U.S. patent application number 14/039912 was filed with the patent office on 2014-06-26 for hepatocyte growth factor (hgf) binding proteins.
This patent application is currently assigned to AVEO PHARMACEUTICALS, INC.. The applicant listed for this patent is AVEO Pharmaceuticals, Inc.. Invention is credited to Lyne Breault, Bijan Etemad-Gilbertson, Jeno Gyuris, May Han, Christine Knuehl, Jie Lin, William M. Winston, JR., S. Kirk Wright.
Application Number | 20140178934 14/039912 |
Document ID | / |
Family ID | 38802082 |
Filed Date | 2014-06-26 |
United States Patent
Application |
20140178934 |
Kind Code |
A1 |
Han; May ; et al. |
June 26, 2014 |
HEPATOCYTE GROWTH FACTOR (HGF) BINDING PROTEINS
Abstract
The present invention provides a family of binding proteins that
bind and neutralize the activity of hepatocyte growth factor (HGF),
in particular human HGF. The binding proteins can be used as
diagnostic and/or therapeutic agents. With regard to their
therapeutic activity, the binding proteins can be used to treat
certain HGF responsive disorders, for example, certain HGF
responsive tumors.
Inventors: |
Han; May; (Brookline,
MA) ; Wright; S. Kirk; (Waltham, MA) ;
Winston, JR.; William M.; (Marlborough, MA) ;
Breault; Lyne; (Roslindale, MA) ; Lin; Jie;
(West Roxbury, MA) ; Etemad-Gilbertson; Bijan;
(Jamaica Plain, MA) ; Knuehl; Christine; (Natick,
MA) ; Gyuris; Jeno; (Lincoln, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AVEO Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
AVEO PHARMACEUTICALS, INC.
Cambridge
MA
|
Family ID: |
38802082 |
Appl. No.: |
14/039912 |
Filed: |
September 27, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13589652 |
Aug 20, 2012 |
8575318 |
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14039912 |
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13051474 |
Mar 18, 2011 |
8268315 |
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13589652 |
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12632758 |
Dec 7, 2009 |
7935502 |
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13051474 |
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11757094 |
Jun 1, 2007 |
7659378 |
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12632758 |
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60810714 |
Jun 2, 2006 |
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60860461 |
Nov 21, 2006 |
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Current U.S.
Class: |
435/69.6 ;
435/320.1; 435/336; 536/23.53 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2317/73 20130101; C07K 2317/24 20130101; C07K 2317/56
20130101; A61P 35/00 20180101; C07K 2317/76 20130101; C07K 2317/92
20130101; C07K 2317/565 20130101; C07K 16/22 20130101 |
Class at
Publication: |
435/69.6 ;
536/23.53; 435/320.1; 435/336 |
International
Class: |
C07K 16/22 20060101
C07K016/22 |
Claims
1-100. (canceled)
101. An isolated nucleic acid comprising a nucleotide sequence
encoding an immunoglobulin heavy chain variable region comprising
the amino acid sequence of SEQ ID NO: 169, and encoding an
immunoglobulin light chain variable region comprising the amino
acid sequence of SEQ ID NO: 179, wherein the immunoglobulin heavy
chain variable region and the immunoglobulin light chain variable
region together bind human hepatocyte growth factor (HGF).
102. The isolated nucleic acid of claim 101, wherein the nucleotide
sequence encodes an immunoglobulin heavy chain sequence comprising
the amino acid sequence of SEQ ID NO: 171.
103. The isolated nucleic acid of claim 101, wherein the nucleotide
sequence encodes an immunoglobulin heavy chain sequence comprising
the amino acid sequence of SEQ ID NO: 210.
104. The isolated nucleic acid of claim 101, wherein the nucleotide
sequence encodes an immunoglobulin light chain sequence comprising
the amino acid sequence of SEQ ID NO: 181.
105. An expression vector comprising the nucleic acid of claim
101.
106. A host cell comprising the expression vector of claim 105.
107. A host cell comprising: a first expression vector comprising a
nucleic acid sequence encoding an immunoglobulin heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:
169; and a second expression vector comprising a nucleic acid
sequence encoding an immunoglobulin light chain variable region
comprising the amino acid sequence of SEQ ID NO: 179; wherein the
immunoglobulin light chain variable region and the immunoglobulin
heavy chain variable region together bind human hepatocyte growth
factor (HGF).
108. A method of producing a monoclonal antibody that binds human
hepatocyte growth factor (HGF) or an antigen binding fragment of
the antibody, the method comprising: (a) growing the host cell of
claim 107 under conditions that permit the expression of a
polypeptide comprising the immunoglobulin heavy chain variable
region and a polypeptide comprising the immunoglobulin light chain
variable region, thereby producing the antibody or antigen binding
fragment of the antibody; and (b) purifying the antibody or the
antigen binding fragment of the antibody.
109. A method of producing a monoclonal antibody that binds human
hepatocyte growth factor (HGF) or an antigen binding fragment of
the antibody, the method comprising: (a) growing the host cell of
claim 106 under conditions that permit the expression of a
polypeptide comprising the immunoglobulin heavy chain variable
region and a polypeptide comprising the immunoglobulin light chain
variable region, thereby producing the antibody or the
antigen-binding fragment of the antibody; and (b) purifying the
antibody or the antigen-binding fragment of the antibody.
110. An isolated nucleic acid comprising a nucleotide sequence
encoding an immunoglobulin heavy chain comprising the amino acid
sequence of SEQ ID NO: 171, and encoding an immunoglobulin light
chain comprising the amino acid sequence of SEQ ID NO: 181, wherein
the immunoglobulin heavy chain and the immunoglobulin light chain
together bind human hepatocyte growth factor (HGF).
111. An expression vector comprising the nucleic acid of claim
110.
112. A host cell comprising the expression vector of claim 111.
113. A host cell comprising: a first expression vector comprising a
nucleic acid sequence encoding an immunoglobulin heavy chain
comprising the amino acid sequence of SEQ ID NO: 171; and a second
expression vector comprising a nucleic acid sequence encoding an
immunoglobulin light chain comprising the amino acid sequence of
SEQ ID NO: 181; wherein the immunoglobulin light chain and the
immunoglobulin heavy chain together bind human hepatocyte growth
factor (HGF).
114. A method of producing a monoclonal antibody that binds human
hepatocyte growth factor (HGF) or an antigen binding fragment of
the antibody, the method comprising: (a) growing the host cell of
claim 113 under conditions that permit the expression of a
polypeptide comprising the immunoglobulin heavy chain and a
polypeptide comprising the immunoglobulin light chain, thereby
producing the antibody or antigen binding fragment of the antibody;
and (b) purifying the antibody or the antigen binding fragment of
the antibody.
115. A method of producing a monoclonal antibody that binds human
hepatocyte growth factor (HGF) or an antigen binding fragment of
the antibody, the method comprising: (a) growing the host cell of
claim 112 under conditions that permit the expression of a
polypeptide comprising the immunoglobulin heavy chain and a
polypeptide comprising the immunoglobulin light chain, thereby
producing the antibody or the antigen-binding fragment of the
antibody; and (b) purifying the antibody or the antigen-binding
fragment of the antibody.
116. An isolated nucleic acid comprising a nucleotide sequence
encoding an immunoglobulin heavy chain comprising the amino acid
sequence of SEQ ID NO: 210, and encoding an immunoglobulin light
chain comprising the amino acid sequence of SEQ ID NO: 181, wherein
the immunoglobulin light chain and the immunoglobulin heavy chain
together bind human hepatocyte growth factor (HGF).
117. An expression vector comprising the nucleic acid of claim
116.
118. A host cell comprising the expression vector of claim 117.
119. A host cell comprising: a first expression vector comprising a
nucleic acid sequence encoding an immunoglobulin heavy chain
comprising the amino acid sequence of SEQ ID NO: 210; and a second
expression vector comprising a nucleic acid sequence encoding an
immunoglobulin light chain comprising the amino acid sequence of
SEQ ID NO: 181; wherein the immunoglobulin light chain and the
immunoglobulin heavy chain together bind human hepatocyte growth
factor (HGF).
120. A method of producing a monoclonal antibody that binds human
hepatocyte growth factor (HGF) or an antigen binding fragment of
the antibody, the method comprising: (a) growing the host cell of
claim 119 under conditions that permit the expression of a
polypeptide comprising the immunoglobulin heavy chain and a
polypeptide comprising the immunoglobulin light chain, thereby
producing the antibody or antigen binding fragment of the antibody;
and (b) purifying the antibody or the antigen binding fragment of
the antibody.
121. A method of producing a monoclonal antibody that binds human
hepatocyte growth factor (HGF) or an antigen binding fragment of
the antibody, the method comprising: (a) growing the host cell of
claim 118 under conditions that permit the expression of a
polypeptide comprising the immunoglobulin heavy chain and a
polypeptide comprising the immunoglobulin light chain, thereby
producing the antibody or the antigen-binding fragment of the
antibody; and (b) purifying the antibody or the antigen-binding
fragment of the antibody.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit and priority to U.S.
Provisional Application Nos. 60/810,714, filed Jun. 2, 2006, and
60/860,461, filed Nov. 21, 2006, the disclosures of which are
incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The field of the invention is molecular biology, immunology
and oncology. More particularly, the field is antibody-based
binding proteins that bind human hepatocyte growth factor
(HGF).
BACKGROUND
[0003] Hepatocyte Growth Factor (HGF), also known as Scatter Factor
(SF), is a multi-functional heterodimeric protein produced
predominantly by mesenchymal cells, and is an effector of cells
expressing the Met tyrosine kinase receptor (Bottaro et al. (1991)
SCIENCE 251: 802-804, Rubin et al. (1993) BIOCHIM. BIOPHYS. ACTA
1155: 357-371). The human Met receptor is also known as "c-Met."
Mature HGF contains two polypeptide chains, the .alpha.-chain and
the .beta.-chain. Published studies suggest it is the .alpha.-chain
that contains HGF's c-Met receptor binding domain.
[0004] When it binds to its cognate receptor, HGF mediates a number
of cellular activities. The HGF-Met signaling pathway plays a role
in liver regeneration, wound healing, neural regeneration,
angiogenesis and malignancies. See, e.g., Cao et al. (2001) PROC.
NATL. ACAD. SCI. USA 98: 7443-7448, Burgess et al. (2006) CANCER
RES. 66: 1721-1729, and U.S. Pat. Nos. 5,997,868 and 5,707,624.
Investigators have been developing a number of HGF modulators,
including antibodies, to treat various disorders that involve HGF
activity, for example, certain HGF responsive cancers. See, e.g.,
International Application Publication No. WO 2005/017107.
[0005] The basic structure common to all antibodies is shown
schematically in FIG. 1. Antibodies are multimeric proteins that
contain four polypeptide chains. Two of the polypeptide chains are
called heavy or H chains and two of the polypeptide chains are
called light or L chains. The immunoglobulin heavy and light chains
are connected by an interchain disulfide bond. The immunoglobulin
heavy chains are connected by a number of interchain disulfide
bonds. A light chain is composed of one variable region (V.sub.L in
FIG. 1) and one constant region (C.sub.L in FIG. 1), while the
heavy chain is composed of one variable region (V.sub.H in FIG. 1)
and at least three constant regions (CH.sub.1, CH.sub.2 and
CH.sub.3 in FIG. 1). The variable regions determine the specificity
of the antibody and the constant regions have other functions.
[0006] Amino acid and structural information indicate that each
variable region comprises three hypervariable regions (also known
as complementarity determining regions or CDRs) flanked by four
relatively conserved framework regions or FRs. The three CDRs,
referred to as CDR.sub.1, CDR.sub.2, and CDR.sub.3, are responsible
for the binding specificity of individual antibodies. When
antibodies are to be used as diagnostic and therapeutic agents,
typically it is desirable to create antibodies that have the
highest binding specificity and affinity to the target molecule. It
is believed that differences in the variable regions can have
profound effects on the specificity and affinity of the
antibody.
[0007] U.S. Pat. No. 5,707,624 describes the use of anti-HGF
antibodies in the treatment of Kaposi's sarcoma. Similarly, U.S.
Pat. No. 5,997,868 describes treating a tumor by administering an
anti-HGF antibody to the patient to be treated so as to block the
ability of endogeneous HGF to promote angiogenesis in the tumor.
More recently, investigators propose that antibodies that bind the
.beta.-chain of HGF may have potential as therapeutic agents in
patients with HGF-dependent tumors (Burgess (2006) supra).
[0008] Notwithstanding, there is still a need for additional HGF
modulators that can be used as therapeutic and diagnostic
agents.
SUMMARY OF THE INVENTION
[0009] The invention is based, in part, upon the discovery of a
family of binding proteins that specifically bind HGF, in
particular, human HGF. The binding proteins are antibody-based in
so far as they contain antigen (i.e., HGF) binding sites based on
the CDRs of a family of antibodies that specifically bind HGF. The
CDRs confer the binding specificity of the binding proteins to HGF.
The binding proteins can be used as diagnostic and therapeutic
agents. When used as a therapeutic agent, the binding proteins are
engineered (e.g., humanized) so as to reduce or eliminate the risk
of inducing an immune response against the binding protein when
administered to the recipient (e.g., a human).
[0010] The binding proteins neutralize the activity of HGF and,
therefore, can be used as a therapeutic agent. In certain
embodiments, the binding proteins prevent HGF from binding to its
cognate receptor, c-Met, thereby neutralizing HGF activity. In
other embodiments, the binding proteins bind to HGF and neutralize
its biological activity but without preventing HGF from binding to
the c-Met receptor. Because HGF has been implicated in the growth
and proliferation of cancer cells, the binding proteins can be used
to inhibit the proliferation of cancer cells. Furthermore, when
administered to a mammal, the binding proteins can inhibit or
reduce tumor growth in the mammal.
[0011] These and other aspects and advantages of the invention will
become apparent upon consideration of the following figures,
detailed description, and claims.
DESCRIPTION OF THE DRAWINGS
[0012] The invention can be more completely understood with
reference to the following drawings.
[0013] FIG. 1 is a schematic representation of a typical
antibody.
[0014] FIG. 2 is a schematic diagram showing the amino acid
sequence defining the complete immunoglobulin heavy chain variable
region of the antibodies denoted as 1A3, 1D3, 1F3, 2B8, 2F8, 3A12,
3B6 and 3D11. The amino acid sequences for each antibody are
aligned against one another and the regions defining the signal
peptide, CDR.sub.1, CDR.sub.2, and CDR.sub.3 are identified in
boxes. The unboxed sequences represent FR sequences.
[0015] FIG. 3 is a schematic diagram showing the CDR.sub.1,
CDR.sub.2, and CDR.sub.3 sequences for each of the immunoglobulin
heavy chain variable region sequences presented in FIG. 2.
[0016] FIG. 4 is a schematic diagram showing the amino acid
sequence defining the complete immunoglobulin light chain variable
region of the antibodies 1A3, 1D3, 1F3, 2B8, 2F8, 3A12, 3B6, and
3D11. The amino acid sequences for each antibody are aligned
against one another and the regions defining the signal peptide,
CDR.sub.1, CDR.sub.2, and CDR.sub.3 are identified in boxes. The
unboxed sequences represent FR sequences.
[0017] FIG. 5 is a schematic diagram showing the CDR.sub.1,
CDR.sub.2, and CDR.sub.3 sequences for each of the immunoglobulin
light chain variable region sequences presented in FIG. 4.
[0018] FIG. 6 is a graph summarizing results from an experiment to
measure tumor inhibitory activity of anti-HGF antibodies 1D3, 1F3,
1A3 and 2B8 in a U87MG xenograft model. Diamonds correspond to PBS;
triangles correspond to anti-HGF antibody 1A3; X corresponds to
anti-HGF antibody 1D3; squares correspond to anti-HGF antibody 1F3,
and circles correspond to anti-HGF antibody 2B8.
