions of the invention
are used in combination with surgical and/or radiation
procedures.
[0066] In another aspect, the invention features methods for
detecting the presence of an ET2 protein, in a sample, in vitro
(e.g., a biological sample, a tissue biopsy, e.g., a cancerous
lesion). The subject method can be used to evaluate, e.g., diagnose
or stage a disorder described herein, e.g. a cancerous disorder or
other disorder characterized by unwanted angiogenesis. The method
includes: (i) contacting the sample (and optionally, a reference,
e.g., control, sample) with an ET2-ligand, as described herein,
under conditions that allow interaction of the ET2-ligand and the
ET2 protein to occur; and (ii) detecting formation of a complex
between the ET2-ligand, and the sample (and optionally, the
reference, e.g., control, sample). Formation of the complex is
indicative of the presence of ET2 protein, and can indicate the
suitability or need for a treatment described herein. E.g., a
statistically significant change in the formation of the complex in
the sample relative to the reference sample, e.g., the control
sample, is indicative of the presence and/or level of ET2 in the
sample. In one embodiment, the ET2-ligand may recognize and/or
distinguish between a complex containing active ET2 and a complex
containing an inactive (e.g., zymogen) form of ET2.
[0067] In yet another aspect, the invention provides a method for
detecting the presence of ET2 in vivo (e.g., in vivo imaging in a
subject). The subject method can be used to evaluate, e.g.,
diagnose, localize, or stage a disorder described herein, e.g., a
cancerous disorder or other disorder characterized by unwanted
angiogenesis. The method includes: (i) administering to a subject
(and optionally a control subject) an ET2-ligand (e.g., an antibody
or antigen binding fragment thereof), under conditions that allow
interaction of the ET2-ligand and the ET2 protein to occur; and
(ii) detecting formation of a complex between the ligand and ET2,
wherein a statistically significant change in the formation of the
complex in the subject relative to the reference, e.g., the control
subject or subject's baseline, is indicative of the presence and/or
level of the ET2. In other embodiments, a method of diagnosing or
staging, a disorder as described herein (e.g., a cancerous disorder
or other disorder characterized by unwanted angiogenesis), is
provided. The method includes: (i) identifying a subject having, or
at risk of having, the disorder; (ii) obtaining a sample of a
tissue or cell affected with the disorder; (iii) contacting said
sample or a control sample with an ET2-ligand, under conditions
that allow interaction of the binding agent and the ET2 protein to
occur, and (iv) detecting formation of a complex. A statistically
significant alteration in the formation of the complex between the
ligand with respect to a reference sample, e.g., a control sample,
is indicative of the disorder or the stage of the disorder.
[0068] Preferably, the ET2-ligand used in the in vivo and in vitro
diagnostic methods is directly or indirectly labeled with a
detectable substance to facilitate detection of the bound or
unbound binding agent. Suitable detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials and radioactive materials. In one embodiment,
the ET2-ligand is coupled to a radioactive ion, e.g., indium
(.sup.111In), iodine (.sup.131 or .sup.125I), yttrium (.sup.90Y),
actinium (.sup.225Ac), bismuth (.sup.213Bi), sulfur (.sup.35S),
carbon (.sup.14C), tritium (.sup.3H), rhodium (.sup.188Rh), or
phosphorous (.sup.32P). In another embodiment, the ligand is
labeled with an NMR contrast agent.
[0069] The invention also provides polypeptides and nucleic acids
that encompass a range of amino acid and nucleic acid
sequences.
[0070] A ET2-binding ligand can be used to treat or prevent
angiogenesis-related disorders, particularly angiogenesis-dependent
cancers and tumors.
[0071] Angiogenesis-related disorders include, but are not limited
to, solid tumors; blood born tumors such as leukemias; tumor
metastasis; benign tumors (e.g., hemangiomas, acoustic neuromas,
neurofibromas, trachomas, and pyogenic granulomas; rheumatoid
arthritis); psoriasis; ocular angiogenic diseases, for example,
diabetic retinopathy, retinopathy of prematurity, macular
degeneration, corneal graft rejection, neovascular glaucoma,
retrolental fibroplasia, rubeosis; Osler-Webber Syndrome;
myocardial angiogenesis; plaque neovascularization; telangiectasia;
hemophiliac joints; angiofibroma; and wound granulation.
[0072] "Angiogenesis-dependent cancers and tumors" are cancers
tumors that require, for their growth (expansion in volume and/or
mass), an increase in the number and density of the blood vessels
supplying then with blood. In one embodiment a ET2-binding ligand
causes regression of such cancers and tumors. "Regression" refers
to the reduction of tumor mass and size, e.g., a reduction of at
least 2, 5, 10, or 25%.
[0073] In another aspect, the invention features a method of
contacting a cell (in vitro, ex vivo, or in vivo), e.g., an
endothelial cell, e.g., an endothelial cell in the vicinity of a
cancer, e.g., a tumor. The method can include providing a ligand
that interacts with ET2, e.g., a ligand described herein, and
contacting the cell with the ligand, in an amount sufficient to
form at least one detectable ligand-cell complex. The ligand can
include, for example, a label or cytotoxic entity, e.g., an
immunoglobulin Fc domain or a cytotoxic drug.
[0074] The invention also provides methods for identifying protein
ligands (e.g., antibody ligands) of ET2. In one embodiment, a
method includes: providing a library and screening the library to
identify a member that encodes a protein that binds to the ET2. The
screening can be performed in a number of ways. For example, the
library can be a display library, e.g., a phage display library or
a phagemid library. The phage/phagemid library can be an antibody
(e.g., Fab) or Kunitz domain library. Methods utilizing phage
display libraries can further include the steps of: recovering
phage that bind ET2 and isolating a nucleic acid from the phage,
wherein the nucleic acid encodes the protein or polypeptide ligand
of ET2. The phage may be eluted from ET2 using a competitor peptide
or by altering buffer conditions (e.g., pH).
[0075] The ET2 can be recombinantly expressed and can be tagged.
The ET2 is purified and attached to a support, e.g., to
paramagnetic beads or other magnetically responsive particle. The
ET2 can also be expressed on the surface of a cell. The display
library can be screened to identify members that specifically bind
to the cell, e.g., only if the ET2 is expressed. The ET2 can be
human ET2. The ET2 can be treated or mutated to remove
glycosylation. Also, a fragment of ET2 may be used, e.g., a serine
protease domain.
[0076] As used herein, the term "substantially identical" (or
"substantially homologous") is used herein to refer to a first
amino acid or nucleotide sequence that contains a sufficient number
of identical or equivalent (e.g., with a similar side chain, e.g.,
conserved amino acid substitutions) amino acid residues or
nucleotides to a second amino acid or nucleotide sequence such that
the first and second amino acid or nucleotide sequences have
similar activities. In the case of antibodies, the second antibody
has the same specificity and has at least 5%, 10%, 25%, or 50% of
the affinity of the first antibody.
[0077] Sequences similar or homologous (e.g., at least about 60%,
70%, 80%, 85%, 90%, 95% sequence identity) to the sequences
disclosed herein are also part of this application. In some
embodiments, the sequence identity can be about 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively,
substantial identity exists when the nucleic acid segments will
hybridize under selective hybridization conditions (e.g., highly
stringent hybridization conditions), to the complement of the
strand encoding the ET2 ligand. The nucleic acids may be present in
whole cells, in a cell lysate, or in a partially purified or
substantially pure form.
[0078] Calculations of "homology" or "sequence identity" between
two sequences (the terms are used interchangeably herein) are
performed as follows. The sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in one or both of
a first and a second amino acid or nucleic acid sequence for
optimal alignment and non-homologous sequences can be disregarded
for comparison purposes). In one embodiment, the length of a
reference sequence aligned for comparison purposes is at least 30%,
preferably at least 40%, more preferably at least 50%, even more
preferably at least 60%, and even more preferably at least 70%,
80%, 90%, 100% of the length of the reference sequence. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, which need to be introduced for optimal
alignment of the two sequences.
[0079] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In one embodiment, the percent identity
between two amino acid sequences is determined using the Needleman
and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has
been incorporated into the GAP program in the GCG software package
(Accelrys, San Diego, Calif.), using either a Blossum 62 matrix or
a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and
a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred
embodiment, the percent identity between two nucleotide sequences
is determined using the GAP program in the GCG software package,
using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or
80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly
preferred set of parameters (and the one that should be used if the
practitioner is uncertain about what parameters should be applied
to determine if a molecule is within a sequence identity or
homology limitation of the invention) are a Blossum 62 scoring
matrix with a gap penalty of 12, a gap extend penalty of 4, and a
frameshift gap penalty of 5.
[0080] As used herein, the term "homologous" is synonymous with
"similarity" and means that a sequence of interest differs from a
reference sequence by the presence of one or more amino acid
substitutions (although modest amino acid insertions or deletions)
may also be present. Presently preferred means of calculating
degrees of homology or similarity to a reference sequence are
through the use of BLAST algorithms (available from the National
Center of Biotechnology Information (NCBI), National Institutes of
Health, Bethesda Md.), in each case, using the algorithm default or
recommended parameters for determining significance of calculated
sequence relatedness. The percent identity between two amino acid
or nucleotide sequences can also be determined using the algorithm
of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4.
[0081] As used herein, the term "hybridizes under low stringency,
medium stringency, high stringency, or very high stringency
conditions" describes conditions for hybridization and washing.
Guidance for performing hybridization reactions can be found in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described
in that reference and either can be used. Specific hybridization
conditions referred to herein are as follows: 1) low stringency
hybridization conditions in 6.times. sodium chloride/sodium citrate
(SSC) at about 45.degree. C., followed by two washes in
0.2.times.SSC, 0.1% SDS at least at 50.degree. C. (the temperature
of the washes can be increased to 55.degree. C. for low stringency
conditions); 2) medium stringency hybridization conditions in
6.times.SSC at about 45.degree. C., followed by one or more washes
in 0.2.times.SSC, 0.1% SDS at 60.degree. C.; 3) high stringency
hybridization conditions in 6.times.SSC at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
65.degree. C.; and preferably 4) very high stringency hybridization
conditions are 0.5M sodium phosphate, 7% SDS at 65.degree. C.,
followed by one or more washes at 0.2.times.SSC, 1% SDS at
65.degree. C. Very high stringency conditions (4) are the preferred
conditions and the ones that should be used unless otherwise
specified.
[0082] It is understood that the binding agent polypeptides of the
invention may have additional conservative or non-essential amino
acid substitutions, which do not have a substantial effect on the
polypeptide functions. Whether or not a particular substitution
will be tolerated, i.e., will not adversely affect desired
biological properties, such as binding activity can be determined
as described in Bowie, et al. (1990) Science 247:1306-1310. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0083] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of the binding agent, e.g.,
the antibody, without abolishing or more preferably, without
substantially altering a biological activity, whereas an
"essential" amino acid residue results in such a change.
[0084] Binding affinity can be determined by a variety of methods
including equilibrium dialysis, equilibrium binding, gel
filtration, ELISA, or spectroscopy (e.g., using a fluorescence
assay). These techniques can be used to measure the concentration
of bound and free ligand as a function of ligand (or target)
concentration. The concentration of bound ligand ([Bound]) is
related to the concentration of free ligand ([Free]) and the
concentration of binding sites for the ligand on the target where
(N) is the number of binding sites per target molecule by the
following equation: [Bound]=N[Free]/((1/K.sub.a)+[Free])
[0085] It is not always necessary to make an exact determination of
K.sub.a, though, since sometimes it is sufficient to obtain a
quantitative measurement of affinity, e.g., determined using a
method such as ELISA or FACS analysis, is proportional to K.sub.a,
and thus can be used for comparisons, such as determining whether a
higher affinity is, e.g., 2 fold higher. Better binding can be
indicated by a greater numerical K.sub.a, or a lesser numerical
K.sub.d than a reference. Unless otherwise noted, binding
affinities are determined in phosphate buffered saline at pH7.
[0086] The details of one or more non-limiting embodiments of the
invention are set forth in the accompanying drawings and the
description below. Other features, objects, and advantages of the
invention will be apparent from the description and drawings, and
from the claims.
DESCRIPTION OF DRAWINGS
[0087] FIGS. 1A and 1B provide the nucleotide and amino acid
sequence of human ET-2S (SEQ ID NO:93 and SEQ ID NO:94,
respectively).
[0088] FIGS. 2A and 2B provide the nucleotide and amino acid
sequence of human ET-2L (SEQ ID NO:1 and SEQ ID NO:2,
respectively).
[0089] FIGS. 3A and 3B depict distribution of tumor volumes (5A)
and tumor weights (5B) on day 39 for a treatment with the H10
antibody in a mouse model.
DETAILED DESCRIPTION
[0090] Endotheliases are an attractive target for the treatment of
diseases characterized by unwanted angiogenesis due to the role of
these enzymes in the proteolytic processing of extracellular matrix
components during new blood vessel formation. Endotheliase-2 (ET2)
is a transmembrane serine protease expressed on the surface of
endothelial cells. Exemplary nucleic acid and amino acid sequence
of two forms of human ET2, ET2-S, and ET-2L (for short and long
forms, respectively) are provided in FIGS. 1 and 2. See also WO
01/36604.
[0091] This disclosure provides, inter alia, ligands that bind to
ET2, e.g., immunoglobulins that inhibit ET2 with high affinity and
selectivity. The disclosure also provides methods for identifying
proteins, e.g., antibodies, that bind to ET2. In many cases, the
identified proteins are at least partially specific.
[0092] ET2 is a type-II membrane-type serine protease and a member
of the endotheliase class of angiogenesis-associated proteases. ET2
RNA is expressed in endothelial cells and some tumor cell lines (WO
01/36604). ET2 RNA has also been detected in other tissues. The ET2
protein has a transmembrane region at the N-terminus, followed by a
single low density lipoprotein-A (LDR-A) receptor domain and a
single scavenger-receptor cysteine-rich domain (WO 01/36604). The
C-terminus contains a trypsin-like serine protease domain
characterized by the presence of the catalytic triad residues
histidine, aspartate, and serine, in 3 conserved regions of the
protease domain. Three repetitive sequences having the sequence
ASPAGTPPGRASP (SEQ ID NO:144) are present near the transmembrane
domain and contain a sequence motif for N-myristoylation (WO
01/36604).
Display Libraries
[0093] In one embodiment, a display library can be used to identify
proteins that bind to the ET2. A display library is a collection of
entities; each entity includes an accessible polypeptide component
and a recoverable component that encodes or identifies the
polypeptide component. The polypeptide component can be of any
length, e.g. from three amino acids to over 300 amino acids. In a
selection, the polypeptide component of each member of the library
is probed with the ET2 and if the polypeptide component binds to
the ET2, the display library member is identified, typically by
retention on a support.
[0094] Retained display library members are recovered from the
support and analyzed. The analysis can include amplification and a
subsequent selection under similar or dissimilar conditions. For
example, positive and negative selections can be alternated. The
analysis can also include determining the amino acid sequence of
the polypeptide component and purification of the polypeptide
component for detailed characterization.
[0095] A variety of formats can be used for display libraries.
Examples include the following.
[0096] Phage Display. One format utilizes viruses, particularly
bacteriophages. This format is termed "phage display." The
polypeptide component is typically covalently linked to a
bacteriophage coat protein. The linkage results form translation of
a nucleic acid encoding the polypeptide component fused to the coat
protein. The linkage can include a flexible peptide linker, a
protease site, or an amino acid incorporated as a result of
suppression of a stop codon. Phage display is described, for
example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985)
Science 228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO
92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; de
Haard et al. (1999) J. Biol. Chem 274:18218-30; Hoogenboom et al.
(1998) Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol
Today 2:371-8; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay
et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989)
Science 246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734;
Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al.
(1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580;
Garrard et al. (1991) Bio/Technology 9:1373-1377; Rebar et al.
(1996) Methods Enzymol. 267:129-49; Hoogenboom et al. (1991) Nuc
Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS
88:7978-7982.
[0097] Phage display systems have been developed for filamentous
phage (phage fl, fd, and M13) as well as other bacteriophage (e.g.
T7 bacteriophage and lambdoid phages; see, e.g., Santini (1998) J.
Mol. Biol. 282:125-135; Rosenberg et al. (1996) Innovations 6:1-6;
Houshmet al. (1999) Anal Biochem 268:363-370). The filamentous
phage display systems typically use fusions to a minor coat
protein, such as gene III protein, and gene VIII protein, a major
coat protein, but fusions to other coat proteins such as gene VI
protein, gene VII protein, gene 1.times. protein, or domains
thereof can also been used (see, e.g., WO 00/71694). In one
embodiment, the fusion is to a domain of the gene III protein,
e.g., the anchor domain or "stump," (see, e.g., U.S. Pat. No.
5,658,727 for a description of the gene III protein anchor domain).
It is also possible to physically associate the protein being
displayed to the coat using a non-peptide linkage, e.g., a
non-covalent bond or a non-peptide covalent bond. For example, a
disulfide bond and/or c-fos and c-jun coiled-coils can be used for
physical associations (see, e.g., Crameri et al. (1993) Gene 137:69
and WO 01/05950).
[0098] The valency of the polypeptide component can also be
controlled. For example, cloning of the sequence encoding the
polypeptide component into the complete phage genome results in
multivariant display since all replicates of the gene III protein
are fused to the polypeptide component. For reduced valency, a
phagemid system can be utilized. In this system, the nucleic acid
encoding the polypeptide component fused to gene III is provided on
a plasmid, typically of length less than 7000 nucleotides. The
plasmid includes a phage origin of replication so that the plasmid
is incorporated into bacteriophage particles when bacterial cells
bearing the plasmid are infected with helper phage, e.g. M13K01.
The helper phage provides an intact copy of gene III and other
phage genes required for phage replication and assembly. The helper
phage has a defective origin such that the helper phage genome is
not efficiently incorporated into phage particles relative to the
plasmid that has a wild type origin.
[0099] Bacteriophage displaying the polypeptide component can be
grown and harvested using standard phage preparatory methods, e.g.
PEG precipitation from growth media.
[0100] After selection of individual display phages, the nucleic
acid encoding the selected peptide components is amplified by
infecting cells using the selected phages. Individual colonies or
plaques can be picked, the corresponding nucleic acid can be
isolated and sequenced.
[0101] Cell-based Display. In still another format the library is a
cell-display library. Proteins are displayed on the surface of a
cell, e.g., a eukaryotic or prokaryotic cell. Exemplary prokaryotic
cells include E. coli cells, B. subtilis cells, spores (see, e.g.,
Lu et al. (1995) Biotechnology 13:366). Exemplary eukaryotic cells
include yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces
pombe, Hanseula, or Pichia pastoris). Yeast surface display is
described, e.g., in Boder and Wittrup (1997) Nat. Biotechnol.
15:553-557 and WO 03/029,456. This application describes a yeast
display system that can be used to display immunoglobulin proteins
such as Fab fragments, and the use of mating to generate
combinations of heavy and light chains.
[0102] In one embodiment, variegated nucleic acid sequences are
cloned into a vector for yeast display. The cloning joins the
variegated sequence with a domain (or complete) yeast cell surface
protein, e.g., Aga2, Aga1, Flo1, or Gas1. A domain of these
proteins can anchor the polypeptide encoded by the variegated
nucleic acid sequence by a transmembrane domain (e.g., Flo1) or by
covalent linkage to the phospholipid bilayer (e.g., Gas 1). The
vector can be configured to express two polypeptide chains on the
cell surface such that one of the chains is linked to the yeast
cell surface protein. For example, the two chains can be
immunoglobulin chains.
[0103] Ribosome Display. RNA and the polypeptide encoded by the RNA
can be physically associated by stabilizing ribosomes that are
translating the RNA and have the nascent polypeptide still
attached. Typically, high divalent Mg.sup.2+ concentrations and low
temperature are used. See, e.g., Mattheakis et al. (1994) Proc.
Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol
18:1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30. and
Schaffitzel et al. (1999) J Immunol Methods. 231(1-2):119-35.
[0104] Peptide-Nucleic Acid Fusions. Another format utilizes
peptide-nucleic acid fusions. Polypeptide-nucleic acid fusions can
be generated by the in vitro translation of mRNA that include a
covalently attached puromycin group, e.g., as described in Roberts
and Szostak (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302, and
U.S. Pat. No. 6,207,446. The mRNA can then be reverse transcribed
into DNA and crosslinked to the polypeptide.
[0105] Other Display Formats. Yet another display format is a
non-biological display in which the polypeptide component is
attached to a non-nucleic acid tag that identifies the polypeptide.
For example, the tag can be a chemical tag attached to a bead that
displays the polypeptide or a radiofrequency tag (see, e.g., U.S.
Pat. No. 5,874,214).
[0106] Scaffolds. Scaffolds for display can include: antibodies
(e.g., Fab fragments, single chain Fv molecules (scFV), single
domain antibodies, camelid antibodies, and camelized antibodies);
T-cell receptors; MHC proteins; extracellular domains (e.g.,
fibronectin Type III repeats, EGF repeats); protease inhibitors
(e.g., Kunitz domains, ecotin, BPTI, and so forth); TPR repeats;
trifoil structures; zinc finger domains; DNA-binding proteins;
particularly monomeric DNA binding proteins; RNA binding proteins;
enzymes, e.g., proteases (particularly inactivated proteases),
RNase; chaperones, e.g., thioredoxin, and heat shock proteins; and
intracellular signaling domains (such as SH2 and SH3 domains).
[0107] Appropriate criteria for evaluating a scaffolding domain can
include: (1) amino acid sequence, (2) sequences of several
homologous domains, (3) 3-dimensional structure, and/or (4)
stability data over a range of pH, temperature, salinity, organic
solvent, oxidant concentration. In one embodiment, the scaffolding
domain is a small, stable protein domains, e.g., a protein of less
than 100, 70, 50, 40 or 30 amino acids. The domain may include one
or more disulfide bonds or may chelate a metal, e.g., zinc.
[0108] Examples of small scaffolding domains include: Kunitz
domains (58 amino acids, 3 disulfide bonds), Cucurbida maxima
trypsin inhibitor domains (31 amino acids, 3 disulfide bonds),
domains related to guanylin (14 amino acids, 2 disulfide bonds),
domains related to heat-stable enterotoxin IA from gram negative
bacteria (18 amino acids, 3 disulfide bonds), EGF domains (50 amino
acids, 3 disulfide bonds), kringle domains (60 amino acids, 3
disulfide bonds), fungal carbohydrate-binding domains (35 amino
acids, 2 disulfide bonds), endothelin domains (18 amino acids, 2
disulfide bonds), and Streptococcal G IgG-binding domain (35 amino
acids, no disulfide bonds).
[0109] Examples of small intracellular scaffolding domains include
SH2, SH3, and EVH domains. Generally, any modular domain,
intracellular or extracellular, can be used.
[0110] Another useful type of scaffolding domain is the
immunoglobulin (Ig) domain. Methods using immunoglobulin domains
for display are described below (see, e.g., "Antibody Display
Libraries").
[0111] Display technology can also be used to obtain ligands, e.g.,
antibody ligands that bind particular epitopes of a target. This
can be done, for example, by using competing non-target molecules
that lack the particular epitope or are mutated within the epitope,
e.g., with alanine. Such non-target molecules can be used in a
negative selection procedure as described below, as competing
molecules when binding a display library to the target, or as a
pre-elution agent, e.g., to capture in a wash solution dissociating
display library members that are not specific to the target.
[0112] Iterative Selection. In one preferred embodiment, display
library technology is used in an iterative mode. A first display
library is used to identify one or more ligands for a target. These
identified ligands are then varied using a mutagenesis method to
form a second display library. Higher affinity ligands are then
selected from the second library, e.g., by using higher stringency
or more competitive binding and washing conditions.
[0113] In some implementations, the mutagenesis is targeted to
regions known or likely to be at the binding interface. If, for
example, the identified ligands are antibodies, then mutagenesis
can be directed to the CDR regions of the heavy or light chains as
described herein. Further, mutagenesis can be directed to framework
regions near or adjacent to the CDRs. In the case of antibodies,
mutagenesis can also be limited to one or a few of the CDRs, e.g.,
to make precise step-wise improvements. Likewise, if the identified
ligands are enzymes, mutagenesis can be directed to the active site
and vicinity.
[0114] Some exemplary mutagenesis techniques include: error-prone
PCR (Leung et al. (1989) Technique 1:11-15), recombination, DNA
shuffling using random cleavage (Stemmer (1994) Nature 389-391;
termed "nucleic acid shuffling"), random chimeragenesis on
transient templates (RACHITT.TM.) (Coco et al. (2001) Nature
Biotech. 19:354), site-directed mutagenesis (Zoller et al. (1987)
Nucl Acids Res 10:6487-6504), cassette mutagenesis (Reidhaar-Olson
(1991) Methods Enzymol. 208:564-586) and incorporation of
degenerate oligonucleotides (Griffiths et al. (1994) EMBO J
13:3245).
[0115] In one example of iterative selection, the methods described
herein are used to first identify a protein ligand from a display
library that binds a ET2 with at least a minimal binding
specificity for a target or a minimal activity, e.g., an
equilibrium dissociation constant for binding of less than 1 nM, 10
nM, or 100 nM. The nucleic acid sequence encoding the initial
identified protein ligands are used as a template nucleic acid for
the introduction of variations, e.g., to identify a second protein
ligand that has enhanced properties (e.g., binding affinity,
kinetics, or stability) relative to the initial protein ligand.
[0116] Off-Rate Selection. Since a slow dissociation rate can be
predictive of high affinity, particularly with respect to
interactions between polypeptides and their targets, the methods
described herein can be used to isolate ligands with a desired
kinetic dissociation rate (i.e. reduced) for a binding interaction
to a target.
[0117] To select for slow dissociating ligands from a display
library, the library is contacted to an immobilized target. The
immobilized target is then washed with a first solution that
removes non-specifically or weakly bound biomolecules. Then the
bound ligands are eluted with a second solution that includes a
saturating amount of free target, i.e., replicates of the target
that are not attached to the particle. The free target binds to
biomolecules that dissociate from the target. Rebinding is
effectively prevented by the saturating amount of free target
relative to the much lower concentration of immobilized target.
[0118] The second solution can have solution conditions that are
substantially physiological or that are stringent. Typically, the
solution conditions of the second solution are identical to the
solution conditions of the first solution. Fractions of the second
solution are collected in temporal order to distinguish early from
late fractions. Later fractions include biomolecules that
dissociate at a slower rate from the target than biomolecules in
the early fractions.
[0119] Further, it is also possible to recover display library
members that remain bound to the target even after extended
incubation. These can either be dissociated using chaotropic
conditions or can be amplified while attached to the target. For
example, phage bound to the target can be contacted to bacterial
cells.
[0120] Selecting or Screening for Specificity. The display library
screening methods described herein can include a selection or
screening process that discards display library members that bind
to a non-target molecule. Examples of non-target molecules include,
e.g., the Fc domain of the anti-ET2 antibody.
[0121] In one implementation, a so-called "negative selection" step
is used to discriminate between the target and related non-target
molecule and a related, but distinct non-target molecules. The
display library or a pool thereof is contacted to the non-target
molecule. Members of the sample that do not bind the non-target are
collected and used in subsequent selections for binding to the
target molecule or even for subsequent negative selections. The
negative selection step can be prior to or after selecting library
members that bind to the target molecule.
[0122] In another implementation, a screening step is used. After
display library members are isolated for binding to the target
molecule, each isolated library member is tested for its ability to
bind to a non-target molecule (e.g., a non-target listed above).
For example, a high-throughput ELISA screen can be used to obtain
this data. The ELISA screen can also be used to obtain quantitative
data for binding of each library member to the target. The
non-target and target binding data are compared (e.g., using a
computer and software) to identify library members that
specifically bind to the target.
Other Expression Libraries
[0123] Other types of collections of proteins (e.g., expression
libraries) can be used to identify proteins with a particular
property (e.g., ability to bind ET2 and/or ability to inhibit ET2),
including, e.g., protein arrays of antibodies (see, e.g., De Wildt
et al. (2000) Nat. Biotechnol. 18:989-994), lambda gt11 libraries,
two-hybrid libraries and so forth.
[0124] Protein Arrays. Different proteins can be immobilized on a
solid support, for example, on a bead or an array. For a protein
array, each of the proteins is immobilized at a unique address on a
support. Typically, the address is a two-dimensional address.
[0125] In some implementations, cells or phage that express the
protein can be grown directly on a filter that is used as the
array. In other implementations, recombinant protein production is
used to produce at least partially purified samples of the protein.
The partially purified or pure samples are disposed on the
array.
[0126] Methods of producing protein arrays are described, e.g., in
De Wildt et al. (2000) Nat. Biotechnol. 18:989-994; Lueking et al.
(1999) Anal. Biochem. 270:103-111; Ge (2000) Nucleic Acids Res. 28,
e3, I-VII; MacBeath and Schreiber (2000) Science 289:1760-1763; WO
01/40803 and WO 99/51773A1. Proteins for the array can be spotted
at high speed, e.g., using commercially available robotic apparati,
e.g., from Genetic MicroSystems or BioRobotics. The array substrate
can be, for example, nitrocellulose, plastic, glass, e.g.,
surface-modified glass. For example, the array can be an array of
antibodies, e.g., as described in De Wildt, supra.
Diversity
[0127] Display libraries include variation at one or more positions
in the displayed polypeptide. The variation at a given position can
be synthetic or natural. For some libraries, both synthetic and
natural diversity are included.
[0128] Synthetic Diversity. Libraries can include regions of
diverse nucleic acid sequence that originate from artificially
synthesized sequences. Typically, these are formed from degenerate
oligonucleotide populations that include a distribution of
nucleotides at each given position. The inclusion of a given
sequence is random with respect to the distribution. One example of
a degenerate source of synthetic diversity is an oligonucleotide
that includes NNN wherein N is any of the four nucleotides in equal
proportion.
[0129] Synthetic diversity can also be more constrained, e.g., to
limit the number of codons in a nucleic acid sequence at a given
trinucleotide to a distribution that is smaller than NNN. For
example, such a distribution can be constructed using less than
four nucleotides at some positions of the codon. In addition,
trinucleotide addition technology can be used to further constrain
the distribution.
[0130] So-called "trinucleotide addition technology" is described,
e.g., in Wells et al. (1985) Gene 34:315-323, U.S. Pat. Nos.
4,760,025 and 5,869,644. Oligonucleotides are synthesized on a
solid phase support, one codon (i.e., trinucleotide) at a time. The
support includes many functional groups for synthesis such that
many oligonucleotides are synthesized in parallel. The support is
first exposed to a solution containing a mixture of the set of
codons for the first position. The unit is protected so additional
units are not added. The solution containing the first mixture is
washed away and the solid support is deprotected so a second
mixture containing a set of codons for a second position can be
added to the attached first unit. The process is iterated to
sequentially assemble multiple codons. Trinucleotide addition
technology enables the synthesis of a nucleic acid that at a given
position can encode a number of amino acids. The frequency of these
amino acids can be regulated by the proportion of codons in the
mixture. Further the choice of amino acids at the given position is
not restricted to quadrants of the codon table as is the case if
mixtures of single nucleotides are added during the synthesis.