[0019] FIG. 7 is a graph summarizing results from an experiment to
measure tumor inhibitory activity of anti-HGF antibodies 1D3, 1F3,
1A3 and 2B8 in a U118 xenograft model. Diamonds correspond to IgG;
squares correspond to anti-HGF antibody 1F3, triangles to anti-HGF
antibody 1D3; X corresponds to anti-HGF antibody 1A3; and circles
correspond to anti-HGF antibody 2B8.
[0020] FIG. 8 is a table summarizing surface plasmon resonance data
on antigen-binding affinity and kinetics of interaction between
human HGF and chimeric, chimeric/humanized, or humanized 2B8
antibodies. The table lists the pairs of Kappa light chain and IgG1
heavy chain tested. Those antibodies with standard deviations
(STDEV) listed were analyzed in three independent experiments.
[0021] FIG. 9 is a bar chart summarizing experimental data
indicating that Hu2B8 binds an epitope mutually exclusive to murine
monoclonal antibody 2B8. Humanized or chimeric 2B8 was captured on
an anti-human Fc chip. HGF then was bound to the humanized or
chimeric 2B8. The ability of mouse 2B8 or the control antibody
(polyclonal goat anti-HGF antibody) to bind the captured HGF was
measured. Both humanized 2B8 antibodies and chimeric 2B8 prevent
murine 2B8 from binding HGF. White bars correspond to the chimeric
2B8 antibody; gray bars correspond to the humanized Hu2B8 antibody
(kappa variable region Kv1-39.1 and heavy chain variable region
Hv5-51.1); black bars correspond to the humanized Hu2B8 antibody
(kappa variable region Kv3-15.1 and heavy chain variable region
Hv5-51.1).
DETAILED DESCRIPTION OF THE INVENTION
[0022] The invention is based, in part, upon the discovery of a
family of binding proteins that specifically bind, and neutralize
the activity of, HGF, in particular, human HGF. The binding
proteins can be used in a variety of diagnostic and therapeutic
applications. The binding proteins are based upon the antigen
binding sites of certain monoclonal antibodies that have been
selected for their ability to bind, and neutralize the activity of,
HGF. In particular, the binding proteins contain immunoglobulin
variable region CDR sequences that together define a binding site
for HGF.
[0023] In view of the neutralizing activity of these antibodies,
they are particularly useful in modulating the growth and/or
proliferation of HGF responsive cells, for example, cancer cells.
When used as a therapeutic agent, the binding proteins can be
engineered so as to minimize or eliminate the risk of inducing an
immune response against the binding proteins when administered to
the recipient. Furthermore, depending upon the particular
application, it is contemplated that the binding proteins can be
conjugated to other moieties, for example, detectable labels, for
example, radiolabels, and effector molecules, for example, other
protein and small molecule-based therapeutics. Each of these
features and aspects of the invention are discussed in more detail
below.
I--Binding Proteins that Bind HGF
[0024] In one aspect, the invention provides an isolated binding
protein that binds human HGF. The binding protein comprises (i) an
immunoglobulin light chain variable region comprising the structure
CDR.sub.L1-CDR.sub.L2-CDR.sub.L3, and (ii) an immunoglobulin heavy
chain variable region comprising three complementarity determining
regions (CDRs), wherein the immunoglobulin light chain variable
region and the immunoglobulin heavy chain variable region together
define a single binding site for binding human HGF. CDR.sub.L1
comprises the amino acid sequence X.sub.1 X.sub.2 Ser X.sub.4
X.sub.5 X.sub.6 X.sub.7 X.sub.8 X.sub.9 X.sub.10 X.sub.11 X.sub.12
X.sub.13 X.sub.14 X.sub.15, wherein amino acid X.sub.1 is Arg, Lys,
or Ser, X.sub.2 is Ala or Thr, X.sub.4 is Glu, Gln, or Ser, X.sub.5
is Asn, Asp, or Ser, X.sub.6 is Ile or Val, X.sub.7 is Asp, Lys,
Ser, Val, or Tyr, X.sub.8 is a peptide bond or Tyr, X.sub.9 is a
peptide bond or Asp, X.sub.10 is a peptide bond or Gly, X.sub.11 is
a peptide bond or Asn, X.sub.12 is a peptide bond, Ile, or Ser,
X.sub.13 is Asn or Tyr, X.sub.14 is Ile, Leu, Met, or Val, X.sub.15
is Ala, Asn, His, or Ser. CDR.sub.L2 comprises the amino acid
sequence X.sub.16 X.sub.17 X.sub.18 X.sub.19 X.sub.20 X.sub.21
X.sub.22, wherein amino acid X.sub.16 is Ala, Asp, Arg, Gly, or
Val, X.sub.17 is Ala, Thr, or Val, X.sub.18 is Asn, Ser, or Thr,
X.sub.19 is Arg, Asn, Lys, or His, X.sub.20 is Leu or Arg, X.sub.21
is Ala, Asn, Glu, Val, or Pro, X.sub.22 is Asp, Ser, or Thr.
CDR.sub.L3 comprises the amino acid sequence X.sub.23 X.sub.24
X.sub.25 X.sub.26 X.sub.27 X.sub.28 Pro X.sub.30 Thr, wherein amino
acid X.sub.23 is Leu, Gly, or Gln, X.sub.24 is His or Gln, X.sub.25
is Phe, Ser, Trp, or Tyr, X.sub.26 is Asp, Ile, Ser, Trp, or Tyr,
X.sub.27 is Gly, Glu, Asn, or Ser, X.sub.28 is Asp, Asn, Phe, Thr,
or Tyr, X.sub.30 is Leu, Phe, Pro, or Tyr.
[0025] In another aspect, the invention provides an isolated
binding protein that binds human HGF comprising (i) an
immunoglobulin heavy chain variable region comprising the structure
CDR.sub.H1-CDR.sub.H2-CDR.sub.H3 and (ii) an immunoglobulin light
chain variable region comprising three complementarity determining
regions (CDRs), wherein the immunoglobulin heavy chain variable
region and the immunoglobulin light chain variable region together
define a single binding site for binding human HGF. CDR.sub.H1
comprises the amino acid sequence X.sub.1 Tyr X.sub.3 X.sub.4
X.sub.5, wherein amino acid X.sub.1 is Asp, Asn, Ser, or Thr,
X.sub.3 is Phe, Ser, Trp, or Tyr, X.sub.4 is Ile, Leu, or Met,
X.sub.5 is Asn, His, or Ser. CDR.sub.H2 comprises the amino acid
sequence X.sub.6 Ile X.sub.8 X.sub.9 X.sub.10 X.sub.11 Gly X.sub.13
X.sub.14 X.sub.15 Tyr X.sub.17 X.sub.18 X.sub.19 X.sub.20 X.sub.21
X.sub.22, wherein amino acid X.sub.6 is Lys, Gln, Glu, Val, or Tyr,
X.sub.8 is Asn, Gly, Ser, Trp, or Tyr, X.sub.9 is Ala, Pro or Ser,
X.sub.10 is Gly or Thr, X.sub.11 is a peptide bond, Asp, Asn, Gly,
or Ser, X.sub.13 is Asp, Asn, His, or Ser, X.sub.14 is Ser or Thr,
X.sub.15 is Asn or Tyr, X.sub.17 is Asn or Pro, X.sub.18 is Ala,
Asp, Gly, Gln, Glu, Pro, or Ser, X.sub.19 is Asn, Lys, Met, or Ser,
X.sub.20 is Leu, Phe or Val, X.sub.21 is Lys, Met, or Gln, X.sub.22
is Asp, Gly or Ser. CDR.sub.H3 comprises the amino acid sequence
X.sub.23 X.sub.24 X.sub.25 X.sub.26 X.sub.27 X.sub.28 X.sub.29
X.sub.30 X.sub.31 X.sub.32 X.sub.33 X.sub.34 Tyr, wherein amino
acid X.sub.23 is Arg, Asn, Gln, or Glu, X.sub.24 is Gly, Leu, Arg,
or Tyr, X.sub.25 is a peptide bond, Asp, or Gly, X.sub.26 is a
peptide bond or Gly, X.sub.27 is a peptide bond or Tyr, X.sub.28 is
a peptide bond, Leu, or Tyr, X.sub.29 is a peptide bond, Gly, Leu,
Arg, or Val, X.sub.30 is a peptide bond, Asp, Gly, or Glu, X.sub.31
is a peptide bond, Asn, Arg, Ser, or Tyr, X.sub.32 is peptide bond,
Ala, Gly, Ile, or Tyr, X.sub.33 is Met or Phe, X.sub.34 is Ala or
Asp.
[0026] It is understood that the binding protein can comprise both
the immunoglobulin light chain and the immunoglobulin heavy chain
sequences or the fragments thereof, noted above. Furthermore, it is
understood that the binding protein can be an intact antibody or an
antigen binding fragment thereof, or a biosynthetic antibody
site.
[0027] In certain embodiments, the CDR sequences of the
immunoglobulin light chain and the immunoglobulin heavy chain are
interposed with framework regions (FR).
[0028] In certain other embodiments, the CDR sequences of the
immunoglobulin light chain and the immunoglobulin heavy chain are
interposed between human or humanized framework regions.
[0029] In another aspect, the invention provides an isolated
binding protein that specifically binds human HGF. The binding
protein comprises: (a) an immunoglobulin light chain variable
region comprising the structure CDR.sub.L1-CDR.sub.L2-CDR.sub.L3
and (b) immunoglobulin heavy chain variable region, wherein the
immunoglobulin light chain variable region and the immunoglobulin
heavy chain variable region together define a single binding site
for binding human HGF. The CDR.sub.L1 comprises a sequence selected
from the group consisting of SEQ ID NO. 8 (1A3), SEQ ID NO. 18
(2B8), SEQ ID NO. 28 (2F8), SEQ ID NO. 38 (3B6), SEQ ID NO. 48
(3D11), SEQ ID NO. 58 (1D3), SEQ ID NO. 68 (1F3), and SEQ ID NO. 78
(3A12). The CDR.sub.L2 comprises a sequence selected from the group
consisting of SEQ ID NO. 9 (1A3), SEQ ID NO. 19 (2B8), SEQ ID NO.
29 (2F8), SEQ ID NO. 39 (3B6), SEQ ID NO. 49 (3D11), SEQ ID NO. 59
(1D3), SEQ ID NO. 69 (1F3), SEQ ID NO. 79 (3A12) and SEQ ID NO. 206
(LRMR2B8LC). The CDR.sub.L3 comprises a sequence selected from the
group consisting of SEQ ID NO. 10 (1A3), SEQ ID NO. 20 (2B8), SEQ
ID NO. 30 (2F8), SEQ ID NO. 40 (3B6), SEQ ID NO. 50 (3D11), SEQ ID
NO. 60 (1D3), SEQ ID NO. 70 (1F3), and SEQ ID NO. 80 (3A12).
Throughout the specification and claims, the sequences denoted by a
particular SEQ ID NO. are followed in parentheses by the antibody
that was the origin of the particular sequence. By way of example,
SEQ ID NO. 8 (1A3) indicates that the sequence of SEQ ID NO. 8 is
based upon the sequence present in antibody 1A3.
[0030] In one embodiment, the binding protein comprises an
immunoglobulin light chain variable region comprising a CDR.sub.L1
comprising the sequence of SEQ ID NO. 8 (1A3), a CDR.sub.L2
comprising the sequence of SEQ ID NO. 9 (1A3), and a CDR.sub.L3
comprising the sequence of SEQ ID NO. 10 (1A3).
[0031] In another embodiment, the binding protein comprises an
immunoglobulin light chain variable region comprising a CDR.sub.L1
comprising the sequence of SEQ ID NO. 18 (2B8), a CDR.sub.L2
comprising the sequence of SEQ ID NO. 19 (2B8) or SEQ ID NO. 206
(LRMR2B8LC), and a CDR.sub.L3 comprising the sequence of SEQ ID NO.
20 (2B8).
[0032] In another embodiment, the binding protein comprises an
immunoglobulin light chain variable region comprising a CDR.sub.L1
comprising the sequence of SEQ ID NO. 28 (2F8), a CDR.sub.L2
comprising the sequence of SEQ ID NO. 29 (2F8), and a CDR.sub.L3
comprising the sequence of SEQ ID NO. 30 (2F8).
[0033] In another embodiment, the binding protein comprises an
immunoglobulin light chain variable region comprising a CDR.sub.L1
comprising the sequence of SEQ ID NO. 38 (3B6), a CDR.sub.L2
comprising the sequence of SEQ ID NO. 39 (3B6), and a CDR.sub.L3
comprising the sequence of SEQ ID NO. 40 (3B6).
[0034] In another embodiment, the binding protein comprises an
immunoglobulin light chain variable region comprising a CDR.sub.L1
comprising the sequence of SEQ ID NO. 48 (3D11), a CDR.sub.L2
comprising the sequence of SEQ ID NO. 49 (3D11), and a CDR.sub.L3
comprising the sequence of SEQ ID NO. 50 (3D11).
[0035] In another embodiment, the binding protein comprises an
immunoglobulin light chain variable region comprising a CDR.sub.L1
comprising the sequence of SEQ ID NO. 58 (1D3), a CDR.sub.L2
comprising the sequence of SEQ ID NO. 59 (1D3), and a CDR.sub.L3
comprising the sequence of SEQ ID NO. 60 (1D3).
[0036] In another embodiment, the binding protein comprises an
immunoglobulin light chain variable region comprising a CDR.sub.L1
comprising the sequence of SEQ ID NO. 68 (1F3), a CDR.sub.L2
comprising the sequence of SEQ ID NO. 69 (1F3), and a CDR.sub.L3
comprising the sequence of SEQ ID NO. 70 (1F3).
[0037] In another embodiment, the binding protein comprises an
immunoglobulin light chain variable region comprising a CDR.sub.L1
comprising the sequence of SEQ ID NO. 78 (3A12), a CDR.sub.L2
comprising the sequence of SEQ ID NO. 79 (3A12), and a CDR.sub.L3
comprising the sequence of SEQ ID NO. 80 (3A12).
[0038] In each of the foregoing embodiments, the CDR.sub.L1,
CDR.sub.L2, and CDR.sub.L3 sequences preferably are interposed
between human or humanized immunoglobulin FRs. It is understood
that the binding protein can be an intact antibody, an antigen
binding fragment thereof, or a biosynthetic antibody site.
[0039] In another aspect, the invention provides an isolated
binding protein that binds human HGF. The binding protein comprises
(a) an immunoglobulin heavy chain variable region comprising the
structure CDR.sub.H1-CDR.sub.H2-CDR.sub.H3, and (b) an
immunoglobulin light chain variable region, wherein the
immunoglobulin heavy chain variable region and the immunoglobulin
light chain variable region together define a single binding site
for binding human HGF. The CDR.sub.H1 comprises a sequence selected
from the group consisting of SEQ ID NO. 5 (1A3), SEQ ID NO. (2B8),
SEQ ID NO. 25 (2F8), SEQ ID NO. 35 (3B6), SEQ ID NO. 45 (3D11), SEQ
ID NO. 55 (1D3), SEQ ID NO. 65 (1F3), and SEQ ID NO. 75 (3A12); the
CDR.sub.H2 comprises a sequence selected from the group consisting
of SEQ ID NO. 6 (1A3), SEQ ID NO. 16 (2B8), SEQ ID NO. 26 (2F8),
SEQ ID NO. 36 (3B6), SEQ ID NO. 46 (3D11), SEQ ID NO. 56 (1D3), SEQ
ID NO. 66 (1F3), SEQ ID NO. 76 (3A12), SEQ ID NO. 202 (Hu2B8
Hv1f.1), SEQ ID NO. 203 (Hu2B8 Hv5a.1 or Hu2B8 Hv5-51.1), SEQ ID
NO. 204 (LR2B8HC) and SEQ ID NO. 205 (LRMR2B8HC); and the
CDR.sub.H3 comprises a sequence selected from the group consisting
of SEQ ID NO. 7 (1A3), SEQ ID NO. 17 (2B8), SEQ ID NO. 27 (2F8),
SEQ ID NO. 37 (3B6), SEQ ID NO. 47 (3D11), SEQ ID NO. 57 (1D3), SEQ
ID NO. 67 (1F3), and SEQ ID NO. 77 (3A12).