[0131] Natural Diversity. Libraries can include regions of diverse
nucleic acid sequence that originate (or are synthesized based on)
from different naturally-occurring sequences. An example of natural
diversity that can be included in a display library is the sequence
diversity present in immune cells (see also below). Nucleic acids
are prepared from these immune cells and are manipulated into a
format for polypeptide display. Another example of naturally
occurring diversity is the diversity of sequences among different
species of organisms. For example, diverse nucleic acid sequences
can be amplified from environmental samples, such as soil, and used
to construct a display library.
Antibody Display Libraries
[0132] In one embodiment, the display library presents a diverse
pool of polypeptides, each of which includes an immunoglobulin
domain, e.g., an immunoglobulin variable domain. Display libraries
are particularly useful, for example for identifying human or
"humanized" antibodies that recognize human antigens. Such
antibodies can be used as therapeutics to treat human disorders
such as cancer. Since the constant and framework regions of the
antibody are human, these therapeutic antibodies may avoid
themselves being recognized and targeted as antigens. The constant
regions may also be optimized to recruit effector functions of the
human immune system. The in vitro display selection process
surmounts the inability of a normal human immune system to generate
antibodies against self-antigens. Other types of antibody
expression libraries can be used, including, e.g., protein arrays
of antibodies (see, e.g., De Wildt et al. (2000) Nat. Biotechnol.
18:989-994), lambda gt11 libraries, and so forth.
[0133] A typical antibody display library displays a polypeptide
that includes a VH domain and a VL domain. An "immunoglobulin
domain" refers to a domain from the variable or constant domain of
immunoglobulin molecules. Immunoglobulin domains typically contain
two .beta.-sheets formed of about seven .beta.-strands, and a
conserved disulphide bond (see, e.g., A. F. Williams and A. N.
Barclay 1988 Ann. Rev Immunol. 6:381-405). The display library can
display the antibody as a Fab fragment (e.g., using two polypeptide
chains) or a single chain Fv (e.g., using a single polypeptide
chain). Other formats can also be used.
[0134] As in the case of the Fab and other formats, the displayed
antibody can include one or more constant regions as part of a
light and/or heavy chain. In one embodiment, each chain includes
one constant region, e.g., as in the case of a Fab. In other
embodiments, additional constant regions are displayed.
[0135] Antibody libraries can be constructed by a number of
processes (see, e.g., de Haard et al. (1999) J. Biol. Chem
274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20. and
Hoogenboom et al. (2000) Immunol Today 21:371-8. Further, elements
of each process can be combined with those of other processes. The
processes can be used such that variation is introduced into a
single immunoglobulin domain (e.g., VH or VL) or into multiple
immunoglobulin domains (e.g., VH and VL). The variation can be
introduced into an immunoglobulin variable domain, e.g., in the
region of one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4,
referring to such regions of either and both of heavy and light
chain variable domains. In one embodiment, variation is introduced
into all three CDRs of a given variable domain. In another
preferred embodiment, the variation is introduced into CDR1 and
CDR2, e.g., of a heavy chain variable domain. Any combination is
feasible. In one process, antibody libraries are constructed by
inserting diverse oligonucleotides that encode CDRs into the
corresponding regions of the nucleic acid. The oligonucleotides can
be synthesized using monomeric nucleotides or trinucleotides. For
example, Knappik et al. (2000) J. Mol. Biol. 296:57-86 describe a
method for constructing CDR encoding oligonucleotides using
trinucleotide synthesis and a template with engineered restriction
sites for accepting the oligonucleotides.
[0136] In another process, an animal, e.g., a rodent, is immunized
with the ET2. The animal is optionally boosted with the antigen to
further stimulate the response. Then spleen cells are isolated from
the animal, and nucleic acid encoding VH and/or VL domains is
amplified and cloned for expression in the display library.
[0137] In yet another process, antibody libraries are constructed
from nucleic acid amplified from naive germline immunoglobulin
genes. The amplified nucleic acid includes nucleic acid encoding
the VH and/or VL domain. Sources of immunoglobulin-encoding nucleic
acids are described below. Amplification can include PCR, e.g.,
with primers that anneal to the conserved constant region, or
another amplification method.
[0138] Nucleic acid encoding immunoglobulin domains can be obtained
from the immune cells of, e.g., a human, a primate, mouse, rabbit,
camel, or rodent. In one example, the cells are selected for a
particular property. B cells at various stages of maturity can be
selected. In another example, the B cells are naive.
[0139] In one embodiment, fluorescent-activated cell sorting (FACS)
is used to sort B cells that express surface-bound IgM, IgD, or IgG
molecules. Further, B cells expressing different isotypes of IgG
can be isolated. In another preferred embodiment, the B or T cell
is cultured in vitro. The cells can be stimulated in vitro, e.g.,
by culturing with feeder cells or by adding mitogens or other
modulatory reagents, such as antibodies to CD40, CD40 ligand or
CD20, phorbol myristate acetate, bacterial lipopolysaccharide,
concanavalin A, phytohemagglutinin or pokeweed mitogen.
[0140] In still another embodiment, the cells are isolated from a
subject that has an immunological disorder, e.g., systemic lupus
erythematosus (SLE), rheumatoid arthritis, vasculitis, Sjogren
syndrome, systemic sclerosis, or anti-phospholipid syndrome. The
subject can be a human, or an animal, e.g., an animal model for the
human disease, or an animal having an analogous disorder. In yet
another embodiment, the cells are isolated from a transgenic
non-human animal that includes a human immunoglobulin locus.
[0141] In one preferred embodiment, the cells have activated a
program of somatic hypermutation. Cells can be stimulated to
undergo somatic mutagenesis of immunoglobulin genes, for example,
by treatment with anti-immunoglobulin, anti-CD40, and anti-CD38
antibodies (see, e.g., Bergthorsdottir et al. (2001) J Immunol.
166:2228). In another embodiment, the cells are naive.
[0142] The nucleic acid encoding an immunoglobulin variable domain
can be isolated from a natural repertoire by the following
exemplary method. First, RNA is isolated from the immune cell. Full
length (i.e., capped) mRNAs are separated (e.g. by degrading
uncapped RNAs with calf intestinal phosphatase). The cap is then
removed with tobacco acid pyrophosphatase and reverse transcription
is used to produce the cDNAs.
[0143] The reverse transcription of the first (antisense) strand
can be done in any manner with any suitable primer. See, e.g., de
Haard et al. (1999) J. Biol. Chem. 274:18218-30. The primer binding
region can be constant among different immunoglobulins, e.g., in
order to reverse transcribe different isotypes of immunoglobulin.
The primer binding region can also be specific to a particular
isotype of immunoglobulin. Typically, the primer is specific for a
region that is 3' to a sequence encoding at least one CDR. In
another embodiment, poly-dT primers may be used (and may be
preferred for the heavy-chain genes).
[0144] A synthetic sequence can be ligated to the 3' end of the
reverse transcribed strand. The synthetic sequence can be used as a
primer binding site for binding of the forward primer during PCR
amplification after reverse transcription. The use of the synthetic
sequence can obviate the need to use a pool of different forward
primers to fully capture the available diversity.
[0145] The variable domain-encoding gene is then amplified, e.g.,
using one or more rounds. If multiple rounds are used, nested
primers can be used for increased fidelity. The amplified nucleic
acid is then cloned into a display library vector.
[0146] Any method for amplifying nucleic acid sequences may be used
for amplification. Methods that maximize, and do not bias,
diversity are preferred. A variety of techniques can be used for
nucleic acid amplification. The polymerase chain reaction (PCR;
U.S. Pat. Nos. 4,683,195 and 4,683,202, Saiki, et al. (1985)
Science 230, 1350-1354) utilizes cycles of varying temperature to
drive rounds of nucleic acid synthesis. Transcription-based methods
utilize RNA synthesis by RNA polymerases to amplify nucleic acid
(U.S. Pat. No. 6,066,457; U.S. Pat. No. 6,132,997; U.S. Pat. No.
5,716,785; Sarkar et. al., Science (1989) 244: 331-34; Stofler et
al., Science (1988) 239: 491). NASBA (U.S. Pat. Nos. 5,130,238;
5,409,818; and 5,554,517) utilizes cycles of transcription,
reverse-transcription, and RnaseH-based degradation to amplify a
DNA sample. Still other amplification methods include rolling
circle amplification (RCA; U.S. Pat. Nos. 5,854,033 and 6,143,495)
and strand displacement amplification (SDA; U.S. Pat. Nos.
5,455,166 and 5,624,825).
Secondary Screening Methods
[0147] After selecting candidate display library members that bind
to a target, each candidate display library member can be further
analyzed, e.g., to further characterize its binding properties for
the target. Each candidate display library member can be subjected
to one or more secondary screening assays. The assay can be for a
binding property, a catalytic property, an inhibitory property, a
physiological property (e.g., cytotoxicity, renal clearance,
immunogenicity), a structural property (e.g., stability,
conformation, oligomerization state) or another functional
property. The same assay can be used repeatedly, but with varying
conditions, e.g., to determine pH, ionic, or thermal
sensitivities.
[0148] As appropriate, the assays can use the display library
member directly, a recombinant polypeptide produced from the
nucleic acid encoding a displayed polypeptide, or a synthetic
peptide synthesized based on the sequence of a displayed peptide.
Exemplary assays for binding properties include the following.
[0149] ELISA. Polypeptides encoded by a display library can also be
screened for a binding property using an ELISA assay. For example,
each polypeptide is contacted to a microtitre plate whose bottom
surface has been coated with the target, e.g., a limiting amount of
the target. The plate is washed with buffer to remove
non-specifically bound polypeptides. Then the amount of the
polypeptide bound to the plate is determined by probing the plate
with an antibody that can recognize the polypeptide, e.g., a tag or
constant portion of the polypeptide. The antibody is linked to an
enzyme such as alkaline phosphatase, which produces a calorimetric
product when appropriate substrates are provided. The polypeptide
can be purified from cells or assayed in a display library format,
e.g., as a fusion to a filamentous bacteriophage coat. In another
version of the ELISA assay, each polypeptide of a diversity strand
library is used to coat a different well of a microtitre plate. The
ELISA then proceeds using a constant target molecule to query each
well.
[0150] Homogeneous Binding Assays. The binding interaction of
candidate polypeptide with a target can be analyzed using a
homogenous assay, i.e., after all components of the assay are
added, additional fluid manipulations are not required. For
example, fluorescence resonance energy transfer (FRET) can be used
as a homogenous assay (see, for example, Lakowicz et al., U.S. Pat.
No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A
fluorophore label on the first molecule (e.g., the molecule
identified in the fraction) is selected such that its emitted
fluorescent energy can be absorbed by a fluorescent label on a
second molecule (e.g., the target) if the second molecule is in
proximity to the first molecule. The fluorescent label on the
second molecule fluoresces when it absorbs to the transferred
energy. Since the efficiency of energy transfer between the labels
is related to the distance separating the molecules, the spatial
relationship between the molecules can be assessed. In a situation
in which binding occurs between the molecules, the fluorescent
emission of the `acceptor` molecule label in the assay should be
maximal. A binding event that is configured for monitoring by FRET
can be conveniently measured through standard fluorometric
detection means well known in the art (e.g., using a fluorimeter).
By titrating the amount of the first or second binding molecule, a
binding curve can be generated to estimate the equilibrium binding
constant.
[0151] Another example of a homogenous assay is Alpha Screen
(Packard Bioscience, Meriden Conn.). Alpha Screen uses two labeled
beads. One bead generates singlet oxygen when excited by a laser.
The other bead generates a light signal when singlet oxygen
diffuses from the first bead and collides with it. The signal is
only generated when the two beads are in proximity. One bead can be
attached to the display library member, the other to the target.
Signals are measured to determine the extent of binding.
[0152] The homogenous assays can be performed while the candidate
polypeptide is attached to the display library vehicle, e.g., a
bacteriophage.
[0153] Surface Plasmon Resonance (SPR). The binding interaction of
a molecule isolated from a display library and a target can be
analyzed using SPR. SPR or Biomolecular Interaction Analysis (BIA)
detects biospecific interactions in real time, without labeling any
of the interactants. Changes in the mass at the binding surface
(indicative of a binding event) of the BIA chip result in
alterations of the refractive index of light near the surface (the
optical phenomenon of surface plasmon resonance (SPR)). The changes
in the refractivity generate a detectable signal, which are
measured as an indication of real-time reactions between biological
molecules. Methods for using SPR are described, for example, in
U.S. Pat. No. 5,641,640; Raether (1988) Surface Plasmons Springer
Verlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345;
Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705 and on-line
resources provide by BIAcore International AB (Uppsala,
Sweden).
[0154] Information from SPR can be used to provide an accurate and
quantitative measure of the equilibrium dissociation constant
(K.sub.d), and kinetic parameters, including K.sub.on and
K.sub.off, for the binding of a biomolecule to a target. Such data
can be used to compare different biomolecules. For example,
proteins encoded by nucleic acid selected from a library of
diversity strands can be compared to identify individuals that have
high affinity for the target or that have a slow K.sub.off. This
information can also be used to develop structure-activity
relationships (SAR). For example, the kinetic and equilibrium
binding parameters of matured versions of a parent protein can be
compared to the parameters of the parent protein. Variant amino
acids at given positions can be identified that correlate with
particular binding parameters, e.g., high affinity and slow
K.sub.off. This information can be combined with structural
modeling (e.g., using homology modeling, energy minimization, or
structure determination by x-ray crystallography or NMR). As a
result, an understanding of the physical interaction between the
protein and its target can be formulated and used to guide other
design processes.
[0155] Protein Arrays. Polypeptides identified from the display
library can be immobilized on a solid support, for example, on a
bead or an array. For a protein array, each of the polypeptides is
immobilized at a unique address on a support. Typically, the
address is a two-dimensional address. Protein arrays are described
below (see, e.g., Diagnostics).
[0156] Cellular Assays. A library of candidate polypeptides (e.g.,
previously identified by a display library or otherwise) can be
screened by transforming the library into a host cell. For example,
the library can include vector nucleic acid sequences that include
segments that encode the polypeptides and that direct expression,
e.g., such that the polypeptides are produced within the cell,
secreted from the cell, or attached to the cell surface. The cells
can be screened for polypeptides that bind to the ET2, e.g., as
detected by a change in a cellular phenotype or a cell-mediated
activity. For example, in the case of an antibody that binds to the
ET2, the activity may be cell or complement-mediated
cytotoxicity.
Automation
[0157] In one embodiment, at least some aspects of the screening
method are automated. Automated methods can be used for a high
throughput screen, e.g., to detect interactions with ET2 such as
binding interactions or enzymatic interaction (e.g., inhibition of
ET2 activity). For example, clones isolated from a primary screen
and encoding candidate ligands are stored in an arrayed format
(e.g., microtitre plates). A robotic device can be automatically
controlled to set up assays for each of the candidate ligands in a
variety of formats, e.g., ELISA (using purified ligands or phage
displaying the ligand), enzyme assays, cell based assays, and so
forth. Enzymatic activity, for example, can be detected by any of a
variety of methods, including spectroscopically, colorimetrically,
using mass spectroscopy, and so forth.
[0158] Data indicating the performance of each clone for a
particular assay, e.g., a binding assay, an activity assay, or a
cell-based assay, can be stored in database. Software can be used
to access the database and select clones that meet particular
criteria, e.g., exceed a threshold for an assay. The software can
then direct a robotic arm to pick the selected clones from the
stored array, prepare nucleic acid encoding the ligand, prepare the
ligand itself, and/or produce and screen secondary libraries whose
members are mutated variants of the initially picked ligand.
[0159] Various robotic devices that can be employed in the
automation process include multi-well plate conveyance systems,
magnetic bead particle processors, liquid handling units, colony
picking units. These devices can be built on custom specifications
or purchased from commercial sources, such as Autogen (Framingham
Mass.), Beckman Coulter (USA), Biorobotics (Woburn Mass.), Genetix
(New Milton, Hampshire UK), Hamilton (Reno Nev.), Hudson
(Springfield N.J.), Labsystems (Helsinki, Finland), Perkin Elmer
Lifesciences (Wellseley Mass.), Packard Bioscience (Meriden Conn.),
and Tecan (Mannedorf, Switzerland).
Methods for Obtaining ET2-Binding Antibodies
[0160] In addition to the use of display libraries, other methods
can be used to obtain a ET2-binding antibody. For example, the ET2
protein or a region thereof can be used as an antigen in a
non-human animal, e.g., a rodent.
[0161] In one embodiment, the non-human animal includes at least a
part of a human immunoglobulin gene. For example, it is possible to
engineer mouse strains deficient in mouse antibody production with
large fragments of the human Ig loci. Using the hybridoma
technology, antigen-specific Mabs derived from the genes with the
desired specificity may be produced and selected. See, e.g.,
XENOMOUSE.TM., Green et al. Nature Genetics 7:13-21 (1994), U.S.
2003-0070185, WO 96/34096, published Oct. 31, 1996, and PCT
Application No. PCT/US96/05928, filed Apr. 29, 1996.
[0162] In another embodiment, a monoclonal antibody is obtained
from the non-human animal, and then modified, e.g., humanized or
deimmunized. Winter describes a CDR-grafting method that may be
used to prepare the humanized antibodies of the present invention
(UK Patent Application GB 2188638A, filed on Mar. 26, 1987; U.S.
Pat. No. 5,225,539. All of the CDRs of a particular human antibody
may be replaced with at least a portion of a non-human CDR or only
some of the CDRs may be replaced with non-human CDRs. It is only
necessary to replace the number of CDRs required for binding of the
humanized antibody to a predetermined antigen.
[0163] Humanized antibodies can be generated by replacing sequences
of the Fv variable region that are not directly involved in antigen
binding with equivalent sequences from human Fv variable regions.
General methods for generating humanized antibodies are provided by
Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986,
BioTechniques 4:214, and by Queen et al. U.S. Pat. No. 5,585,089,
U.S. Pat. No. 5,693,761 and U.S. Pat. No. 5,693,762. Those methods
include isolating, manipulating, and expressing the nucleic acid
sequences that encode all or part of immunoglobulin Fv variable
regions from at least one of a heavy or light chain. Sources of
such nucleic acid are well known to those skilled in the art and,
for example, may be obtained from a hybridoma producing an antibody
against a predetermined target, as described above. The recombinant
DNA encoding the humanized antibody, or fragment thereof, can then
be cloned into an appropriate expression vector.
[0164] A ET2-binding antibody may also be modified by specific
deletion of human T cell epitopes or "deimmunization" by the
methods disclosed in WO 98/52976 and WO 00/34317, the contents of
which are specifically incorporated by reference herein. Briefly,
the heavy and light chain variable regions of an antibody can be
analyzed for peptides that bind to MHC Class II; these peptides
represent potential T-cell epitopes (as defined in WO 98/52976 and
WO 00/34317). For detection of potential T-cell epitopes, a
computer modeling approach termed "peptide threading" can be
applied, and in addition a database of human MHC class II binding
peptides can be searched for motifs present in the VH and VL
sequences, as described in WO 98/52976 and WO 00/34317. These
motifs bind to any of the 18 major MHC class II DR allotypes, and
thus constitute potential T cell epitopes. Potential T-cell
epitopes detected can be eliminated by substituting small numbers
of amino acid residues in the variable regions, or preferably, by
single amino acid substitutions. As far as possible conservative
substitutions are made, often but not exclusively, an amino acid
common at this position in human germline antibody sequences may be
used. Human germline sequences are disclosed in Tomlinson, I. A. et
al. (1992) J. Mol. Biol. 227:776-798; Cook, G. P. et al. (1995)
Immunol. Today Vol. 16 (5): 237-242; Chothia, D. et al. (1992) J.
Mol. Bio. 227:799-817. The V BASE directory provides a
comprehensive directory of human immunoglobulin variable region
sequences (compiled by Tomlinson, I. A. et al. MRC Centre for
Protein Engineering, Cambridge, UK). After the deimmunizing changes
are identified, nucleic acids encoding V.sub.H and V.sub.L can be
constructed by mutagenesis or other synthetic methods (e.g., de
novo synthesis, cassette replacement, and so forth). Mutagenized
variable sequence can, optionally, be fused to a human constant
region, e.g., human IgG1 or K constant regions.
[0165] In some cases a potential T cell epitope will include
residues which are known or predicted to be important for antibody
function. For example, potential T cell epitopes are usually biased
towards the CDRs. In addition, potential T cell epitopes can occur
in framework residues important for antibody structure and binding.
Changes to eliminate these potential epitopes will in some cases
require more scrutiny, e.g., by making and testing chains with and
without the change. Where possible, potential T cell epitopes that
overlap the CDRs were eliminated by substitutions outside the CDRs.
In some cases, an alteration within a CDR is the only option, and
thus variants with and without this substitution should be tested.
In other cases, the substitution required to remove a potential T
cell epitope is at a residue position within the framework that
might be critical for antibody binding. In these cases, variants
with and without this substitution should be tested. Thus, in some
cases several variant deimmunized heavy and light chain variable
regions were designed and various heavy/light chain combinations
tested in order to identify the optimal deimmunized antibody. The
choice of the final deimmunized antibody can then be made by
considering the binding affinity of the different variants in
conjunction with the extent of deimmunization, i.e., the number of
potential T cell epitopes remaining in the variable region.
Deimmunization can be used to modify any antibody, e.g., an
antibody that includes a non-human sequence, e.g., a synthetic
antibody, a murine antibody other non-human monoclonal antibody, or
an antibody isolated from a display library.
Germlining Antibodies
[0166] It is possible to modify an antibody that binds ET2, e.g.,
an antibody described herein, in order to make the variable regions
of the antibody more similar to one or more germline sequences. For
example, an antibody can include one, two, three or more amino acid
substitutions, e.g., in a framework or CDR region, to make it more
similar to a reference germline sequence. One exemplary germlining
method can include: identifying one or more germline sequences that
are similar (e.g., most similar in a particular database) to the
sequence of the isolated antibody. Then mutations (at the amino
acid level) can be made in the isolated antibody, either
incrementally, in combination, or both. For example, a nucleic acid
library that includes sequences encoding some or all possible
germline mutations is made. The mutated antibodies are then
evaluated, e.g., to identify an antibody that has one or more
additional germline residues relative to the isolated antibody and
that is still useful (e.g., has a functional activity). In one
embodiment, as many germline residues are introduced into an
isolated antibody as possible.
[0167] In one embodiment, mutagenesis is used to substitute or
insert one or more germline residues into a CDR region. For
example, the germline CDR residue can be from a germline sequence
that is similar (e.g., most similar) to the variable region being
modified. After mutagenesis, activity (e.g., binding or other
functional activity) of the antibody can be evaluated to determine
if the germline residue or residues are tolerated. Similar
mutagenesis can be performed in the framework regions.
[0168] Selecting a germline sequence can be performed in different
ways. For example, a germline sequence can be selected if it meets
a predetermined criteria for selectivity or similarity, e.g., at
least a certain percentage identity, e.g., at least 75, 80, 85, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity. The
selection can be performed using at least 2, 3, 5, or 10 germline
sequences. In the case of CDR1 and CDR2, identifying a similar
germline sequence can include selecting one such sequence. In the
case of CDR3, identifying a similar germline sequence can include
selecting one such sequence, but may including using two germline
sequences that separately contribute to the amino-terminal portion
and the carboxy-terminal portion. In other implementations more
than one or two germline sequences are used, e.g., to form a
consensus sequence.
[0169] In one embodiment, with respect to a particular reference
variable domain sequence, e.g., a sequence described herein, a
related variable domain sequence has at least 30, 40, 50, 60, 70,
80, 90, 95 or 100% of the CDR amino acid positions that are not
identical to residues in the reference CDR sequences, residues that
are identical to residues at corresponding positions in a human
germline sequence (i.e., an amino acid sequence encoded by a human
germline nucleic acid).
[0170] In one embodiment, with respect to a particular reference
variable domain sequence, e.g., a sequence described herein, a
related variable domain sequence has at least 30, 50, 60, 70, 80,
90 or 100% of the FR regions are identical to FR sequence from a
human germline sequence, e.g., a germline sequence related to the
reference variable domain sequence.
[0171] Accordingly, it is possible to isolate an antibody which has
similar activity to a given antibody of interest, but is more
similar to one or more germline sequences, particularly one or more
human germline sequences. For example, an antibody can be at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% identical to a
germline sequence in a region outside the CDRs (e.g., framework
regions). Further an antibody can include at least 1, 2, 3, 4, or 5
germline residues in a CDR region, the germline residue being from
a germline sequence of similar (e.g., most similar) to the variable
region being modified. Germline sequences of primary interest are
human germline sequences. The activity of the antibody (e.g., the
binding activity) can be within a factor or 100, 10, 5, 2, 0.5,
0.1, and 0.001 of the original antibody.
[0172] Exemplary germline reference sequences for Vkappa include:
O12/O2, O18/O8, A20, A30, L14, L1, L15, L4/18a, L5/L19, L8, L23,
L9, L24, L11, L12, O11/O1, A17, A1, A18, A2, A19/A3, A23, A27, A11,
L2/L16, L6, L20, L25, B3, B2, A26/A10, and A14. See, e.g.,
Tomlinson et al. (1995) EMBO J. 14(18):4628-3.
[0173] A germline reference sequence for the HC variable domain can
be based on a sequence that has particular canonical structures,
e.g., 1-3 structures in the H1 and H2 hypervariable loops. The
canonical structures of hypervariable loops of an immunoglobulin
variable domain can be inferred from its sequence, as described in
Chothia et al. (1992) J. Mol. Biol. 227:799-817; Tomlinson et al.
(1992) J. Mol. Biol. 227:776-798); and Tomlinson et al. (1995) EMBO
J. 14(18):4628-38. Exemplary sequences with a 1-3 structure
include: DP-1, DP-8, DP-12, DP-2, DP-25, DP-15, DP-7, DP-4, DP-31,
DP-32, DP-33, DP-35, DP-40, 7-2, hv3005, hv3005f3, DP-46, DP-47,
DP-58, DP-49, DP-50, DP-51, DP-53, and DP-54.
Ligand Production
[0174] Standard recombinant nucleic acid methods can be used to
express a protein ligand that binds to ET2. Generally, a nucleic
acid sequence encoding the protein ligand is cloned into a nucleic
acid expression vector. Of course, if the protein includes multiple
polypeptide chains, each chain must be cloned into an expression
vector, e.g., the same or different vectors, that are expressed in
the same or different cells.
[0175] Antibody Production. Some antibodies, e.g., Fabs, can be
produced in bacterial cells, e.g., E. coli cells. For example, if
the Fab is encoded by sequences in a phage display vector that
includes a suppressible stop codon between the display entity and a
bacteriophage protein (or fragment thereof), the vector nucleic
acid can be transferred into a bacterial cell that cannot suppress
a stop codon. In this case, the Fab is not fused to the gene III
protein and is secreted into the periplasm and/or media.
[0176] Antibodies can also be produced in eukaryotic cells. In one
embodiment, the antibodies (e.g., scFv's) are expressed in a yeast
cell such as Pichia (see, e.g., Powers et al. (2001) J Immunol
Methods. 251:123-35), Hanseula, or Saccharomyces.
[0177] In one preferred embodiment, antibodies are produced in
mammalian cells. Preferred mammalian host cells for expressing the
clone antibodies or antigen-binding fragments thereof include
Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells,
described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA
77:4216-4220, used with a DHFR selectable marker, e.g., as
described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621),
lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, COS
cells, and a cell from a transgenic animal, e.g., a transgenic
mammal. For example, the cell is a mammary epithelial cell.
[0178] In addition to the nucleic acid sequence encoding the
diversified immunoglobulin domain, the recombinant expression
vectors may carry additional sequences, such as sequences that
regulate replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017). For example, typically the selectable marker gene
confers resistance to drugs, such as G418, hygromycin or
methotrexate, on a host cell into which the vector has been
introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr.sup.- host
cells with methotrexate selection/amplification) and the neo gene
(for G418 selection).
[0179] In an exemplary system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr-CHO
cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to enhancer/promoter regulatory
elements (e.g., derived from SV40, CMV, adenovirus and the like,
such as a CMV enhancer/AdMLP promoter regulatory element or an SV40
enhancer/AdMLP promoter regulatory element) to drive high levels of
transcription of the genes. The recombinant expression vector also
carries a DHFR gene, which allows for selection of CHO cells that
have been transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are
cultured to allow for expression of the antibody heavy and light
chains and intact antibody is recovered from the culture medium.
Standard molecular biology techniques are used to prepare the
recombinant expression vector, transfect the host cells, select for
transformants, culture the host cells and recover the antibody from
the culture medium. For example, some antibodies can be isolated by
affinity chromatography with a Protein A or Protein G coupled
matrix.
[0180] For antibodies that include an Fc domain, the antibody
production system preferably synthesizes antibodies in which the Fc
region is glycosylated. For example, the Fc domain of IgG molecules
is glycosylated at asparagine 297 in the CH2 domain. This
asparagine is the site for modification with biantennary-type
oligosaccharides. It has been demonstrated that this glycosylation
is required for effector functions mediated by Fc.gamma. receptors
and complement Clq (Burton and Woof (1992) Adv. Immunol. 51:1-84;
Jefferis et al. (1998) Immunol. Rev. 163:59-76). In one embodiment,
the Fc domain is produced in a mammalian expression system that
appropriately glycosylates the residue corresponding to asparagine
297. The Fc domain can also include other eukaryotic
post-translational modifications.
[0181] Antibodies can also be produced by a transgenic animal. For
example, U.S. Pat. No. 5,849,992 describes a method of expressing
an antibody in the mammary gland of a transgenic mammal. A
transgene is constructed that includes a milk-specific promoter and
nucleic acids encoding the antibody of interest and a signal
sequence for secretion. The milk produced by females of such
transgenic mammals includes, secreted-therein, the antibody of
interest. The antibody can be purified from the milk, or for some
applications, used directly.
[0182] Generation of transgenic animals are well known in the art.
One method for producing a transgenic mouse is as follows. Briefly,
a targeting construct that encodes the antibody is microinjected
into the male pronucleus of fertilized oocytes. The oocytes are
injected into the uterus of a pseudopregnant foster mother for the
development into viable pups. Some offspring will have incorproted
the transgene.
Assay Systems for ET2 Ligands
[0183] Potential ET2 ligands can be further characterized in assays
that measure their modulatory activity toward ET2 or fragments
thereof in vitro or in vivo. For example, ET2 can be combined with
a substrate under assay conditions permitting reaction of the ET2
with the substrate. The assay is performed in the absence of the
potential ET2 ligand, and in the presence of increasing
concentrations of the potential ET2 ligand. The concentration of
ligand at which 50% of the ET2 activity is inhibited by the test
compound is the IC.sub.50 value (Inhibitory Concentration) or
EC.sub.50 (Effective Concentration) value for that compound. Within
a series or group of test ligands, those having lower IC.sub.50 or
EC.sub.50 values are considered more potent inhibitors of ET2 than
those compounds having higher IC.sub.50 or EC.sub.50 values.
Preferred ligands have an IC.sub.50 value of 100 nM or less as
measured in an in vitro assay for inhibition of ET2 activity.
[0184] The ligands can also be evaluated for selectivity toward
ET2. For example, a potential ET2 ligand can be assayed for its
potency toward ET2 and a panel of serine proteases and other
enzymes and an IC.sub.50 value or EC.sub.50 value can be determined
for each enzymatic target. In one embodiment, a compound that
demonstrates a low IC.sub.50 value or EC.sub.50 value for the ET2,
and a higher IC.sub.50 value or EC.sub.50 value for other enzymes
within the test panel (e.g., urokinase, tissue plasminogen
activator, thrombin, Factor Xa) is considered to be selective
toward ET2. In one embodiment, a compound that demonstrates a low
IC.sub.50 value or EC.sub.50 value for the ET2, and a higher
IC.sub.50 value or EC.sub.50 value for ET1 than ET2 is considered
to be selective toward ET2.
[0185] Potential ET2 ligands can also be evaluated for their
activity in vivo. For example, to evaluate the activity of a ligand
to reduce tumor growth through inhibition of endotheliase, the
procedures described by Jankun et al., Canc. Res., 57: 559-563
(1997) to evaluate PAI-1 can be employed. Briefly, the ATCC cell
lines DU145 and LnCaP are injected into SCID mice. After tumors are
established, the mice are administered the test ligand. Tumor
volume measurements are taken twice a week for about five weeks. A
ligand can be deemed active in this assay if an animal to which the
ligand was administered exhibited decreased tumor volume, as
compared to animals receiving appropriate control compounds (e.g.,
non-specific antibody molecules).
[0186] To evaluate the ability of a ligand to reduce the occurrence
of, or inhibit, metastasis, the procedures described by Kobayashi
et al., Int. J. Canc., 57: 727-733d (1994) can be employed.
Briefly, a murine xenograft selected for high lung colonization
potential in injected into C57B1/6 mice i.v. (experimental
metastasis) or s.c. into the abdominal wall (spontaneous
metastasis). Various concentrations of the compound to be tested
can be admixed with the tumor cells in Matrigel prior to injection.
Daily i.p. injections of the test compound are made either on days
1-6 or days 7-13 after tumor inoculation. The animals are
sacrificed about three or four weeks after tumor inoculation, and
the lung tumor colonies are counted. Evaluation of the resulting
data permits a determination as to efficacy of the test compound,
optimal dosing and route of administration.
[0187] The activity of the ligands toward decreasing tumor volume
and metastasis can be evaluated in model described in Rabbani et
al., Int. J. Cancer 63: 840-845 (1995). See also Xing et al., Canc.
Res., 57: 3585-3593 (1997). There, Mat LyLu tumor cells were
injected into the flank of Copenhagen rats. The animals were
implanted with osmotic minipumps to continuously administer various
doses of test compound for up to three weeks. The tumor mass and
volume of experimental and control animals were evaluated during
the experiment, as were metastatic growths. Evaluation of the
resulting data permits a determination as to efficacy of the test
compound, optimal dosing, and route of administration. Some of
these authors described a related protocol in Xing et al., Canc.
Res., 57: 3585-3593 (1997).
[0188] To evaluate the inhibitory activity of a ligand toward
neovascularization, a rabbit cornea neovascularization model can be
employed. See, e.g., Avery et al., Arch. Opthalmol., 108: 1474-1475
(1990). In this model, New Zealand albino rabbits are anesthetized.
A central corneal incision is made, forming a radial corneal
pocket. A slow release prostaglandin pellet is placed in the pocket
to induce neovascularization. The test ligand is administered i. p.
for five days, then the animals are sacrificed. The effect of the
test ligand is evaluated by review of periodic photographs taken of
the limbus, which can be used to calculate the area of neovascular
response and, therefore, limbal neovascularization. A decreased
area of neovascularization as compared with appropriate controls
indicates the test ligand was effective at decreasing or inhibiting
neovascularization.
[0189] An exemplary angiogenesis model used to evaluate the effect
of a test compound in preventing angiogenesis is described by Min
et al., Canc. Res., 56: 2428-2433 (1996). In this model, C57BL6
mice receive subcutaneous injections of a Matrigel mixture
containing bFGF, as the angiogenesis-inducing agent, with and
without the test ligand. After five days, the animals are
sacrificed and the Matrigel plugs, in which neovascularization can
be visualized, are photographed. An experimental animal receiving
Matrigel and an effective dose of test ligand will exhibit less
vascularization than a control animal or an experimental animal
receiving a less- or non-effective does of ligand.
[0190] An in vivo system designed to test compound for their
ability to limit the spread of primary tumors is described by
Crowley et al., Proc. Natl. Acad. Sci., 90: 5021-5025 (1993). Nude
mice are injected with tumor cells (PC3) engineered to express CAT
(chloramphenicol acetyltransferase). Compounds to be tested for
their ability to decrease tumor size and/or metastases are
administered to the animals, and subsequent measurements of tumor
size and/or metastatic growths are made. In addition, the level of
CAT detected in various organs provides an indication of the
ability of the test compound to inhibit metastasis; detection of
less CAT in tissues of a treated animal versus a control animal
indicates less CAT-expressing cells migrated to that tissue.
[0191] In vivo experimental modes designed to evaluate the
inhibitory potential of a test serine protease inhibitors, using a
tumor cell line F311, are described by Alonso et al., Breast Canc.
Res. Treat., 40:209-223 (1996). This group describes in vivo
studies for toxicity determination, tumor growth, invasiveness,
spontaneous metastasis, experimental lung metastasis, and an
angiogenesis assay.
[0192] The CAM model (chick embryo chorioallantoic membrane model),
first described by L. Ossowski (J. Cell. Biol., 107: 2437-2445
(1988)), provides another method for evaluating the protease
inhibitory activity of a test compound. In the CAM model, tumor
cells invade through the chorioallantoic membrane containing CAM
with tumor cells in the presence of several serine protease
inhibitors results in less or no invasion of the tumor cells
through the membrane. Thus, the CAM assay is performed with CAM and
tumor cells in the presence and absence of various concentrations
of test compound. The invasiveness of tumor cells is measured under
such conditions to provide an indication of the compound's
inhibitory activity. A compound having inhibitory activity
correlates with less tumor invasion.
[0193] The CAM model is also used in to assay angiogenesis (i.e.,
effect on formation of new blood vessels (Brooks et al., Methods in
Molecular Biology, 129: 257-269 (1999)). According to this model, a
filter disc containing an angiogenesis inducer, such as basic
fibroblast growth factor (bFDG) is placed onto the CAM. Diffusion
of the cytokine into the CAM induces local angiogenesis, which may
be measured in several ways such as by counting the number of blood
vessel branch points within the CAM directly below the filter disc.
The ability of identified compounds to inhibit cytokine-induced
angiogenesis can be tested using this model. A test compound can
either be added to the filter disc that contains the angiogenesis
inducer, be placed directly on the membrane or be administered
systemically. The extent of new blood vessel formation in the
presence and/or absence of test compound can be compared using this
model. The formation of fewer new blood vessels in the presence of
a test compound would be indicative of anti-angiogenesis
activity.
[0194] Endothelial cell proliferation. A candidate ET2-binding
ligand can be tested for endothelial proliferation inhibiting
activity using a biological activity assay such as the bovine
capillary endothelial cell proliferation assay, the chick CAM
assay, the mouse corneal assay, and evaluating the effect of the
ligand on implanted tumors. The chick CAM assay is described, e.g.,
by O'Reilly, et al. in "Angiogenic Regulation of Metastatic Growth"
Cell, vol. 79 (2), Oct. 21, 1994, pp. 315-328. Briefly, three day
old chicken embryos with intact yolks are separated from the egg
and placed in a petri dish. After three days of incubation a
methylcellulose disc containing the protein to be tested is applied
to the CAM of individual embryos. After 48 hours of incubation, the
embryos and CAMs are observed to determine whether endothelial
growth has been inhibited. The mouse corneal assay involves
implanting a growth factor-containing pellet, along with another
pellet containing the suspected endothelial growth inhibitor, in
the cornea of a mouse and observing the pattern of capillaries that
are elaborated in the cornea.
[0195] Angiogenesis. Angiogenesis may be assayed, e.g., using
various human endothelial cell systems, such as umbilical vein,
coronary artery, or dermal cells. Suitable assays include Alamar
Blue based assays (available from Biosource International) to
measure proliferation; migration assays using fluorescent
molecules, such as the use of Becton Dickinson Falcon HTS
FluoroBlock cell culture inserts to measure migration of cells
through membranes in presence or absence of angiogenesis enhancer
or suppressors; and tubule formation assays based on the formation
of tubular structures by endothelial cells on MATRIGEL.TM. (Becton
Dickinson).
[0196] Cell adhesion. Cell adhesion assays measure adhesion of
cells to purified adhesion proteins or adhesion of cells to each
other, in presence or absence of candidate ET2 binding ligands.
Cell-protein adhesion assays measure the ability of agents to
modulate the adhesion of cells to purified proteins. For example,
recombinant proteins are produced, diluted to 2.5 g/mL in PBS, and
used to coat the wells of a microtiter plate. The wells used for
negative control are not coated. Coated wells are then washed,
blocked with 1% BSA, and washed again. Compounds are diluted to
2.times.final test concentration and added to the blocked, coated
wells. Cells are then added to the wells, and the unbound cells are
washed off. Retained cells are labeled directly on the plate by
adding a membrane-permeable fluorescent dye, such as calcein-AM,
and the signal is quantified in a fluorescent microplate
reader.
[0197] Cell-cell adhesion assays can be used to measure the ability
of candidate ET2 binding ligands to modulate binding of cells to
each other. These assays can use cells that naturally or
recombinantly express an adhesion protein of choice. In an
exemplary assay, cells expressing the cell adhesion protein are
plated in wells of a multiwell plate together with other cells
(either more of the same cell type, or another type of cell to
which the cells adhere). The cells that can adhere are labeled with
a membrane-permeable fluorescent dye, such as BCECF, and allowed to
adhere to the monolayers in the presence of candidate ligands.
Unbound cells are washed off, and bound cells are detected using a
fluorescence plate reader. High-throughput cell adhesion assays
have also been described. See, e.g., Falsey J R et al., Bioconjug
Chem. May-June 2001; 12(3):346-53.
[0198] Tubulogenesis. Tubulogenesis assays can be used to monitor
the ability of cultured cells, generally endothelial cells, to form
tubular structures on a matrix substrate, which generally simulates
the environment of the extracellular matrix. Exemplary substrates
include MATRIGEL.TM. (Becton Dickinson), an extract of basement
membrane proteins containing laminin, collagen IV, and heparin
sulfate proteoglycan, which is liquid at 4.degree. C. and forms a
solid gel at 37.degree. C. Other suitable matrices comprise
extracellular components such as collagen, fibronectin, and/or
fibrin. Cells are stimulated with a pro-angiogenic stimulant, and
their ability to form tubules is detected by imaging. Tubules can
generally be detected after an overnight incubation with stimuli,
but longer or shorter time frames may also be used. Tube formation
assays are well known in the art (e.g., Jones M K et al., 1999,
Nature Medicine 5:1418-1423). These assays have traditionally
involved stimulation with serum or with the growth factors FGF or
VEGF. In one embodiment, the assay is performed with cells cultured
in serum free medium. In one embodiment, the assay is performed in
the presence of one or more pro-angiogenic agents, e.g.,
inflammatory angiogenic factors, such as TNF-.alpha., FGF, VEGF,
phorbol myristate acetate (PMA), TNF-alpha, ephrin, etc.
[0199] Cell Migration. An exemplary assay for endothelial cell
migration is the human microvascular endothelial (HMVEC) migration
assay. See, e.g., Tolsma et al. (1993) J. Cell Biol 122, 497-511.
Migration assays are known in the art (e.g., Paik J H et al., 2001,
J Biol Chem 276:11830-11837). In one example, cultured endothelial
cells are seeded onto a matrix-coated porous lamina, with pore
sizes generally smaller than typical cell size. The lamina is
typically a membrane, such as the transwell polycarbonate membrane
(Corning Costar Corporation, Cambridge, Mass.), and is generally
part of an upper chamber that is in fluid contact with a lower
chamber containing pro-angiogenic stimuli. Migration is generally
assayed after an overnight incubation with stimuli, but longer or
shorter time frames may also be used. Migration is assessed as the
number of cells that crossed the lamina, and may be detected by
staining cells with hemotoxylin solution (VWR Scientific.), or by
any other method for determining cell number. In another exemplary
set up, cells are fluorescently labeled and migration is detected
using fluorescent readings, for instance using the Falcon HTS
FluoroBlok (Becton Dickinson). While some migration is observed in
the absence of stimulus, migration is greatly increased in response
to pro-angiogenic factors. The assay can be used to test the effect
of a ET2-binding ligand on endothelial cell migration.
[0200] Sprouting assay. An exemplary sprouting assay is a
three-dimensional in vitro angiogenesis assay that uses a
cell-number defined spheroid aggregation of endothelial cells
("spheroid"), embedded in a collagen gel-based matrix. The spheroid
can serve as a starting point for the sprouting of capillary-like
structures by invasion into the extracellular matrix (termed "cell
sprouting") and the subsequent formation of complex anastomosing
networks (Korff and Augustin, 1999, J Cell Sci 112:3249-58). In an
exemplary experimental set-up, spheroids are prepared by pipetting
400 human umbilical vein endothelial cells (HUMVECs) into
individual wells of a nonadhesive 96-well plates to allow overnight
spheroidal aggregation (Korff and Augustin, J Cell Biol 143:
1341-52, 1998). Spheroids are harvested and seeded in 900 .mu.l of
methocel-collagen solution and pipetted into individual wells of a
24 well plate to allow collagen gel polymerization. Test agents are
added after 30 min by pipetting 100 .mu.l of 10-fold concentrated
working dilution of the test substances on top of the gel. Plates
are incubated at 37.degree. C. for 24 h. Dishes are fixed at the
end of the experimental incubation period by addition of
paraformaldehyde. Sprouting intensity of endothelial cells can be
quantitated by an automated image analysis system to determine the
cumulative sprout length per spheroid.
[0201] In some embodiments, an ET2 binding ligand has a
statistically significant effect in an assay described herein,
e.g., a cellular assay described herein.
Pharmaceutical Compositions
[0202] In another aspect, the present invention provides
compositions, e.g., pharmaceutically acceptable compositions, which
include an ET2-ligand, e.g., an antibody molecule, other
polypeptide or peptide identified as binding to ET2, or described
herein, formulated together with a pharmaceutically acceptable
carrier. As used herein, "pharmaceutical compositions" encompass
labeled ligands for in vivo imaging as well as therapeutic
compositions.
[0203] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Preferably, the carrier is suitable for intravenous, intramuscular,
subcutaneous, parenteral, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of
administration, the active compound, i.e., protein ligand may be
coated in a material to protect the compound from the action of
acids and other natural conditions that may inactivate the
compound.
[0204] A "pharmaceutically acceptable salt" refers to a salt that
retains the desired biological activity of the parent compound and
does not impart any undesired toxicological effects (see e.g.,
Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of
such salts include acid addition salts and base addition salts.
Acid addition salts include those derived from nontoxic inorganic
acids, such as hydrochloric, nitric, phosphoric, sulfuric,
hydrobromic, hydroiodic, phosphorous and the like, as well as from
nontoxic organic acids such as aliphatic mono- and dicarboxylic
acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,
aromatic acids, aliphatic and aromatic sulfonic acids and the like.
Base addition salts include those derived from alkaline earth
metals, such as sodium, potassium, magnesium, calcium and the like,
as well as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0205] The compositions of this invention may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends on
the intended mode of administration and therapeutic application.
Typical preferred compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
administration of humans with antibodies. The preferred mode of
administration is parenteral (e.g., intravenous, subcutaneous,
intraperitoneal, intramuscular). In one embodiment, the ET2-ligand
is administered by intravenous infusion or injection. In another
preferred embodiment, the ET2-ligand is administered by
intramuscular or subcutaneous injection.
[0206] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0207] Pharmaceutical compositions typically must be sterile and
stable under the conditions of manufacture and storage. A
pharmaceutical composition can also be tested to insure it meets
regulatory and industry standards for administration. For example,
endotoxin levels in the preparation can be tested using the Limulus
amebocyte lysate assay (e.g., using the kit from Bio Whittaker lot
# 7L3790, sensitivity 0.125 EU/mL) according to the USP 24/NF 19
methods. Sterility of pharmaceutical compositions can be determined
using thioglycollate medium according to the USP 24/NF 19 methods.
For example, the preparation is used to inoculate the
thioglycollate medium and incubated at 35.degree. C. for 14 or more
days. The medium is inspected periodically to detect growth of a
microorganism.
[0208] The composition can be formulated as a solution,
microemulsion, dispersion, liposome, or other ordered structure
suitable to high drug concentration. Sterile injectable solutions
can be prepared by incorporating the active compound (i.e., the
ligand) in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of
sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying that yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin.
[0209] The anti-ET2 protein ligands of the present invention can be
administered by a variety of methods known in the art, although for
many applications, the preferred route/mode of administration is
intravenous injection or infusion. For example, for therapeutic
applications, the ET2-ligand can be administered by intravenous
infusion at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach
a dose of about 1 to 100 mg/m.sup.2 or 7 to 25 mg/m.sup.2. The
route and/or mode of administration will vary depending upon the
desired results. In certain embodiments, the active compound may be
prepared with a carrier that will protect the compound against
rapid release, such as a controlled release formulation, including
implants, and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known. See, e.g., Sustained
and Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978.
[0210] In certain embodiments, the ligand may be orally
administered, for example, with an inert diluent or an assimilable
edible carrier. The compound (and other ingredients, if desired)
may also be enclosed in a hard or soft shell gelatin capsule,
compressed into tablets, or incorporated directly into the
subject's diet. For oral therapeutic administration, the compounds
may be incorporated with excipients and used in the form of
ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, and the like. To administer a compound
of the invention by other than parenteral administration, it may be
necessary to coat the compound with, or co-administer the compound
with, a material to prevent its inactivation.
[0211] Pharmaceutical compositions can be administered with medical
devices known in the art. For example, in one embodiment, a
pharmaceutical composition of the invention can be administered
with a needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335,
5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of
well-known implants and modules useful in the present invention
include: U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Pat. No. 4,486,194, which discloses a therapeutic device for
administering medicants through the skin; U.S. Pat. No. 4,447,233,
which discloses a medication infusion pump for delivering
medication at a precise infusion rate; U.S. Pat. No. 4,447,224,
which discloses a variable flow implantable infusion apparatus for
continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses
an osmotic drug delivery system having multi-chamber compartments;
and U.S. Pat. No. 4,475,196, which discloses an osmotic drug
delivery system. Of course, many other such implants, delivery
systems, and modules are also known.
[0212] In certain embodiments, the compounds of the invention can
be formulated to ensure proper distribution in vivo. For example,
the blood-brain barrier (BBB) excludes many highly hydrophilic
compounds. To ensure that the therapeutic compounds of the
invention cross the BBB (if desired), they can be formulated, for
example, in liposomes. For methods of manufacturing liposomes, see,
e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The
liposomes may comprise one or more moieties that are selectively
transported into specific cells or organs, thus enhance targeted
drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol.
29:685).
[0213] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0214] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody of the invention
is 0.1-20 mg/kg, more preferably 1-10 mg/kg. The anti-ET2 antibody
can be administered by intravenous infusion at a rate of less than
30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100
mg/m.sup.2 or about 5 to 30 mg/m.sup.2. For ligands smaller in
molecular weight than an antibody, appropriate amounts can be
proportionally less. It is to be noted that dosage values may vary
with the type and severity of the condition to be alleviated. It is
to be further understood that for any particular subject, specific
dosage regimens should be adjusted over time according to the
individual need and the professional judgment of the person
administering or supervising the administration of the
compositions, and that dosage ranges set forth herein are exemplary
only and are not intended to limit the scope or practice of the
claimed composition.
[0215] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of an ET2-ligand of the invention. A
"therapeutically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired therapeutic result. A therapeutically effective amount of
the composition may vary according to factors such as the disease
state, age, sex, and weight of the individual, and the ability of
the protein ligand to elicit a desired response in the individual.
A therapeutically effective amount is also one in which any toxic
or detrimental effects of the composition is outweighed by the
therapeutically beneficial effects. A "therapeutically effective
dosage" preferably inhibits a measurable parameter, e.g., tumor
growth rate by at least about 20%, more preferably by at least
about 40%, even more preferably by at least about 60%, and still
more preferably by at least about 80% relative to untreated
subjects. The ability of a compound to inhibit a measurable
parameter, e.g., cancer, can be evaluated in an animal model system
predictive of efficacy in human tumors. Alternatively, this
property of a composition can be evaluated by examining the ability
of the compound to inhibit, such inhibition in vitro by assays
known to the skilled practitioner.
[0216] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically, since a prophylactic
dose is used in subjects prior to or at an earlier stage of
disease, the prophylactically effective amount will be less than
the therapeutically effective amount.
[0217] Also within the scope of the invention are kits comprising
the protein ligand that binds to ET2 and instructions for use,
e.g., treatment, prophylactic, or diagnostic use. In one
embodiment, the instructions for diagnostic applications include
the use of the ET2-ligand (e.g., antibody or antigen-binding
fragment thereof, or other polypeptide or peptide) to detect ET2,
in vitro, e.g., in a sample, e.g., a biopsy or cells from a patient
having a cancer or neoplastic disorder, or in vivo. In another
embodiment, the instructions for therapeutic applications include
suggested dosages and/or modes of administration in a patient with
a cancer or neoplastic disorder. The kit can further contain a
least one additional reagent, such as a diagnostic or therapeutic
agent, e.g., a diagnostic or therapeutic agent as described herein,
and/or one or more additional ET2-ligands, formulated as
appropriate, in one or more separate pharmaceutical
preparations.
Stabilization and Retention
[0218] In one embodiment, an ET2-ligand is physically associated
with a moiety that improves its stabilization and/or retention in
circulation, e.g., in blood, serum, lymph, or other tissues, e.g.,
by at least 1.5, 2, 5, 10, or 50 fold.
[0219] For example, an ET2-ligand can be associated with a polymer,
e.g., a substantially non-antigenic polymers, such as polyalkylene
oxides or polyethylene oxides. Suitable polymers will vary
substantially by weight. Polymers having molecular number average
weights ranging from about 200 to about 35,000 (or about 1,000 to
about 15,000, and 2,000 to about 12,500) can be used.
[0220] For example, an ET2-ligand can be conjugated to a water
soluble polymer, e.g., hydrophilic polyvinyl polymers, e.g.
polyvinylalcohol and polyvinylpyrrolidone. A non-limiting list of
such polymers include polyalkylene oxide homopolymers such as
polyethylene glycol (PEG) or polypropylene glycols,
polyoxyethylenated polyols, copolymers thereof and block copolymers
thereof, provided that the water solubility of the block copolymers
is maintained. Additional useful polymers include polyoxyalkylenes
such as polyoxyethylene, polyoxypropylene, and block copolymers of
polyoxyethylene and polyoxypropylene (Pluronics);
polymethacrylates; carbomers; branched or unbranched
polysaccharides which comprise the saccharide monomers D-mannose,
D- and L-galactose, fucose, fructose, D-xylose, L-arabinose,
D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic
acid (e.g. polymannuronic acid, or alginic acid), D-glucosamine,
D-galactosamine, D-glucose and neuraminic acid including
homopolysaccharides and heteropolysaccharides such as lactose,
amylopectin, starch, hydroxyethyl starch, amylose, dextrane
sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit
of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of
sugar alcohols such as polysorbitol and polymannitol; heparin or
heparon.
[0221] Other compounds can also be attached to the same polymer,
e.g., a cytotoxin, a label, or another targeting agent, e.g.,
another ET2-ligand or an unrelated ligand. Mono-activated,
alkoxy-terminated polyalkylene oxides (PAO's), e.g.,
monomethoxy-terminated polyethylene glycols (mPEG's); C.sub.1-4
alkyl-terminated polymers; and bis-activated polyethylene oxides
(glycols) can be used for crosslinking. See, e.g., U.S. Pat. No.
5,951,974
[0222] In one embodiment, the polymer prior to cross-linking to the
ligand need not be, but preferably is, water soluble. Generally,
after crosslinking, the product is water soluble, e.g., exhibits a
water solubility of at least about 0.01 mg/ml, and more preferably
at least about 0.1 mg/ml, and still more preferably at least about
1 mg/ml. In addition, the polymer should not be highly immunogenic
in the conjugate form, nor should it possess viscosity that is
incompatible with intravenous infusion or injection if the
conjugate is intended to be administered by such routes.
[0223] In one embodiment, the polymer contains only a single group
which is reactive. This helps to avoid cross-linking of ligand
molecules to one another. However, it is within the scope herein to
maximize reaction conditions to reduce cross-linking between ligand
molecules, or to purify the reaction products through gel
filtration or ion exchange chromatography to recover substantially
homogenous derivatives. In other embodiments, the polymer contains
two or more reactive groups for the purpose of linking multiple
ligands to the polymer backbone. Again, gel filtration or ion
exchange chromatography can be used to recover the desired
derivative in substantially homogeneous form.
[0224] The molecular weight of the polymer can range up to about
500,000 D, and preferably is at least about 20,000 D, or at least
about 30,000 D, or at least about 40,000 D. The molecular weight
chosen can depend upon the effective size of the conjugate to be
achieved, the nature (e.g. structure, such as linear or branched)
of the polymer, and the degree of derivatization.
[0225] A covalent bond can be used to attach an ET2-ligand to a
polymer, for example, crosslinking to the N-terminal amino group of
the ligand and epsilon amino groups found on lysine residues of the
ligand, as well as other amino, imino, carboxyl, sulfhydryl,
hydroxyl or other hydrophilic groups. The polymer may be covalently
bonded directly to the ET2-ligand without the use of a
multifunctional (ordinarily bifunctional) crosslinking agent.
Covalent binding to amino groups is accomplished by known
chemistries based upon cyanuric chloride, carbonyl diimidazole,
aldehyde reactive groups (PEG alkoxide plus diethyl acetal of
bromoacetaldehyde; PEG plus DMSO and acetic anhydride, or PEG
chloride plus the phenoxide of 4-hydroxybenzaldehyde, activated
succinimidyl esters, activated dithiocarbonate PEG,
2,4,5-trichlorophenylcloroformate or P-nitrophenylcloroformate
activated PEG.) Carboxyl groups can be derivatized by coupling
PEG-amine using carbodiimide. Sulfhydryl groups can be derivatized
by coupling to maleimido-substituted PEG (e.g. alkoxy-PEG amine
plus sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate) WO 97/10847 or
PEG-maleimide commercially available from Shearwater Polymers,
Inc., Huntsville, Ala.). Alternatively, free amino groups on the
ligand (e.g. epsilon amino groups on lysine residues) can be
thiolated with 2-imino-thiolane (Traut's reagent) and then coupled
to maleimide-containing derivatives of PEG, e.g., as described in
Pedley et al., Br. J. Cancer, 70: 1126-1130 (1994).
[0226] Functionalized PEG polymers that can be attached to an
ET2-ligand are available, e.g., from Shearwater Polymers, Inc.
(Huntsville, Ala.). Such commercially available PEG derivatives
include, e.g., amino-PEG, PEG amino acid esters, PEG-hydrazide,
PEG-thiol, PEG-succinate, carboxymethylated PEG, PEG-propionic
acid, PEG amino acids, PEG succinimidyl succinate, PEG succinimidyl
propionate, succinimidyl ester of carboxymethylated PEG,
succinimidyl carbonate of PEG, succinimidyl esters of amino acid
PEGs, PEG-oxycarbonylimidazole, PEG-nitrophenyl carbonate, PEG
tresylate, PEG-glycidyl ether, PEG-aldehyde, PEG vinylsulfone,
PEG-maleimide, PEG-orthopyridyl-disulfide, heterofunctional PEGs,
PEG vinyl derivatives, PEG silanes, and PEG phospholides. The
reaction conditions for coupling these PEG derivatives may vary
depending on the ET2-ligand, the desired degree of PEGylation, and
the PEG derivative utilized. Some factors involved in the choice of
PEG derivatives include: the desired point of attachment (such as
lysine or cysteine R-groups), hydrolytic stability and reactivity
of the derivatives, stability, toxicity and antigenicity of the
linkage, suitability for analysis, etc. Specific instructions for
the use of any particular derivative are available from the
manufacturer.
[0227] The conjugates of an ET2-ligand and a polymer can be
separated from the unreacted starting materials, e.g., by gel
filtration or ion exchange chromatography, e.g., HPLC. Heterologous
species of the conjugates are purified from one another in the same
fashion. Resolution of different species (e.g. containing one or
two PEG residues) is also possible due to the difference in the
ionic properties of the unreacted amino acids. See, e.g., WO
96/34015.
Kits
[0228] An ET2 ligand described herein can be provided in a kit,
e.g., as a component of a kit. For example, the kit includes (a) an
ET2 ligand, e.g., a composition that includes an ET2 ligand, and,
optionally (b) informational material. The informational material
can be descriptive, instructional, marketing or other material that
relates to the methods described herein and/or the use of an ET2
ligand for the methods described herein.
[0229] The informational material of the kits is not limited in its
form. In one embodiment, the informational material can include
information about production of the compound, molecular weight of
the compound, concentration, date of expiration, batch or
production site information, and so forth. In one embodiment, the
informational material relates to using the ligand to treat,
prevent, or diagnosis a disorder described herein, e.g., an
angiogenesis or an endothelial-cell related disorder.
[0230] In one embodiment, the informational material can include
instructions to administer an ET2 ligand in a suitable manner to
perform the methods described herein, e.g., in a suitable dose,
dosage form, or mode of administration (e.g., a dose, dosage form,
or mode of administration described herein). In another embodiment,
the informational material can include instructions to administer
an ET2 ligand to a suitable subject, e.g., a human, e.g., a human
having, or at risk for, increased angiogenesis (e.g., cancer or
metastatic cancer. For example, the material can include
instructions to administer an ET2 ligand to a cancer patient, a
patient with an inflammatory disorder, or a patient with excessive
endothelial cell activity.
[0231] The informational material of the kits is not limited in its
form. In many cases, the informational material, e.g.,
instructions, is provided in printed matter, e.g., a printed text,
drawing, and/or photograph, e.g., a label or printed sheet.