[0040] In one embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising: a CDR.sub.H1
comprising the sequence of SEQ ID NO. 5 (1A3); a CDR.sub.H2
comprising the sequence of SEQ ID NO. 6 (1A3); and a CDR.sub.H3
comprising the sequence of SEQ ID NO. 7 (1A3).
[0041] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising: a CDR.sub.H1
comprising the sequence of SEQ ID NO. 15 (2B8); a CDR.sub.H2
comprising the sequence of SEQ ID NO. 16 (2B8), SEQ ID NO. 202
(Hu2B8 Hv1f.1), SEQ ID NO. 203 (Hu2B8 Hv5a.1 or Hu2B8 Hv5-51.1),
SEQ ID NO. 204 (LR2B8HC) or SEQ ID NO. 205 (LRMR2B8HC); and a
CDR.sub.H3 comprising the sequence of SEQ ID NO. 17 (2B8).
[0042] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising: a CDR.sub.H1
comprising the sequence of SEQ ID NO. 25 (2F8); a CDR.sub.H2
comprising the sequence of SEQ ID NO. 26 (2F8); and a CDR.sub.H3
comprising the sequence of SEQ ID NO. 27 (2F8).
[0043] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising a CDR.sub.H1
comprising the sequence of SEQ ID NO. 35 (3B6); a CDR.sub.H2
comprising the sequence of SEQ ID NO. 36 (3B6); and a CDR.sub.H3
comprising the sequence of SEQ ID NO. 37 (3B6).
[0044] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising: a CDR.sub.H1
comprising the sequence of SEQ ID NO. 45 (3D11); a CDR.sub.H2
comprising the sequence of SEQ ID NO. 46 (3D11); and a CDR.sub.H3
comprising the sequence of SEQ ID NO. 47 (3D11).
[0045] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising: a CDR.sub.H1
comprising the sequence of SEQ ID NO. 55 (1D3); a CDR.sub.H2
comprising the sequence of SEQ ID NO. 56 (1D3); and a CDR.sub.H3
comprising the sequence of SEQ ID NO. 57 (1D3).
[0046] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising: a CDR.sub.H1
comprising the sequence of SEQ ID NO. 65 (1F3); a CDR.sub.H2
comprising the sequence of SEQ ID NO. 66 (1F3); and a CDR.sub.H3
comprising the sequence of SEQ ID NO. 67 (1F3).
[0047] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising: a CDR.sub.H1
comprising the sequence of SEQ ID NO. 75 (3A12); a CDR.sub.H2
comprising the sequence of SEQ ID NO. 76 (3A12); and a CDR.sub.H3
comprising the sequence of SEQ ID NO. 77 (3A12).
[0048] In each of the foregoing embodiments, the CDR.sub.H1,
CDR.sub.H2, and CDR.sub.H3 sequences preferably are interposed
between human or humanized immunoglobulin FRs. It is understood
that the binding protein can be an intact antibody, an antigen
binding fragment thereof, or a biosynthetic antibody site.
[0049] In another aspect, the invention provides a binding protein
that binds human HGF. The binding protein comprises an
immunoglobulin heavy chain variable region selected from the group
consisting of residues 20-141 of SEQ ID NO. 2 (1A3), residues
20-137 of SEQ ID NO. 12 (2B8), residues 20-137 of SEQ ID NO. 22
(2F8), residues 20-139 of SEQ ID NO. 32 (3B6), residues 20-132 of
SEQ ID NO. 42 (3D11), residues 20-141 of SEQ ID NO. 52 (1D3),
residues 20-141 of SEQ ID NO. 62 (1F3), and residues 20-141 of SEQ
ID NO. 72 (3A12) and an immunoglobulin light chain variable region
selected from the group consisting of residues 21-127 of SEQ ID NO.
4 (1A3), residues 21-127 of SEQ ID NO. 14 (2B8), residues 20-131 of
SEQ ID NO. 24 (2F8), residues 23-129 of SEQ ID NO. 34 (3B6),
residues 23-128 of SEQ ID NO. 44 (3D11), residues 21-127 of SEQ ID
NO. 54 (1D3), residues 21-127 of SEQ ID NO. 64 (1F3), and residues
21-127 of SEQ ID NO. 74 (3A12).
[0050] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising the amino
acid sequence of residues 20-141 of SEQ ID NO. 2 (1A3), and an
immunoglobulin light chain variable region comprising the amino
acid sequence of residues 21-127 of SEQ ID NO. 4 (1A3).
[0051] In one embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising the amino
acid sequence of residues 20-137 of SEQ ID NO. 12 (2B8), and an
immunoglobulin light chain variable region comprising the amino
acid sequence of residues 21-127 of SEQ ID NO. 14 (2B8).
[0052] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising the amino
acid sequence of residues 20-137 of SEQ ID NO. 22 (2F8), and an
immunoglobulin light chain variable region comprising the amino
acid sequence of residues 20-131 of SEQ ID NO. 24 (2F8).
[0053] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising the amino
acid sequence of residues 20-139 of SEQ ID NO. 32 (3B6), and an
immunoglobulin light chain variable region comprising the amino
acid sequence of residues 23-129 of SEQ ID NO. 34 (3B6).
[0054] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising the amino
acid sequence of residues 20-132 of SEQ ID NO. 42 (3D11), and an
immunoglobulin light chain variable region comprising the amino
acid sequence of residues 23-128 of SEQ ID NO. 44 (3D11).
[0055] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising the amino
acid sequence of residues 20-141 of SEQ ID NO. 52 (1D3), and an
immunoglobulin light chain variable region comprising the amino
acid sequence of residues 21-127 of SEQ ID NO. 54 (1D3).
[0056] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising the amino
acid sequence of residues 20-141 of SEQ ID NO. 62 (1F3), and an
immunoglobulin light chain variable region comprising the amino
acid sequence of residues 21-127 of SEQ ID NO. 64 (1F3).
[0057] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising the amino
acid sequence of residues 20-141 of SEQ ID NO. 72 (3A12), and an
immunoglobulin light chain variable region comprising the amino
acid sequence of residues 21-127 of SEQ ID NO. 74 (3A12).
[0058] In each of the foregoing embodiments, the binding protein
can be an intact antibody, an antigen binding fragment thereof, or
a biosynthetic antibody site.
[0059] In another aspect, the invention provides an isolated
binding protein that binds human HGF. The binding protein comprises
(i) an immunoglobulin light chain variable region selected from the
group consisting of SEQ ID NO. 173 (Hu2B8 Kv1-39.1 light chain
variable region), SEQ ID NO. 179 (Hu2B8 Kv3-15.1 light chain
variable region), SEQ ID NO. 193 (LR2B8LC light chain variable
region), and SEQ ID NO. 199 (LRMR2B8LC light chain variable
region); and (ii) an immunoglobulin heavy chain variable region
selected from the group consisting of SEQ ID NO. 159 (Hu2B8 Hv1f.1
heavy chain variable region), SEQ ID NO. 165 (Hu2B8 Hv5a.1 heavy
chain variable region), SEQ ID NO. 169 (Hu2B8 Hv5-51.1 heavy chain
variable region), SEQ ID NO. 183 (LR2B8HC heavy chain variable
region), and SEQ ID NO. 189 (LRMR2B8LC light chain variable
region). The binding protein can be an intact antibody, an antigen
binding fragment thereof, or a biosynthetic antibody site.
[0060] In another aspect, the invention provides an isolated
binding protein that binds human HGF. The binding protein comprises
(i) an immunoglobulin light chain selected from the group
consisting of SEQ ID NO. 177 (Hu2B8 Kv1-39.1+kappa constant (Km(3)
allotype (allele 2)), SEQ ID NO. 181 (Hu2B8 Kv3-15.1+Kappa constant
(Km(3) allotype (allele 2)), SEQ ID NO. 197 (LR2B8LC+Kappa constant
(Km(3) allotype (allele 1)), and SEQ ID NO. 201 (LRMR2B8LC+Kappa
constant (Km(3) allotype (allele 1)); and (ii) an immunoglobulin
heavy chain selected from the group consisting of SEQ ID NO. 163
(Hu2B8 Hv1f.1+IgG1 Constant (G1m(17,1) allotype)), SEQ ID NO. 167
(Hu2B8 Hv5a.1+IgG1 Constant (G1m(17,1) allotype)), SEQ ID NO. 171
(Hu2B8 Hv5-51.1+IgG1 Constant (G1m(17,1) allotype)), SEQ ID NO. 187
(LR2B8HC+IgG1 Constant (G1m(3) allotype) (allele 1)), and SEQ ID
NO. 191 (LRMR2B8HC+IgG1 Constant (G1m(3) allotype) (allele 1)). The
binding protein can be an intact antibody, an antigen binding
fragment thereof, or a biosynthetic antibody site.
[0061] In another aspect, the invention provides an isolated
binding protein that binds reduced human HGF. The binding protein
comprises (i) an immunoglobulin light chain variable region
comprising three CDRs, and (ii) an immunoglobulin heavy chain
variable region comprising three CDRs. The CDRs typically are
interposed between FRs. The CDRs of the immunoglobulin light chain
and the immunoglobulin heavy chain together define a binding site
that binds reduced human HGF, for example, the .alpha.-chain of
reduced HGF. Reduced HGF refers to HGF treated with an amount of
reducing agent, for example, dithiothreitol (DTT),
2-mercaptoethanol, or glutathione sufficient to reduce the
disulfide linkage between the .alpha.-chain and the .beta.-chain.
Exemplary concentrations include, for example, 100 mM DTT and 5%
2-mercaptoethanol.
[0062] In certain embodiments, the binding protein comprises an
immunoglobulin light chain variable region comprising at least one
CDR selected from the group consisting of CDR.sub.L1, CDR.sub.L2
and CDR.sub.L3. Optionally, the binding protein comprises two CDRs,
for example, CDR.sub.L1 and CDR.sub.L2, or CDR.sub.L1 and
CDR.sub.L3, or CDR.sub.L1 and CDR.sub.L3. Optionally, the binding
protein comprises all three CDRs, i.e., CDR.sub.L1, CDR.sub.L2 and
CDR.sub.L3. CDR.sub.L1 comprises the amino acid sequence X.sub.1
X.sub.2 Ser X.sub.4 X.sub.5 X.sub.6 X.sub.7 X.sub.8 X.sub.9
X.sub.10 X.sub.11 X.sub.12 X.sub.13 X.sub.14 X.sub.15, wherein
amino acid X.sub.1 is Arg or Lys, X.sub.2 is Ala or Thr, X.sub.4 is
Glu or Gln, X.sub.5 is Asn, Ser, or Asp, X.sub.6 is Ile or Val,
X.sub.7 is Tyr, Asp, or Lys, X.sub.8 is a peptide bond or Tyr,
X.sub.9 is a peptide bond or Asp, X.sub.10 is a peptide bond or
Gly, X.sub.11 is a peptide bond or Asn, X.sub.12 is a peptide bond
or Ser, X.sub.13 is Asn or Tyr, X.sub.14 is Ile or Leu, X.sub.15 is
Ala, Asn, or Ser. CDR.sub.L2 comprises the amino acid sequence
X.sub.16 X.sub.17 X.sub.18 X.sub.19 Leu X.sub.21 X.sub.22, wherein
amino acid X.sub.16 is Ala, Asp, Val, or Arg, X.sub.17 is Ala or
Val, X.sub.18 is Asn, Ser, or Thr, X.sub.19 is Arg, Asn, or His,
X.sub.21 is Ala, Glu, Val, or Pro, X.sub.22 is Asp or Ser.
CDR.sub.L3 comprises the amino acid sequence X.sub.23 X.sub.24
X.sub.25 X.sub.26 X.sub.27 X.sub.28 Pro X.sub.30 Thr, wherein amino
acid X.sub.23 is Leu or Gln, X.sub.24 is His or Gln, X.sub.25 is
Phe, Ser, or Tyr, X.sub.26 is Asp, Ile, or Trp, X.sub.27 is Gly or
Glu, X.sub.28 is Asp, Phe, or Thr, X.sub.30 is Phe, Pro, or
Tyr.
[0063] In another embodiment, the binding protein comprises an
immunoglobulin heavy chain variable region comprising at least one
CDR selected from the group consisting of CDR.sub.H1, CDR.sub.H2,
and CDR.sub.H3. Optionally, the binding protein comprises two CDRs,
for example, CDR.sub.H1 and CDR.sub.H2, or CDR.sub.H1 and
CDR.sub.H3, or CDR.sub.H1 and CDR.sub.H3. Optionally, the binding
protein comprises all three CDRs, i.e., CDR.sub.H1, CDR.sub.H2 and
CDR.sub.H3. CDR.sub.H1 comprises the amino acid sequence X.sub.1
Tyr X.sub.3 X.sub.4 X.sub.5, wherein amino acid X.sub.1 is Asp,
Asn, Ser, or Thr, X.sub.3 is Phe, Trp, or Tyr, X.sub.4 is Ile or
Met, X.sub.5 is Asn, His, or Ser. CDR.sub.H2 comprises the amino
acid sequence X.sub.6 Ile X.sub.8 X.sub.9 Gly X.sub.11 Gly X.sub.13
X.sub.14 X.sub.15 Tyr X.sub.17 X.sub.18 X.sub.19 X.sub.20 Lys
X.sub.22, wherein amino acid X.sub.6 is Lys, Gln, or Tyr, X.sub.8
is Gly, Ser, or Tyr, X.sub.9 is Pro or Ser, X.sub.11 is Asp, Gly,
or Ser, X.sub.13 is Asp or Ser, X.sub.14 is Ser or Thr, X.sub.15 is
Asn or Tyr, X.sub.17 is Asn or Pro, X.sub.18 is Ala, Asp, Gly, or
Glu, X.sub.19 is Asn, Met, or Ser, X.sub.20 is Phe or Val, X.sub.22
is Asp or Gly. CDR.sub.H3 comprises the amino acid sequence
X.sub.23 X.sub.24 X.sub.25 X.sub.26 X.sub.27 X.sub.28 X.sub.29
X.sub.30 X.sub.31 X.sub.32 X.sub.33 Asp Tyr, wherein amino acid
X.sub.23 is Arg or Gln, X.sub.24 is Gly or Leu, X.sub.25 is Asp,
Gly, or a peptide bond, X.sub.26 is Gly or a peptide bond, X.sub.27
is a peptide bond or Tyr, X.sub.28 is Leu, a peptide bond or Tyr,
X.sub.29 is a Gly, Arg or Leu, X.sub.30 is Asp, Gly or Glu,
X.sub.31 is a Tyr, Arg or Asn, X.sub.32 is Ala, Gly or Tyr,
X.sub.33 is Met or Phe.
[0064] It is understood that the binding protein can comprise both
the immunoglobulin heavy chain and the immunoglobulin light chain
sequences or the fragments thereof, noted above. Furthermore, it is
understood that the binding protein can be an intact antibody or an
antigen binding fragment thereof, or a biosynthetic antibody
site.