However, the informational material can also be provided in other
formats, such as computer readable material, video recording, or
audio recording. In another embodiment, the informational material
of the kit is contact information, e.g., a physical address, email
address, website, or telephone number, where a user of the kit can
obtain substantive information about an ET2 ligand and/or its use
in the methods described herein. Of course, the informational
material can also be provided in any combination of formats.
[0232] In addition to an ET2 ligand, the composition of the kit can
include other ingredients, such as a solvent or buffer, a
stabilizer, a preservative, a flavoring agent (e.g., a bitter
antagonist or a sweetener), a fragrance or other cosmetic
ingredient, and/or a second agent for treating a condition or
disorder described herein, e.g., cancer or inflammation.
Alternatively, the other ingredients can be included in the kit,
but in different compositions or containers than an ET2 ligand. In
such embodiments, the kit can include instructions for admixing an
ET2 ligand and the other ingredients, or for using an ET2 ligand
together with the other ingredients.
[0233] An ET2 ligand can be provided in any form, e.g., liquid,
dried or lyophilized form. It is preferred that an ET2 ligand be
substantially pure and/or sterile. When an ET2 ligand is provided
in a liquid solution, the liquid solution preferably is an aqueous
solution, with a sterile aqueous solution being preferred. When an
ET2 ligand is provided as a dried form, reconstitution generally is
by the addition of a suitable solvent. The solvent, e.g., sterile
water or buffer, can optionally be provided in the kit.
[0234] The kit can include one or more containers for the
composition containing an ET2 ligand. In some embodiments, the kit
contains separate containers, dividers or compartments for the
composition and informational material. For example, the
composition can be contained in a bottle, vial, or syringe, and the
informational material can be contained in a plastic sleeve or
packet. In other embodiments, the separate elements of the kit are
contained within a single, undivided container. For example, the
composition is contained in a bottle, vial or syringe that has
attached thereto the informational material in the form of a label.
In some embodiments, the kit includes a plurality (e.g., a pack) of
individual containers, each containing one or more unit dosage
forms (e.g., a dosage form described herein) of an ET2 ligand. For
example, the kit includes a plurality of syringes, ampules, foil
packets, or blister packs, each containing a single unit dose of an
ET2 ligand. The containers of the kits can be air tight, waterproof
(e.g., impermeable to changes in moisture or evaporation), and/or
light-tight.
[0235] The kit optionally includes a device suitable for
administration of the composition, e.g., a syringe, inhalant,
pipette, forceps, measured spoon, dropper (e.g., eye dropper), swab
(e.g., a cotton swab or wooden swab), or any such delivery device.
In a preferred embodiment, the device is an implantable device that
dispenses metered doses of the ligand.
Treatments
[0236] Protein ligands that bind to ET2 and identified by the
method described herein and/or detailed herein have therapeutic and
prophylactic utilities. For example, these ligands can be
administered to cells in culture, e.g. in vitro or ex vivo, or in a
subject, e.g., in vivo, to treat, prevent, and/or diagnose a
variety of disorders, such as diseases characterized by unwanted
angiogenesis, e.g., cancers.
[0237] As used herein, the term "treat" or "treatment" is defined
as the application or administration of an anti-ET2 antibody, alone
or in combination with, a second agent to a subject, e.g., a
patient, or application or administration of the agent to an
isolated tissue or cell, e.g., cell line, from a subject, e.g., a
patient, who has a disorder (e.g., a disorder as described herein),
a symptom of a disorder or a predisposition toward a disorder, with
the purpose to cure, heal, alleviate, relieve, alter, remedy,
ameliorate, improve or affect the disorder, the symptoms of the
disorder or the predisposition toward the disorder. Treating a cell
refers to the inhibition, ablation or killing of a cell in vitro or
in vivo, or otherwise reducing capacity of a cell, e.g., an
aberrant cell, to mediate a disorder, e.g., a disorder as described
herein (e.g., a cancerous disorder). In one embodiment, "treating a
cell" refers to a reduction in the activity and/or proliferation of
a cell, e.g., a hyperproliferative cell. Such reduction does not
necessarily indicate a total elimination of the cell, but a
reduction, e.g., a statistically significant reduction, in the
activity or the growth rate of the cell.
[0238] As used herein, an amount of an ET2-ligand effective to
treat a disorder, or a "therapeutically effective amount" refers to
an amount of the ligand which is effective, upon single or multiple
dose administration to a subject, in treating a cell, e.g., a
cancer cell (e.g., a ET2-expressing cancer cell), or in prolonging
life of, curing, alleviating, relieving or improving the condition
of a subject with a disorder as described herein beyond that
expected in the absence of such treatment. As used herein,
"inhibiting the growth" of the neoplasm refers to slowing,
interrupting, arresting or stopping its growth and metastases and
does not necessarily indicate a total elimination of the neoplastic
growth.
[0239] As used herein, an amount of an ET2-ligand effective to
prevent a disorder, or a "a prophylactically effective amount" of
the ligand refers to an amount of an ET2-ligand, e.g., an anti-ET2
antibody described herein, which is effective, upon single- or
multiple-dose administration to the subject, in preventing or
delaying the occurrence of the onset or recurrence of a disorder,
e.g., a cancer.
[0240] The terms "induce", "inhibit", "potentiate", "elevate",
"increase", "decrease" or the like, e.g., which denote quantitative
differences between two states, refer to a difference, e.g., a
statistically significant difference, between the two states. For
example, "an amount effective to inhibit the proliferation of the
ET2-expressing hyperproliferative cells" means that the rate of
growth of the cells will be different, e.g., statistically
significantly different, from the untreated cells.
[0241] As used herein, the term "subject" is intended to include
human and non-human animals. Preferred human animals include a
human patient having a disorder characterized by abnormal cell
proliferation or cell differentiation. The term "non-human animals"
of the invention includes all vertebrates, e.g., non-mammals (such
as chickens, amphibians, reptiles) and mammals, such as non-human
primates, sheep, dog, cow, pig, etc.
[0242] In one embodiment, the subject is a human subject.
Alternatively, the subject can be a mammal expressing a ET2-like
antigen with which an antibody of the invention cross-reacts. A
protein ligand of the invention can be administered to a human
subject for therapeutic purposes (discussed further below).
Moreover, an ET2-ligand can be administered to a non-human mammal
expressing the ET2-like antigen to which the ligand binds (e.g., a
primate, pig or mouse) for veterinary purposes or as an animal
model of human disease. Regarding the latter, such animal models
may be useful for evaluating the therapeutic efficacy of the ligand
(e.g., testing of dosages and time courses of administration).
[0243] In one embodiment, the invention provides a method of
treating (e.g., ablating, killing, reducing growth of cell division
of) a cell (e.g., a non-cancerous cell, e.g., a normal, benign or
hyperplastic cell, or a cancerous cell, e.g., a malignant cell,
e.g., cell found in a solid tumor, a soft tissue tumor, or a
metastatic lesion (e.g., a cell found in renal, urothelial,
colonic, rectal, pulmonary, breast or hepatic, cancers and/or
metastasis))s. Methods of the invention include the steps of
contacting the cell with an ET2-ligand, e.g., an anti-ET2 antibody
described herein, in an amount sufficient to treat, e.g., inhibit
cell growth or division, or ablate or kill the cell.
[0244] The subject method can be used on cells in culture, e.g. in
vitro or ex vivo. For example, cancerous or metastatic cells (e.g.,
renal, urothelial, colon, rectal, lung, breast, ovarian, prostatic,
or liver cancerous or metastatic cells) can be cultured in vitro in
culture medium and the contacting step can be effected by adding
the ET2-ligand to the culture medium. The method can be performed
on cells (e.g., cancerous or metastatic cells) present in a
subject, as part of an in vivo (e.g., therapeutic or prophylactic)
protocol. For in vivo embodiments, the contacting step is effected
in a subject and includes administering the ET2-ligand to the
subject under conditions effective to permit both binding of the
ligand to the cell and the treating, e.g., the inhibition of growth
or division, or the killing or ablating of the cell.
[0245] The inhibitors of ET2 can reduce angiogenesis (e.g.,
uncontrolled or unwanted angiogenesis)--such as angiogenesis
associated with vascular malformations and cardiovascular disorders
(e.g., atherosclerosis, restenosis and arteriovenous
malformations), chronic inflammatory diseases (e.g., diabetes
mellitus, inflammatory bowel disease, psoriasis and rheumatoid
arthritis), aberrant wound repairs (e.g., those that are observed
following excimer laser eye surgery), circulatory disorders (e.g.,
Raynaud's phenomenon), crest syndromes (e.g., calcinosis,
esophageal and dyomotiloty), dermatological disorders (e.g.,
Port-wine stains, arterial ulcers, systemic vasculitis and
scleroderma), or ocular disorders (e.g., blindness caused by
neovascular disease, neovascular glaucoma, corneal
neovascularization, trachoma, diabetic retinopathy and myopic
degeneration). See, e.g., Carmeliet and Jain, Nature, 407: 249-257,
2000.
[0246] The method can be used to treat a cancer. As used herein,
the terms "cancer", "hyperproliferative", "malignant", and
"neoplastic" are used interchangeably, and refer to those cells in
an abnormal state or condition characterized by rapid proliferation
or neoplasm. The terms include all types of cancerous growths or
oncogenic processes, metastatic tissues or malignantly transformed
cells, tissues, or organs, irrespective of histopathologic type or
stage of invasiveness. "Pathologic hyperproliferative" cells occur
in disease states characterized by malignant tumor growth.
[0247] The common medical meaning of the term "neoplasia" refers to
"new cell growth" that results as a loss of responsiveness to
normal growth controls, e.g. to neoplastic cell growth. A
"hyperplasia" refers to cells undergoing an abnormally high rate of
growth. However, as used herein, the terms neoplasia and
hyperplasia can be used interchangeably, as their context will
reveal, referring generally to cells experiencing abnormal cell
growth rates. Neoplasias and hyperplasias include "tumors," which
may be benign, premalignant or malignant.
[0248] Examples of cancerous disorders include, but are not limited
to, solid tumors, soft tissue tumors, and metastatic lesions.
Examples of solid tumors include malignancies, e.g., sarcomas,
adenocarcinomas, and carcinomas, of the various organ systems, such
as those affecting lung, breast, lymphoid, gastrointestinal (e.g.,
colon), and genitourinary tract (e.g., renal, urothelial cells),
pharynx, prostate, ovary as well as adenocarcinomas which include
malignancies such as most colon cancers, rectal cancer, renal-cell
carcinoma, liver cancer, non-small cell carcinoma of the lung,
cancer of the small intestine and so forth. Metastatic lesions of
the aforementioned cancers can also be treated or prevented using
the methods and compositions of the invention.
[0249] The subject method can be useful in treating malignancies of
the various organ systems, such as those affecting lung, breast,
lymphoid, gastrointestinal (e.g., colon), and genitourinary tract,
prostate, ovary, pharynx, as well as adenocarcinomas which include
malignancies such as most colon cancers, renal-cell carcinoma,
prostate cancer and/or testicular tumors, non-small cell carcinoma
of the lung, cancer of the small intestine and cancer of the
esophagus. Exemplary solid tumors that can be treated include:
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, non-small cell lung
carcinoma, bladder carcinoma, epithelial carcinoma, glioma,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,
pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
meningioma, melanoma, neuroblastoma, and retinoblastoma.
[0250] The term "carcinoma" is recognized by those skilled in the
art and refers to malignancies of epithelial or endocrine tissues
including respiratory system carcinomas, gastrointestinal system
carcinomas, genitourinary system carcinomas, testicular carcinomas,
breast carcinomas, prostatic carcinomas, endocrine system
carcinomas, and melanomas. Exemplary carcinomas include those
forming from tissue of the cervix, lung, prostate, breast, head and
neck, colon and ovary. The term also includes carcinosarcomas,
e.g., which include malignant tumors composed of carcinomatous and
sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma
derived from glandular tissue or in which the tumor cells form
recognizable glandular structures.
[0251] The term "sarcoma" is recognized by those skilled in the art
and refers to malignant tumors of mesenchymal derivation.
[0252] The subject method can also be used to inhibit the
proliferation of hyperplastic/neoplastic cells of hematopoietic
origin, e.g., arising from myeloid, lymphoid or erythroid lineages,
or precursor cells thereof. For instance, the present invention
contemplates the treatment of various myeloid disorders including,
but not limited to, acute promyeloid leukemia (APML), acute
myelogenous leukemia (AML) and chronic myelogenous leukemia (CML)
(reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol.
11:267-97). Lymphoid malignancies which may be treated by the
subject method include, but are not limited to acute lymphoblastic
leukemia (ALL), which includes B-lineage ALL and T-lineage ALL,
chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),
hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
Additional forms of malignant lymphomas contemplated by the
treatment method of the present invention include, but are not
limited to, non-Hodgkin's lymphoma and variants thereof, peripheral
T-cell lymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous
T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF)
and Hodgkin's disease.
[0253] ET2 ligands that are agonists can be used to stimulate
angiogenesis, e.g., aid wound healing, burns, and other disorders
which require increased angiogenesis.
[0254] Methods of administering ET2-ligands are described in
"Pharmaceutical Compositions". Suitable dosages of the molecules
used will depend on the age and weight of the subject and the
particular drug used. The ligands can be used as competitive agents
to inhibit, reduce an undesirable interaction, e.g., between a
natural or pathological agent and the ET2.
[0255] In one embodiment, the ET2-ligands are used to kill, ablate,
or inhibit the growth of cancerous cells and normal, benign
hyperplastic, and cancerous cells in vivo. The ligands can be used
by themselves or conjugated to an agent, e.g., a cytotoxic drug,
radioisotope. This method includes: administering the ligand alone
or attached to a cytotoxic drug, to a subject requiring such
treatment.
[0256] The terms "cytotoxic agent" and "cytostatic agent" and
"anti-tumor agent" are used interchangeably herein and refer to
agents that have the property of inhibiting the growth or
proliferation (e.g., a cytostatic agent), or inducing the killing,
of hyperproliferative cells, e.g., an aberrant cancer cell. In
cancer therapeutic embodiment, the term "cytotoxic agent" is used
interchangeably with the terms "anti-cancer" or "anti-tumor" to
mean an agent, which inhibits the development or progression of a
neoplasm, particularly a solid tumor, a soft tissue tumor, or a
metastatic lesion.
[0257] Nonlimiting examples of anti-cancer agents include, e.g.,
antimicrotubule agents, topoisomerase inhibitors, antimetabolites,
mitotic inhibitors, alkylating agents, intercalating agents, agents
capable of interfering with a signal transduction pathway, agents
that promote apoptosis, radiation, and antibodies against other
tumor-associated antigens (including naked antibodies, immunotoxins
and radioconjugates). Examples of the particular classes of
anti-cancer agents are provided in detail as follows:
antitubulin/antimicrotubule, e.g., paclitaxel, vincristine,
vinblastine, vindesine, vinorelbin, taxotere; topoisomerase I
inhibitors, e.g., topotecan, camptothecin, doxorubicin, etoposide,
mitoxantrone, daunorubicin, idarubicin, teniposide, amsacrine,
epirubicin, merbarone, piroxantrone hydrochloride; antimetabolites,
e.g., 5-fluorouracil (5-FU), methotrexate, 6-mercaptopurine,
6-thioguanine, fludarabine phosphate, cytarabine/Ara-C,
trimetrexate, gemcitabine, acivicin, alanosine, pyrazofurin,
N-Phosphoracetyl-L-Asparate=PALA, pentostatin, 5-azacitidine, 5-Aza
2'-deoxycytidine, ara-A, cladribine, 5-fluorouridine, FUDR,
tiazofurin,
N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]--
2-thenoyl]-L-glutamic acid; alkylating agents, e.g., cisplatin,
carboplatin, mitomycin C, BCNU=Carmustine, melphalan, thiotepa,
busulfan, chlorambucil, plicamycin, dacarbazine, ifosfamide
phosphate, cyclophosphamide, nitrogen mustard, uracil mustard,
pipobroman, 4-ipomeanol; agents acting via other mechanisms of
action, e.g., dihydrolenperone, spiromustine, and desipeptide;
biological response modifiers, e.g., to enhance anti-tumor
responses, such as interferon; apoptotic agents, such as
actinomycin D; and anti-hormones, for example anti-estrogens such
as tamoxifen or, for example antiandrogens such as
4'-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3'-(trifluorometh-
yl)propionanilide.
[0258] ET2-ligands can recognize normal, endothelial cells. The
ligands can also bind to cells in the vicinity of the cancerous
cells. The ligands can inhibit the growth of, and/or kill these
cells. In this manner, the ligands may indirectly attack the
cancerous cells which may rely on surrounding cells for nutrients,
growth signals and so forth. Thus, the ET2-ligands (e.g., modified
with a cytotoxin) can selectively target cells in cancerous tissue
(including the cancerous cells themselves).
[0259] The ligands may be used to deliver a variety of cytotoxic
drugs including therapeutic drugs, a compound emitting radiation,
molecules of plants, fungal, or bacterial origin, biological
proteins, and mixtures thereof. The cytotoxic drugs can be
intracellularly acting cytotoxic drugs, such as short-range
radiation emitters, including, for example, short-range,
high-energy .alpha.-emitters, as described herein.
[0260] Enzymatically active toxins and fragments thereof are
exemplified by diphtheria toxin A fragment, nonbinding active
fragments of diphtheria toxin, exotoxin A (from Pseudomonas
aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
.alpha.-sacrin, certain Aleurites fordii proteins, certain Dianthin
proteins, Phytolacca americana proteins (PAP, PAPII and PAP-S),
Morodica charantia inhibitor, curcin, crotin, Saponaria officinalis
inhibitor, gelonin, mitogillin, restrictocin, phenomycin, and
enomycin. Procedures for preparing enzymatically active
polypeptides of the immunotoxins are described in WO84/03508 and
WO85/03508. Examples of cytotoxic moieties that can be conjugated
to the antibodies include adriamycin, chlorambucil, daunomycin,
methotrexate, neocarzinostatin, and platinum.
[0261] In the case of polypeptide toxins, recombinant nucleic acid
techniques can be used to construct a nucleic acid that encodes the
ligand (e.g., antibody or antigen-binding fragment thereof) and the
cytotoxin (or a polypeptide component thereof) as translational
fusions. The recombinant nucleic acid is then expressed, e.g., in
cells and the encoded fusion polypeptide isolated.
[0262] Procedures for conjugating protein ligands (e.g.,
antibodies) with the cytotoxic agents have been previously
described. Procedures for conjugating chlorambucil with antibodies
are described by Flechner (1973) European Journal of Cancer,
9:741-745; Ghose et al. (1972) British Medical Journal, 3:495-499;
and Szekerke, et al. (1972) Neoplasma, 19:211-215. Procedures for
conjugating daunomycin and adriamycin to antibodies are described
by Hurwitz, E. et al. (1975) Cancer Research, 35:1175-1181 and Amon
et al. (1982) Cancer Surveys, 1:429-449. Procedures for preparing
antibody-ricin conjugates are described in U.S. Pat. No. 4,414,148
and by Osawa, T., et al. (1982) Cancer Surveys, 1:373-388 and the
references cited therein. Coupling procedures as also described in
EP 86309516.2.
[0263] To kill or ablate normal, benign hyperplastic, or cancerous
cells, a first protein ligand is conjugated with a prodrug which is
activated only when in close proximity with a prodrug activator.
The prodrug activator is conjugated with a second protein ligand,
preferably one which binds to a non-competing site on the target
molecule. Whether two protein ligands bind to competing or
non-competing binding sites can be determined by conventional
competitive binding assays. Drug-prodrug pairs suitable for use in
the practice of the present invention are described in Blakey et
al., (1996) Cancer Research, 56:3287-3292.
[0264] Alternatively, the ET2-ligand can be coupled to high energy
radiation emitters, for example, a radioisotope, such as .sup.131I,
a .gamma.-emitter, which, when localized at the tumor site, results
in a killing of several cell diameters. See, e.g., S. E. Order,
"Analysis, Results, and Future Prospective of the Therapeutic Use
of Radiolabeled Antibody in Cancer Therapy", Monoclonal Antibodies
for Cancer Detection and Therapy, R. W. Baldwin et al. (eds.), pp
303-316 (Academic Press 1985). Other suitable radioisotopes include
.alpha.-emitters, such as .sup.212Bi, .sup.213Bi, and .sup.211At,
and .beta.-emitters, such as .sup.186Re and .sup.90Y. Moreover,
Lu.sup.117 may also be used as both an imaging and cytotoxic
agent.
[0265] Radioimmunotherapy (RIT) using antibodies labeled with
.sup.131I, .sup.90Y, and .sup.177Lu is under intense clinical
investigation. There are significant differences in the physical
characteristics of these three nuclides and as a result, the choice
of radionuclide is very critical in order to deliver maximum
radiation dose to the tumor. The higher beta energy particles of
.sup.90Y may be good for bulky tumors. The relatively low energy
beta particles of .sup.131I are ideal, but in vivo dehalogenation
of radioiodinated molecules is a major disadvantage for
internalizing antibody. In contrast, .sup.177Lu has low energy beta
particle with only 0.2-0.3 mm range and delivers much lower
radiation dose to bone marrow compared to .sup.90Y. In addition,
due to longer physical half-life (compared to .sup.90Y), the tumor
residence times are higher. As a result, higher activities (more
mCi amounts) of .sup.177Lu labeled agents can be administered with
comparatively less radiation dose to marrow. There have been
several clinical studies investigating the use of .sup.177Lu
labeled antibodies in the treatment of various cancers. (Mulligan T
et al. (1995) Clin Cancer Res. 1: 1447-1454; Meredith R F, et al.
(1996) J Nucl Med 37:1491-1496; Alvarez R D, et al. (1997)
Gynecologic Oncology 65: 94-101).
[0266] The ET2-ligands can be used directly in vivo to eliminate
antigen-expressing cells via natural complement-dependent
cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity
(ADCC). The protein ligands of the invention, can include
complement binding effector domain, such as the Fc portions from
IgG1, -2, or -3 or corresponding portions of IgM which bind
complement. In one embodiment, a population of target cells is ex
vivo treated with a binding agent of the invention and appropriate
effector cells. The treatment can be supplemented by the addition
of complement or serum containing complement. Further, phagocytosis
of target cells coated with a protein ligand of the invention can
be improved by binding of complement proteins. In another
embodiment target, cells coated with the protein ligand that
includes a complement binding effector domain are lysed by
complement.
[0267] Also encompassed by the present invention is a method of
killing or ablating which involves using the ET2-ligand for
prophylaxis. For example, these materials can be used to prevent or
delay development or progression of cancers.
[0268] Use of the therapeutic methods of the present invention to
treat cancers has a number of benefits. Since the protein ligands
specifically recognize ET2, other tissue is spared and high levels
of the agent are delivered directly to the site where therapy is
required. Treatment in accordance with the present invention can be
effectively monitored with clinical parameters. Alternatively,
these parameters can be used to indicate when such treatment should
be employed.
[0269] ET2-ligands of the invention can be administered in
combination with one or more of the existing modalities for
treating cancers, including, but not limited to: surgery; radiation
therapy, and chemotherapy.
Diagnostic Uses
[0270] Protein ligands that bind to ET2 and identified by the
method described herein and/or detailed herein have in vitro and in
vivo diagnostic, therapeutic and prophylactic utilities.
[0271] In one aspect, the present invention provides a diagnostic
method for detecting the presence of a ET2, in vitro (e.g., a
biological sample, such as tissue, biopsy, e.g., a cancerous
tissue) or in vivo (e.g., in vivo imaging in a subject).
[0272] The method includes: (i) contacting a sample with
ET2-ligand; and (ii) detecting formation of a complex between the
ET2-ligand and the sample. The method can also include contacting a
reference sample (e.g., a control sample) with the ligand, and
determining the extent of formation of the complex between the
ligand an the sample relative to the same for the reference sample.
A change, e.g., a statistically significant change, in the
formation of the complex in the sample or subject relative to the
control sample or subject can be indicative of the presence of ET2
in the sample.
[0273] Another method includes: (i) administering the ET2-ligand to
a subject; and (iii) detecting formation of a complex between the
ET2-ligand, and the subject. The detecting can include determining
location or time of formation of the complex.
[0274] The ET2-ligand can be directly or indirectly labeled with a
detectable substance to facilitate detection of the bound or
unbound antibody. Suitable detectable substances include various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials and radioactive materials.
[0275] Complex formation between the ET2-ligand and ET2 can be
detected by measuring or visualizing either the ligand bound to the
ET2 or unbound ligand. Conventional detection assays can be used,
e.g., an enzyme-linked immunosorbent assays (ELISA), a
radioimmunoassay (RIA) or tissue immunohistochemistry. Further to
labeling the ET2-ligand, the presence of ET2 can be assayed in a
sample by a competition immunoassay utilizing standards labeled
with a detectable substance and an unlabeled ET2-ligand. In one
example of this assay, the biological sample, the labeled standards
and the ET2 binding agent are combined and the amount of labeled
standard bound to the unlabeled ligand is determined. The amount of
ET2 in the sample is inversely proportional to the amount of
labeled standard bound to the ET2 binding agent.
[0276] Fluorophore and chromophore labeled protein ligands can be
prepared. Since antibodies and other proteins absorb light having
wavelengths up to about 310 nm, the fluorescent moieties should be
selected to have substantial absorption at wavelengths above 310 nm
and preferably above 400 nm. A variety of suitable fluorescers and
chromophores are described by Stryer (1968) Science, 162:526 and
Brand, L. et al. (1972) Annual Review of Biochemistry, 41:843-868.
The protein ligands can be labeled with fluorescent chromophore
groups by conventional procedures such as those disclosed in U.S.
Pat. Nos. 3,940,475, 4,289,747, and 4,376,110. One group of
fluorescers having a number of the desirable properties described
above is the xanthene dyes, which include the fluoresceins and
rhodamines. Another group of fluorescent compounds are the
naphthylamines. Once labeled with a fluorophore or chromophore, the
protein ligand can be used to detect the presence or localization
of the ET2 in a sample, e.g., using fluorescent microscopy (such as
confocal or deconvolution microscopy).
[0277] Histological Analysis. Immunohistochemistry can be performed
using the protein ligands described herein. For example, in the
case of an antibody, the antibody can synthesized with a label
(such as a purification or epitope tag), or can be detectably
labeled, e.g., by conjugating a label or label-binding group. For
example, a chelator can be attached to the antibody. The antibody
is then contacted to a histological preparation, e.g., a fixed
section of tissue that is on a microscope slide. After an
incubation for binding, the preparation is washed to remove unbound
antibody. The preparation is then analyzed, e.g., using microscopy,
to identify if the antibody bound to the preparation.
[0278] Of course, the antibody (or other polypeptide or peptide)
can be unlabeled at the time of binding. After binding and washing,
the antibody is labeled in order to render it detectable.
[0279] Protein Arrays. The ET2-ligand can also be immobilized on a
protein array. The protein array can be used as a diagnostic tool,
e.g., to screen medical samples (such as isolated cells, blood,
sera, biopsies, and the like). Of course, the protein array can
also include other ligands, e.g., that bind to ET2 or to other
target molecules.
[0280] Methods of producing polypeptide arrays are described, e.g.,
in De Wildt et al. (2000) Nat. Biotechnol. 18:989-994; Lueking et
al. (1999) Anal. Biochem. 270:103-111; Ge (2000) Nucleic Acids Res.
28, e3, I-VII; MacBeath and Schreiber (2000) Science 289:1760-1763;
WO 01/40803 and WO 99/51773A1. Polypeptides for the array can be
spotted at high speed, e.g., using commercially available robotic
apparati, e.g., from Genetic MicroSystems or BioRobotics. The array
substrate can be, for example, nitrocellulose, plastic, glass,
e.g., surface-modified glass. The array can also include a porous
matrix, e.g., acrylamide, agarose, or another polymer.
[0281] For example, the array can be an array of antibodies, e.g.,
as described in De Wildt, supra. Cells that produce the protein
ligands can be grown on a filter in an arrayed format. Polypeptide
production is induced, and the expressed polypeptides are
immobilized to the filter at the location of the cell.
[0282] A protein array can be contacted with a labeled target to
determine the extent of binding of the target to each immobilized
polypeptide from the diversity strand library. If the target is
unlabeled, a sandwich method can be used, e.g., using a labeled
probed, to detect binding of the unlabeled target.
[0283] Information about the extent of binding at each address of
the array can be stored as a profile, e.g., in a computer database.
The protein array can be produced in replicates and used to compare
binding profiles, e.g., of a target and a non-target. Thus, protein
arrays can be used to identify individual members of the diversity
strand library that have desired binding properties with respect to
one or more molecules.
[0284] FACS. (Fluorescent Activated Cell Sorting). The ET2-ligand
can be used to label cells, e.g., cells in a sample (e.g., a
patient sample). The ligand is also attached (or attachable) to a
fluorescent compound. The cells can then be sorted using
fluorescent activated cell sorted (e.g., using a sorter available
from Becton Dickinson Immunocytometry Systems, San Jose Calif.; see
also U.S. Pat. Nos. 5,627,037; 5,030,002; and 5,137,809). As cells
pass through the sorter, a laser beam excites the fluorescent
compound while a detector counts cells that pass through and
determines whether a fluorescent compound is attached to the cell
by detecting fluorescence. The amount of label bound to each cell
can be quantified and analyzed to characterize the sample.
[0285] The sorter can also deflect the cell and separate cells
bound by the ligand from those cells not bound by the ligand. The
separated cells can be cultured and/or characterized.
[0286] In vivo Imaging. In still another embodiment, the invention
provides a method for detecting the presence of a ET2-expressing
cancerous tissues in vivo. The method includes (i) administering to
a subject (e.g., a patient having a cancer or neoplastic disorder)
an anti-ET2 antibody, conjugated to a detectable marker; (ii)
exposing the subject to a means for detecting said detectable
marker to the ET2-expressing tissues or cells. For example, the
subject is imaged, e.g., by NMR or other tomographic means.
[0287] Examples of labels useful for diagnostic imaging in
accordance with the present invention include radiolabels such as
.sup.131I, .sup.111In, .sup.123I, .sup.99mTc, .sup.32P, .sup.125I,
.sup.3H, .sup.14C, and .sup.188Rh, fluorescent labels such as
fluorescein and rhodamine, nuclear magnetic resonance active
labels, positron emitting isotopes detectable by a positron
emission tomography ("PET") scanner, chemiluminescers such as
luciferin, and enzymatic markers such as peroxidase or phosphatase.
Short-range radiation emitters, such as isotopes detectable by
short-range detector probes can also be employed. The protein
ligand can be labeled with such reagents using known techniques.
For example, see Wensel and Meares (1983) Radioimmunoimaging and
Radioimmunotherapy, Elsevier, New York for techniques relating to
the radiolabeling of antibodies and D. Colcher et al. (1986) Meth.
Enzymol. 121: 802-816.
[0288] A radiolabeled ligand of this invention can also be used for
in vitro diagnostic tests. The specific activity of a
isotopically-labeled ligand depends upon the half-life, the
isotopic purity of the radioactive label, and how the label is
incorporated into the antibody.
[0289] Procedures for labeling polypeptides with the radioactive
isotopes (such as .sup.14C, .sup.3H, .sup.35S, .sup.125I, .sup.32P,
.sup.131I) are generally known. For example, tritium labeling
procedures are described in U.S. Pat. No. 4,302,438. Iodinating,
tritium labeling, and .sup.35S labeling procedures, e.g., as
adapted for murine monoclonal antibodies, are described, e.g., by
Goding, J. W. (Monoclonal antibodies: principles and practice:
production and application of monoclonal antibodies in cell
biology, biochemistry, and immunology 2nd ed. London; Orlando:
Academic Press, 1986. pp 124-126) and the references cited therein.
Other procedures for iodinating polypeptides, such as antibodies,
are described by Hunter and Greenwood (1962) Nature 144:945, David
et al. (1974) Biochemistry 13:1014-1021, and U.S. Pat. Nos.
3,867,517 and 4,376,110. Radiolabeling elements which are useful in
imaging include .sup.123I, .sup.131I, .sup.111In, and .sup.99mTc,
for example. Procedures for iodinating antibodies are described by
Greenwood, F. et al. (1963) Biochem. J. 89:114-123; Marchalonis, J.
(1969) Biochem. J. 113:299-305; and Morrison, M. et al. (1971)
Immunochemistry 289-297. Procedures for .sup.99mTc-labeling are
described by Rhodes, B. et al. in Burchiel, S. et al. (eds.), Tumor
Imaging: The Radioimmunochemical Detection of Cancer, New York:
Masson 111-123 (1982) and the references cited therein. Procedures
suitable for .sup.111In-labeling antibodies are described by
Hnatowich, D. J. et al. (1983) J. Immul. Methods, 65:147-157,
Hnatowich, D. et al. (1984) J. Applied Radiation, 35:554-557, and
Buckley, R. G. et al. (1984) F.E.B.S. 166:202-204.
[0290] In the case of a radiolabeled ligand, the ligand is
administered to the patient, is localized to the tumor bearing the
antigen with which the ligand reacts, and is detected or "imaged"
in vivo using known techniques such as radionuclear scanning using
e.g., a gamma camera or emission tomography. See e.g., A. R.
Bradwell et al., "Developments in Antibody Imaging", Monoclonal
Antibodies for Cancer Detection and Therapy, R. W. Baldwin et al.,
(eds.), pp 65-85 (Academic Press 1985). Alternatively, a positron
emission transaxial tomography scanner, such as designated Pet VI
located at Brookhaven National Laboratory, can be used where the
radiolabel emits positrons (e.g., .sup.11C, .sup.18F, .sup.15O, and
.sup.13N).
[0291] MRI Contrast Agents. Magnetic Resonance Imaging (MRI) uses
NMR to visualize internal features of living subject, and is useful
for prognosis, diagnosis, treatment, and surgery. MRI can be used
without radioactive tracer compounds for obvious benefit. Some MRI
techniques are summarized in EP-A-0 502 814. Generally, the
differences related to relaxation time constants T1 and T2 of water
protons in different environments is used to generate an image.
However, these differences can be insufficient to provide sharp
high resolution images.
[0292] The differences in these relaxation time constants can be
enhanced by contrast agents. Examples of such contrast agents
include a number of magnetic agents paramagnetic agents (which
primarily alter T1) and ferromagnetic or superparamagnetic (which
primarily alter T2 response). Chelates (e.g., EDTA, DTPA and NTA
chelates) can be used to attach (and reduce toxicity) of some
paramagnetic substances (e.g., Fe.sup.+3, Mn.sup.+2, Gd.sup.+3).
Other agents can be in the form of particles, e.g., less than 10
.mu.m to about 10 nM in diameter). Particles can have
ferromagnetic, antiferromagnetic or superparamagnetic properties.
Particles can include, e.g., magnetite (Fe.sub.3O.sub.4),
.gamma.-Fe.sub.2O.sub.3, ferrites, and other magnetic mineral
compounds of transition elements. Magnetic particles may include:
one or more magnetic crystals with and without nonmagnetic
material. The nonmagnetic material can include synthetic or natural
polymers (such as sepharose, dextran, dextrin, starch and the
like.
[0293] The ET2-ligands can also be labeled with an indicating group
containing of the NMR-active .sup.19F atom, or a plurality of such
atoms inasmuch as (i) substantially all of naturally abundant
fluorine atoms are the .sup.19F isotope and, thus, substantially
all fluorine-containing compounds are NMR-active; (ii) many
chemically active polyfluorinated compounds such as trifluoracetic
anhydride are commercially available at relatively low cost, and
(iii) many fluorinated compounds have been found medically
acceptable for use in humans such as the perfluorinated polyethers
utilized to carry oxygen as hemoglobin replacements. After
permitting such time for incubation, a whole body MRI is carried
out using an apparatus such as one of those described by Pykett
(1982) Scientific American, 246:78-88 to locate and image cancerous
tissues.
[0294] Also within the scope of the invention are kits comprising
the protein ligand that binds to ET2 and instructions for
diagnostic use, e.g., the use of the ET2-ligand (e.g., antibody or
antigen-binding fragment thereof, or other polypeptide or peptide)
to detect ET2, in vitro, e.g., in a sample, e.g., a biopsy or cells
from a patient having a cancer or neoplastic disorder, or in vivo,
e.g., by imaging a subject. The kit can further contain a least one
additional reagent, such as a label or additional diagnostic agent.
For in vivo use the ligand can be formulated as a pharmaceutical
composition.
[0295] The following invention is further illustrated by the
following examples, which should not be construed as further
limiting. The contents of all references, pending patent
applications and published patents, cited throughout this
application are hereby expressly incorporated by reference.
EXAMPLE 1
Selection and Primary Screening
[0296] In order to isolate antibodies that bind ET2, a phagemid Fab
library was screened against the protease domain of ET2.
[0297] The biotinylated protease domain of ET2 was captured on
streptavidin coated magnetic beads (M280-DYNAL). The ET2 coated
beads were washed three times with 2% non-fat milk in PBS prior to
addition of library phage. Library phage (10.sup.12 particles) were
added to the magnetic beads in a final volume of 100 .mu.l. The mix
was allowed to incubate at room temperature with end over end
mixing for two hours. After this time, the supernatant was removed
and the beads washed three times with 0.1% Tween 2% non-fat milk in
PBS. After the final wash, the beads were transferred to a new
tube. Phage were eluted from the beads by addition of 1 ml of 100
mM Triethanolamine buffer (TEA). After a 10 min incubation at room
temperature the supernatant was removed and added to 5001 of
Tris-HCl pH 7.5. The eluted phage were then amplified and used for
a further round of selection. After three rounds of selection the
output was analyzed as described below. (For methods, see also
Chames et al. (2002) Methods Mol Biol. 178:147-57).
[0298] Library members recovered from the selections were tested
for ET2 binding by phage ELISA (FIG. 3). Each isolate was tested
for binding to ET2, and a blank streptavidin well. Isolates that
gave an ELISA signal for ET2 twice that for streptavidin binding
were considered `positives` and selected for small scale soluble
Fab production. A total of 184 isolates were tested in the phage
ELISA, of which 171 tested positive for ET-2 binding, according to
this method. Exemplary data is provided in Table 4 below:
TABLE-US-00004 TABLE 4 Exemplary Phage ELISA data BSA-Strept-rET2
BSA-STrept 0.342 0.120 0.323 0.090 0.320 0.086 0.278 0.082 0.261
0.090 0.280 0.086 0.247 0.091 0.244 0.088 0.263 0.131 0.264 0.102
0.172 0.087 0.223 0.088 0.200 0.100 0.272 0.083 0.263 0.087 0.233
0.097 0.158 0.129 0.490 0.111 0.225 0.092 0.191 0.092 0.193 0.113
0.210 0.089 0.186 0.103 0.259 0.098 0.198 0.143 0.177 0.116 0.197
0.097 0.173 0.094 0.198 0.148 0.202 0.102 0.270 0.108 0.204 0.095
0.189 0.164 0.202 0.128 0.163 0.110 0.188 0.106 0.199 0.122 0.187
0.109 0.246 0.120 0.215 0.102 0.178 0.162 0.169 0.158 0.189 0.114
0.210 0.125 0.192 0.134 0.182 0.151 0.251 0.115 0.185 0.114
EXAMPLE 2
Fab Production and Screening
[0299] Small scale amounts of soluble Fab were produced in a
96-well format and tested for binding to ET2. To help further
characterize the Fabs, the ELISA was performed in the presence and
absence of a competing ligand that binds to the active site of ET2.
When the Fab ELISA signal is reduced in the presence of the
competing ligand, it is likely that these Fabs bind at or close to
the active site. Exemplary data is provided in Table 5 below:
TABLE-US-00005 TABLE 5 Exemplary Soluble Fab ELISA data Soluble
Soluable Fab + rET Fab - rET pept. Inhib. pept. Inhib. A1 0.21 0.31
B1 0.89 0.947 C1 0.135 0.143 D1 0.267 0.351 E1 0.118 0.204 F1 0.124
0.239 G1 0.22 0.392 H1 0.271 0.472 A2 0.872 0.992 B2 0.172 0.23 C2
0.154 0.191 D2 0.611 0.599 E2 0.128 0.205 F2 0.872 1.248 G2 0.126
0.192 H2 0.128 0.232 A3 0.241 0.435 B3 0.132 0.168 C3 0.114 0.145
D3 0.822 0.83 E3 0.118 0.143 F3 0.224 0.388 G3 0.332 0.591 H3 0.173
0.304 A4 0.168 0.167 B4 0.173 0.229 C4 0.112 0.155 D4 0.134 0.172
E4 0.119 0.168 F4 0.138 0.189 G4 0.652 0.735 H4 0.321 0.42 A5 0.182
0.26 B5 0.184 0.325 C5 0.236 0.419 D5 0.958 0.758 E5 0.154 0.169 F5
0.127 0.219 G5 0.315 0.322 H5 0.225 0.277 A6 0.133 0.128 B6 0.155
0.146 C6 1.091 1.063 D6 0.122 0.163 E6 0.137 0.15 F6 0.186
0.224
[0300] A total of 64 soluble Fabs were identified that bound ET2 in
this assay. Of these, 31 were strongly competed by the peptide, a
further 8 showed weak competition in the presence of the peptide.
We found that competition of the Fab binding to the target enzyme
by a peptide inhibitor was a useful method to identify inhibitors.
This was done by examining the inhibition by the Fabs by another
type of assay. Soluble Fabs that bound ET2 were prepared on a large
scale (450 ml cultures) and used to determine inhibition of ET2 in
a continuous in vitro enzyme assay.
[0301] An assay for evaluating inhibitors of ET2 can be performed
as follows: Test compounds for inhibition of the protease activity
of the protease domain of ET2 are assayed in Costar 96 well tissue
culture plates (Corning N.Y.). Approximately 2-3 nM ET2 is mixed
with varying concentrations of inhibitor in 29.2 mM Tris, pH 8.4,
29.2 mM imidazole, 217 mM NaCl (100 mL final volume), and allowed
to incubate at room temperature for 30 minutes. 400 mM substrate S
2765 (DiaPharma, Westchester, Ohio) is added, and the reaction is
monitored in a SpectraMAX Plus microplate reader (Molecular
Devices, Sunnyvale Calif.) by following the change in absorbance at
405 nm for 1 hour at 37.degree. C. All reagents unless indicated
were obtained from Sigma Chemical Co. (St. Louis, Mo.). Additional
details can be in accordance with the ET1 assay provided further
below.
[0302] An exemplary structure of S 2765 is: ##STR1##
[0303] We showed that those Fabs that show strong competition by
the peptide are good enzyme inhibitors of ET2. The Ki values for
the best inhibitors are shown in Table 6. These clones were
subsequently sequenced. TABLE-US-00006 TABLE 6 Inhibition Data for
Fab Inhibitors Clone Coding Library Ki D5: R3 rET2 MP3 CJ Phagemid
70 .+-. 10 pM D2: R3 rET2 MP1 CJ Phagemid 90 .+-. 30 pM A2: R3 rET2
MP1 CJ Phagemid 160 .+-. 50 pM H10: R3 rET2 MP1 CJ Phagemid 190
.+-. 10 pM F8: R3 rET2 MP1 CJ Phagemid 240 .+-. 20 pM B5: R3 rET2
MP3 CJ Phagemid 250 .+-. 60 pM C9: R3 rET2 MP3 CJ Phagemid 260 .+-.
50 pM
[0304] The following is an assay for ET1 activity. The assay buffer
for assaying ET1 activity was HBSA (10 mM Hepes, 150 mM sodium
chloride, pH 7.4, 0.1% bovine serum albumin). All reagents were
from Sigma Chemical Co. (St. Louis, Mo.), unless otherwise
indicated. Two IC.sub.50 assays at 30-minute (a 30-minute
preincubation of test Fab and enzyme) and at O-minutes (no
preincubation of test Fab and enzyme) were conducted. For the
IC.sub.50 assay at 30-minute, the following reagents were combined
in appropriate wells of a Corning microtiter plate: 50 microliters
of HBSA, 50 microliters of the test compound, diluted (covering a
broad concentration range) in HBSA (or HBSA alone for uninhibited
velocity measurement), and 50 microliters of the rET1 (Corvas
International) diluted in buffer, yielding a final enzyme
concentration of 250 pM. Following a 30-minute incubation at
ambient temperature, the assay was initiated by the addition of 50
microliters of the substrate Spectrozyme tPA
(Methylsulfonyl-D-cyclohexyltyrosyl-L-glycyl-L-arginine-p-nitroanilin-
e acetate, obtained from American Diagnostica, Inc. (Greenwich,
Conn.) and reconstituted in deionized water, followed by dilution
in HBSA prior to the assay) were added to the wells, yielding a
final volume of 200 microliters and a final substrate concentration
of 300 .mu.M (about 1.5-times Km).
[0305] For the IC.sub.50 assay at O-minute, the same reagents were
combined: 50 microliters of HBSA, 50 microliters of the test
compound, diluted (covering the identical concentration range) in
HBSA (or HBSA alone for uninhibited velocity measurement), and 50
microliters of the substrate Spectrozyme tPA. The assay was
initiated by the addition of 50 microliters of rET2. The final
concentrations of all components were identical in both IC.sub.50
assays (at 30- and 0-minute incubations).
[0306] The initial velocity of chromogenic substrate hydrolysis was
measured in both assays by the change of absorbance at 405 nM using
a Thermo Max Kinetic Microplate Reader (Molecular Devices) over a 5
minute period, in which less than 5% of the added substrate was
used. The concentration of added inhibitor, which caused a 50%
decrease in the initial rate of hydrolysis was defined as the
respective IC.sub.50 value in each of the two assays (30- and
0-minute).
EXAMPLE 3
Selectivity of Fab Inhibitors
[0307] The sequence data for the Fab inhibitors is shown in Table 1
(above, in the Summary section). Four clones (A2, B5, D2 & H10)
share the same heavy chain sequence. This sequence contains a
lysine to amber stop codon mutation. Although one would normally
expect such a mutation to result in truncation of the heavy chain,
and consequently result in a non-functional Fab, all propagations
were performed in a supE mutant of E. coli. This mutant strain
inserts a glutamine residue, shown as q in the sequence data, at
the amber stop codon thus allowing production of the mature
Fab.
[0308] The seven Fabs described above were reformatted into IgG1
antibodies. Fab reformatting is a two step process in which the Fab
is first cloned into the IgG1 expression vector (pRRV) which
provides a eukaryotic promoter to drive expression of the heavy and
light chains and the heavy chain constant sequence. In the second
step, the E. coli promoter used to drive expression of the heavy
chain is replaced with a eukaryotic internal ribosome entry
sequence (IRES). To allow expression in the mammalian system the
four clones that had amber stop mutations, A2, B5, D2 & H10,
had the amber mutation replaced with a lysine, the naturally
occurring amino acid at this position.
[0309] Once expression vector construction was complete the
antibodies were transiently expressed in HEK 293T cells and
subsequently purified from the cell culture media using protein A
affinity chromatography. The purified antibody was tested in the
same continuous in vitro assay previously used for analysis of the
Fabs. The Ki values are shown in Table 7.
[0310] In a selectivity screen all IgG's demonstrated <5%
activity at 100 nM against proteases Trypsinogen-IV, MTSP-1,
MTSP-6, MTSP-7, MTSP-10 and ET1. TABLE-US-00007 TABLE 7 Comparison
of Inhibition Data for Fab & IgG Inhibitors Clone Target Ki
(Fab) Ki (IgG) D5 rET2 70 pM 86 pM D2 rET2 95 pM 44 pM A2 rET2 150
pM 53 pM H10 rET2 315 pM 136 pM F8 rET2 410 pM 840 pM B5 rET2 325
pM 102 pM C9 rET2 310 pM 110 pM
EXAMPLE 4
Reduction in Tumor Growth
[0311] One antibody that binds to ET-2 was evaluated in a small
animal efficacy study.
[0312] DU-145 tumor cells injected subcutaneously into the animal's
flank 6-8 week old SCID mice (Charles River). Five to 10 days after
tumor implantation the animals were randomized into groups of 10-15
animals. Treatment was by IP injection, either once a day with Fab
(0, 200 or 400 .mu.g/animal), or once every other day with IgG (0,
10, 50 or 500 .mu.g/animal). The study was allowed to continue
until the tumors reached the maximal allowable size. Tumor sizes
were measured vernier calipers (Mitutoyo Model 573) and tumor
volumes calculated. At the end of the study tumors were excised and
weighed. Animal health was assessed during the study by regular
weighing. Treatment with 400 .mu.g of Fab H10 reduced the rate of
tumor growth relative to the rate in animals given the control
treatments. For example, 35 days after the first dose, average
tumor volumes (FIG. 3A) and tumor weights (FIG. 3B) were reduced
for animals treated with 400 .mu.g of Fab H10. Other useful
antibodies can similarly reduce tumor growth, e.g., reduce tumor
weight by at least 10, 20, 30, 40, 50% relative to a control, e.g.,
after 35 days.
EXAMPLE 5
Exemplary Sequences--A10
[0313] TABLE-US-00008 Translation of A10 HC (1-344) 1 EVQLLESGGG
LVQPGGSLRL SCAASGFTFS RYRMWWVRQA PGKGLEWVSY (SEQ ID NO:25) 51
ISSSGGFTNY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKNA 101
RRALPSMDVW GKGT Translation of A10 LC (1-354) 1 QSALTQPPSA
SGTPGQRVTI SCSGSSSNIG SNYVYWYQQL PGTAPKLLIY (SEQ ID NO:26) 51
SNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEADYYCA AWDDSLSGPV 101
FGGGTKLTVL GQPKAAPS A10 HC Nucleic Acid Sequence
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTC (SEQ ID
NO:27) TTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTATGTGGTGGGTTCGCCA
AGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTATATCTCTTCTTCTGGTGGCTTTACT
AATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATA
CTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGC
GAAAAACGCGCGAAGAGCTCTTCCCTCCATGGACGTCTGGGGCAAAGGGACCAC A10 LC
Nucleic Acid Sequence
CAGAGCGCTTTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCA (SEQ ID
NO:28) TCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATACTGGTACCAGCA
GCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTAATAATCAGCGGCCCTCAGGG
GTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTG
GGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAG
TGGTCCGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCC
CCCTCG
EXAMPLE 6
Exemplary Sequences--G3
[0314] TABLE-US-00009 Translation of G3 HC (1-342) 1 EVQLLESGGG
LVQPGGSLRL SCAASGFTFS RYGMSWVRQA PGKGLEWVSV (SEQ ID NO:29) 51
IYSSGGITRY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARRA 101
PRGEVAFDIW GQGT Translation of G3 LC (1-345) 1 QDIQMTQSPS
FLSASIGDRV TITCWASQGI SNYLAWYQQK PGKAPKLLIS (SEQ ID NO:30) 51
SASTLQSGVP SRFSGSGSGT EFTLTISSLQ PEDSATYYCQ QANSFPWTFG 101
QGTRVEIRRT VAAPS G3 HC Nucleic Acid Sequence
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTC (SEQ ID
NO:31) TTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGGTATGTCTTGGGTTCGCCA
AGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTATCTATTCTTCTGGTGGCATTACT
CGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATA
CTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTACTGTGC
GAGACGGGCCCCGAGGGGGGAGGTCGCTTTTGATATCTGGGGCCAAGGGACA G3 LC Nucleic
Acid Sequence
CAAGACATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTATAGGAGACAGAG (SEQ ID
NO:32) TCACCATCACTTGCTGGGCCAGTCAGGGCATTAGTAATTATTTAGCCTGGTATCAGCA
AAAACCAGGGAAAGCCCCTAAGCTCCTGATCTCTTCTGCATCCACTTTGCAAAGTGGG
GTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCA
GCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCGTG
GACGTTCGGCCAAGGGACCAGGGTGGAAATCAGACGAACTGTGGCTGCACCATCT
EXAMPLE 7
Exemplary Sequences--A6
[0315] TABLE-US-00010 Translation of A6 HC (1-344) 1 EVQLLESGGG
LVQPGGSLRL SCAASGFTFS RYKMWWVRQA PGKGLEWVSY (SEQ ID NO:33) 51
ISPSGGYTGY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKNA 101
RRAFPSMDVW GKGT Translation of A6 LC (1-345) 1 QSALTQDPAV
SVALGQTVRI TCRGDRLRSY YSSWYQQKPR QAPVLVMFGR (SEQ ID NO:34) 51
NNRPSGIPDR FSGSTSGSTA SLTITATQAD DEADYFCSSR DGSGNFLFGG 101
GTKLTVLGQP KAAPS A6 HC Nucleic Acid Sequence
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTC (SEQ ID
NO:35) TTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGTGGTGGGTTCGCCA
AGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTATATCTCTCCTTCTGGTGGCTATACT
GGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATA
CTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGC
GAAAAACGCGCGAAGAGCTTTTCCCTCCATGGACGTCTGGGGCAAAGGGACCAC A6 LC
Nucleic Acid Sequence
CAGAGCGCTTTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGGCAGACAGTCAGGA (SEQ ID
NO:36) TCACATGCCGAGGAGACAGACTCAGAAGTTATTATTCAAGTTGGTACCAGCAGAAGCC
ACGACAGGCCCCTGTTCTTGTCATGTTTGGTAGAAACAACCGGCCCTCAGGGATCCCA
GACCGATTCTCTGGCTCCACCTCAGGAAGCACAGCTTCCTTGACCATCACTGCGACTC
AGGCGGACGATGAGGCTGACTATTTCTGTAGTTCCCGGGACGGCAGTGGTAATTTCCT
CTTCGGCGGAGGGACCAAACTGACCGTCCTTGGTCAGCCCAAGGCTGCCCCCTCG
EXAMPLE 8
Exemplary Sequences--A7
[0316] TABLE-US-00011 Translation of A7 HC (1-342) 1 EVQLLESGGG
LVQPGGSLRL SCAASGFTFS RYRMSWVRQA PGKGLEWVSS (SEQ ID NO:37) 51
ISSSGGITTY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED AAIYYCAKNA 101
RRAFPSMDVW GKGT Translation of A7 LC (1-348) 1 QDIQMTQSPS
SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY (SEQ ID NO:38) 51
AASSLQSGVP SRFSGSGSGT EFTLTINSLQ PEDFATYYCQ QLTGYPSITF 101
GQGTRLDIKR TVAAPS A7 HC Nucleic Acid Sequence
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTC (SEQ ID
NO:39) TTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTATGTCTTGGGTTCGCCA
AGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCTCTTCTTCTGGTGGCATTACT
ACTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATA
CTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACGCTGCAATCTACTATTGTGC
GAAAAACGCGCGAAGAGCTTTTCCCTCCATGGACGTCTGGGGCAAAGGGACC A7 LC Nucleic
Acid Sequence
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAG (SEQ ID
NO:40) TCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCA
GAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGG
GTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAACA
GCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAACTTACTGGTTACCCCTC
GATCACCTTCGGCCAAGGGACACGACTGGACATTAAACGAACTGTGGCTGCACCATCT
EXAMPLE 9
Exemplary Sequences--C8
[0317] TABLE-US-00012 Translation of C8 HC (1-342) 1 EVQLLESGGG
LVQPGGSLRL SCAASGFTFS RYTMSWVRQA PGKGLEWVSY (SEQ ID NO:41) 51
IVPSGGMTKY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARRA 101
PRGEVAFDIW GQGT Translation of C8 LC (1-354) 1 QSVLTQPASV
SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMI (SEQ ID NO:42) 51
YDVSKRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYC TSYTSSSTWV 101
FGGGTKLTVL GQPKAAPS C8 HC Nucleic Acid Sequence
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTC (SEQ ID
NO:43) TTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACACTATGTCTTGGGTTCGCCA
AGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTATATCGTTCCTTCTGGTGGCATGACT
AAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATA
CTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGC
GAGACGGGCCCCGAGGGGGGAGGTCGCTTTTGATATCTGGGGCCAAGGGACA C8 LC Nucleic
Acid Sequence
CAGAGCGTCTTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCA (SEQ ID
NO:44) TCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCA
ACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAAGCGGCCCTCA
GGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCT
CTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCACCTCATATACAAGTAGCAG
CACTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCC
CCCTCG
EXAMPLE 10
Exemplary Sequences--H9
[0318] TABLE-US-00013 Translation of H9 HC (1-344) 1 EVQLLESGGG
LVQPGGSLRL SCAASGFTFS RYSMHWVRQA PGKGLEWVSS (SEQ ID NO:45) 51
IGPSGGKTKY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARPF 101
RGSYYYFDYW GQGT Translation of H9 LC (1-345) 1 QDIQMTQSPS
SLSASIGDRV TITCQASQDT YNRLHWYQQK SGKAPKLLIY (SEQ ID NO:46) 51
DAVNLKRGVP SRFRGSGSGT NFILTITNLQ PEDTATYFCQ HSDDLSLAFG 101
GGTKVEIKRT VAAPS H9 HC Nucleic Acid Sequence
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTC (SEQ ID
NO:47) TTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACTCTATGCATTGGGTTCGCCA
AGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCGGTCCTTCTGGTGGCAAGACT
AAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATA
CTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGC
GAGACCCTTCCGTGGGAGCTACTACTACTTTGACTACTGGGGCCAGGGAACCCT H9 LC
Nucleic Acid Sequence
CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTATAGGAGACAGAG (SEQ ID
NO:48) TCACCATAACTTGCCAGGCGAGTCAGGACACTTACAACCGTCTACATTGGTATCAGCA
GAAATCAGGGAAAGCCCCTAAACTCCTCATCTACGATGCAGTCAATTTGAAAAGGGGG
GTCCCTTCAAGGTTCCGTGGAAGTGGATCTGGGACAAATTTTATTTTGACCATCACCA
ACCTGCAGCCTGAAGATACTGCAACATATTTCTGTCAACATTCTGATGATCTGTCACT
CGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCT
EXAMPLE 11
Exemplary Sequences--G10-R2
[0319] TABLE-US-00014 Translation of G10-R2 HC (1-382) 1 EVQLLESGGG
LVQPGGSLRL SCAASGFTFS RYKMWWVRQA PGKGLEWVSY (SEQ ID NO:49) 51
ISPSGGYTGY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKNA 101
RRAFPSMDVW GKGTTVTVSS ASTKGPS Translation of G10-APSR2 LC (1-360) 1
QDIQMTQSPL SLPVTPGEPA SISCRSSQSL LYSNGYNYLD WYLQRPGQSP (SEQ ID
NO:50) 51 QLLIYLGSNR ASGVPDRFSG SGSGTDFTLK ISRVEAKDVG VYYCMQALQI
101 PRTFGQGTKV EIKRTVAAPS G1 HC Coding Sequence0-R2
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTC (SEQ ID
NO:51) TTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACAAGATGTGGTGGGTTCGCCA
AGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTATATCTCTCCTTCTGGTGGCTATACT
GGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATA
CTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGC
GAAAAACGCGCGAAGAGCTTTTCCCTCCATGGACGTCTGGGGCAAAGGGACCACGGTC
ACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGG G1 LC Coding Sequence0-R2
CAAGACATCCAGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGG (SEQ ID
NO:52) CCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGTATAGTAATGGATACAACTATTT
GGATTGGTACCTGCAGAGACCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCT
AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTCA
CACTGAAAATCAGCAGAGTGGAGGCTAAGGATGTTGGGGTTTATTACTGCATGCAAGC
TCTACAAATTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTG
GCTGCACCATCT
EXAMPLE 12
Exemplary Sequences--F3-R2
[0320] TABLE-US-00015 Translation of F3-R2 HC (1-382) (SEQ ID
NO:53) 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYRMHWVRQA PGKGLEWVSG 51
ISSSGGDTNY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKNA 101
RRAFPSMDVW GKGTTVTVSS ASTKGPS Translation of F3-R2 LC (1-345) (SEQ
ID NO:54) 1 QDIQMTQSPS SVSASVGDTV TITCRASLPV NTWLAWYQQK PGKAPKLLLY
51 AASRLQSGVP SRFSGSGSGT DFTLNISSLQ PEDFATYYCQ QANTFPYTFG 101
QGTKVDIKRT VAAPS F3 HC Coding Sequence-R2 (SEQ ID NO:55)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTA
TGCATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGT
ATCTCTTCTTCTGGTGGCGATACTAATTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAAAAACGCG
CGAAGAGCTTTTCCCTCCATGGACGTCTGGGGCAAAGGGACCACGGTCAC
CGTCTCAAGCGCCTCCACCAAGGGCCCATCGG F3 LC Coding Sequence-R2 (SEQ ID
NO:56) CAAGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGG
AGACACAGTCACCATCACTTGTCGGGCGAGTCTGCCTGTTAACACCTGGT
TAGCCTGGTATCAGCAGAAACCCGGGAAAGCCCCTAAACTCCTGCTCTAT
GCTGCATCCAGATTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGG
CTCTGGGACAGATTTCACTCTCAACATCAGCAGTCTGCAGCCTGAGGATT
TTGCAACCTACTATTGTCAACAGGCGAACACTTTCCCGTACACTTTTGGC
CAGGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCT
EXAMPLE 13
Exemplary Sequences--C6-R2
[0321] TABLE-US-00016 Translation of C6-R2 HC (1-382) (SEQ ID
NO:57) 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYSMHWVRQA PGKGLEWVSR 51
IVPSGGTTFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKNA 101
RRAFPSMDVW GKGTTVTVSS ASTKGPS Translation of C6-R2 LC (1-348) (SEQ
ID NO:58) 1 QSALTQDPAV SVALGQTVRI TCQGDSLRSY YASWYQQKPG QAPVLVIYSK
51 SNRPSGIPDR FSGSSSGSTA SLTITGAQAE DEADYYCNSR DSSGNHLVFG 101
GGTKLTVLGQ PKAAPS C6 HC Coding Sequence-R (SEQ ID NO:59)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACTCTA
TGCATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTCGT
ATCGTTCCTTCTGGTGGCACTACTTTTTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAAAAACGCG
CGAAGAGCTTTTCCCTCCATGGACGTCTGGGGCAAAGGGACCACGGTCAC
CGTCTCAAGCGCCTCCACCAAGGGCCCATCGG C6 LC Coding Sequence-R2 (SEQ ID
NO:60) CAGAGCGCTTTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGAC
AGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCT
GGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATATATAGTAAA
AGTAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGG
AAGCACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTG
ACTATTATTGTAACTCCCGGGACAGCAGTGGTAACCATCTGGTATTCGGC
GGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCG
EXAMPLE 14
Exemplary Sequences--A4-R3
[0322] TABLE-US-00017 Translation of A4-R3 HC (1-382) (SEQ ID
NO:61) 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYNMYWVRQA PGKGLEWVSG 51
IRPSGGSTQY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKNA 101
RRAFPSMDVW GKGTTVTVSS ASTKGPS Translation of A4-R3 LC (1-345) (SEQ
ID NO:62) 1 QSELTQDPAV SVALGQTVRI TCRGDRLRSY YSSWYQQKPR QAPVLVMFGR
51 KNRPSGIPDR FSGSTSGSTA SLTITATQAD DEADYFCSSR DGSGNFLFGG 101
GTKLTVLGQP KAAPS A4 HC Coding Sequence-R3 (SEQ ID NO:63)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACAATA
TGTATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGT
ATCCGTCCTTCTGGTGGCTCTACTCAGTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAAAAACGCG
CGAAGAGCTTTTCCCTCCATGGACGTCTGGGGCAAAGGGACCACGGTCAC
CGTCTCAAGCGCCTCCACCAAGGGCCCATCGG A4 LC Coding Sequence-R3 (SEQ ID
NO:64) CAGAGCGAATTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGGCAGAC
AGTCAGGATTACATGCCGAGGAGACAGACTCAGAAGTTATTATTCAAGTT
GGTACCAGCAGAAGCCACGACAGGCCCCTGTTCTTGTCATGTTTGGTAGA
AAGAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCACCTCAGG
AAGCACAGCTTCCTTGACCATCACTGCGACTCAGGCGGACGATGAGGCTG
ACTATTTCTGTAGTTCCCGGGACGGCAGTGGTAATTTCCTCTTCGGCGGA
GGGACCAAACTGACCGTCCTTGGTCAGCCCAAGGCTGCCCCCTCG
EXAMPLE 15
Exemplary Sequences--C1-R3
[0323] TABLE-US-00018 Translation of C1-R3 HC (1-382) (SEQ ID
NO:65) 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYSMHWVRQA PGKGLEWVSG 51
IRPSGGSTKY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKNA 101
RRAFPSMDVW GKGTTVTVSS ASTKGPS Translation of C1-R3 LC (1-345) (SEQ
ID NO:66) 1 QDIQMTQSPS SLSASVGDRV TITCRASQSI STYLNWYQQR PGEAPKLLIY
51 GASSLVSGVP SRFSGSGSGT DFTLTISSLQ PEDFATYYCH QSYITSWTFG 101
QGTKVEIKRT VA C1 HC Coding Sequence-R3 (SEQ ID NO:67)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACTCTA
TGCATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGT
ATCCGTCCTTCTGGTGGCTCTACTAAGTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAAAAACGCG
CGAAGAGCTTTTCCCTCCATGGACGTCTGGGGCAAAGGGACCACGGTCAC
CGTCTCAAGCGCCTCCACCAAGGGCCCATCGG C1 LC Coding Sequence-R3 (SEQ ID
NO:68) CAAGACATCCAGATGACCCAGTCTCCTTCCTCCCTGTCTGCATCTGTAGG
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTACT
TAAACTGGTATCAGCAGAGACCAGGGGAAGCCCCTAAACTCCTGATCTAT
GGTGCATCCAGTTTGGTGAGTGGGGTCCCATCAAGATTTAGTGGCAGCGG
ATCTGGGACAGATTTCACTCTCACCATCTCCAGTCTGCAACCTGAAGATT
TTGCAACTTACTACTGTCACCAGAGTTACATTACCTCGTGGACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCT
EXAMPLE 16
Exemplary Sequences--A2
[0324] TABLE-US-00019 Translation of A2 HC (1-341) (SEQ ID NO:69) 1
EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYRMYWVRQA PGKGLEWVSS 51
ISPSGGDTRY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG 101
PRGNKYYFDY WGQ Translation of A2 LC (1-337) (SEQ ID NO:70) 1
QDIQMTQSPS FLSAFVGDRV TITCRASQDI RSDLAWYQQT PGKAPKLLIY 51
AASTLKDGAP SRFSGSGSGT EFTLTISSLH PEDLATYYCQ HLNGHPAFGP 101
GTKVNIQRTV AA A2 HC coding nucleic acid (SEQ ID NO:71)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTA
TGTATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTCTCCTTCTGGTGGCGATACTCGTTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTTAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGGGGGA
CCGCGGGGTAACAAGTACTACTTTGACTACTGGGGCCAGGG A2 LC coding nucleic acid
(SEQ ID NO:72) CAAGACATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATTTGTAGG
AGACAGGGTCACCATCACTTGCCGGGCCAGTCAGGACATTAGAAGTGATT
TAGCCTGGTATCAGCAAACACCAGGGAAAGCCCCAAAGCTCCTGATCTAT
GCTGCATCCACTTTGAAAGATGGGGCCCCATCAAGATTCAGCGGCAGTGG
ATCTGGGACAGAATTTACTCTCACAATCAGCAGCCTGCACCCTGAAGATC
TTGCGACTTATTACTGTCAACACCTTAATGGTCACCCTGCTTTCGGCCCT
GGGACCAAAGTGAATATCCAAAGAACTGTGGCTGCAC
EXAMPLE 17
Exemplary Sequences--B5
[0325] TABLE-US-00020 Translation of B5 HC (1-341) (SEQ ID NO:73) 1
EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYRMYWVRQA PGKGLEWVSS 51
ISPSGGDTRY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG 101
PRGNKYYFDY WGQ Translation of B5 LC (1-334) (SEQ ID NO:74) 1
QYELTQPPSV SVSLGQAANI SCSGDRLGDK YTSWYQQQSG QSPVLVIYQD 51
KKRPSGIPER FSGSSSGNTA TLTISGAQAI DEAAYYCQAW ATNVVFGAGT 101
KLTVLGQPKA A B5 HC coding nucleic acid (SEQ ID NO:75)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTA
TGTATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTCTCCTTCTGGTGGCGATACTCGTTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTTAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGGGGGA
CCGCGGGGTAACAAGTACTACTTTGACTACTGGGGCCAGGG B5 LC coding nucleic acid
(SEQ ID NO:76) CAGTACGAATTGACTCAGCCACCCTCAGTGTCCGTGTCCCTAGGACAGGC
AGCCAACATCTCCTGCTCTGGAGATAGATTGGGGGATAAATATACTTCCT
GGTATCAACAACAGTCAGGACAGTCCCCTGTCCTGGTCATCTATCAAGAT
AAGAAGCGACCCTCAGGGATCCCCGAGCGATTCTCTGGCTCCTCCTCTGG
GAACACAGCCACTCTGACCATCAGCGGGGCCCAGGCCATAGATGAGGCTG
CCTATTACTGTCAGGCGTGGGCCACCAATGTGGTTTTCGGCGCTGGGACC
AAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCC
EXAMPLE 18
Exemplary Sequences--D2
[0326] TABLE-US-00021 Translation of D2 HC (1-341) (SEQ ID NO:77) 1
EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYRMYWVRQA PGKGLEWVSS 51
ISPSGGDTRY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG 101
PRGNKYYFDY WGQ Translation of D2 LC (1-340) (SEQ ID NO:78) 1
QDIQMTQSPS SLSASVGDRV TITCRASQTI DNYLNWYQQK PGKAPKLVVY 51
AASTLQTRVP SRFSGSGSGT DFTLTIDSLK PEDFATYFCQ QGFSNPWTFG 101
QGTTVAMIRT VAA D2 HC coding nucleic acid (SEQ ID NO:79)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTA
TGTATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTCTCCTTCTGGTGGCGATACTCGTTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTTAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGGGGGA
CCGCGGGGTAACAAGTACTACTTTGACTACTGGGGCCAGGG D2 LC coding nucleic acid
(SEQ ID NO:80) CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCTTCTGTTGG
AGACAGAGTCACCATCACTTGCCGGGCAAGCCAGACCATTGACAATTATT
TGAATTGGTATCAGCAGAAACCAGGGAAAGCCCCCAAACTCGTGGTCTAT
GCTGCATCCACTTTGCAAACTAGGGTCCCATCAAGGTTCAGTGGCAGTGG
GTCTGGGACAGACTTCACTCTCACCATCGACAGTCTGAAACCTGAAGATT
TTGCAACTTACTTCTGTCAACAGGGTTTCAGTAATCCTTGGACGTTCGGC
CAAGGGACCACGGTGGCAATGATACGAACTGTGGCTGCAC
EXAMPLE 19
Exemplary Sequences--D5
[0327] TABLE-US-00022 Translation of D5 HC (1-332) (SEQ ID NO:81) 1
EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYDMHWVRQA PGKGLEWVSS 51
ISSSGGYTAY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGA 101
RGTSQGYWGQ Translation of D5 LC (1-346) (SEQ ID NO:82) 1 QDIQMTQSPG
TLSLSPGERG TLSCRASQFV SYSYLAWYQQ KPGQAPRLLI 51 YGASSRAKGI
PDRFSGSGSG TDFTLTITRL EPEDFAVYYC QQYVPSVPWT 101 FGQGTKVEVK RTVAA D5
HC coding nucleic acid (SEQ ID NO:83)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGATA
TGCATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTCTTCTTCTGGTGGCTATACTGCTTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGGCGCC
CGAGGTACCAGCCAAGGCTACTGGGGCCAGGG D5 LC coding nucleic acid (SEQ ID
NO:84) CAAGACATCCAGATGACTCAGTCTCCAGGCACCCTGTCATTGTCTCCAGG
GGAAAGAGGCACCCTCTCCTGCAGGGCCAGTCAGTTTGTTAGTTACAGCT
ACTTAGCCTGGTACCAGCAGAAGCCTGGCCAGGCTCCCCGGCTCCTCATC
TATGGCGCATCCAGCAGGGCCAAAGGCATCCCAGACAGGTTCAGTGGCAG
TGGGTCTGGGACAGACTTCACTCTCACCATCACCAGACTGGAGCCTGAAG
ACTTTGCAGTTTATTACTGTCAGCAGTATGTTCCCTCAGTTCCGTGGACG
TTCGGCCAAGGGACCAAGGTGGAAGTCAAACGAACTGTGGCTGCAC
EXAMPLE 20
Exemplary Sequences--F8
[0328] TABLE-US-00023 Translation of F8 HC (1-341) (SEQ ID NO:85) 1
EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYHMWWVRQA PGKGLEWVSG 51
ISSSRGITKY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG 101
PRGNKYYFDY WGQ Translation of F8 LC (1-343) (SEQ ID NO:86) 1
QDIQMTQSPG TLSLSPGERV TLSCRASQSV TSSDLAWYQQ KPGQAPRLLI 51
SGASSRATGI PDRFSGSGSG TDFTLTISRL EPEDFAVYYC QQYGNSPGTF 101
GQGTKVEIKR TVAA F8 HC coding nucleic acid (SEQ ID NO:87)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCATA
TGTGGTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGT
ATCTCTTCTTCTCGTGGCATTACTAAGTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGGGGGA
CCGCGGGGTAACAAGTACTACTTTGACTACTGGGGCCAGGG F8 LC coding nucleic acid
(SEQ ID NO:88) CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGG
GGAAAGAGTCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAGCAGCG
ACTTAGCCTGGTACCAGCAGAAACCTGGTCAGGCTCCCAGGCTCCTCATT
TCTGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAG
TGGGTCTGGGACAGACTTCACCCTCACCATCAGCAGACTGGAACCTGAAG
ATTTTGCAGTGTATTACTGTCAGCAGTATGGTAACTCACCTGGGACGTTC
GGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCAC
EXAMPLE 21
Exemplary Sequences--H10
[0329] TABLE-US-00024 Translation of H10 HC (1-341) (SEQ ID NO:89)
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYRMYWVRQA PGKGLEWVSS 51
ISPSGGDTRY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG 101
PRGNKYYFDY WGQ Translation of H10 LC (1-343) 1 QDIQMTQSPG
TLSLSPGERA TLSCRASQSV SSSYLAWYQQ KPGQAPRLLI 51 YGASSRATGI
PDRFSGSGSG TDFTLTISRL EPEDFAVYYC QQYGSSTWTF 101 GQGTKVEIKR TVAA
(SEQ ID NO:90) H10 HC coding nucleic acid (SEQ ID NO:91)
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACCGTA
TGTATTGGGTTCGCCAAGCTCCTGGTAZkAGGTTTGGAGTGGGTTTCTTC
TATCTCTCCTTCTGGTGGCGATACTCGTTATGCTGACTCCGTTAkAGGTC
GCTTCACTATCTCTAGAGACAACTCTTAGAATACTCTCTACTTGCAGATG
AACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGGGGG
ACCGCGGGGTAACAAGTACTACTTTGACTACTGGGGCCAGGG H10 LC coding nucleic
acid (SEQ ID NO:92)
CAAGACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGG
GGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCT
ACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAG
TGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAG
ATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCAACGTGGACGTTC
GGCCAAGGGACCAAAGTGGAAATCAAACGAACTGTGGCTGCAC
[0330] The stop codon in the middle of a coding nucleic acid can be
replaced by another codon, e.g., a codon that encodes lysine.
Alternatively, a bacterial strain with a tRNA suppressor can be
used to introduce a lysine or other amino acid at this
position.
[0331] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
Sequence CWU 1
1
114 1 2067 DNA Homo sapiens CDS (1)...(2064) 1 atg gag agg gac agc
cac ggg aat gca tct cca gca aga aca cct tca 48 Met Glu Arg Asp Ser
His Gly Asn Ala Ser Pro Ala Arg Thr Pro Ser 1 5 10 15 gct gga gca
tct cca gcc cag gca tct cca gct ggg aca cct cca ggc 96 Ala Gly Ala
Ser Pro Ala Gln Ala Ser Pro Ala Gly Thr Pro Pro Gly 20 25 30 cgg
gca tct cca gcc cag gca tct cca gcc cag gca tct cca gct ggg 144 Arg
Ala Ser Pro Ala Gln Ala Ser Pro Ala Gln Ala Ser Pro Ala Gly 35 40
45 aca cct ccg ggc cgg gca tct cca gcc cag gca tct cca gct ggt aca
192 Thr Pro Pro Gly Arg Ala Ser Pro Ala Gln Ala Ser Pro Ala Gly Thr
50 55 60 cct cca ggc cgg gca tct cca ggc cgg gca tct cca gcc cag
gca tct 240 Pro Pro Gly Arg Ala Ser Pro Gly Arg Ala Ser Pro Ala Gln
Ala Ser 65 70 75 80 cca gcc cgg gca tct ccg gct ctg gca tca ctt tcc
agg tcc tca tcc 288 Pro Ala Arg Ala Ser Pro Ala Leu Ala Ser Leu Ser
Arg Ser Ser Ser 85 90 95 ggc agg tca tca tcc gcc agg tca gcc tcg
gtg aca acc tcc cca acc 336 Gly Arg Ser Ser Ser Ala Arg Ser Ala Ser
Val Thr Thr Ser Pro Thr 100 105 110 aga gtg tac ctt gtt aga gca aca
cca gtg ggg gct gta ccc atc cga 384 Arg Val Tyr Leu Val Arg Ala Thr
Pro Val Gly Ala Val Pro Ile Arg 115 120 125 tca tct cct gcc agg tca
gca cca gca acc agg gcc acc agg gag agc 432 Ser Ser Pro Ala Arg Ser
Ala Pro Ala Thr Arg Ala Thr Arg Glu Ser 130 135 140 cca ggt acg agc
ctg ccc aag ttc acc tgg cgg gag ggc cag aag cag 480 Pro Gly Thr Ser
Leu Pro Lys Phe Thr Trp Arg Glu Gly Gln Lys Gln 145 150 155 160 cta
ccg ctc atc ggg tgc gtg ctc ctc ctc att gcc ctg gtg gtt tcg 528 Leu
Pro Leu Ile Gly Cys Val Leu Leu Leu Ile Ala Leu Val Val Ser 165 170
175 ctc atc atc ctc ttc cag ttc tgg cag ggc cac aca ggg atc agg tac
576 Leu Ile Ile Leu Phe Gln Phe Trp Gln Gly His Thr Gly Ile Arg Tyr
180 185 190 aag gag cag agg gag agc tgt ccc aag cac gct gtt cgc tgt
gac ggg 624 Lys Glu Gln Arg Glu Ser Cys Pro Lys His Ala Val Arg Cys
Asp Gly 195 200 205 gtg gtg gac tgc aag ctg aag agt gac gag ctg ggc
tgc gtg agg ttt 672 Val Val Asp Cys Lys Leu Lys Ser Asp Glu Leu Gly
Cys Val Arg Phe 210 215 220 gac tgg gac aag tct ctg ctt aaa atc tac
tct ggg tcc tcc cat cag 720 Asp Trp Asp Lys Ser Leu Leu Lys Ile Tyr
Ser Gly Ser Ser His Gln 225 230 235 240 tgg ctt ccc atc tgt agc agc
aac tgg aat gac tcc tac tca gag aag 768 Trp Leu Pro Ile Cys Ser Ser
Asn Trp Asn Asp Ser Tyr Ser Glu Lys 245 250 255 acc tgc cag cag ctg
ggt ttc gag agt gct cac cgg aca acc gag gtt 816 Thr Cys Gln Gln Leu
Gly Phe Glu Ser Ala His Arg Thr Thr Glu Val 260 265 270 gcc cac agg
gat ttt gcc aac agc ttc tca atc ttg aga tac aac tcc 864 Ala His Arg
Asp Phe Ala Asn Ser Phe Ser Ile Leu Arg Tyr Asn Ser 275 280 285 acc
atc cag gaa agc ctc cac agg tct gaa tgc cct tcc cag cgg tat 912 Thr
Ile Gln Glu Ser Leu His Arg Ser Glu Cys Pro Ser Gln Arg Tyr 290 295
300 atc tcc ctc cag tgt tcc cac tgc gga ctg agg gcc atg acc ggg cgg
960 Ile Ser Leu Gln Cys Ser His Cys Gly Leu Arg Ala Met Thr Gly Arg
305 310 315 320 atc gtg gga ggg gcg ctg gcc tcg gat agc aag tgg cct
tgg caa gtg 1008 Ile Val Gly Gly Ala Leu Ala Ser Asp Ser Lys Trp
Pro Trp Gln Val 325 330 335 agt ctg cac ttc ggc acc acc cac atc tgt
gga ggc acg ctc att gac 1056 Ser Leu His Phe Gly Thr Thr His Ile
Cys Gly Gly Thr Leu Ile Asp 340 345 350 gcc cag tgg gtg ctc act gcc
gcc cac tgc ttc ttc gtg acc cgg gag 1104 Ala Gln Trp Val Leu Thr
Ala Ala His Cys Phe Phe Val Thr Arg Glu 355 360 365 aag gtc ctg gag
ggc tgg aag gtg tac gcg ggc acc agc aac ctg cac 1152 Lys Val Leu
Glu Gly Trp Lys Val Tyr Ala Gly Thr Ser Asn Leu His 370 375 380 cag
ttg cct gag gca gcc tcc att gcc gag atc atc atc aac agc aat 1200
Gln Leu Pro Glu Ala Ala Ser Ile Ala Glu Ile Ile Ile Asn Ser Asn 385
390 395 400 tac acc gat gag gag gac gac tat gac atc gcc ctc atg cgg
ctg tcc 1248 Tyr Thr Asp Glu Glu Asp Asp Tyr Asp Ile Ala Leu Met
Arg Leu Ser 405 410 415 aag ccc ctg acc ctg tcc gct cac atc cac cct
gct tgc ctc ccc atg 1296 Lys Pro Leu Thr Leu Ser Ala His Ile His
Pro Ala Cys Leu Pro Met 420 425 430 cat gga cag acc ttt agc ctc aat
gag acc tgc tgg atc aca ggc ttt 1344 His Gly Gln Thr Phe Ser Leu
Asn Glu Thr Cys Trp Ile Thr Gly Phe 435 440 445 ggc aag acc agg gag
aca gat gac aag aca tcc ccc ttc ctc cgg gag 1392 Gly Lys Thr Arg
Glu Thr Asp Asp Lys Thr Ser Pro Phe Leu Arg Glu 450 455 460 gtg cag
gtc aat ctc atc gac ttc aag aaa tgc aat gac tac ttg gtc 1440 Val
Gln Val Asn Leu Ile Asp Phe Lys Lys Cys Asn Asp Tyr Leu Val 465 470
475 480 tat gac agt tac ctt acc cca agg atg atg tgt gct ggg gac ctt
cgt 1488 Tyr Asp Ser Tyr Leu Thr Pro Arg Met Met Cys Ala Gly Asp
Leu Arg 485 490 495 ggg ggc aga gac tcc tgc cag gga gac agc ggg ggg
cct ctt gtc tgt 1536 Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly
Gly Pro Leu Val Cys 500 505 510 gag cag aac aac cgc tgg tac ctg gca
ggt gtc acc agc tgg ggc aca 1584 Glu Gln Asn Asn Arg Trp Tyr Leu
Ala Gly Val Thr Ser Trp Gly Thr 515 520 525 ggc tgt ggc cag aga aac
aaa cct ggt gtg tac acc aaa gtg aca gaa 1632 Gly Cys Gly Gln Arg
Asn Lys Pro Gly Val Tyr Thr Lys Val Thr Glu 530 535 540 gtt ctt ccc
tgg att tac agc aag atg gag aac aga gct cag cgg gtt 1680 Val Leu
Pro Trp Ile Tyr Ser Lys Met Glu Asn Arg Ala Gln Arg Val 545 550 555
560 gaa aaa gcg tgg acc tac agg cca ggc agg cag ttg ctg ggc aga tgt
1728 Glu Lys Ala Trp Thr Tyr Arg Pro Gly Arg Gln Leu Leu Gly Arg
Cys 565 570 575 tct ccc aga agt att ttt ttg tgt aag gtt gca atg gac
ttt gaa aac 1776 Ser Pro Arg Ser Ile Phe Leu Cys Lys Val Ala Met
Asp Phe Glu Asn 580 585 590 gtt tca gtt tct gca gag gat ttt gtg ata
gtt ttt gtt atc aag cat 1824 Val Ser Val Ser Ala Glu Asp Phe Val
Ile Val Phe Val Ile Lys His 595 600 605 tta tgc atg gga atc cgc tct
tca tgg cct ttc cca gct ctg ttt gtt 1872 Leu Cys Met Gly Ile Arg
Ser Ser Trp Pro Phe Pro Ala Leu Phe Val 610 615 620 tta gtc ttt ttg
att ttc ttt ttg ttg ttg ttg ttg tct ttt tta aaa 1920 Leu Val Phe
Leu Ile Phe Phe Leu Leu Leu Leu Leu Ser Phe Leu Lys 625 630 635 640
aac aca agt gac tcc att ttg act ctg aca act ttc aca gct gtc acc
1968 Asn Thr Ser Asp Ser Ile Leu Thr Leu Thr Thr Phe Thr Ala Val
Thr 645 650 655 aga atg ctc cct gag aac tac cat tct ttc cct ttc cca
ctt aaa ata 2016 Arg Met Leu Pro Glu Asn Tyr His Ser Phe Pro Phe
Pro Leu Lys Ile 660 665 670 ttt cat cag aac ctc act act atc ata aaa
gag tat aaa gta ata aaa 2064 Phe His Gln Asn Leu Thr Thr Ile Ile
Lys Glu Tyr Lys Val Ile Lys 675 680 685 taa 2067 2 688 PRT Homo
sapiens 2 Met Glu Arg Asp Ser His Gly Asn Ala Ser Pro Ala Arg Thr
Pro Ser 1 5 10 15 Ala Gly Ala Ser Pro Ala Gln Ala Ser Pro Ala Gly
Thr Pro Pro Gly 20 25 30 Arg Ala Ser Pro Ala Gln Ala Ser Pro Ala
Gln Ala Ser Pro Ala Gly 35 40 45 Thr Pro Pro Gly Arg Ala Ser Pro
Ala Gln Ala Ser Pro Ala Gly Thr 50 55 60 Pro Pro Gly Arg Ala Ser
Pro Gly Arg Ala Ser Pro Ala Gln Ala Ser 65 70 75 80 Pro Ala Arg Ala
Ser Pro Ala Leu Ala Ser Leu Ser Arg Ser Ser Ser 85 90 95 Gly Arg
Ser Ser Ser Ala Arg Ser Ala Ser Val Thr Thr Ser Pro Thr 100 105 110
Arg Val Tyr Leu Val Arg Ala Thr Pro Val Gly Ala Val Pro Ile Arg 115
120 125 Ser Ser Pro Ala Arg Ser Ala Pro Ala Thr Arg Ala Thr Arg Glu
Ser 130 135 140 Pro Gly Thr Ser Leu Pro Lys Phe Thr Trp Arg Glu Gly
Gln Lys Gln 145 150 155 160 Leu Pro Leu Ile Gly Cys Val Leu Leu Leu
Ile Ala Leu Val Val Ser 165 170 175 Leu Ile Ile Leu Phe Gln Phe Trp
Gln Gly His Thr Gly Ile Arg Tyr 180 185 190 Lys Glu Gln Arg Glu Ser
Cys Pro Lys His Ala Val Arg Cys Asp Gly 195 200 205 Val Val Asp Cys
Lys Leu Lys Ser Asp Glu Leu Gly Cys Val Arg Phe 210 215 220 Asp Trp
Asp Lys Ser Leu Leu Lys Ile Tyr Ser Gly Ser Ser His Gln 225 230 235
240 Trp Leu Pro Ile Cys Ser Ser Asn Trp Asn Asp Ser Tyr Ser Glu Lys
245 250 255 Thr Cys Gln Gln Leu Gly Phe Glu Ser Ala His Arg Thr Thr
Glu Val 260 265 270 Ala His Arg Asp Phe Ala Asn Ser Phe Ser Ile Leu
Arg Tyr Asn Ser 275 280 285 Thr Ile Gln Glu Ser Leu His Arg Ser Glu
Cys Pro Ser Gln Arg Tyr 290 295 300 Ile Ser Leu Gln Cys Ser His Cys
Gly Leu Arg Ala Met Thr Gly Arg 305 310 315 320 Ile Val Gly Gly Ala
Leu Ala Ser Asp Ser Lys Trp Pro Trp Gln Val 325 330 335 Ser Leu His
Phe Gly Thr Thr His Ile Cys Gly Gly Thr Leu Ile Asp 340 345 350 Ala
Gln Trp Val Leu Thr Ala Ala His Cys Phe Phe Val Thr Arg Glu 355 360
365 Lys Val Leu Glu Gly Trp Lys Val Tyr Ala Gly Thr Ser Asn Leu His
370 375 380 Gln Leu Pro Glu Ala Ala Ser Ile Ala Glu Ile Ile Ile Asn
Ser Asn 385 390 395 400 Tyr Thr Asp Glu Glu Asp Asp Tyr Asp Ile Ala
Leu Met Arg Leu Ser 405 410 415 Lys Pro Leu Thr Leu Ser Ala His Ile
His Pro Ala Cys Leu Pro Met 420 425 430 His Gly Gln Thr Phe Ser Leu
Asn Glu Thr Cys Trp Ile Thr Gly Phe 435 440 445 Gly Lys Thr Arg Glu
Thr Asp Asp Lys Thr Ser Pro Phe Leu Arg Glu 450 455 460 Val Gln Val
Asn Leu Ile Asp Phe Lys Lys Cys Asn Asp Tyr Leu Val 465 470 475 480
Tyr Asp Ser Tyr Leu Thr Pro Arg Met Met Cys Ala Gly Asp Leu Arg 485
490 495 Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val
Cys 500 505 510 Glu Gln Asn Asn Arg Trp Tyr Leu Ala Gly Val Thr Ser
Trp Gly Thr 515 520 525 Gly Cys Gly Gln Arg Asn Lys Pro Gly Val Tyr
Thr Lys Val Thr Glu 530 535 540 Val Leu Pro Trp Ile Tyr Ser Lys Met
Glu Asn Arg Ala Gln Arg Val 545 550 555 560 Glu Lys Ala Trp Thr Tyr
Arg Pro Gly Arg Gln Leu Leu Gly Arg Cys 565 570 575 Ser Pro Arg Ser
Ile Phe Leu Cys Lys Val Ala Met Asp Phe Glu Asn 580 585 590 Val Ser
Val Ser Ala Glu Asp Phe Val Ile Val Phe Val Ile Lys His 595 600 605
Leu Cys Met Gly Ile Arg Ser Ser Trp Pro Phe Pro Ala Leu Phe Val 610
615 620 Leu Val Phe Leu Ile Phe Phe Leu Leu Leu Leu Leu Ser Phe Leu
Lys 625 630 635 640 Asn Thr Ser Asp Ser Ile Leu Thr Leu Thr Thr Phe
Thr Ala Val Thr 645 650 655 Arg Met Leu Pro Glu Asn Tyr His Ser Phe
Pro Phe Pro Leu Lys Ile 660 665 670 Phe His Gln Asn Leu Thr Thr Ile
Ile Lys Glu Tyr Lys Val Ile Lys 675 680 685 3 657 DNA Artificial
Sequence Synthetically generated oligonucleotide 3 cagagcgtct
tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60
tcctgcactg gaaccagtag tgacgttggt cattataatt atgtctcctg gtaccaacag
120 cacccaggca aagcccccaa agtcatgatt tatgatgtca gtagtcggcc
ctccggggtt 180 tctgatcgct tctctgggtc caagtctggc aacacggcct
ccctggccat ctctgggctc 240 caggctgagg acgaggctga ttattactgc
agttcgtata caagcggtga cactctttat 300 gtcttcggaa ctgggaccaa
ggtcaccgtc ctaggtcagc ccaaggccaa ccccactgtc 360 actctgttcc
cgccctcctc tgaggagctc caagccaaca aggccacact agtgtgtctg 420
atcagtgact tctacccggg agctgtgaca gtggcctgga aggcagatgg cagccccgtc
480 aaggcgggag tggagaccac caaaccctcc aaacagagca acaacaagta
cgcggccagc 540 agctacctga gcctgacgcc cgagcagtgg aagtcccaca
gaagctacag ctgccaggtc 600 acgcatgaag ggagcaccgt ggagaagaca
gtggcccctg cagaatgctc ttaataa 657 4 217 PRT Artificial Sequence
Synthetically generated peptide 4 Gln Ser Val Leu Thr Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys
Thr Gly Thr Ser Ser Asp Val Gly His Tyr 20 25 30 Asn Tyr Val Ser
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Val 35 40 45 Met Ile
Tyr Asp Val Ser Ser Arg Pro Ser Gly Val Ser Asp Arg Phe 50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Ala Ile Ser Gly Leu 65
70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr
Ser Gly 85 90 95 Asp Thr Leu Tyr Val Phe Gly Thr Gly Thr Lys Val
Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Asn Pro Thr Val Thr Leu
Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr
Val Ala Trp Lys Ala Asp Gly Ser Pro Val 145 150 155 160 Lys Ala Gly
Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185
190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205 Lys Thr Val Ala Pro Ala Glu Cys Ser 210 215 5 398 DNA
Artificial Sequence Synthetically generated oligonucleotide 5
gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60 tcttgcgctg cttccggatt cactttctct cgttacccta tgttttgggt
tcgccaagct 120 cctggtaaag gtttggagtg ggtttcttat atctcttctt
ctggtggctt tactggttat 180 gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240 ttgcagatga acagcttaag
ggctgaggac actgcagtct actattgtgc gagaggggga 300 ccgcggggta
acaagtacta ctttgactac tggggccagg gaaccctggt caccgtctca 360
agcgcctcca ccaagggccc atcggtcttc ccgctagc 398 6 132 PRT Artificial
Sequence Synthetically generated peptide 6 Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Pro Met
Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Tyr Ile Ser Ser Ser Gly Gly Phe Thr Gly Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Pro Arg Gly Asn Lys Tyr Tyr
Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu 130 7 639
DNA Artificial Sequence Synthetically generated oligonucleotide 7
agctacgaat tgactcagcc accctcagtg tccgtgtccc taggacaggc agccaacatc
60 tcctgctctg gagatagatt gggggataaa tatacttcct ggtatcaaca
acagtcagga 120 cagtcccctg tcctggtcat ctatcaagat aagaagcgac
cctcagggat ccccgagcga 180 ttctctggct cctcctctgg gaacacagcc
actctgacca tcagcggggc ccaggccata 240 gatgaggctg cctattactg
tcaggcgtgg gccaccaatg tggttttcgg cgctgggacc 300 aagctgaccg