[0065] In certain embodiments, the binding protein comprises an
immunoglobulin light chain variable region comprising (i) a
CDR.sub.L1 having a sequence selected from the group consisting of
SEQ ID NO. 8 (1A3), SEQ ID NO. 28 (2F8), SEQ ID NO. 38 (3B6), SEQ
ID NO. 58 (1D3), and SEQ ID NO. 68 (1F3), (ii) a CDR.sub.L2 having
a sequence selected from the group consisting of SEQ ID NO. 9
(1A3), SEQ ID NO. 29 (2F8), SEQ ID NO. 39 (3B6), SEQ ID NO. 59
(1D3), and SEQ ID NO. 69 (1F3), and (iii) a CDR.sub.L3 having a
sequence selected from the group consisting of SEQ ID NO. 10 (1A3),
SEQ ID NO. 30 (2F8), SEQ ID NO. 40 (3B6), SEQ ID NO. 60 (1D3), and
SEQ ID NO. 70 (1F3). The CDR sequences can be interposed between
human or humanized FRs. In other embodiments, the binding protein
comprises an immunoglobulin light chain variable region comprising
an amino acid sequence selected from the group consisting of
residues 21-127 of SEQ ID NO. 4 (1A3), residues 20-131 of SEQ ID
NO. 24 (2F8), residues 23-129 of SEQ ID NO. 34 (3B6), residues
21-127 of SEQ ID NO. 54 (1D3), and residues 21-127 of SEQ ID NO. 64
(1F3).
[0066] In certain other embodiments, the binding protein comprises
an immunoglobulin heavy chain variable region comprising (i) a
CDR.sub.H1 having a sequence selected from the group consisting of
SEQ ID NO. 5 (1A3), SEQ ID NO. 25 (2F8), SEQ ID NO. 35 (3B6), SEQ
ID NO. 55 (1D3), and SEQ ID NO. 65 (1F3), (ii) a CDR.sub.H2 having
a sequence selected from the group consisting of SEQ ID NO. 6
(1A3), SEQ ID NO. 26 (2F8), SEQ ID NO. 36 (3B6), SEQ ID NO. 56
(1D3), and SEQ ID NO. 66 (1F3), and (iii) a CDR.sub.H3 having a
sequence selected from the group consisting of SEQ ID NO. 7 (1A3),
SEQ ID NO. 27 (2F8), SEQ ID NO. 37 (3B6), SEQ ID NO. 57 (1D3), and
SEQ ID NO. 67 (1F3). The CDR sequences can be interposed between
human or humanized FRs. In another embodiment, the immunoglobulin
heavy chain variable region comprises an amino acid sequence
selected from the group consisting of residues 20-141 of SEQ ID NO.
2 (1A3), residues 20-137 of SEQ ID NO. 22 (2F8), residues 20-139 of
SEQ ID NO. 32 (3B6), residues 20-141 of SEQ ID NO. 52 (1D3), and
residues 20-141 of SEQ ID NO. 62 (1F3).
[0067] In another aspect, the invention provides an isolated
binding protein that binds human HGF and comprises an
immunoglobulin light chain variable region and an immunoglobulin
heavy chain variable region. The isolated binding protein competes
for binding to HGF with at least one reference antibody selected
from the group consisting of (i) an antibody having an
immunoglobulin light chain variable region of residues 20-131 of
SEQ ID NO. 24 (2F8), and an immunoglobulin heavy chain variable
region of residues 20-137 of SEQ ID NO. 22 (2F8), (ii) an antibody
having an immunoglobulin light chain variable region of residues
23-129 of SEQ ID NO. 34 (3B6), and an immunoglobulin heavy chain
variable region of residues 20-139 of SEQ ID NO. 32 (3B6), and
(iii) an antibody having an immunoglobulin light chain variable
region of residues 23-128 of SEQ ID NO. 44 (3D11), and an
immunoglobulin heavy chain variable region of residues 20-132 of
SEQ ID NO. 42 (3D11). Under certain circumstances, the binding
protein binds the same epitope of HGF as one of the reference
antibodies.
[0068] It is understood that each of the binding proteins discussed
above can be an intact antibody, for example, a monoclonal
antibody. Alternatively, the binding protein can be an antigen
binding fragment of an antibody, or can be a biosynthetic antibody
binding site. Antibody fragments include Fab, Fab', (Fab').sub.2 or
Fv fragments. Techniques for making such antibody fragments are
known to those skilled in the art. A number of biosynthetic
antibody binding sites are known in the art and include, for
example, single Fv or sFv molecules, described, for example, in
U.S. Pat. No. 5,476,786. Other biosynthetic antibody binding sites
include bispecific or bifunctional binding proteins, for example,
bispecific or bifunctional antibodies, which are antibodies or
antibody fragments that bind at least two different antigens. For
example, bispecific binding proteins can bind HGF, for example,
human HGF, and another antigen of interest. Methods for making
bispecific antibodies are known in art and, include, for example,
by fusing hybridomas or by linking Fab' fragments. See, e.g.,
Songsivilai et al. (1990) CLIN. EXP. IMMUNOL. 79: 315-325; Kostelny
et al. (1992) J. IMMUNOL. 148: 1547-1553.
[0069] The binding proteins of the invention can bind hHGF
containing a cysteine to arginine substitution at position 561 or a
glycine to glutamate substitution at position 555.
[0070] In another aspect, the invention provides an isolated
binding protein that binds human HGF with a k.sub.d of
4.0.times.10.sup.-5 s.sup.-1 or lower, 3.0.times.10.sup.-5 s.sup.-1
or lower, or 2.0.times.10.sup.-5 s.sup.-1 or lower. The isolated
binding proteins can bind human HGF with a k.sub.d from
5.0.times.10.sup.-5 s.sup.-1 to 0.5.times.10.sup.-5s.sup.-1, or
from 4.0.times.10.sup.-5 s.sup.-1 to 1.0.times.10.sup.-5s.sup.-1,
or from 3.0.times.10.sup.-5 s.sup.-1 to 1.5.times.10.sup.-5
s.sup.-1. In another aspect, the invention provides an isolated
binding protein that binds human HGF with a K.sub.D of 100 pM or
lower, or 20 pM or lower, or 10 pM or lower, or 5 pM or lower. The
isolated binding proteins can bind human HGF with a K.sub.D from
100 pM to 5 pM, or from 20 pM to 5 pM, or from 15 pM to 10 pM, or
from 20 pM to 10 pM, or from 15 pM to 5 pM. Unless otherwise
specified, K.sub.D values are determined by the methods, and under
the conditions, described in Example 6.
[0071] In another aspect, the invention provides an isolated
binding protein that binds human HGF, wherein the antibody binds to
human HGF with lower K.sub.D at 37.degree. C. than at 25.degree. C.
The binding protein binding optionally binds human HGF with a
K.sub.D less than 5 pM at 37.degree. C.
[0072] In other aspects and embodiments, the binding proteins can
inhibit hHGF from binding to c-Met. For example, the binding
proteins can have an IC.sub.50 (concentration at 50% of maximum
inhibition) of at least about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0
nM when assayed using the protocol described in Example 7(a). In
certain other embodiments, the binding proteins can neutralize HGF
BrdU incorporation in 4 MBr-5 cells (ATCC, Catalog No. CCL208)
using the method described in Example 7(b).
[0073] The binding proteins have an IC.sub.50 of 50 nM or lower,
preferably 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.5 nM or lower,
when assayed using the protocol described in Example 7(b). In
certain other embodiments, the binding proteins can be used to
inhibit HGF stimulated c-Met phosphorylation in PC-3 cells (ATCC,
Manassas, Va. Catalog No. CRL-1435) using the assay described in
Example 9. The binding proteins inhibit HGF-stimulated (1.25 nM)
c-Met phosphorylation in PC-3 cells with an IC.sub.50 of 2 nM or
less (Table 8), using the assay described in Example 9.
II--Production of Binding Proteins
[0074] Binding proteins of the invention can be produced in various
ways using approaches know in the art. For example, DNA molecules
encoding light chain variable regions and heavy chain variable
regions can be chemically synthesized, using a commercial
synthesizer and sequence information provided herein. Such
synthetic DNA molecules can be ligated to other appropriate
nucleotide sequences, including, e.g., constant region coding
sequences, and expression control sequences, to produce
conventional gene expression constructs encoding the desired
binding proteins. Production of defined gene constructs is within
routine skill in the art. Alternatively, the sequences provided
herein can be cloned out of hybridomas by conventional
hybridization techniques or PCR techniques, using synthetic nucleic
acid probes whose sequences are based on sequence information
provided herein or prior art sequence information regarding genes
encoding the heavy and light chains of murine antibodies in
hybridoma cells. Production and use of such probes is within
ordinary skill in the art.
[0075] The nucleic acids encoding the desired binding proteins can
be introduced (ligated) into expression vectors, which can be
introduced into a host cell via standard transfection or
transformation techniques known in the art. Exemplary host cells
include, for example, E. coli cells, Chinese hamster ovary (CHO)
cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney
cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),
and myeloma cells that do not otherwise produce immunoglobulin
protein. Transfected host cells can be grown under conditions that
permit the host cells to express the genes of interest, for
example, the genes that encode the immunoglobulin light or heavy
chain variable regions. The resulting expression products can be
harvested using techniques known in the art.
[0076] The particular expression and purification conditions will
vary depending upon what expression system is employed. For
example, if the gene is to be expressed in E. coli, it is first
cloned into an expression vector. This is accomplished by
positioning the engineered gene downstream from a suitable
bacterial promoter, e.g., Trp or Tac, and a signal sequence, e.g.,
a sequence encoding fragment B of protein A (FB). The resulting
expressed fusion protein typically accumulates in refractile or
inclusion bodies in the cytoplasm of the cells, and may be
harvested after disruption of the cells by French press or
sonication. The refractile bodies then are solubilized, and the
expressed proteins refolded and cleaved by the methods already
established for many other recombinant proteins.
[0077] If the engineered gene is to be expressed in eukayotic host
cells, for example, myeloma cells or CHO cells, it is first
inserted into an expression vector containing a suitable eukaryotic
promoter, a secretion signal, immunoglobulin enhancers, and various
introns. This expression vector optionally can contain sequences
encoding all or part of a constant region, enabling an entire, or a
part of, a heavy or light chain to be expressed. The gene construct
can be transfected into myeloma cells or CHO cells using
established transfection protocols. Such transfected cells can
express V.sub.L or V.sub.H fragments, V.sub.L-V.sub.H heterodimers,
V.sub.H-V.sub.L or V.sub.L-V.sub.H single chain polypeptides,
complete heavy or light immunoglobulin chains, or portions thereof,
each of which may be attached to a protein domain having another
function (e.g., cytotoxicity).
III--Modifications to the Binding Proteins
[0078] It is understood that the binding proteins can be modified
to optimize performance depending upon the intended use of the
binding proteins. For example, when the binding protein is being
used as a therapeutic agent, the binding protein can be modified to
reduce its immunogenicity in the intended recipient. Alternatively
or in addition, the binding protein can be fused or coupled to
another protein or peptide, for example, a growth factor, cytokine,
or cytotoxin. Such modifications can be achieved by using routine
gene manipulation techniques known in the art.
[0079] Various techniques for reducing the antigenicity of
antibodies and antibody fragments are known in the art. These
techniques can be used to reduce or eliminate the antigenicity of
the binding proteins of the invention. For example, when the
binding proteins are to be administered to a human, the binding
proteins preferably are engineered to reduce their antigenicity in
humans. This process often is referred to as humanization.
Preferably, the humanized binding proteins have the same or
substantially the same affinity for the antigen as the original
non-humanized binding protein it was derived from.
[0080] In one well known humanization approach, chimeric proteins
are created in which immunoglobulin constant regions of antibodies
from one species, e.g., mouse, are replaced with immunoglobulin
constant regions from a second, different species, e.g., a human.
In this example, the resulting antibody is a mouse-human chimera,
where the human constant region sequences, in principle, are less
immunogenic than the counterpart murine sequences. This type of
antibody engineering is described, for example, Morrison, et al.
(1984) PROC. NAT. ACAD. SCI. 81: 6851-6855, Neuberger et al. (1984)
NATURE 312: 604-608; U.S. Pat. No. 6,893,625 (Robinson); U.S. Pat.
No. 5,500,362 (Robinson); and U.S. Pat. No. 4,816,567
(Cabilly).
[0081] In another approach, known as CDR grafting, the CDRs of the
light and heavy chain variable regions of an antibody of interest
are grafted into frameworks (FRs) from another species. For
example, murine CDRs can be grafted into human FR sequences. In
some embodiments, the CDRs of the light and heavy chain variable
regions of an anti-HGF antibody are grafted into human FRs or
consensus human FRs. In order to create consensus human FRs, FRs
from several human heavy chain or light chain amino acid sequences
are aligned to identify a consensus amino acid sequence. CDR
grafting is described, for example, in U.S. Pat. No. 7,022,500
(Queen); U.S. Pat. No. 6,982,321 (Winter); U.S. Pat. No. 6,180,370
(Queen); U.S. Pat. No. 6,054,297 (Carter); U.S. Pat. No. 5,693,762
(Queen); U.S. Pat. No. 5,859,205 (Adair); U.S. Pat. No. 5,693,761
(Queen); U.S. Pat. No. 5,565,332 (Hoogenboom); U.S. Pat. No.
5,585,089 (Queen); U.S. Pat. No. 5,530,101 (Queen); Jones et al.
(1986) NATURE 321: 522-525; Riechmann et al. (1988) NATURE 332:
323-327; Verhoeyen et al. (1988) SCIENCE 239: 1534-1536; and Winter
(1998) FEBS LETT 430: 92-94.
[0082] In an approach called "superhumanization," antibodies in
which human immunogenicity is reduced or eliminated are created by
an alternative form of grafting. In superhumanization, human FR
sequences are chosen from a set of human germline genes based on
the structural similarity of the human CDRs to those of the mouse
antibody to be humanized. This approach is described, for example,
in U.S. Pat. No. 6,881,557 (Foote) and in Tan et al. (2002) J.
IMMUNOL 169:1119-1125.
[0083] Other approaches to reduce immunogenicity include,
techniques are known as "reshaping," "hyperchimerization," or
"veneering/resurfacing" to produce humanized antibodies. See, e.g.,
Vaswami et al. (1998) ANNALS OF ALLERGY, ASTHMA, & IMMUNOL. 81:
105; Roguska et al. (1996) PROT. ENGINEER 9: 895-904; and U.S. Pat.
No. 6,072,035 (Hardman). In the veneering/resurfacing approach, the
surface accessible amino acid residues in the murine antibody are
replaced by amino acid residues more frequently found at the same
positions in a human antibody. This type of antibody resurfacing is
described, for example, in U.S. Pat. No. 5,639,641 (Pedersen).
[0084] One exemplary approach for converting a mouse antibody into
a form suitable for medical use in humans is known as ACTIVMAB.TM.
technology (Vaccinex, Inc., Rochester, N.Y.), which involves a
vaccinia virus-based vector to express antibodies in mammalian
cells. High levels of combinatorial diversity of immunoglobulin
heavy and light chains are said to be produced. See, e.g., U.S.
Pat. No. 6,706,477 (Zauderer); U.S. Pat. No. 6,800,442 (Zauderer);
and U.S. Pat. No. 6,872,518 (Zauderer).
[0085] Another exemplary approach for converting a mouse antibody
into a form suitable for use in humans is technology practiced
commercially by KaloBios Pharmaceuticals, Inc. (Palo Alto, Calif.).
This technology involves the use of a proprietary human "acceptor"
library to produce an "epitope focused" library for antibody
selection.
[0086] Another exemplary approach for modifying a mouse antibody
into a form suitable for medical use in humans is HUMAN
ENGINEERING.TM. (HE.TM.) technology, which is practiced
commercially by XOMA (US) LLC. See, e.g., International Application
Publication No. WO 93/11794 and U.S. Pat. Nos. 5,766,886;
5,770,196; 5,821,123; and 5,869,619.