tcctaggtca gcccaaggct gccccctcgg tcactctgtt cccgccctcc 360
tctgaggagc ttcaagccaa caaggccaca ctggtgtgtc tcataagtga
cttctacccg 420 ggagccgtga cagtggcctg gaaggcagat agcagccccg
tcaaggcggg agtggagacc 480 accacaccct ccaaacaaag caacaacaag
tacgcggcca gcagctatct gagcctgacg 540 cctgagcagt ggaagtccca
cagaagctac agctgccagg tcacgcatga agggagcacc 600 gtggagaaga
cagtggcccc tacaggatgt tcataataa 639 8 211 PRT Artificial Sequence
Synthetically generated peptide 8 Ser Tyr Glu Leu Thr Gln Pro Pro
Ser Val Ser Val Ser Leu Gly Gln 1 5 10 15 Ala Ala Asn Ile Ser Cys
Ser Gly Asp Arg Leu Gly Asp Lys Tyr Thr 20 25 30 Ser Trp Tyr Gln
Gln Gln Ser Gly Gln Ser Pro Val Leu Val Ile Tyr 35 40 45 Gln Asp
Lys Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60
Ser Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Ile 65
70 75 80 Asp Glu Ala Ala Tyr Tyr Cys Gln Ala Trp Ala Thr Asn Val
Val Phe 85 90 95 Gly Ala Gly Thr Lys Leu Thr Val Leu Gly Gln Pro
Lys Ala Ala Pro 100 105 110 Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
Glu Leu Gln Ala Asn Lys 115 120 125 Ala Thr Leu Val Cys Leu Ile Ser
Asp Phe Tyr Pro Gly Ala Val Thr 130 135 140 Val Ala Trp Lys Ala Asp
Ser Ser Pro Val Lys Ala Gly Val Glu Thr 145 150 155 160 Thr Thr Pro
Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr 165 170 175 Leu
Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys 180 185
190 Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr
195 200 205 Gly Cys Ser 210 9 398 DNA Artificial Sequence
Synthetically generated oligonucleotide 9 gaagttcaat tgttagagtc
tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60 tcttgcgctg
cttccggatt cactttctct cgttaccgta tgtattgggt tcgccaagct 120
cctggtaaag gtttggagtg ggtttcttct atctctcctt ctggtggcga tactcgttat
180 gctgactccg ttaaaggtcg cttcactatc tctagagaca actcttagaa
tactctctac 240 ttgcagatga acagcttaag ggctgaggac actgcagtct
actattgtgc gagaggggga 300 ccgcggggta acaagtacta ctttgactac
tggggccagg gaaccctggt caccgtctca 360 agcgcctcca ccaagggccc
atcggtcttc ccgctagc 398 10 131 PRT Artificial Sequence
Synthetically generated peptide 10 Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Arg Met Tyr Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser
Ile Ser Pro Ser Gly Gly Asp Thr Arg Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Asn Thr Leu Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg Gly Gly Pro Arg Gly Asn Lys Tyr Tyr Phe Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu 130 11 651 DNA
Artificial Sequence Synthetically generated oligonucleotide 11
gacatccaga tgacccagtc tccaggcacc ctgtctttgt ctccagggga aagagtcacc
60 ctctcctgca gggccagtca gagtgttacc agcagcgact tagcctggta
ccagcagaaa 120 cctggtcagg ctcccaggct cctcatttct ggtgcatcca
gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca
gacttcaccc tcaccatcag cagactggaa 240 cctgaagatt ttgcagtgta
ttactgtcag cagtatggta actcacctgg gacgttcggc 300 caagggacca
aggtggaaat caaacgaact gtggctgcac catctgtctt catcttcccg 360
ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc
420 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc
gggtaactcc 480 caggagagtg tcacagagca ggacagcaag gacagcacct
acagcctcag cagcaccctg 540 acgctgagca aagcagacta cgagaaacac
aaagtctacg cctgcgaagt cacccatcag 600 ggcctgagct cgcccgtcac
aaagagcttc aacaggggag agtgttaata a 651 12 215 PRT Artificial
Sequence Synthetically generated peptide 12 Asp Ile Gln Met Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Val Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser 20 25 30 Asp Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45
Ile Ser Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50
55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu
Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly
Asn Ser Pro 85 90 95 Gly Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180
185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 13 398 DNA
Artificial Sequence Synthetically generated oligonucleotide 13
gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60 tcttgcgctg cttccggatt cactttctct cgttaccata tgtggtgggt
tcgccaagct 120 cctggtaaag gtttggagtg ggtttctggt atctcttctt
ctcgtggcat tactaagtat 180 gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240 ttgcagatga acagcttaag
ggctgaggac actgcagtct actattgtgc gagaggggga 300 ccgcggggta
acaagtacta ctttgactac tggggccagg gaaccctggt caccgtctca 360
agcgcctcca ccaagggccc atcggtcttc ccgctagc 398 14 132 PRT Artificial
Sequence Synthetically generated peptide 14 Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 His Met
Trp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Gly Ile Ser Ser Ser Arg Gly Ile Thr Lys Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Pro Arg Gly Asn Lys Tyr Tyr
Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu 130 15 651
DNA Artificial Sequence Synthetically generated oligonucleotide 15
gacatccaga tgacccagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta
ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta
ttactgtcag cagtatggta gctcaacgtg gacgttcggc 300 caagggacca
aagtggaaat caaacgaact gtggctgcac catctgtctt catcttcccg 360
ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc
420 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc
gggtaactcc 480 caggagagtg tcacagagca ggacagcaag gacagcacct
acagcctcag cagcaccctg 540 acgctgagca aagcagacta cgagaaacac
aaagtctacg cctgcgaagt cacccatcag 600 ggcctgagct cgcccgtcac
aaagagcttc aacaggggag agtgttaata a 651 16 215 PRT Artificial
Sequence Synthetically generated peptide 16 Asp Ile Gln Met Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50
55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu
Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly
Ser Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180
185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 17 645 DNA
Artificial Sequence Synthetically generated oligonucleotide 17
gacatccaga tgacccagtc tccatccttc ctgtctgcat ttgtaggaga cagggtcacc
60 atcacttgcc gggccagtca ggacattaga agtgatttag cctggtatca
gcaaacacca 120 gggaaagccc caaagctcct gatctatgct gcatccactt
tgaaagatgg ggccccatca 180 agattcagcg gcagtggatc tgggacagaa
tttactctca caatcagcag cctgcaccct 240 gaagatcttg cgacttatta
ctgtcaacac cttaatggtc accctgcttt cggccctggg 300 accaaagtga
atatccaaag aactgtggct gcaccatctg tcttcatctt cccgccatct 360
gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc
420 agagaagcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa
ctcccaggag 480 agtgtcacag agcaggacag caaagacagc acctacagcc
tcagcagcac cctgacgctg 540 agcaaagcag actacgagaa acacaaagtc
tacgcctgcg aagtcaccca tcagggcctg 600 agctcgcccg tcacaaagag
cttcaacagg ggagagtgtt aataa 645 18 213 PRT Artificial Sequence
Synthetically generated peptide 18 Asp Ile Gln Met Thr Gln Ser Pro
Ser Phe Leu Ser Ala Phe Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Ile Arg Ser Asp 20 25 30 Leu Ala Trp Tyr
Gln Gln Thr Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala
Ala Ser Thr Leu Lys Asp Gly Ala Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu His Pro 65
70 75 80 Glu Asp Leu Ala Thr Tyr Tyr Cys Gln His Leu Asn Gly His
Pro Ala 85 90 95 Phe Gly Pro Gly Thr Lys Val Asn Ile Gln Arg Thr
Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185
190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205 Asn Arg Gly Glu Cys 210 19 648 DNA Artificial Sequence
Synthetically generated oligonucleotide 19 gacatccaga tgacccagtc
tccatcctcc ctgtctgctt ctgttggaga cagagtcacc 60 atcacttgcc
gggcaagcca gaccattgac aattatttga attggtatca gcagaaacca 120
gggaaagccc ccaaactcgt ggtctatgct gcatccactt tgcaaactag ggtcccatca
180 aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcgacag
tctgaaacct 240 gaagattttg caacttactt ctgtcaacag ggtttcagta
atccttggac gttcggccaa 300 gggaccacgg tggcaatgat acgaactgtg
gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc
tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg
ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg
540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac
ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttaataa
648 20 214 PRT Artificial Sequence Synthetically generated peptide
20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Asp
Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Val Val 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Thr Arg Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Asp Ser Leu Lys Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Phe Cys Gln Gln Gly Phe Ser Asn Pro Trp 85 90 95 Thr Phe Gly Gln
Gly Thr Thr Val Ala Met Ile Arg Thr Val Ala Ala 100 105 110 Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130
135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys
210 21 654 DNA Artificial Sequence Synthetically generated
oligonucleotide 21 gacatccaga tgacccagtc tccaggcacc ctgtcattgt
ctccagggga aagaggcacc 60 ctctcctgca gggccagtca gtttgttagt
tacagctact tagcctggta ccagcagaag 120 cctggccagg ctccccggct
cctcatctat ggcgcatcca gcagggccaa aggcatccca 180 gacaggttca
gtggcagtgg gtctgggaca gacttcactc tcaccatcac cagactggag 240
cctgaagact ttgcagttta ttactgtcag cagtatgttc cctcagttcc gtggacgttc
300 ggccaaggga ccaaggtgga agtcaaacga actgtggctg caccatctgt
cttcatcttc 360 ccgccatctg atgagcagtt gaaatctgga actgcctctg
ttgtgtgcct gctgaataac 420 ttctatccca gagaggccaa agtacagtgg
aaggtggata acgccctcca atcgggtaac 480 tcccaggaga gtgtcacaga
gcaggacggc aaggacagca cctacagcct cagcagcacc 540 ctgacgctga
gcaaagcaga ctacgaggaa cacaaagtct acgcctgcga agtcacccat 600
cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta ataa 654 22
216 PRT Artificial Sequence Synthetically generated peptide 22 Asp
Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Gly Thr Leu Ser Cys Arg Ala Ser Gln Phe Val Ser Tyr Ser
20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Lys Gly Ile Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Thr Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Tyr Val Pro Ser Val 85 90 95 Pro Trp Thr Phe Gly Gln
Gly Thr Lys Val Glu Val Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145
150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Gly Lys Asp Ser Thr
Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Glu His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys
210 215 23 389 DNA Artificial Sequence Synthetically generated
oligonucleotide 23 gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60 tcttgcgctg cttccggatt cactttctct
cgttacgata tgcattgggt tcgccaagct 120 cctggtaaag gtttggagtg
ggtttcttct atctcttctt ctggtggcta tactgcttat 180
gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240 ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc
gagaggcgcc 300 cgaggtacca gccaaggcta ctggggccag ggaaccctgg
tcaccgtctc aagcgcctcc 360 accaagggcc catcggtctt cccgctagc 389 24
129 PRT Artificial Sequence Synthetically generated peptide 24 Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
20 25 30 Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Gly Gly Tyr Thr Ala Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Arg Gly
Thr Ser Gln Gly Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu 25
114 PRT Artificial Sequence Synthetically generated peptide 25 Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
20 25 30 Arg Met Trp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Gly Phe Thr Asn Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Ala Arg Arg
Ala Leu Pro Ser Met Asp Val Trp Gly Lys 100 105 110 Gly Thr 26 118
PRT Artificial Sequence Synthetically generated peptide 26 Gln Ser
Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20
25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys
Ala Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Pro Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro
Ser 115 27 344 DNA Artificial Sequence Synthetically generated
oligonucleotide 27 gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60 tcttgcgctg cttccggatt cactttctct
cgttaccgta tgtggtgggt tcgccaagct 120 cctggtaaag gtttggagtg
ggtttcttat atctcttctt ctggtggctt tactaattat 180 gctgactccg
ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac 240
ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gaaaaacgcg
300 cgaagagctc ttccctccat ggacgtctgg ggcaaaggga ccac 344 28 354 DNA
Artificial Sequence Synthetically generated oligonucleotide 28
cagagcgctt tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc
60 tcttgttctg gaagcagctc caacatcgga agtaattatg tatactggta
ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat agtaataatc
agcggccctc aggggtccct 180 gaccgattct ctggctccaa gtctggcacc
tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta
ttactgtgca gcatgggatg acagcctgag tggtccggtg 300 ttcggcggag
ggaccaagct gaccgtccta ggtcagccca aggctgcccc ctcg 354 29 114 PRT
Artificial Sequence Synthetically generated peptide 29 Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25
30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Val Ile Tyr Ser Ser Gly Gly Ile Thr Arg Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Ala Pro Arg Gly Glu
Val Ala Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr 30 115 PRT
Artificial Sequence Synthetically generated protein 30 Gln Asp Ile
Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Ile 1 5 10 15 Gly
Asp Arg Val Thr Ile Thr Cys Trp Ala Ser Gln Gly Ile Ser Asn 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
35 40 45 Ile Ser Ser Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Ser Ala Thr Tyr Tyr Cys Gln
Gln Ala Asn Ser Phe Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Arg
Val Glu Ile Arg Arg Thr Val Ala 100 105 110 Ala Pro Ser 115 31 342
DNA Artificial Sequence Synthetically generated oligonucleotide 31
gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60 tcttgcgctg cttccggatt cactttctct cgttacggta tgtcttgggt
tcgccaagct 120 cctggtaaag gtttggagtg ggtttctgtt atctattctt
ctggtggcat tactcgttat 180 gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240 ttgcagatga acagcttaag
ggctgaggac actgcagtct actactgtgc gagacgggcc 300 ccgagggggg
aggtcgcttt tgatatctgg ggccaaggga ca 342 32 345 DNA Artificial
Sequence Synthetically generated oligonucleotide 32 caagacatcc
agatgaccca gtctccatcc ttcctgtctg catctatagg agacagagtc 60
accatcactt gctgggccag tcagggcatt agtaattatt tagcctggta tcagcaaaaa
120 ccagggaaag cccctaagct cctgatctct tctgcatcca ctttgcaaag
tggggtccca 180 tcaaggttca gcggcagtgg atctgggaca gaattcactc
tcacaatcag cagcctgcag 240 cctgaagatt ctgcaactta ctattgtcaa
caggctaaca gtttcccgtg gacgttcggc 300 caagggacca gggtggaaat
cagacgaact gtggctgcac catct 345 33 114 PRT Artificial Sequence
Synthetically generated peptide 33 Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Lys Met Trp Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr
Ile Ser Pro Ser Gly Gly Tyr Thr Gly Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Asn Ala Arg Arg Ala Phe Pro Ser Met Asp
Val Trp Gly Lys 100 105 110 Gly Thr 34 115 PRT Artificial Sequence
Synthetically generated peptide 34 Gln Ser Ala Leu Thr Gln Asp Pro
Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys
Arg Gly Asp Arg Leu Arg Ser Tyr Tyr Ser 20 25 30 Ser Trp Tyr Gln
Gln Lys Pro Arg Gln Ala Pro Val Leu Val Met Phe 35 40 45 Gly Arg
Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60
Thr Ser Gly Ser Thr Ala Ser Leu Thr Ile Thr Ala Thr Gln Ala Asp 65
70 75 80 Asp Glu Ala Asp Tyr Phe Cys Ser Ser Arg Asp Gly Ser Gly
Asn Phe 85 90 95 Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
Gln Pro Lys Ala 100 105 110 Ala Pro Ser 115 35 344 DNA Artificial
Sequence Synthetically generated oligonucleotide 35 gaagttcaat
tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60
tcttgcgctg cttccggatt cactttctct cgttacaaga tgtggtgggt tcgccaagct
120 cctggtaaag gtttggagtg ggtttcttat atctctcctt ctggtggcta
tactggttat 180 gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240 ttgcagatga acagcttaag ggctgaggac
actgcagtct actattgtgc gaaaaacgcg 300 cgaagagctt ttccctccat
ggacgtctgg ggcaaaggga ccac 344 36 345 DNA Artificial Sequence
Synthetically generated oligonucleotide 36 cagagcgctt tgactcagga
ccctgctgtg tctgtggcct tggggcagac agtcaggatc 60 acatgccgag
gagacagact cagaagttat tattcaagtt ggtaccagca gaagccacga 120
caggcccctg ttcttgtcat gtttggtaga aacaaccggc cctcagggat cccagaccga
180 ttctctggct ccacctcagg aagcacagct tccttgacca tcactgcgac
tcaggcggac 240 gatgaggctg actatttctg tagttcccgg gacggcagtg
gtaatttcct cttcggcgga 300 gggaccaaac tgaccgtcct tggtcagccc
aaggctgccc cctcg 345 37 114 PRT Artificial Sequence Synthetically
generated peptide 37 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Arg Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser
Ser Gly Gly Ile Thr Thr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Ile Tyr Tyr Cys 85 90
95 Ala Lys Asn Ala Arg Arg Ala Phe Pro Ser Met Asp Val Trp Gly Lys
100 105 110 Gly Thr 38 116 PRT Artificial Sequence Synthetically
generated peptide 38 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Asn Ser Leu Gln 65 70 75 80 Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Thr Gly Tyr Pro 85 90
95 Ser Ile Thr Phe Gly Gln Gly Thr Arg Leu Asp Ile Lys Arg Thr Val
100 105 110 Ala Ala Pro Ser 115 39 342 DNA Artificial Sequence
Synthetically generated oligonucleotide 39 gaagttcaat tgttagagtc
tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60 tcttgcgctg
cttccggatt cactttctct cgttaccgta tgtcttgggt tcgccaagct 120
cctggtaaag gtttggagtg ggtttcttct atctcttctt ctggtggcat tactacttat
180 gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa
tactctctac 240 ttgcagatga acagcttaag ggctgaggac gctgcaatct
actattgtgc gaaaaacgcg 300 cgaagagctt ttccctccat ggacgtctgg
ggcaaaggga cc 342 40 348 DNA Artificial Sequence Synthetically
generated oligonucleotide 40 caagacatcc agatgaccca gtctccatcc
tccctgtctg catctgtagg agacagagtc 60 accatcactt gccgggcaag
tcagagcatt agcagctatt taaattggta tcagcagaaa 120 ccagggaaag
cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 180
tcaaggttca gcggcagtgg atctgggaca gaattcactc tcacaatcaa cagcctgcag
240 cctgaagatt ttgcaactta ttactgtcaa caacttactg gttacccctc
gatcaccttc 300 ggccaaggga cacgactgga cattaaacga actgtggctg caccatct
348 41 114 PRT Artificial Sequence Synthetically generated peptide
41 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Arg Tyr 20 25 30 Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Tyr Ile Val Pro Ser Gly Gly Met Thr
Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Ala
Pro Arg Gly Glu Val Ala Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr
42 118 PRT Artificial Sequence Synthetically generated peptide 42
Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5
10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly
Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly
Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Tyr Cys Thr Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala
Ala Pro Ser 115 43 342 DNA Artificial Sequence Synthetically
generated oligonucleotide 43 gaagttcaat tgttagagtc tggtggcggt
cttgttcagc ctggtggttc tttacgtctt 60 tcttgcgctg cttccggatt
cactttctct cgttacacta tgtcttgggt tcgccaagct 120 cctggtaaag
gtttggagtg ggtttcttat atcgttcctt ctggtggcat gactaagtat 180
gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240 ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc
gagacgggcc 300 ccgagggggg aggtcgcttt tgatatctgg ggccaaggga ca 342
44 354 DNA Artificial Sequence Synthetically generated
oligonucleotide 44 cagagcgtct tgactcagcc tgcctccgtg tctgggtctc
ctggacagtc gatcaccatc 60 tcctgcactg gaaccagcag tgacgttggt
ggttataact atgtctcctg gtaccaacag 120 cacccaggca aagcccccaa
actcatgatt tatgatgtca gtaagcggcc ctcaggggtt 180 tctaatcgct
tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240
caggctgagg acgaggctga ttattactgc acctcatata caagtagcag cacttgggtg
300 ttcggcggag ggaccaagct gaccgtccta ggtcagccca aggctgcccc ctcg 354
45 114 PRT Artificial Sequence Synthetically generated peptide 45
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg
Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Gly Pro Ser Gly Gly Lys Thr Lys
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Pro Phe Arg
Gly Ser Tyr Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr 46
115 PRT Artificial Sequence Synthetically generated peptide 46 Gln
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Ile 1 5 10
15 Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Thr Tyr Asn
20 25 30 Arg Leu His Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys
Leu Leu 35 40 45 Ile Tyr Asp Ala Val Asn Leu Lys Arg Gly Val Pro
Ser Arg Phe Arg 50 55 60 Gly Ser Gly Ser Gly Thr Asn Phe Ile Leu
Thr Ile Thr Asn Leu Gln 65 70 75 80 Pro Glu Asp Thr Ala Thr Tyr Phe
Cys Gln His Ser Asp Asp Leu Ser 85 90 95 Leu Ala Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser 115
47 344 DNA Artificial Sequence Synthetically generated
oligonucleotide 47 gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60 tcttgcgctg cttccggatt cactttctct
cgttactcta tgcattgggt tcgccaagct 120 cctggtaaag gtttggagtg
ggtttcttct atcggtcctt ctggtggcaa gactaagtat 180 gctgactccg
ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac 240
ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gagacccttc
300 cgtgggagct actactactt tgactactgg ggccagggaa ccct 344 48 345 DNA
Artificial Sequence Synthetically generated oligonucleotide 48
caagacatcc agatgaccca gtctccatcc tccctgtctg catctatagg agacagagtc
60 accataactt
gccaggcgag tcaggacact tacaaccgtc tacattggta tcagcagaaa 120
tcagggaaag cccctaaact cctcatctac gatgcagtca atttgaaaag gggggtccct
180 tcaaggttcc gtggaagtgg atctgggaca aattttattt tgaccatcac
caacctgcag 240 cctgaagata ctgcaacata tttctgtcaa cattctgatg
atctgtcact cgctttcggc 300 ggagggacca aggtggagat caaacgaact
gtggctgcac catct 345 49 127 PRT Artificial Sequence Synthetically
generated peptide 49 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Lys Met Trp Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Pro
Ser Gly Gly Tyr Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Asn Ala Arg Arg Ala Phe Pro Ser Met Asp Val Trp Gly Lys
100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser 115 120 125 50 120 PRT Artificial Sequence Synthetically
generated peptide 50 Gln Asp Ile Gln Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro 1 5 10 15 Gly Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Leu Tyr 20 25 30 Ser Asn Gly Tyr Asn Tyr Leu
Asp Trp Tyr Leu Gln Arg Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu
Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile
Ser Arg Val Glu Ala Lys Asp Val Gly Val Tyr Tyr Cys Met Gln 85 90
95 Ala Leu Gln Ile Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105 110 Lys Arg Thr Val Ala Ala Pro Ser 115 120 51 382 DNA
Artificial Sequence Synthetically generated oligonucleotide 51
gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60 tcttgcgctg cttccggatt cactttctct cgttacaaga tgtggtgggt
tcgccaagct 120 cctggtaaag gtttggagtg ggtttcttat atctctcctt
ctggtggcta tactggttat 180 gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240 ttgcagatga acagcttaag
ggctgaggac actgcagtct actattgtgc gaaaaacgcg 300 cgaagagctt
ttccctccat ggacgtctgg ggcaaaggga ccacggtcac cgtctcaagc 360
gcctccacca agggcccatc gg 382 52 360 DNA Artificial Sequence
Synthetically generated oligonucleotide 52 caagacatcc agatgaccca
gtctccactc tccctgcccg tcacccctgg agagccggcc 60 tccatctcct
gcaggtctag tcagagcctc ctgtatagta atggatacaa ctatttggat 120
tggtacctgc agagaccagg gcagtctcca cagctcctga tctatttggg ttctaatcgg
180 gcctccgggg tccctgacag gttcagtggc agtggatcag gcacagattt
cacactgaaa 240 atcagcagag tggaggctaa ggatgttggg gtttattact
gcatgcaagc tctacaaatt 300 cctcggacgt tcggccaagg gaccaaggtg
gaaatcaaac gaactgtggc tgcaccatct 360 53 127 PRT Artificial Sequence
Synthetically generated peptide 53 Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Arg Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly
Ile Ser Ser Ser Gly Gly Asp Thr Asn Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Asn Ala Arg Arg Ala Phe Pro Ser Met Asp
Val Trp Gly Lys 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser 115 120 125 54 115 PRT Artificial Sequence
Synthetically generated peptide 54 Gln Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Val Ser Ala Ser Val 1 5 10 15 Gly Asp Thr Val Thr Ile
Thr Cys Arg Ala Ser Leu Pro Val Asn Thr 20 25 30 Trp Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Leu Tyr
Ala Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile Ser Ser Leu Gln 65
70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Thr
Phe Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys
Arg Thr Val Ala 100 105 110 Ala Pro Ser 115 55 382 DNA Artificial
Sequence Synthetically generated oligonucleotide 55 gaagttcaat
tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60
tcttgcgctg cttccggatt cactttctct cgttaccgta tgcattgggt tcgccaagct
120 cctggtaaag gtttggagtg ggtttctggt atctcttctt ctggtggcga
tactaattat 180 gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240 ttgcagatga acagcttaag ggctgaggac
actgcagtct actattgtgc gaaaaacgcg 300 cgaagagctt ttccctccat
ggacgtctgg ggcaaaggga ccacggtcac cgtctcaagc 360 gcctccacca
agggcccatc gg 382 56 345 DNA Artificial Sequence Synthetically
generated oligonucleotide 56 caagacatcc agatgaccca gtctccatct
tccgtgtctg catctgtagg agacacagtc 60 accatcactt gtcgggcgag
tctgcctgtt aacacctggt tagcctggta tcagcagaaa 120 cccgggaaag
cccctaaact cctgctctat gctgcatcca gattacaaag tggggtccca 180
tcaaggttca gcggcagtgg ctctgggaca gatttcactc tcaacatcag cagtctgcag
240 cctgaggatt ttgcaaccta ctattgtcaa caggcgaaca ctttcccgta
cacttttggc 300 caggggacca aagtggatat caaacgaact gtggctgcac catct
345 57 127 PRT Artificial Sequence Synthetically generated peptide
57 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Arg Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Arg Ile Val Pro Ser Gly Gly Thr Thr
Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Ala
Arg Arg Ala Phe Pro Ser Met Asp Val Trp Gly Lys 100 105 110 Gly Thr
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 58
116 PRT Artificial Sequence Synthetically generated peptide 58 Gln
Ser Ala Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10
15 Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala
20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val
Ile Tyr 35 40 45 Ser Lys Ser Asn Arg Pro Ser Gly Ile Pro Asp Arg
Phe Ser Gly Ser 50 55 60 Ser Ser Gly Ser Thr Ala Ser Leu Thr Ile
Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Asn
Ser Arg Asp Ser Ser Gly Asn His 85 90 95 Leu Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser
115 59 382 DNA Artificial Sequence Synthetically generated
oligonucleotide 59 gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60 tcttgcgctg cttccggatt cactttctct
cgttactcta tgcattgggt tcgccaagct 120 cctggtaaag gtttggagtg
ggtttctcgt atcgttcctt ctggtggcac tactttttat 180 gctgactccg
ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac 240
ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gaaaaacgcg
300 cgaagagctt ttccctccat ggacgtctgg ggcaaaggga ccacggtcac
cgtctcaagc 360 gcctccacca agggcccatc gg 382 60 348 DNA Artificial
Sequence Synthetically generated oligonucleotide 60 cagagcgctt
tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60
acatgccaag gagacagcct cagaagctat tatgcaagct ggtaccagca gaagccagga
120 caggcccctg tacttgtcat atatagtaaa agtaaccggc cctcagggat
cccagaccga 180 ttctctggct ccagctcagg aagcacagct tccttgacca
tcactggggc tcaggcggaa 240 gatgaggctg actattattg taactcccgg
gacagcagtg gtaaccatct ggtattcggc 300 ggagggacca agctgaccgt
cctaggtcag cccaaggctg ccccctcg 348 61 127 PRT Artificial Sequence
Synthetically generated peptide 61 Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Asn Met Tyr Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly
Ile Arg Pro Ser Gly Gly Ser Thr Gln Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Asn Ala Arg Arg Ala Phe Pro Ser Met Asp
Val Trp Gly Lys 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser 115 120 125 62 115 PRT Artificial Sequence
Synthetically generated peptide 62 Gln Ser Glu Leu Thr Gln Asp Pro
Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys
Arg Gly Asp Arg Leu Arg Ser Tyr Tyr Ser 20 25 30 Ser Trp Tyr Gln
Gln Lys Pro Arg Gln Ala Pro Val Leu Val Met Phe 35 40 45 Gly Arg
Lys Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60
Thr Ser Gly Ser Thr Ala Ser Leu Thr Ile Thr Ala Thr Gln Ala Asp 65
70 75 80 Asp Glu Ala Asp Tyr Phe Cys Ser Ser Arg Asp Gly Ser Gly
Asn Phe 85 90 95 Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
Gln Pro Lys Ala 100 105 110 Ala Pro Ser 115 63 382 DNA Artificial
Sequence Synthetically generated oligonucleotide 63 gaagttcaat
tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60
tcttgcgctg cttccggatt cactttctct cgttacaata tgtattgggt tcgccaagct
120 cctggtaaag gtttggagtg ggtttctggt atccgtcctt ctggtggctc
tactcagtat 180 gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240 ttgcagatga acagcttaag ggctgaggac
actgcagtct actattgtgc gaaaaacgcg 300 cgaagagctt ttccctccat
ggacgtctgg ggcaaaggga ccacggtcac cgtctcaagc 360 gcctccacca
agggcccatc gg 382 64 345 DNA Artificial Sequence Synthetically
generated oligonucleotide 64 cagagcgaat tgactcagga ccctgctgtg
tctgtggcct tggggcagac agtcaggatt 60 acatgccgag gagacagact
cagaagttat tattcaagtt ggtaccagca gaagccacga 120 caggcccctg
ttcttgtcat gtttggtaga aagaaccggc cctcagggat cccagaccga 180
ttctctggct ccacctcagg aagcacagct tccttgacca tcactgcgac tcaggcggac
240 gatgaggctg actatttctg tagttcccgg gacggcagtg gtaatttcct
cttcggcgga 300 gggaccaaac tgaccgtcct tggtcagccc aaggctgccc cctcg
345 65 127 PRT Artificial Sequence Synthetically generated peptide
65 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Arg Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Gly Ile Arg Pro Ser Gly Gly Ser Thr
Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Ala
Arg Arg Ala Phe Pro Ser Met Asp Val Trp Gly Lys 100 105 110 Gly Thr
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 66
112 PRT Artificial Sequence Synthetically generated peptide 66 Gln
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10
15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr
20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Arg Pro Gly Glu Ala Pro Lys
Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Leu Val Ser Gly Val Pro
Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys His Gln Ser Tyr Ile Thr Ser 85 90 95 Trp Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 67 382 DNA
Artificial Sequence Synthetically generated oligonucleotide 67
gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60 tcttgcgctg cttccggatt cactttctct cgttactcta tgcattgggt
tcgccaagct 120 cctggtaaag gtttggagtg ggtttctggt atccgtcctt
ctggtggctc tactaagtat 180 gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240 ttgcagatga acagcttaag
ggctgaggac actgcagtct actattgtgc gaaaaacgcg 300 cgaagagctt
ttccctccat ggacgtctgg ggcaaaggga ccacggtcac cgtctcaagc 360
gcctccacca agggcccatc gg 382 68 345 DNA Artificial Sequence
Synthetically generated oligonucleotide 68 caagacatcc agatgaccca
gtctccttcc tccctgtctg catctgtagg agacagagtc 60 accatcactt
gccgggcaag tcagagcatt agcacctact taaactggta tcagcagaga 120
ccaggggaag cccctaaact cctgatctat ggtgcatcca gtttggtgag tggggtccca
180 tcaagattta gtggcagcgg atctgggaca gatttcactc tcaccatctc
cagtctgcaa 240 cctgaagatt ttgcaactta ctactgtcac cagagttaca
ttacctcgtg gacgttcggc 300 caagggacca aggtggaaat caaacgaact
gtggctgcac catct 345 69 112 PRT Artificial Sequence Synthetically
generated peptide 69 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Arg Met Tyr Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Pro
Ser Gly Gly Asp Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Asn Thr Leu Tyr Leu 65 70 75 80 Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95 Arg Gly Gly Pro Arg Gly Asn Lys Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110 70 112 PRT Artificial Sequence Synthetically generated
peptide 70 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala
Phe Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Arg Ser 20 25 30 Asp Leu Ala Trp Tyr Gln Gln Thr Pro Gly
Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Ala Ser Thr Leu Lys
Asp Gly Ala Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu His 65 70 75 80 Pro Glu Asp Leu
Ala Thr Tyr Tyr Cys Gln His Leu Asn Gly His Pro 85 90 95 Ala Phe
Gly Pro Gly Thr Lys Val Asn Ile Gln Arg Thr Val Ala Ala 100 105 110
71 341 DNA Artificial Sequence Synthetically generated
oligonucleotide 71 gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60 tcttgcgctg cttccggatt cactttctct
cgttaccgta tgtattgggt tcgccaagct 120 cctggtaaag gtttggagtg
ggtttcttct atctctcctt ctggtggcga tactcgttat 180 gctgactccg
ttaaaggtcg cttcactatc tctagagaca actcttagaa tactctctac 240
ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gagaggggga
300 ccgcggggta acaagtacta ctttgactac tggggccagg g 341 72 337 DNA
Artificial Sequence Synthetically generated oligonucleotide 72
caagacatcc agatgaccca gtctccatcc ttcctgtctg catttgtagg agacagggtc
60 accatcactt gccgggccag tcaggacatt agaagtgatt tagcctggta
tcagcaaaca 120 ccagggaaag ccccaaagct cctgatctat gctgcatcca
ctttgaaaga tggggcccca 180 tcaagattca gcggcagtgg atctgggaca
gaatttactc tcacaatcag cagcctgcac 240 cctgaagatc ttgcgactta
ttactgtcaa caccttaatg gtcaccctgc tttcggccct 300 gggaccaaag
tgaatatcca aagaactgtg gctgcac 337 73 112 PRT Artificial Sequence
Synthetically generated peptide 73 Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Arg Met Tyr Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser
Ile Ser Pro Ser Gly Gly Asp Thr Arg Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Asn Thr Leu Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg Gly Gly Pro Arg Gly Asn Lys Tyr Tyr Phe Asp
Tyr Trp Gly Gln 100 105 110 74 111 PRT Artificial Sequence
Synthetically generated peptide 74 Gln Tyr Glu Leu Thr Gln Pro Pro
Ser Val Ser Val Ser Leu Gly Gln 1 5 10 15 Ala Ala Asn Ile Ser Cys
Ser Gly Asp Arg Leu Gly Asp Lys Tyr Thr 20 25 30 Ser Trp Tyr Gln
Gln Gln Ser Gly Gln Ser Pro Val Leu Val Ile Tyr 35 40 45 Gln Asp
Lys Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60
Ser Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Ile 65
70 75 80 Asp Glu Ala Ala Tyr Tyr Cys Gln Ala Trp Ala Thr Asn Val
Val Phe 85 90 95 Gly Ala Gly Thr Lys Leu Thr Val Leu Gly Gln Pro
Lys Ala Ala 100 105 110 75 341 DNA Artificial Sequence
Synthetically generated oligonucleotide 75 gaagttcaat tgttagagtc
tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60 tcttgcgctg
cttccggatt cactttctct cgttaccgta tgtattgggt tcgccaagct 120
cctggtaaag gtttggagtg ggtttcttct atctctcctt ctggtggcga tactcgttat
180 gctgactccg ttaaaggtcg cttcactatc tctagagaca actcttagaa
tactctctac 240 ttgcagatga acagcttaag ggctgaggac actgcagtct
actattgtgc gagaggggga 300 ccgcggggta acaagtacta ctttgactac
tggggccagg g 341 76 334 DNA Artificial Sequence Synthetically
generated oligonucleotide 76 cagtacgaat tgactcagcc accctcagtg
tccgtgtccc taggacaggc agccaacatc 60 tcctgctctg gagatagatt
gggggataaa tatacttcct ggtatcaaca acagtcagga 120 cagtcccctg
tcctggtcat ctatcaagat aagaagcgac cctcagggat ccccgagcga 180
ttctctggct cctcctctgg gaacacagcc actctgacca tcagcggggc ccaggccata
240 gatgaggctg cctattactg tcaggcgtgg gccaccaatg tggttttcgg
cgctgggacc 300 aagctgaccg tcctaggtca gcccaaggct gccc 334 77 112 PRT
Artificial Sequence Synthetically generated peptide 77 Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25
30 Arg Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Ser Ile Ser Pro Ser Gly Gly Asp Thr Arg Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Asn
Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Pro Arg Gly Asn Lys
Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 78 113 PRT Artificial
Sequence Synthetically generated peptide 78 Gln Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Asp Asn 20 25 30 Tyr Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Val 35 40 45
Val Tyr Ala Ala Ser Thr Leu Gln Thr Arg Val Pro Ser Arg Phe Ser 50
55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Ser Leu
Lys 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Phe
Ser Asn Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Thr Val Ala Met
Ile Arg Thr Val Ala 100 105 110 Ala 79 341 DNA Artificial Sequence
Synthetically generated oligonucleotide 79 gaagttcaat tgttagagtc
tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60 tcttgcgctg
cttccggatt cactttctct cgttaccgta tgtattgggt tcgccaagct 120
cctggtaaag gtttggagtg ggtttcttct atctctcctt ctggtggcga tactcgttat
180 gctgactccg ttaaaggtcg cttcactatc tctagagaca actcttagaa
tactctctac 240 ttgcagatga acagcttaag ggctgaggac actgcagtct
actattgtgc gagaggggga 300 ccgcggggta acaagtacta ctttgactac
tggggccagg g 341 80 340 DNA Artificial Sequence Synthetically
generated oligonucleotide 80 caagacatcc agatgaccca gtctccatcc
tccctgtctg cttctgttgg agacagagtc 60 accatcactt gccgggcaag
ccagaccatt gacaattatt tgaattggta tcagcagaaa 120 ccagggaaag
cccccaaact cgtggtctat gctgcatcca ctttgcaaac tagggtccca 180
tcaaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcga cagtctgaaa
240 cctgaagatt ttgcaactta cttctgtcaa cagggtttca gtaatccttg
gacgttcggc 300 caagggacca cggtggcaat gatacgaact gtggctgcac 340 81
110 PRT Artificial Sequence Synthetically generated peptide 81 Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
20 25 30 Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Gly Gly Tyr Thr Ala Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Arg Gly
Thr Ser Gln Gly Tyr Trp Gly Gln 100 105 110 82 115 PRT Artificial
Sequence Synthetically generated peptide 82 Gln Asp Ile Gln Met Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro 1 5 10 15 Gly Glu Arg Gly
Thr Leu Ser Cys Arg Ala Ser Gln Phe Val Ser Tyr 20 25 30 Ser Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 35 40 45
Leu Ile Tyr Gly Ala Ser Ser Arg Ala Lys Gly Ile Pro Asp Arg Phe 50
55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg
Leu 65 70 75 80 Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
Val Pro Ser 85 90 95 Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val
Glu Val Lys Arg Thr 100 105 110 Val Ala Ala 115 83 332 DNA
Artificial Sequence Synthetically generated oligonucleotide 83
gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60 tcttgcgctg cttccggatt cactttctct cgttacgata tgcattgggt
tcgccaagct 120 cctggtaaag gtttggagtg ggtttcttct atctcttctt
ctggtggcta tactgcttat 180 gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240 ttgcagatga acagcttaag
ggctgaggac actgcagtct actattgtgc gagaggcgcc 300 cgaggtacca
gccaaggcta ctggggccag gg 332 84 346 DNA Artificial Sequence
Synthetically generated oligonucleotide 84 caagacatcc agatgactca
gtctccaggc accctgtcat tgtctccagg ggaaagaggc 60 accctctcct
gcagggccag tcagtttgtt agttacagct acttagcctg gtaccagcag 120
aagcctggcc aggctccccg gctcctcatc tatggcgcat ccagcagggc caaaggcatc
180 ccagacaggt tcagtggcag tgggtctggg acagacttca ctctcaccat
caccagactg 240 gagcctgaag actttgcagt ttattactgt cagcagtatg
ttccctcagt tccgtggacg 300 ttcggccaag ggaccaaggt ggaagtcaaa
cgaactgtgg ctgcac 346 85 113 PRT Artificial Sequence Synthetically
generated peptide 85 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Arg Tyr 20 25 30 His Met Trp Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser
Ser Arg Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Pro Arg Gly Asn Lys Tyr Tyr Phe Asp Tyr Trp Gly
100 105 110 Gln 86 114 PRT Artificial Sequence Synthetically
generated peptide 86 Gln Asp Ile Gln Met Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro 1 5 10 15 Gly Glu Arg Val Thr Leu Ser Cys Arg
Ala Ser Gln Ser Val Thr Ser 20 25 30 Ser Asp Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu 35 40 45 Leu Ile Ser Gly Ala
Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu 65 70 75 80 Glu
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Asn Ser 85 90
95 Pro Gly Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val
100 105 110 Ala Ala 87 341 DNA Artificial Sequence Synthetically
generated oligonucleotide 87 gaagttcaat tgttagagtc tggtggcggt
cttgttcagc ctggtggttc tttacgtctt 60 tcttgcgctg cttccggatt
cactttctct cgttaccata tgtggtgggt tcgccaagct 120 cctggtaaag
gtttggagtg ggtttctggt atctcttctt ctcgtggcat tactaagtat 180
gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240 ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc
gagaggggga 300 ccgcggggta acaagtacta ctttgactac tggggccagg g 341 88
343 DNA Artificial Sequence Synthetically generated oligonucleotide
88 caagacatcc agatgaccca gtctccaggc accctgtctt tgtctccagg
ggaaagagtc 60 accctctcct gcagggccag tcagagtgtt accagcagcg
acttagcctg gtaccagcag 120 aaacctggtc aggctcccag gctcctcatt
tctggtgcat ccagcagggc cactggcatc 180 ccagacaggt tcagtggcag
tgggtctggg acagacttca ccctcaccat cagcagactg 240 gaacctgaag
attttgcagt gtattactgt cagcagtatg gtaactcacc tgggacgttc 300
ggccaaggga ccaaggtgga aatcaaacga actgtggctg cac 343 89 113 PRT
Artificial Sequence Synthetically generated peptide 89 Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25
30 Arg Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Ser Ile Ser Pro Ser Gly Gly Asp Thr Arg Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Pro Arg Gly Asn
Lys Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln 90 114 PRT
Artificial Sequence Synthetically generated peptide 90 Gln Asp Ile
Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro 1 5 10 15 Gly
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser 20 25
30 Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
35 40 45 Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp
Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Arg Leu 65 70 75 80 Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Tyr Gly Ser Ser 85 90 95 Thr Trp Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala 91 341 DNA
Artificial Sequence Synthetically generated oligonucleotide 91
gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60 tcttgcgctg cttccggatt cactttctct cgttaccgta tgtattgggt
tcgccaagct 120 cctggtaaag gtttggagtg ggtttcttct atctctcctt
ctggtggcga tactcgttat 180 gctgactccg ttaaaggtcg cttcactatc
tctagagaca actcttagaa tactctctac 240 ttgcagatga acagcttaag
ggctgaggac actgcagtct actattgtgc gagaggggga 300 ccgcggggta
acaagtacta ctttgactac tggggccagg g 341 92 343 DNA Artificial
Sequence Synthetically generated oligonucleotide 92 caagacatcc
agatgaccca gtctccaggc accctgtctt tgtctccagg ggaaagagcc 60
accctctcct gcagggccag tcagagtgtt agcagcagct acttagcctg gtaccagcag
120 aaacctggcc aggctcccag gctcctcatc tatggtgcat ccagcagggc
cactggcatc 180 ccagacaggt tcagtggcag tgggtctggg acagacttca
ctctcaccat cagcagactg 240 gagcctgaag attttgcagt gtattactgt
cagcagtatg gtagctcaac gtggacgttc 300 ggccaaggga ccaaagtgga
aatcaaacga actgtggctg cac 343 93 1689 DNA Homo sapiens 93
atggagaggg acagccacgg gaatgcatct ccagcaagaa caccttcagc tggagcatct
60 ccagcccagg catctccagc tgggacacct ccaggccggg catctccagc
ccaggcatct 120 ccagcccagg catctccagc tgggacacct ccgggccggg
catctccagc ccaggcatct 180 ccagctggta cacctccagg ccgggcatct
ccaggccggg catctccagc ccaggcatct 240 ccagcccggg catctccggc
tctggcatca ctttccaggt cctcatccgg caggtcatca 300 tccgccaggt
cagcctcggt gacaacctcc ccaaccagag tgtaccttgt tagagcaaca 360
ccagtggggg ctgtacccat ccgatcatct cctgccaggt cagcaccagc aaccagggcc
420 accagggaga gcccaggtac gagcctgccc aagttcacct ggcgggaggg
ccagaagcag 480 ctaccgctca tcgggtgcgt gctcctcctc attgccctgg
tggtttcgct catcatcctc 540 ttccagttct ggcagggcca cacagggatc
aggtacaagg agcagaggga gagctgtccc 600 aagcacgctg ttcgctgtga
cggggtggtg gactgcaagc tgaagagtga cgagctgggc 660 tgcgtgaggt
ttgactggga caagtctctg cttaaaatct actctgggtc ctcccatcag 720
tggcttccca tctgtagcag caactggaat gactcctact cagagaagac ctgccagcag
780 ctgggtttcg agagtgctca ccggacaacc gaggttgccc acagggattt
tgccaacagc 840 ttctcaatct tgagatacaa ctccaccatc caggaaagcc
tccacaggtc tgaatgccct 900 tcccagcggt atatctccct ccagtgttcc
cactgcggac tgagggccat gaccgggcgg 960 atcgtgggag gggcgctggc
ctcggatagc aagtggcctt ggcaagtgag tctgcacttc 1020 ggcaccaccc
acatctgtgg aggcacgctc attgacgccc agtgggtgct cactgccgcc 1080
cactgcttct tcgtgacccg ggagaaggtc ctggagggct ggaaggtgta cgcgggcacc
1140 agcaacctgc accagttgcc tgaggcagcc tccattgccg agatcatcat
caacagcaat 1200 tacaccgatg aggaggacga ctatgacatc gccctcatgc
ggctgtccaa gcccctgacc 1260 ctgtccgctc acatccaccc tgcttgcctc
cccatgcatg gacagacctt tagcctcaat 1320 gagacctgct ggatcacagg
ctttggcaag accagggaga cagatgacaa gacatccccc 1380 ttcctccggg
aggtgcaggt caatctcatc gacttcaaga aatgcaatga ctacttggtc 1440
tatgacagtt accttacccc aaggatgatg tgtgctgggg accttcgtgg gggcagagac
1500 tcctgccagg gagacagcgg ggggcctctt gtctgtgagc agaacaaccg
ctggtacctg 1560 gcaggtgtca ccagctgggg cacaggctgt ggccagagaa
acaaacctgg tgtgtacacc 1620 aaagtgacag aagttcttcc ctggatttac
agcaagatgg agagcgaggt gcgattcata 1680 aaatcctaa 1689 94 562 PRT
Homo sapiens 94 Met Glu Arg Asp Ser His Gly Asn Ala Ser Pro Ala Arg
Thr Pro Ser 1 5 10 15 Ala Gly Ala Ser Pro Ala Gln Ala Ser Pro Ala
Gly Thr Pro Pro Gly 20 25 30 Arg Ala Ser Pro Ala Gln Ala Ser Pro
Ala Gln Ala Ser Pro Ala Gly 35 40 45 Thr Pro Pro Gly Arg Ala Ser
Pro Ala Gln Ala Ser Pro Ala Gly Thr 50 55 60 Pro Pro Gly Arg Ala
Ser Pro Gly Arg Ala Ser Pro Ala Gln Ala Ser 65 70 75 80 Pro Ala Arg
Ala Ser Pro Ala Leu Ala Ser Leu Ser Arg Ser Ser Ser 85 90 95 Gly
Arg Ser Ser Ser Ala Arg Ser Ala Ser Val Thr Thr Ser Pro Thr 100 105
110 Arg Val Tyr Leu Val Arg Ala Thr Pro Val Gly Ala Val Pro Ile Arg
115 120 125 Ser Ser Pro Ala Arg Ser Ala Pro Ala Thr Arg Ala Thr Arg
Glu Ser 130 135 140 Pro Gly Thr Ser Leu Pro Lys Phe Thr Trp Arg Glu
Gly Gln Lys Gln 145 150 155 160 Leu Pro Leu Ile Gly Cys Val Leu Leu
Leu Ile Ala Leu Val Val Ser 165 170 175 Leu Ile Ile Leu Phe Gln Phe
Trp Gln Gly His Thr Gly Ile Arg Tyr 180 185 190 Lys Glu Gln Arg Glu
Ser Cys Pro Lys His Ala Val Arg Cys Asp Gly 195 200 205 Val Val Asp
Cys Lys Leu Lys
Ser Asp Glu Leu Gly Cys Val Arg Phe 210 215 220 Asp Trp Asp Lys Ser
Leu Leu Lys Ile Tyr Ser Gly Ser Ser His Gln 225 230 235 240 Trp Leu
Pro Ile Cys Ser Ser Asn Trp Asn Asp Ser Tyr Ser Glu Lys 245 250 255
Thr Cys Gln Gln Leu Gly Phe Glu Ser Ala His Arg Thr Thr Glu Val 260
265 270 Ala His Arg Asp Phe Ala Asn Ser Phe Ser Ile Leu Arg Tyr Asn
Ser 275 280 285 Thr Ile Gln Glu Ser Leu His Arg Ser Glu Cys Pro Ser
Gln Arg Tyr 290 295 300 Ile Ser Leu Gln Cys Ser His Cys Gly Leu Arg
Ala Met Thr Gly Arg 305 310 315 320 Ile Val Gly Gly Ala Leu Ala Ser
Asp Ser Lys Trp Pro Trp Gln Val 325 330 335 Ser Leu His Phe Gly Thr
Thr His Ile Cys Gly Gly Thr Leu Ile Asp 340 345 350 Ala Gln Trp Val
Leu Thr Ala Ala His Cys Phe Phe Val Thr Arg Glu 355 360 365 Lys Val
Leu Glu Gly Trp Lys Val Tyr Ala Gly Thr Ser Asn Leu His 370 375 380
Gln Leu Pro Glu Ala Ala Ser Ile Ala Glu Ile Ile Ile Asn Ser Asn 385
390 395 400 Tyr Thr Asp Glu Glu Asp Asp Tyr Asp Ile Ala Leu Met Arg
Leu Ser 405 410 415 Lys Pro Leu Thr Leu Ser Ala His Ile His Pro Ala
Cys Leu Pro Met 420 425 430 His Gly Gln Thr Phe Ser Leu Asn Glu Thr
Cys Trp Ile Thr Gly Phe 435 440 445 Gly Lys Thr Arg Glu Thr Asp Asp
Lys Thr Ser Pro Phe Leu Arg Glu 450 455 460 Val Gln Val Asn Leu Ile
Asp Phe Lys Lys Cys Asn Asp Tyr Leu Val 465 470 475 480 Tyr Asp Ser
Tyr Leu Thr Pro Arg Met Met Cys Ala Gly Asp Leu Arg 485 490 495 Gly
Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys 500 505
510 Glu Gln Asn Asn Arg Trp Tyr Leu Ala Gly Val Thr Ser Trp Gly Thr
515 520 525 Gly Cys Gly Gln Arg Asn Lys Pro Gly Val Tyr Thr Lys Val
Thr Glu 530 535 540 Val Leu Pro Trp Ile Tyr Ser Lys Met Glu Ser Glu
Val Arg Phe Ile 545 550 555 560 Lys Ser 95 5 PRT Artificial
Sequence Synthetically generated peptide VARIANT 2, 4 Xaa = any
amino acid 95 Tyr Xaa Met Xaa Trp 1 5 96 5 PRT Artificial Sequence
Synthetically generated peptide VARIANT 3, 5 Xaa = any amino acid
96 Arg Tyr Xaa Met Xaa 1 5 97 5 PRT Artificial Sequence
Synthetically generated peptide VARIANT 3 Xaa = Ser, Arg, Lys
VARIANT 5 Xaa = Ser, Tyr, Trp, His 97 Arg Tyr Xaa Met Xaa 1 5 98 14
PRT Artificial Sequence Synthetically generated peptide VARIANT 4,
5, 7, 8, 9 Xaa = any amino acid VARIANT 1, 2 Xaa = Ile or Ser
VARIANT 10 Xaa = may be absent 98 Xaa Xaa Ser Xaa Xaa Gly Xaa Xaa
Xaa Xaa Tyr Ala Asp Ser 1 5 10 99 12 PRT Artificial Sequence
Synthetically generated peptide VARIANT 1 Xaa = Gly, Asn VARIANT 2,
5 Xaa = Ala, Gly VARIANT 3 Xaa = Arg, Pro VARIANT 6 Xaa = Phe, Asn
VARIANT 7 Xaa = Lys, Pro VARIANT 8 Xaa = Ser, Tyr VARIANT 9 Xaa =
Met, Tyr VARIANT 10 Xaa = Phe, Asp VARIANT 11 Xaa = Val, Asp 99 Xaa
Xaa Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr 1 5 10 100 12 PRT
Artificial Sequence Synthetically generated peptide VARIANT 1 Xaa =
Gly, Arg, Asn VARIANT 2, 5 Xaa = Ala, Gly VARIANT 3 Xaa = Arg, Pro
VARIANT 4 Xaa = Gly, Arg VARIANT 6 Xaa = Phe, Asn, Glu VARIANT 7
Xaa = Val, Lys, Pro VARIANT 8 Xaa = Ala, Ser, Tyr VARIANT 9 Xaa =
Met, Tyr, Phe VARIANT 10 Xaa = Phe, Asp VARIANT 11 Xaa = Ile, Val,
Asp 100 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr 1 5 10 101
8 PRT Artificial Sequence Synthetically generated peptide 101 Gly
Pro Arg Gly Asn Lys Tyr Tyr 1 5 102 6 PRT Artificial Sequence
Synthetically generated peptide 102 Ala Arg Gly Thr Ser Gln 1 5 103
12 PRT Artificial Sequence Synthetically generated peptide VARIANT
6 Xaa = Ile, Val VARIANT 8 Xaa = Ser, Thr VARIANT 9 Xaa = Ser, Tyr
VARIANT 10 Xaa = Leu, Tyr VARIANT 11 Xaa = Ala, Leu, Asn 103 Arg
Ala Ser Gln Ser Xaa Ser Xaa Xaa Xaa Xaa Ala 1 5 10 104 12 PRT
Artificial Sequence Synthetically generated peptide VARIANT 4 Xaa =
Leu, Gln VARIANT 5 Xaa = Ser, Thr, Phe, Asp, Pro VARIANT 6 Xaa =
Ile, Val VARIANT 7 Xaa = Ser, Thr, Arg, Asp, Asn VARIANT 8 Xaa =
Ser, Thr, Tyr, Asn VARIANT 9 Xaa = Ser, Tyr, Trp, Asp VARIANT 10
Xaa = leu, Tyr, Asp VARIANT 11 Xaa = Ala, Leu, Asn 104 Arg Ala Ser
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala 1 5 10 105 7 PRT Artificial
Sequence Synthetically generated peptide VARIANT 1, 6, 7 Xaa = any
amino acid 105 Xaa Ala Ser Ser Leu Xaa Xaa 1 5 106 7 PRT Artificial
Sequence Synthetically generated peptide VARIANT 1 Xaa = Ala, Gly
VARIANT 4 Xaa = Ser, Thr, Arg VARIANT 5 Xaa = Leu, Arg VARIANT 6
Xaa = Ala, Val, Lys, Gln VARIANT 7 Xaa = ser, Thr, Lys, Asp 106 Xaa
Ala Ser Xaa Xaa Xaa Xaa 1 5 107 10 PRT Artificial Sequence
Synthetically generated peptide VARIANT 3, 4, 5, 6, 8, 10 Xaa = any
amino acid 107 Gln Gln Xaa Xaa Xaa Xaa Pro Xaa Thr Xaa 1 5 10 108
10 PRT Artificial Sequence Synthetically generated peptide VARIANT
3 Xaa = Ala, Gly, Ser, Leu, Tyr VARIANT 4 Xaa = Gly, thr, Val, Tyr,
Phe, Asn VARIANT 5 Xaa = Gly, Ser, Thr, Ile, Asn, Pro VARIANT 6 Xaa
= Ser, Thr, Tyr, Phe, Asn VARIANT 7 Xaa = Ser, Thr, Val, Pro
VARIANT 8 Xaa = Ala, Gly, Ser, Tyr, Trp, Pro VARIANT 9 Xaa = Thr,
Ile, Trp 108 Gln Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr 1 5 10 109 12
PRT Artificial Sequence Synthetically generated peptide VARIANT 2,
4, 5, 6, 7, 8, 10 Xaa = any amino acid 109 Ser Xaa Asp Xaa Xaa Xaa
Xaa Xaa Tyr Xaa Ser Trp 1 5 10 110 13 PRT Artificial Sequence
Synthetically generated peptide VARIANT 5, 7, 8, 9 Xaa = any amino
acid VARIANT 6 Xaa = Val or Ile VARIANT 10 Xaa = may be absent
VARIANT 12 Xaa = Ala or Asn 110 Arg Ala Ser Gln Xaa Xaa Xaa Xaa Xaa
Xaa Leu Xaa Trp 1 5 10 111 8 PRT Artificial Sequence Synthetically
generated peptide VARIANT 3 Xaa = Ser or Thr VARIANT 4 Xaa = Arg or
Leu VARIANT 5, 6 Xaa = any amino acid 111 Ala Ser Xaa Xaa Xaa Xaa
Gly Arg 1 5 112 17 PRT Artificial Sequence Synthetically generated
peptide VARIANT 1, 3 Xaa = Gly, Ser, Val, Tyr, Arg VARIANT 4 Xaa =
Ser, Pro VARIANT 6 Xaa = Gly, Arg VARIANT 8 Xaa = Ser, Thr, Ile,
Met, Tyr, Phe, Lys,Asp VARIANT 10 Xaa = Ala, Gly, Thr, Phe, Arg,
Lys, Asn, Gln 112 Xaa Ile Xaa Xaa Ser Xaa Gly Xaa Thr Xaa Tyr Ala
Asp Ser Val Lys 1 5 10 15 Gly 113 17 PRT Artificial Sequence
Synthetically generated peptide VARIANT 1 Xaa = Gly, Ser, Tyr
VARIANT 3 Xaa = Ser, Val, Arg VARIANT 4 Xaa = Ser, Pro VARIANT 8
Xaa = Ser, Ile, Tyr, Asp VARIANT 10 Xaa = Gly, Arg, Lys, Asn 113
Xaa Ile Xaa Xaa Ser Gly Gly Xaa Thr Xaa Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 114 13 PRT Artificial Sequence Synthetically generated
peptide 114 Ala Ser Pro Ala Gly Thr Pro Pro Gly Arg Ala Ser Pro 1 5
10
* * * * *