[0087] Any suitable approach, including any of the above
approaches, can be used to reduce or eliminate human immunogenicity
of a binding protein of interest.
[0088] In addition, it is possible to create fully human antibodies
in mice. In this approach, human antibodies are prepared using a
transgenic mouse in which the mouse's antibody-producing genes have
been replaced by a substantial portion of the human antibody
producing genes. Such mice produce human immunoglobulin instead of
murine immunoglobulin molecules. See, e.g., WO 98/24893 (Jacobovitz
et al.) and Mendez et al. (1997) NATURE GENETICS 15: 146-156. Fully
human anti-HGF monoclonal antibodies can be produced using the
following approach. Transgenic mice containing human immunoglobulin
genes are immunized with the antigen of interest, e.g., HGF.
Lymphatic cells from the mice then are obtained from the mice,
which are then fused with a myeloid-type cell line to prepare
immortal hybridoma cell lines. The hybridoma cell lines are
screened and selected to identify hybridoma cell lines that produce
antibodies specific to HGF.
[0089] Binding proteins of the invention can be conjugated with
other molecules, depending upon their intended use. For example, if
the binding protein is going to be used as a therapeutic, then the
binding protein can be conjugated with another agent, for example,
an effector molecule that modulates or otherwise promotes the
therapy. To the extent that the effector is non-protein based
agent, for example, a small molecule drug, a radiolabel or toxin,
then, the agent can be chemically coupled to the binding protein
using standard in vitro coupling chemistries. If, on the other
hand, the effector molecule is a protein or peptide, for example,
an enzyme, receptor, toxin, growth factor, cytokine or other
immunomodulator, then the binding protein can either be chemically
coupled to the effector using in vitro coupling chemistries or can
be coupled to the effector as a fusion protein. Fusion proteins can
be constructed and expressed using the techniques similar to those
discussed in section II.
IV--Use of Binding Proteins
[0090] The binding proteins described herein can be used as a
diagnostic agent or a therapeutic agent.
[0091] (1) Therapeutic Applications
[0092] Because the binding proteins of the invention neutralize the
activity of HGF, they can be used in various therapeutic
applications. For example, certain binding proteins of the
invention are useful in the prevention or treatment of
hyperproliferative diseases or disorders, e.g., various forms of
cancer.
[0093] The binding proteins can be used to inhibit or reduce the
proliferation of tumor cells. In such an approach, the tumor cells
are exposed to a therapeutically effective amount of the binding
protein so as to inhibit or reduce proliferation of the tumor cell.
In certain embodiments, the binding proteins inhibit tumor cell
proliferation by at least 50%, 60%, 70%, 80%, 90%, 95% or 100%.
[0094] In certain embodiments, the binding protein is used to
inhibit or reduce proliferation of a tumor cell wherein the binding
protein reduces the ability of hHGF to bind to c-Met. In other
embodiments, the binding protein is used to inhibit or reduce the
proliferation of a tumor cell even when the binding protein binds
hHGF but does not substantially inhibit hHGF binding to c-Met, as
shown by antibody 3B6 in Tables 5 and 6.
[0095] In addition, the binding protein can be used to inhibit, or
slow down tumor growth or development in a mammal. In such a
method, an effective amount of the binding protein is administered
to the mammal so as to inhibit or slow down tumor growth in the
mammal. Accordingly, the binding proteins can be used to treat
tumors, for example, in a mammal. The method comprises
administering to the mammal a therapeutically effective amount of
the binding protein. The binding protein can be administered alone
or in combination with another pharmaceutically active molecule, so
as to treat the tumor.
[0096] It is contemplated that the binding proteins of the
invention can be used in the treatment of a variety of HGF
responsive disorders, including, for example, HGF responsive tumor
cells in lung cancer, breast cancer, colon cancer, prostate cancer,
ovarian cancer, head and neck cancer, ovarian cancer, multiple
myeloma, liver cancer, gastric cancer, esophageal cancer, kidney
cancer, nasopharangeal cancer, pancreatic cancer, mesothelioma,
melanoma and glioblastoma.
[0097] As used herein, "treat, "treating" and "treatment" refer to
the treatment of a disease-state in a mammal, particularly in a
human, and include: (a) preventing the disease-state from occurring
in a mammal, in particular, when such mammal is predisposed to the
disease-state but has not yet been diagnosed as having it; (b)
inhibiting the disease-state, i.e., arresting its development;
and/or (c) relieving the disease-state, i.e., causing regression of
the disease state.
[0098] Generally, a therapeutically effective amount of active
component will be in the range of from about 0.1 mg/kg to about 100
mg/kg, optionally from about 1 mg/kg to about 100 mg/kg, optionally
from about 1 mg/kg to 10 mg/kg. The amount administered will depend
on variables such as the type and extent of disease or indication
to be treated, the overall health status of the particular patient,
the relative biological efficacy of the binding protein delivered,
the formulation of the binding protein, the presence and types of
excipients in the formulation, and the route of administration. The
initial dosage administered may be increased beyond the upper level
in order to rapidly achieve the desired blood-level or tissue
level, or the initial dosage may be smaller than the optimum and
the daily dosage may be progressively increased during the course
of treatment depending on the particular situation. Human dosage
can be optimized, e.g., in a conventional Phase I dose escalation
study designed to run from 0.5 mg/kg to 20 mg/kg. Dosing frequency
can vary, depending on factors such as route of administration,
dosage amount and the disease condition being treated. Exemplary
dosing frequencies are once per day, once per week and once every
two weeks. A preferred route of administration is parenteral, e.g.,
intravenous infusion. Formulation of monoclonal antibody-based
drugs is within ordinary skill in the art. In some embodiments of
the invention, the binding protein, e.g., monoclonal antibody, is
lyophilized and reconstituted in buffered saline at the time of
administration.
[0099] The binding proteins may be administered either alone or in
combination with other pharmaceutically active ingredients. The
other active ingredients, e.g., immunomodulators, can be
administered together with the binding protein, or can be
administered before or after the binding protein.
[0100] Formulations containing the binding proteins for therapeutic
use, typically include the binding proteins combined with a
pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" means buffers, carriers, and
excipients, that are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio. The carrier(s) should be "acceptable" in the
sense of being compatible with the other ingredients of the
formulations and not deleterious to the recipient. Pharmaceutically
acceptable carriers, in this regard, are intended to include any
and all buffers, solvents, dispersion media, coatings, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is known in the art.
[0101] The formulations can be conveniently presented in a dosage
unit form and can be prepared by any suitable method, including any
of the methods well known in the pharmacy art. A pharmaceutical
composition of the invention should be formulated to be compatible
with its intended route of administration. Examples of routes of
administration include parenteral administration or non-parenteral
administration, for example, intravenous, intradermal, inhalation,
transdermal (topical), transmucosal, and rectal administration.
Useful solutions for oral or parenteral administration can be
prepared by any of the methods well known in the pharmaceutical
art, described, for example, in Remington's Pharmaceutical
Sciences, 18th ed. (Mack Publishing Company, 1990).
[0102] Formulations suitable for oral administration can be in the
form of: discrete units such as injectables, capsules, gelatin
capsules, sachets, tablets, troches, or lozenges, each containing a
predetermined amount of the binding protein; a powder or granular
composition; a solution or a suspension in an aqueous liquid or
non-aqueous liquid; or an oil-in-water emulsion or a water-in-oil
emulsion.
[0103] Formulations suitable for parenteral administration include,
for example, the following components: a sterile diluent such as
water for injection, saline solution, fixed oils, polyethylene
glycols, glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0104] In general, compositions suitable for injectable use include
aqueous solutions (where water soluble) or dispersions and powders
for the extemporaneous preparation of sterile injectable solutions
or dispersion. For intravenous administration, suitable carriers
include physiological saline, bacteriostatic water, Cremophor ELTM
(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). It
should be stable under the conditions of manufacture and storage
and should be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyetheylene glycol), and suitable mixtures thereof.
[0105] Pharmaceutical formulations preferably are sterile.
Sterilization can be accomplished, for example, by filtration
through sterile filtration membranes. Where the composition is
lyophilized, sterilization using this method can be conducted prior
to or following lyophilization and reconstitution. Once the
pharmaceutical composition has been formulated, it can be stored,
for example, in vials as a solution, suspension, gel, emulsion,
solid, or as a dehydrated or lyophilized powder.
[0106] (2) Diagnostic Applications
[0107] Whenever the binding proteins are used for diagnostic
purposes, either in vitro or in vivo, the binding proteins
typically are labeled either directly or indirectly with a
detectable moiety. The detectable moiety can be any moiety which is
capable of producing, either directly or indirectly, a detectable
signal. For example, the detectable moiety may be a radioisotope,
such as .sup.3Hydrogen (.sup.3H), .sup.14-Carbon (.sup.14C),
.sup.32Phosphorus (.sup.32P), .sup.35Sulfur (.sup.35 S), or
.sup.125Iodine (.sup.125I); a fluorescent or chemiluminescent
compound, such as fluorescein isothiocyanate, rhodamine, or
luciferin; an enzyme, such as alkaline phosphatase,
beta-galactosidase, or horseradish peroxidase; a spin probe, such
as a spin label; or a colored particle, for example, a latex or
gold particle. It is understood that the binding protein can be
conjugated to the detectable moiety using a number of approaches
known in the art, for example, as described in Hunter et al. (1962)
NATURE 144: 945; David et al. (1974) BIOCHEMISTRY 13: 1014; Pain et
al. (1981) J. IMMUNOL. METH. 40: 219; and Nygren (1982) J.
HISTOCHEM. AND CYTOCHEM. 30: 407. The labels may be detected, e.g.,
visually or with the aid of a spectrophotometer or other
detector.
[0108] The binding proteins can be employed in a wide range of
immunoassay techniques available in the art. Exemplary immunoassays
include, for example, sandwich immunoassays, competitive
immunoassays, immunohistochemical procedures.
[0109] In a sandwich immunoassay, two antibodies that bind an
analyte or antigen of interest are used, e.g., one immobilized onto
a solid support, and one free in solution and labeled with a
detectable moiety. When a sample containing the antigen is
introduced into this system, the antigen binds to both the
immobilized antibody and the labeled antibody, to form a "sandwich"
immune complex on the surface of the support. The complexed protein
is detected by washing away non-bound sample components and excess
labeled antibody, and measuring the amount of labeled antibody
complexed to protein on the support's surface. Alternatively, the
antibody free in solution can be detected by a third antibody
labeled with a detectable moiety which binds the free antibody. A
detailed review of immunological assay design, theory and protocols
can be found in numerous texts, including Butt, ed., (1984)
PRACTICAL IMMUNOLOGY, Marcel Dekker, New York; Harlow et al. eds.
(1988) ANTIBODIES, A LABORATORY APPROACH, Cold Spring Harbor
Laboratory; and Diamandis et al., eds. (1996) IMMUNOASSAY, Academic
Press, Boston.
[0110] It is contemplated that the labeled binding proteins are
useful as in vivo imaging agents, whereby the binding proteins can
target the imaging agents to particular tissues of interest in the
recipient. A preferred remotely detectable moiety for in vivo
imaging includes the radioactive atom Technetium.sup.-99m
(.sup.99mTc), a gamma emitter with a half-life of about six hours.
Non-radioactive moieties also useful in in vivo imaging include
nitroxide spin labels as well as lanthanide and transition metal
ions all of which induce proton relaxation in situ. In addition to
immunoimaging, the complexed radioactive moieties may be used in
standard radioimmunotherapy protocols to destroy the targeted cell.
Preferred nucleotides for high dose radioimmunotherapy include the
radioactive atoms .sup.90Yttrium (.sup.90Yt), .sup.131Iodine
(.sup.131I) and .sup.111Indium (.sup.111In). The binding protein
can be labeled with .sup.131I, .sup.111In and .sup.99mTC using
coupling techniques known in the imaging arts. Similarly,
procedures for preparing and administering the imaging agent as
well as capturing and processing images are well known in the
imaging art and so are not discussed in detail herein. Similarly,
methods for performing antibody-based immunotherapies are well
known in the art. See, for example, U.S. Pat. No. 5,534,254.
[0111] Throughout the description, where compositions are described
as having, including, or comprising specific components, it is
contemplated that compositions also consist essentially of, or
consist of, the recited components. Similarly, where processes are
described as having, including, or comprising specific process
steps, the processes also consist essentially of, or consist of,
the recited processing steps. Except where indicated otherwise, the
order of steps or order for performing certain actions are
immaterial so long as the invention remains operable. Moreover,
unless otherwise noted, two or more steps or actions may be
conducted simultaneously.
EXAMPLES
[0112] The following Examples discuss the production and
characterization of a number of anti-hHGF monoclonal
antibodies.
Example 1
Production of Anti-hHGF Monoclonal Antibodies
[0113] This Example describes the production of a number of
anti-hHGF monoclonal antibodies.
[0114] Immunizations, fusions, and primary screens were conducted
at MBS Inc. (Portland, Me.), following the Repetitive Immunization
Multiple Sites (RIMMS) protocol. Five AJ mice and Five Balb/c mice
were immunized with recombinant human HGF (R&D Systems,
Minneapolis, Minn.; Catalog No. 294-HGN-025). Two mice with sera
displaying highest anti-HGF activity by Enzyme Linked Immunosorbent
Assay (ELISA) were chosen for subsequent fusion. Spleens and lymph
nodes from the appropriate mice were harvested. B-cells then were
harvested and fused with an myeloma line. Fusion products were
serially diluted on one or more plates to near clonality.
Supernatants from the resulting fusions were screened for their
binding to hHGF by ELISA. Supernatants identified as containing
antibodies to HGF were further characterized by in vitro functional
testing as discussed in the following examples. A panel of
hybridomas was selected and the hybridomas were subcloned and
expanded. The monoclonal antibodies then were purified by affinity
chromatography on Protein A/G resin under standard conditions.
Example 2
Sequence Analysis of Anti-hHGF Monoclonal Antibodies
[0115] This Example describes isotype and sequence analyses of the
anti-hHGF monoclonal antibodies produced in Example 1.
[0116] a. Determination of HGF Murine Monoclonal Antibody
Isotypes
[0117] The light-chain type and heavy chain isotype of each
monoclonal antibody were determined using the IsoStrip Mouse
Monoclonal Antibody Isotyping Kit in accordance the manufacturer's
instructions (Roche Applied Science).
[0118] All the antibodies were determined to contain a Kappa
immunoglobulin light chain and an IgG1 immunoglobulin heavy
chain.
[0119] b. Determination of Nucleotide Sequences Encoding
Immunoglobulin Heavy and Light Chain Variable Regions
[0120] Total RNA was extracted from each monoclonal hybridoma cell
line using the RNeasy Miniprep kit according to the manufacturer's
instructions (Qiagen Venlo, The Netherlands). Full-length first
strand cDNA was generated using the BD SMART.TM. RACE cDNA
Amplification Kit according to the manufacturer's instructions
(Clontech) using the oligonucleotide primers BD SMART II A (5'
aagcagtggtatcaacgcagagtacgcggg 3') (SEQ ID NO. 85) and 5'-RACE CDS
Primer (5' tttttttttttttttttttttttttvn 3', where v=a, g, or c and
n=a, g, c, or t) (SEQ ID NO. 86) for the purpose of 5' RACE (Rapid
Amplification of cDNA Ends).
[0121] The variable regions of the Kappa and Heavy (IgG1)
immunoglobulin chains were amplified by PCR (Polymerase Chain
Reaction) using the Expand High-Fidelity PCR System (Roche Applied
Science) according to the manufacturer's instructions. Heavy chain
variable regions were amplified with the 5' oligonucleotide primer
mix Universal Primer Mix A (mix of 5'
ctaatacgactcactatagggcaagcagtggtatcaacgcagagt 3' (SEQ ID NO. 87)
and 5' ctaatacgactcactatagggc 3'(SEQ ID NO. 88)) and a 3' IgG1
Constant Region specific primer, either 5' tatgcaaggcttacaaccaca 3'
(SEQ ID NO. 89) or 5' gccagtggatagacagatgggggtgtcg 3' (SEQ ID NO.
90). Kappa chain variable regions were amplified with the 5'
oligonucleotide primer mix Universal Primer Mix A and a 3' Kappa
Constant Region specific primer, either 5'
ctcattcctgttgaagctcttgacaat 3' (SEQ ID NO. 91) or 5'
cgactgaggcacctccagatgtt 3' (SEQ ID NO. 92).
[0122] Individual PCR products were fractionated by agarose gel
electrophoresis and purified using the Qiaquick Gel Purification
kit according to the manufacturer's instructions (Qiagen). The PCR
products were subsequently cloned into the pCR2.1 TOPO plasmid
using the topoisomerase based cloning kit TOPO TA Cloning.RTM. Kit
(with pCR.RTM.2.1-TOPO.RTM. vector) according to the manufacturer's
instructions (Invitrogen, Carlsbad, Calif.) and transformed into
DH5 bacteria using standard transformation techniques. Plasmid DNA
isolated from transformed bacterial clones was sequenced using T7
(5' TAATACGACTCACTATAGGG 3') (SEQ ID NO. 93), M13 Forward (5'
GTAAAACGACGGCCAGT 3') (SEQ ID NO. 94), and M13 Reverse primers (5'
CAGGAAACAGCTATGACC 3') (SEQ ID NO. 95) by Agencourt Bioscience
using standard dideoxy DNA sequencing methods to identify the
sequence of the variable region sequences. The sequences were
analyzed using Vector NTI software (Invitrogen, Carlsbad, Calif.)
and the IMGT/V-Quest webserver (http://imgt.cines.fr/textes/vquest)
to identify and confirm variable region sequences.
[0123] c. Determination of Nucleotide Sequences Encoding
Immunoglobulin Heavy and Light Chain Constant Region Sequences for
1A3, 1D3, 1F3, and 2B8 Kappa and IgG1 Chains
[0124] Full Length cDNAs for the 1A3, 1D3, and 1F3 IgG1 chains were
PCR amplified from the cDNA created above using the forward primer
5' ggggacaagtttgtacaaaaaagcaggctgccaccatgaactttgggctcagattgattttcc
3' (start codon underlined) (SEQ ID NO. 96) and the reverse primer
5' ggggaccactttgtacaagaaagctgggttcatttaccaggagagtgggagagg 3' (stop
codon underlined) (SEQ ID NO. 97). Full Length cDNA for the 2B8
IgG1 chain was amplified from the cDNA created above using the
forward primer 5'
ggggacaagtttgtacaaaaaagcaggctgccaccatgggatggagctatatcatcctcttt 3'
(start codon underlined) (SEQ ID NO. 98) and reverse primer 5'
ggggaccactttgtacaagaaagctgggttcatttaccaggagagtgggagag 3' (stop
codon underlined) (SEQ ID NO. 99).
[0125] Full Length cDNA for the 2B8 Kappa Chain was amplified using
the forward primer 5'
ggggacaagtttgtacaaaaaagcaggctgccaccatggaatcacagactctggtcttcata 3'
(start codon underlined) (SEQ ID NO. 100) and the reverse primer 5'
ggggaccactttgtacaagaaagctgggtctaacactcattcctgttgaagctc 3' (stop
codon underlined) (SEQ ID NO. 101). PCR fragments were subcloned
into pDONR221 (Invitrogen, Carlsbad, Calif.) by Gateway BP
recombination reaction (Invitrogen, Carlsbad, Calif.) and sequenced
by Agencourt Bioscience using standard dideoxy DNA sequencing
methods to identify the sequence of the constant region and further
confirm variable region sequences.
[0126] d. Sequence Analysis
[0127] Variable Regions (normal text) were identified using
IMGT/V-QUEST webserver software
(http://imgt.cines.fr/textes/vquest/). Signal Peptide sequences
were predicted based on identification of the in frame start codon
(ATG) that was upstream of the identified Variable Region. Signal
Peptide sequences were identified and are underlined below.
[0128] The last nucleotide of each variable region is the first
base of the next codon generated by the variable/constant region
junction. This nucleotide is included in the variable region
because it is part of that exon. Amino acid sequences of the
constant regions listed below include the translation of this
junction codon.
[0129] In order to create the complete heavy or kappa chain
antibody sequences, the variable region sequences noted below are
combined with their respective constant region sequences (the
signal sequences are underlined).
TABLE-US-00001 (1) 1A3 Heavy Chain Variable Region (SEQ ID NO. 1) 1
atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa
61 gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct
gaaactctcc 121 tgtgcagcct ctgaattcac tttcagtaac tattacatgt
cttgggttcg ccagactcca 181 gagaagaggc tgcagtgggt cgcatacatt
agtcctggtg gtggtagctc ctactatcca 241 gccagtgtga agggtcgatt
caccatctcc agagacaatg ccaagaacac cctgtacctg 301 caaatgagca
gtctgaagtc tgaggacaca gccatgtatt actgtgcaag acaaggggat 361
ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc
421 tcag (2) 1A3 Kappa Light Chain Variable Region (SEQ ID NO. 3) 1
atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt
61 gacatccaga tgactcagtc tccagcctcc ctatctgttt ctgtgggaga
aactgtcacc 121 atcacatgtc gagcaagtga gaatatttat agtaatttag
catggtatca gcagaaacag 181 ggaaaatctc ctcagctcct ggtctatgct
gcaacaaact tagcagatgg tgtgccatca 241 aggttcagtg gcagtggatc
aggcacacag ttttccctca agatcaacag cctgcagtct 301 gaagattttg
ggacttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg 361
gggaccaagc tggaaataaa ac (3) 2B8 Heavy Chain Variable Region (SEQ
ID NO. 11) 1 atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt
ccactcccag 61 gtccaactgc agcagcctgg ggctgaactg gtgaagcctg
ggacttcagt gaagctgtcc 121 tgcaaggctt ctggctacac cttcaccacc
tactggatgc actgggtgaa tcagaggcct 181 ggacaaggcc ttgagtggat
tggagagatt aatcctacca acggtcatac taactacaat 241 gagaagttca
agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg 301
caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactatgtt
361 ggtagcatct ttgactactg gggccaaggc accactctca cagtctcctc ag (4)
2B8 Kappa Light Chain Variable Region (SEQ ID NO. 13) 1 atggaatcac
agactctggt cttcatatcc atactgctct ggttatatgg tgctgatggg 61
aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc
121 ttgagctgca aggccagtga gaatgtggtt tcttatgtat cctggtatca
acagaaacca 181 gcgcagtctc ctaaactgct gatatacggg gcatccaacc
ggaacactgg ggtccccgat 241 cgcttcacag gcagtggatc tgcaacagat
ttcactctga ccatcagcag tgtgcgggct 301 gaagaccttg cagattatca
ctgtgggcag agttacaact atccgtacac gttcggaggg 361 gggaccaggc
tggaaataaa ac (5) 2F8 Heavy Chain Variable Region (SEQ ID NO. 21) 1
atggaatgga gctgggtctt tctcttcctc ctgtcagtaa ctgcaggtgt ccactgccag
61 gtccagctga agcagtctgg agctgagctg gtgaggcctg ggacttcagt
gaagatgtcc 121 tgcaaggctt ctggctacac cttcactacc tactatatac
actgggtgaa tcagaggcct 181 ggacagggcc ttgagtggat tggaaagatt
ggtcctggaa gtggtagtac ttactacaat 241 gagatgttca aagacaaggc
cacattgact gtagacacat cctccagcac agcctacatg 301 cagctcagca
gcctgacatc tgacgactct gcggtctatt tctgtgcaag aaggggactg 361
ggacgtggct ttgactactg gggccaaggc accactctca cagtctcctc ag (6) 2F8
Kappa Light Chain Variable Region (SEQ ID NO. 23) 1 atggagacag
acacaatcct gctatgggtg ctgctgctct gggttccagg ctccactggt 61
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc
121 atctcctgca aggccagcca aagtgttgat tatgatggta atagttatat
caactggtac 181 caacagaaac caggacagcc acccaaagtc ctcatctatg
ttgcatccaa tctagaatct 241 gggatcccag ccaggtttag tggcagtggg
tctgggacag acttcaccct caacatccat 301 cctgtggagg aggaggatgc
tgcaacctat tactgtcagc aaagtattga ggatcctccc 361 acgttcggtg
ctgggaccaa gctggagctg aaac (7) 3B6 Heavy Chain Variable Region (SEQ
ID NO. 31) 1 atggaatggc cttgtatctt tctcttcctc ctgtcagtaa ctgaaggtgt
ccactcccag 61 gttcagctgc agcagtctgg ggctgaactg gtgaggcctg
ggtcctcagt gaagatttcc 121 tgcaaggctt ctggctatgt attcagtagc
tactggatga actgggtgaa gcagaggcct 181 ggacagggtc ttgagtggat
tggacagatt tatcctggag atggtgatag taactacaat 241 ggaaacttca
agggtaaagc cacactgact gcagacaaat cctccagtac agcctacatg 301
cagctcagca gcctaacatc tgaggactct gcggtctatt tctgtgcatc ccagctcggg
361 ctacgtgaga actactttga ctactggggc caaggcacca ctctcacagt ctcctcag
(8) 3B6 Kappa Light Chain Variable Region (2 possible ATG start
codons (uppercase)) (SEQ ID NO. 33) 1 ATGgacATGa ggacccctgc
tcagtttctt ggaatcttgt tgctctggtt tccaggtatc 61 aaatgtgaca
tcaagatgac ccagtctcca tcttccatgt atgcatctct aggagagaga 121
gtcacaatca cttgcaaggc gagtcaggac attaaaagct atttaagctg gttccagcag
181 aaaccaggga aatctcctaa gaccctgatc tatcgtgtaa acagattggt
agatggggtc 241 ccatcaaggt tcagtggcag tggatctggg caagattctt
ctctcaccat caccagcctg 301 gagaatgaag atatgggaat ttattattgt
ctacagtatg atgagtttcc gttcacgttc 361 ggagggggga ccaagctgga aataaagc
(9) 3D11 Heavy Chain Variable Region (SEQ ID NO. 41) 1 atggctgtcc
cggtgctgtt cctctgcctg gttgcatttc caagctgtgt cctgtcccag 61
gtacagctga aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcact
121 tgcactgtct ctgggttttc attaaccagc tatagtttac actgggttcg
ccagcctcca 181 ggaaagggtc tggaatggct gggagtaata tgggctggtg
gaaacacaaa ttataattcg 241 tctctcatgt ccagactgac catcaggaaa
gacaactcca agagccaagt tttcttaaaa 301 atgaacagtc tgcaaactga
tgacacagcc atgtactact gtgccagaga gaggtttgct 361 tactggggcc
aagggactct ggtcactgtc tctgcag (10) 3D11 Kappa Light Chain Variable
Region (SEQ ID NO. 43) 1 atggattttc aagtgcagat tttcagcttc
ctgctaatca gtgcctcagt caaaatatcc 61 agaggacaaa ttgttctcac
ccagtctcca gcaatcatgt ctgcatatcc aggggagaag 121 gtcaccatga
cctgcagtgc cagctcaagt gtaagttaca tgcactggta ccagcagaag 181
tcaggcacct cccccaaaag atggatttat gacacatcca aactggcttc tggagtccct
241 gctcgcttca gtggcagtgg gtctgggacc tcttactccc tcacaatcag
tagtatggag 301 gctgaagatg ctgccactta ttactgccag cagtggagta
gtaacccact cacgttcggt 361 gctgggacca agctggagct gaaac (11) 1D3
Heavy Chain Variable Region (SEQ ID NO. 51) 1 atgaactttg ggctcagatt
gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa 61 gtgcagctgg
tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc 121
tgtgcagcct ctggattcac tttcagtgac tattacatgt cttgggttcg ccagactcca
181 gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac
ctactatcca 241 gacagtgtga agggtcgatt caccatctcc cgagacaatg
ccaagaacac cctgtacctg 301 caaatgagca gtctgaagtc tgaggacaca
gccatatatt actgtgtgag acaaggggat 361 ggttattacg gggactatgc
tatggactac tggggtcaag gaacctcagt catcgtctcc 421 tcag (12) 1D3 Kappa
Light Chain Variable Region (SEQ ID NO. 53) 1 atgagtgtgc ccactcaggt
cctggggttg ctgctgctgt ggcttacaga tgtcagatgt 61 gacatccaga
tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc 121
atcacatgtc gaacaagtga gaatatttac agtaatttag cgtggtatca gcagaaacag
181 ggaaaatctc ctcagctcct aatctatgct gcaacaaact tagcagatgg
tgtgccatca 241 aggttcagtg gcagtggatc aggcacacag ttttccctca
ggatcaacag cctgcagtct 301 gaagattttg ggaggtatta ctgtcaacat
ttttggggga ctccgtacac gttcggaggg 361 gggaccaaac tggaaataaa ac (13)
1F3 Heavy Chain Variable Region (SEQ ID NO. 61) 1 atgaactttg
ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgag 61
gtgcagctgg tggagtctgg gggaggctta gtgcagtctg gagggtccct gaaactctcc
121 tgtgcggcct ctggattcac tttcagtaac tatttcatgt cttgggttcg
ccagactcca 181 gagaagaggc tggagtgggt cgcatatatt agtagtggtg
gtggtagcac ctactatcca 241 gacagtgtga agggtcgatt caccatctct
agagacaatg ccaagaacac cctgtacctg 301 caaatgagca gtctgaagtc
tgaggacaca gccatgtatt actgtgtaag acaaggggat 361 ggttactacg
gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 421 tcag
(14) 1F3 Kappa Light Chain Variable Region (SEQ ID NO. 63) 1
atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt
61 gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga
aactgtcacc 121 atcacatgtc gagcaagtga gaatatttac agtaatttag
catggtatca gcagaaacag 181 ggaaaatctc ctcagctcct ggtctatgat
gcaacacact taccagatgg tgtgccatca 241 aggttcagtg gcagtggatc
aggcacacag ttttccctca agatcaacag cctgcagtct 301 gaagattttg
ggagttatta ctgtcaacat ttttggggta ctccgtacac gtttggaggg 361
gggaccagac tggaaattaa ac (15) 3A12 Heavy Chain Variable Region (SEQ
ID NO. 71) 1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt
gaagtgtgaa 61 gtgcagctgg tggagtctgg gggaggctta gtgcagcctg
gagggtccct gaaaatctcc 121 tgtgcagcct ctggatttac tttcagtaac
tatttcatgt cttgggttcg ccagactcca 181 gagaagaggc tggagtgggt
cgcatacatt agtagtggtg gtggtagcac ctactatcca 241 gacagtgtga
agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 301
caaatgaaca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggagat
361 ggttactatg gggactatgc tatggactac tggggtcaag gaacctcagt
caccgtctcc 421 tcag (16) 3A12 Kappa Light Chain Variable Region
(SEQ ID NO. 73) 1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt
ggcttacaga tgccagatgt 61 gacatccaga tgactcagtc gccagcctcc
ctatctgtat ctgtgggaga aactgtcacc 121 atcacatgtc gagcaagtga
gaatatttac attaatttag catggtatca gcagaaacag 181 ggaaaatctc
ctcagctcct ggtccatgct gcaacaaagt tagcagatgg tgtgccatca 241
aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag cctgcagtct
301 gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac
gttcggaggg 361 gggaccaaac tagaaataaa ac (17) Reference Mouse IgG1
Heavy Chain Constant Region (J00453) (SEQ ID NO. 81) 1 ccaaaacgac
acccccatct gtctatccac tggcccctgg atctgctgcc caaactaact 61
ccatggtgac cctgggatgc ctggtcaagg gctatttccc tgagccagtg acagtgacct
121 ggaactctgg atccctgtcc agcggtgtgc acaccttccc agctgtcctg
gagtctgacc 181 tctacactct gagcagctca gtgactgtcc cctccagccc
tcggcccagc gagaccgtca 241 cctgcaacgt tgcccacccg gccagcagca
ccaaggtgga caagaaaatt gtgcccaggg 301 attgtggttg taagccttgc
atatgtacag tcccagaagt atcatctgtc ttcatcttcc 361 ccccaaagcc
caaggatgtg ctcaccatta ctctgactcc taaggtcacg tgtgttgtgg 421
tagacatcag caaggatgat cccgaggtcc agttcagctg gtttgtagat gatgtggagg
481 tgcacacagc tcagacgcaa ccccgggagg agcagttcaa cagcactttc
cgctcagtca 541 gtgaacttcc catcatgcac caggactggc tcaatggcaa
ggagttcaaa tgcagggtca 601 acagtgcagc tttccctgcc cccatcgaga
aaaccatctc caaaaccaaa ggcagaccga 661 aggctccaca ggtgtacacc
attccacctc ccaaggagca gatggccaag gataaagtca 721 gtctgacctg
catgataaca gacttcttcc ctgaagacat tactgtggag tggcagtgga 781
atgggcagcc agcggagaac tacaagaaca ctcagcccat catgaacacg aatggctctt
841 acttcgtcta cagcaagctc aatgtgcaga agagcaactg ggaggcagga
aatactttca 901 cctgctctgt gttacatgag ggcctgcaca accaccatac
tgagaagagc ctctcccact 961 ctcctggtaa atga (18) Mouse IgG1 Heavy
Chain Constant Region Determined for 1A3, 1D3, 1F3, and 2B8
(derived from AJ strain mice) (SEQ ID NO. 82) 1 ccaaaacgac
acccccatct gtctatccac tggcccctgg atctgctgcc caaactaact 61
ccatggtgac cctgggatgc ctggtcaagg gctatttccc tgagccagtg acagtgacct
121 ggaactctgg atccctgtcc agcggtgtgc acaccttccc agctgtcctg
cagtctgacc 181 tctacactct gagcagctca gtgactgtcc cctccagcac
ctggcccagc gagaccgtca 241 cctgcaacgt tgcccacccg gccagcagca
ccaaggtgga caagaaaatt gtgcccaggg 301 attgtggttg taagccttgc
atatgtacag tcccagaagt atcatctgtc ttcatcttcc 361 ccccaaagcc
caaggatgtg ctcaccatta ctctgactcc taaggtcacg tgtgttgtgg 421
tagacatcag caaggatgat cccgaggtcc agttcagctg gtttgtagat gatgtggagg
481 tgcacacagc tcagacgcaa ccccgggagg agcagttcaa cagcactttc
cgctcagtca 541 gtgaacttcc catcatgcac caggactggc tcaatggcaa
ggagttcaaa tgcagggtca 601 acagtgcagc tttccctgcc cccatcgaga
aaaccatctc caaaaccaaa ggcagaccga 661 aggctccaca ggtgtacacc
attccacctc ccaaggagca gatggccaag gataaagtca 721 gtctgacctg
catgataaca gacttcttcc ctgaagacat tactgtggag tggcagtgga 781
atgggcagcc agcggagaac tacaagaaca ctcagcccat catggacaca gatggctctt
841 acttcgtcta cagcaagctc aatgtgcaga agagcaactg ggaggcagga
aatactttca 901 cctgctctgt gttacatgag ggcctgcaca accaccatac
tgagaagagc ctctcccact 961 ctcctggtaa atga (19) Reference Mouse
Kappa Light Chain Constant Region (V00807) and Mouse Kappa Light
Chain Constant Region Determined for 1D3, 1F3, and 2B8 (derived
from AJ strain mice) (SEQ ID NO. 83) 1 gggctgatgc tgcaccaact
gtatccatct tcccaccatc cagtgagcag ttaacatctg 61 gaggtgcctc
agtcgtgtgc ttcttgaaca acttctaccc caaagacatc aatgtcaagt 121
ggaagattga tggcagtgaa cgacaaaatg gcgtcctgaa cagttggact gatcaggaca
181 gcaaagacag cacctacagc atgagcagca ccctcacgtt gaccaaggac
gagtatgaac 241 gacataacag ctatacctgt gaggccactc acaagacatc
aacttcaccc attgtcaaga 301 gcttcaacag gaatgagtgt tag (20) Mouse
Kappa Light Chain Constant Region Determined for 1A3 containing one
altered nucleotide compared to 1D3, 1F3, and 2B8 (underlined) (SEQ
ID NO. 84) 1 gggctgatgc tgcaccaact gtatccatct tcccaccatc cagtgagcag
ttaacatctg 61 gaggtgcctc agtcgtgtgc ttcttgaaca acttctaccc
caaagacatc aatgtcaagt 121 ggaagattga tggcagtgaa cgacaaaatg
gcgtcctgaa cagttggact gatcaggaca 181 gcaaagacag cacctacagc
atgagcagca ccctcatgtt gaccaaggac gagtatgaac 241 gacataacag
ctatacctgt gaggccactc acaagacatc aacttcaccc attgtcaaga 301
gcttcaacag gaatgagtgt tag
[0130] Each of the amino acid sequences defining the immunoglobulin
heavy chain variable regions for the antibodies produced in Example
1 are set forth in FIG. 2. Each of the sequences are aligned with
one another and the sequences defining the signal peptide,
CDR.sub.1, CDR.sub.2 and CDR.sub.3 are identified by boxes. FIG. 3
shows an alignment of the separate CDR.sub.1, CDR.sub.2 and
CDR.sub.3 sequences for each of the antibodies.
[0131] Each of the amino acid sequences defining the immunoglobulin
light chain variable regions for each of the antibodies produced in
Example 1 are set forth in FIG. 4. Each of the sequences are
aligned with one another and the sequences defining the signal
peptide, CDR.sub.1, CDR.sub.2 and CDR.sub.3 are identified by
boxes. FIG. 5 shows an alignment of the separate CDR.sub.1,
CDR.sub.2 and CDR.sub.3 sequences for each of the antibodies.
[0132] For convenience, Table 1 provides a concordance chart
showing the correspondence between the antibody sequences discussed
in this Example with those presented in the Sequence Listing.
TABLE-US-00002 TABLE 1 SEQ. ID NO. Protein or Nucleic Acid 1 Heavy
Chain Variable Region 1A3 - nucleic acid 2 Heavy Chain Variable
Region 1A3 - protein 3 Light (kappa) Chain Variable Region 1A3 -
nucleic acid 4 Light (kappa) Chain Variable Region 1A3 - protein 5
Heavy Chain CDR.sub.1 1A3 6 Heavy Chain CDR.sub.2 1A3 7 Heavy Chain
CDR.sub.3 1A3 8 Light (kappa) Chain CDR.sub.1 1A3 9 Light (kappa)
Chain CDR.sub.2 1A3 10 Light (kappa) Chain CDR.sub.3 1A3 11 Heavy
Chain Variable Region 2B8 - nucleic acid 12 Heavy Chain Variable
Region 2B8 - protein 13 Light (kappa) Chain Variable Region 2B8 -
nucleic acid 14 Light (kappa) Chain Variable Region 2B8 - protein
15 Heavy Chain CDR.sub.1 2B8 16 Heavy Chain CDR.sub.2 2B8 17 Heavy
Chain CDR.sub.3 2B8 18 Light (kappa) Chain CDR.sub.1 2B8 19 Light
(kappa) Chain CDR.sub.2 2B8 20 Light (kappa) Chain CDR.sub.3 2B8 21
Heavy Chain Variable Region 2F8 - nucleic acid 22 Heavy Chain
Variable Region 2F8 - protein 23 Light (kappa) Chain Variable
Region 2F8 - nucleic acid 24 Light (kappa) Chain Variable Region
2F8 - protein 25 Heavy Chain CDR.sub.1 2F8 26 Heavy Chain CDR.sub.2
2F8 27 Heavy Chain CDR.sub.3 2F8 28 Light (kappa) Chain CDR.sub.1
2F8 29 Light (kappa) Chain CDR.sub.2 2F8 30 Light (kappa) Chain
CDR.sub.3 2F8 31 Heavy Chain Variable Region 3B6 - nucleic acid 32
Heavy Chain Variable Region 3B6 - protein 33 Light (kappa) Chain
Variable Region 3B6 - nucleic acid 34 Light (kappa) Chain Variable
Region 3B6 - protein 35 Heavy Chain CDR.sub.1 3B6 36 Heavy Chain
CDR.sub.2 3B6 37 Heavy Chain CDR.sub.3 3B6 38 Light (kappa) Chain
CDR.sub.1 3B6 39 Light (kappa) Chain CDR.sub.2 3B6 40 Light (kappa)
Chain CDR.sub.3 3B6 41 Heavy Chain Variable Region 3D11 - nucleic
acid 42 Heavy Chain Variable Region 3D11 - protein 43 Light (kappa)
Chain Variable Region 3D11 - nucleic acid 44 Light (kappa) Chain
Variable Region 3D11 - protein 45 Heavy Chain CDR.sub.1 3D11 46
Heavy Chain CDR.sub.2 3D11 47 Heavy Chain CDR.sub.3 3D11 48 Light
(kappa) Chain CDR.sub.1 3D11 49 Light (kappa) Chain CDR.sub.2 3D11
50 Light (kappa) Chain CDR.sub.3 3D11 51 Heavy Chain Variable
Region 1D3 - nucleic acid 52 Heavy Chain Variable Region 1D3 -
protein 53 Light (kappa) Chain Variable Region 1D3 - nucleic acid
54 Light (kappa) Chain Variable Region 1D3 - protein 55 Heavy Chain
CDR.sub.1 1D3 56 Heavy Chain CDR.sub.2 1D3 57 Heavy Chain CDR.sub.3
1D3 58 Light (kappa) Chain CDR.sub.1 1D3 59 Light (kappa) Chain
CDR.sub.2 1D3 60 Light (kappa) Chain CDR.sub.3 1D3 61 Heavy Chain
Variable Region 1F3 - nucleic acid 62 Heavy Chain Variable Region
1F3 - protein 63 Light (kappa) Chain Variable Region 1F3 - nucleic
acid 64 Light (kappa) Chain Variable Region 1F3 - protein 65 Heavy
Chain CDR.sub.1 1F3 66 Heavy Chain CDR.sub.2 1F3 67 Heavy Chain
CDR.sub.3 1F3 68 Light (kappa) Chain CDR.sub.1 1F3 69 Light (kappa)
Chain CDR.sub.2 1F3 70 Light (kappa) Chain CDR.sub.3 1F3 71 Heavy
Chain Variable Region 3A12 - nucleic acid 72 Heavy Chain Variable
Region 3A12 - protein 73 Light (kappa) Chain Variable Region 3A12 -
nucleic acid 74 Light (kappa) Chain Variable Region 3A12 - protein
75 Heavy Chain CDR.sub.1 3A12 76 Heavy Chain CDR.sub.2 3A12 77
Heavy Chain CDR.sub.3 3A12 78 Light (kappa) Chain CDR.sub.1 3A12 79
Light (kappa) Chain CDR.sub.2 3A12 80 Light (kappa) Chain CDR.sub.3
3A12
[0133] Also, for convenience, the following sequences represent the
actual or contemplated full length heavy and light chain sequences
(i.e., containing both the variable and constant region sequences)
for each of the antibodies described in this Example. It is noted
that the constant regions of the murine antibodies 2F8, 3A12, 3B6,
and 3D11 were not sequenced but are presumed to have the same
constant region sequences as the 1D3, 1F3, and 2B8 antibodies,
which were sequenced, as they were all derived from AJ strain mice.
It is appreciated, however, that the variable region sequences
described herein can be ligated to each of a number of other
constant region sequences known to those skilled in the art to
produce active full length immunoglobulin heavy and light
chains.
TABLE-US-00003 (1) Nucleic Acid Sequence Encoding the Full Length
1A3 Heavy Chain Sequence (1A3 Heavy Chain Variable Region and IgG1
Constant Region) (signal sequence underlined) (SEQ ID NO. 122) 1
atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa
61 gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct
gaaactctcc 121 tgtgcagcct ctgaattcac tttcagtaac tattacatgt
cttgggttcg ccagactcca 181 gagaagaggc tgcagtgggt cgcatacatt
agtcctggtg gtggtagctc ctactatcca 241 gccagtgtga agggtcgatt
caccatctcc agagacaatg ccaagaacac cctgtacctg 301 caaatgagca
gtctgaagtc tgaggacaca gccatgtatt actgtgcaag acaaggggat 361
ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc
421 tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc
tgcccaaact 481 aactccatgg tgaccctggg atgcctggtc aagggctatt
tccctgagcc agtgacagtg 541 acctggaact ctggatccct gtccagcggt
gtgcacacct tcccagctgt cctgcagtct 601 gacctctaca ctctgagcag
ctcagtgact gtcccctcca gcacctggcc cagcgagacc 661 gtcacctgca
acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc 721
agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc
781 ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt
cacgtgtgtt 841 gtggtagaca tcagcaagga tgatcccgag gtccagttca
gctggtttgt agatgatgtg 901 gaggtgcaca cagctcagac gcaaccccgg
gaggagcagt tcaacagcac tttccgctca 961 gtcagtgaac ttcccatcat
gcaccaggac tggctcaatg gcaaggagtt caaatgcagg 1021 gtcaacagtg
cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga 1081
ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa
1141 gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt
ggagtggcag 1201 tggaatgggc agccagcgga gaactacaag aacactcagc
ccatcatgga cacagatggc 1261 tcttacttcg tctacagcaa gctcaatgtg
cagaagagca actgggaggc aggaaatact 1321 ttcacctgct ctgtgttaca
tgagggcctg cacaaccacc atactgagaa gagcctctcc 1381 cactctcctg
gtaaatga (2) Protein Sequence Defining the Full Length 1A3 Heavy
Chain Sequence (1A3 Heavy Chain Variable Region and IgG1 Constant
Region) (without signal sequence) (SEQ ID NO. 123) 1 evqlvesggg
lvqpggslkl scaaseftfs nyymswvrqt pekrlqwvay ispgggssyy 61
pasvkgrfti srdnakntly lqmsslksed tamyycarqg dgyygdyamd ywgqgtsvtv
121 ssakttppsv yplapgsaaq tnsmvtlgcl vkgyfpepvt vtwnsgslss
gvhtfpavlq 181 sdlytlsssv tvpsstwpse tvtcnvahpa sstkvdkkiv
prdcgckpci ctvpevssvf 241 ifppkpkdvl titltpkvtc vvvdiskddp
evqfswfvdd vevhtaqtqp reeqfnstfr 301 syselpimhq dwlngkefkc
rvnsaafpap iektisktkg rpkapqvyti pppkeqmakd 361 kvsltcmitd
ffpeditvew qwngqpaeny kntqpimdtd gsyfvyskln vqksnweagn 421
tftcsvlheg lhnhhteksl shspgk (3) Nucleic Acid Sequence Encoding the
Full Length 1A3 Light Chain Sequence (1A3 Kappa Variable Region and
Constant Region) (signal sequence underlined) (SEQ ID NO. 124) 1
atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt
61 gacatccaga tgactcagtc tccagcctcc ctatctgttt ctgtgggaga
aactgtcacc 121 atcacatgtc gagcaagtga gaatatttat agtaatttag
catggtatca gcagaaacag 181 ggaaaatctc ctcagctcct ggtctatgct
gcaacaaact tagcagatgg tgtgccatca 241 aggttcagtg gcagtggatc
aggcacacag ttttccctca agatcaacag cctgcagtct 301 gaagattttg
ggacttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg 361
gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca
421 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa
caacttctac 481 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg
aacgacaaaa tggcgtcctg 541 aacagttgga ctgatcagga cagcaaagac
agcacctaca gcatgagcag caccctcatg 601 ttgaccaagg acgagtatga
acgacataac agctatacct gtgaggccac tcacaagaca 661 tcaacttcac
ccattgtcaa gagcttcaac aggaatgagt gttag Kappa Variable Region and
Constant Region) (without signal sequence) (SEQ ID NO. 125) 1
diqmtqspas lsysvgetvt itcraseniy snlawyqqkq gkspqllvya atnladgvps
61 rfsgsgsgtq fslkinslqs edfgtyycqh fwgtpytfgg gtkleikrad
aaptvsifpp 121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl
nswtdqdskd stysmsstlm 181 ltkdeyerhn sytceathkt stspivksfn rnec (5)
Nucleic Acid Sequence Encoding the Full Length 2B8 Heavy Chain
Sequence (2B8 Heavy Chain Variable Region and IgG1 Constant Region)
(signal sequence underlined) (SEQ ID NO. 126) 1 atgggatgga
gctatatcat cctctttttg gtagcaacag ctacagatgt ccactcccag 61
gtccaactgc agcagcctgg ggctgaactg gtgaagcctg ggacttcagt gaagctgtcc
121 tgcaaggctt ctggctacac cttcaccacc tactggatgc actgggtgaa
tcagaggcct 181 ggacaaggcc ttgagtggat tggagagatt aatcctacca
acggtcatac taactacaat 241 gagaagttca agagcaaggc cacactgact
gtagacaaat cctccagcac agcctacatg 301 caactcagca gcctgacatc
tgaggactct gcggtctatt actgtgcaag aaactatgtt 361 ggtagcatct
ttgactactg gggccaaggc accactctca cagtctcctc agccaaaacg 421
acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg
481 accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac
ctggaactct 541 ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc
tgcagtctga cctctacact 601 ctgagcagct cagtgactgt cccctccagc
acctggccca gcgagaccgt cacctgcaac 661 gttgcccacc cggccagcag
caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt 721 tgtaagcctt
gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag 781
cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc
841 agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga
ggtgcacaca 901 gctcagacgc aaccccggga ggagcagttc aacagcactt
tccgctcagt cagtgaactt 961 cccatcatgc accaggactg gctcaatggc
aaggagttca aatgcagggt caacagtgca 1021 gctttccctg cccccatcga
gaaaaccatc tccaaaacca aaggcagacc gaaggctcca 1081 caggtgtaca
ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc 1141
tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag
1201 ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc
ttacttcgtc 1261 tacagcaagc tcaatgtgca gaagagcaac tgggaggcag
gaaatacttt cacctgctct 1321 gtgttacatg agggcctgca caaccaccat
actgagaaga gcctctccca ctctcctggt 1381 aaatga (6) Protein Sequence
Defining the Full Length 2B8 Heavy Chain Sequence (2B8 Heavy Chain
Variable Region and IgG1 Constant Region) (without signal sequence)
(SEQ ID NO. 127) 1 qvqlqqpgae lvkpgtsvkl sckasgytft tywmhwvnqr
pgqglewige inptnghtny 61 nekfkskatl tvdkssstay mqlssltsed
savyycarny vgsifdywgq gttltvssak 121 ttppsvypla pgsaaqtnsm
vtlgclvkgy fpepvtvtwn sgslssgvht fpavlqsdly 181 tlsssvtvps
stwpsetvtc nvahpasstk vdkkivprdc gckpcictvp evssvfifpp 241
kpkdvltitl tpkvtcvvvd iskddpevqf swfvddvevh taqtqpreeq fnstfrsvse
301 lpimhqdwln gkefkcrvns aafpapiekt isktkgrpka pqvytipppk
eqmakdkvsl 361 tcmitdffpe ditvewqwng qpaenykntq pimdtdgsyf
vysklnvqks nweagntftc 421 svlheglhnh htekslshsp gk (7) Nucleic Acid
Sequence Encoding the Full Length 2B8 Light Chain Sequence (2B8
Kappa Variable Region and Constant Region) (signal sequence
underlined) (SEQ ID NO. 128) 1 atggaatcac agactctggt cttcatatcc
atactgctct ggttatatgg tgctgatggg 61 aacattgtaa tgacccaatc
tcccaaatcc atgtccatgt cagtaggaga gagggtcacc 121 ttgagctgca
aggccagtga gaatgtggtt tcttatgtat cctggtatca acagaaacca 181
gcgcagtctc ctaaactgct gatatacggg gcatccaacc ggaacactgg ggtccccgat
241 cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag
tgtgcgggct 301 gaagaccttg cagattatca ctgtgggcag agttacaact
atccgtacac gttcggaggg 361 gggaccaggc tggaaataaa acgggctgat
gctgcaccaa ctgtatccat cttcccacca 421 tccagtgagc agttaacatc
tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 481 cccaaagaca
tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 541
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg
601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac
tcacaagaca 661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag
(8) Protein Sequence Defining the Full Length 2B8 Light Chain
Sequence (2B8 Kappa Variable Region and Constant Region) (without
signal sequence) (SEQ ID NO. 129) 1 nivmtqspks msmsvgervt
lsckasenvv syvswyqqkp aqspklliyg asnrntgvpd 61 rftgsgsatd
ftltissvra edladyhcgq synypytfgg gtrleikrad aaptvsifpp 121
sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt
181 ltkdeyerhn sytceathkt stspivksfn rnec (9) Nucleic Acid Sequence
Encoding the Full Length 2F8 Heavy Chain Sequence (2F8 Heavy Chain
Variable Region and IgG1 Constant Region) (signal sequence
underlined) (SEQ ID NO. 130) 1 atggaatgga gctgggtctt tctcttcctc
ctgtcagtaa ctgcaggtgt ccactgccag 61 gtccagctga agcagtctgg
agctgagctg gtgaggcctg ggacttcagt gaagatgtcc 121 tgcaaggctt
ctggctacac cttcactacc tactatatac actgggtgaa tcagaggcct 181
ggacagggcc ttgagtggat tggaaagatt ggtcctggaa gtggtagtac ttactacaat
241 gagatgttca aagacaaggc cacattgact gtagacacat cctccagcac
agcctacatg 301 cagctcagca gcctgacatc tgacgactct gcggtctatt
tctgtgcaag aaggggactg 361 ggacgtggct ttgactactg gggccaaggc
accactctca cagtctcctc agccaaaacg 421 acacccccat ctgtctatcc
actggcccct ggatctgctg cccaaactaa ctccatggtg 481 accctgggat
gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct
541 ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga
cctctacact 601 ctgagcagct cagtgactgt cccctccagc acctggccca
gcgagaccgt cacctgcaac 661 gttgcccacc cggccagcag caccaaggtg
gacaagaaaa ttgtgcccag ggattgtggt 721 tgtaagcctt gcatatgtac
agtcccagaa gtatcatctg tcttcatctt ccccccaaag 781 cccaaggatg
tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc 841
agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca
901 gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt
cagtgaactt 961 cccatcatgc accaggactg gctcaatggc aaggagttca
aatgcagggt caacagtgca 1021 gctttccctg cccccatcga gaaaaccatc
tccaaaacca aaggcagacc gaaggctcca 1081 caggtgtaca ccattccacc
tcccaaggag cagatggcca aggataaagt cagtdtgacc 1141 tgcatgataa
cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag 1201
ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc
1261 tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt
cacctgctct 1321 gtgttacatg agggcctgca caaccaccat actgagaaga
gcctctccca ctctcctggt 1381 aaatga (10) Protein Sequence Defining
the Full Length 2F8 Heavy Chain Sequence (2F8 Heavy Chain Variable
Region and IgG1 Constant Region) (without signal sequence) (SEQ ID
NO. 131) 1 qvqlkqsgae lvrpgtsvkm sckasgytft tyyihwvnqr pgqglewigk
igpgsgstyy 61 nemfkdkatl tvdtssstay mqlssltsdd savyfcarrg
lgrgfdywgq gttltvssak 121 ttppsvypla pgsaaqtnsm vtlgclvkgy
fpepvtvtwn sgslssgvht fpavlqsdly 181 tlsssvtvps stwpsetvtc
nvahpasstk vdkkivprdc gckpcictvp evssvfifpp 241 kpkdvltitl
tpkvtcvvvd iskddpevqf swfvddvevh taqtqpreeq fnstfrsyse 301
lpimhqdwln gkefkcrvns aafpapiekt isktkgrpka pqvytipppk eqmakdkvsl
361 tcmitdffpe ditvewqwng qpaenykntq pimdtdgsyf vysklnvqks
nweagntftc 421 svlheglhnh htekslshsp gk (11) Nucleic Acid Sequence
Encoding the Full Length 2F8 Light Chain Sequence (2F8 Kappa
Variable Region and Constant Region) (signal sequence underlined)
(SEQ ID NO. 132) 1 atggagacag acacaatcct gctatgggtg ctgctgctct
gggttccagg ctccactggt 61 gacattgtgc tgacccaatc tccagcttct
ttggctgtgt ctctagggca gagggccacc 121 atctcctgca aggccagcca
aagtgttgat tatgatggta atagttatat caactggtac 181 caacagaaac
caggacagcc acccaaagtc ctcatctatg ttgcatccaa tctagaatct 241
gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat
301 cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtattga
ggatcctccc 361 acgttcggtg ctgggaccaa gctggagctg aaacgggctg
atgctgcacc aactgtatcc 421 atcttcccac catccagtga gcagttaaca
tctggaggtg cctcagtcgt gtgcttcttg 481 aacaacttct accccaaaga
catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 541 aatggcgtcc
tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 601
agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc
661 actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgttag
(12) Protein Sequence Defining the Full Length 2F8 Light Chain
Sequence (2F8 Kappa Variable Region and Constant Region) (without
signal sequence) (SEQ ID NO. 133) 1 divltqspas lavslgqrat
isckasqsvd ydgnsyinwy qqkpgqppkv liyvasnles 61 giparfsgsg
sgtdftlnih pveeedaaty ycqqsiedpp tfgagtklel kradaaptvs 121
ifppsseqlt sggasvvcfl nnfypkdinv kwkidgserq ngvlnswtdq dskdstysms
181 stltltkdey erhnsytcea thktstspiv ksfnrnec (13) Nucleic Acid
Sequence Encoding the Full Length 3B6 Heavy Chain Sequence f3B6
Heavy Chain Variable Region and IgG1 Constant Region) (signal
sequence underlined) (SEQ ID NO. 134) 1 atggaatggc cttgtatctt
tctcttcctc ctgtcagtaa ctgaaggtgt ccactcccag 61 gttcagctgc
agcagtctgg ggctgaactg gtgaggcctg ggtcctcagt gaagatttcc 121
tgcaaggctt ctggctatgt attcagtagc tactggatga actgggtgaa gcagaggcct
181 ggacagggtc ttgagtggat tggacagatt tatcctggag atggtgatag
taactacaat 241 ggaaacttca agggtaaagc cacactgact gcagacaaat
cctccagtac agcctacatg 301 cagctcagca gcctaacatc tgaggactct
gcggtctatt tctgtgcatc ccagctcggg 361 ctacgtgaga actactttga
ctactggggc caaggcacca ctctcacagt ctcctcagcc 421 aaaacgacac
ccccatctgt ctatccactg gcccctggat ctgctgccca aactaactcc 481
atggtgaccc tgggatgcct ggtcaagggc tatttccctg agccagtgac agtgacctgg
541 aactctggat ccctgtccag cggtgtgcac accttcccag ctgtcctgca
gtctgacctc 601 tacactctga gcagctcagt gactgtcccc tccagcacct
ggcccagcga gaccgtcacc 661 tgcaacgttg cccacccggc cagcagcacc
aaggtggaca agaaaattgt gcccagggat 721 tgtggttgta agccttgcat
atgtacagtc ccagaagtat catctgtctt catcttcccc 781 ccaaagccca
aggatgtgct caccattact ctgactccta aggtcacgtg tgttgtggta 841
gacatcagca aggatgatcc cgaggtccag ttcagctggt ttgtagatga tgtggaggtg
901 cacacagctc agacgcaacc ccgggaggag cagttcaaca gcactttccg
ctcagtcagt 961 gaacttccca tcatgcacca ggactggctc aatggcaagg
agttcaaatg cagggtcaac 1021 agtgcagctt tccctgcccc catcgagaaa
accatctcca aaaccaaagg cagaccgaag 1081 gctccacagg tgtacaccat
tccacctccc aaggagcaga tggccaagga taaagtcagt 1141 ctgacctgca
tgataacaga cttcttccct gaagacatta ctgtggagtg gcagtggaat 1201
gggcagccag cggagaacta caagaacact cagcccatca tggacacaga tggctcttac
1261 ttcgtctaca gcaagctcaa tgtgcagaag agcaactggg aggcaggaaa
tactttcacc 1321 tgctctgtgt tacatgaggg cctgcacaac caccatactg
agaagagcct ctcccactct 1381 cctggtaaat ga (14) Protein Sequence
Defining the Full Length 3B6 Heavy Chain Sequence (3B6 Heavy Chain
Variable Region and IgG1 Constant Region) (without signal sequence)
(SEQ ID NO. 135) 1 qvqlqqsgae lvrpgssvki sckasgyvfs sywmnwvkqr
pgqglewigq iypgdgdsny 61 ngnfkgkatl tadkssstay mqlssltsed
savyfcasql glrenyfdyw gqgttltvss 121 akttppsvyp lapgsaaqtn
smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd 181 lytlsssvtv
psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif 241
ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv
301 selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp
pkeqmakdkv 361 sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs
yfvysklnvq ksnweagntf 421 tcsvlheglh nhhtekslsh spgk (15) Nucleic
Acid Sequence Encoding the Full Length 3B6 Light Chain Sequence
(3B6 Kappa Variable Region and Constant Region) (signal sequence
underlined) (SEQ ID NO. 136) 1 ATGgacATGa ggacccctgc tcagtttctt
ggaatcttgt tgctctggtt tccaggtatc 61 aaatgtgaca tcaagatgac
ccagtctcca tcttccatgt atgcatctct aggagagaga 121 gtcacaatca
cttgcaaggc gagtcaggac attaaaagct atttaagctg gttccagcag 181
aaaccaggga aatctcctaa gaccctgatc tatcgtgtaa acagattggt agatggggtc
241 ccatcaaggt tcagtggcag tggatctggg caagattctt ctctcaccat
caccagcctg 301 gagaatgaag atatgggaat ttattattgt ctacagtatg
atgagtttcc gttcacgttc 361 ggagggggga ccaagctgga aataaagcgg
gctgatgctg caccaactgt atccatcttc 421 ccaccatcca gtgagcagtt
aacatctgga ggtgcctcag tcgtgtgctt cttgaacaac 481 ttctacccca
aagacatcaa tgtcaagtgg aagattgatg gcagtgaacg acaaaatggc 541
gtcctgaaca gttggactga tcaggacagc aaagacagca cctacagcat gagcagcacc
601 ctcacgttga ccaaggacga gtatgaacga cataacagct atacctgtga
ggccactcac 661 aagacatcaa cttcacccat tgtcaagagc ttcaacagga
atgagtgtta g (16) Protein Sequence Defining the Full Length 3B6
Light Chain Sequence (3B6 Kappa Variable Region and Constant
Region) (without signal sequence) (SEQ ID NO. 137) 1 dikmtqspss
myaslgervt itckasqdik sylswfqqkp gkspktliyr vnrlvdgvps 61
rfsgsgsgqd ssltitslen edmgiyyclq ydefpftfgg gtkleikrad aaptvsifpp
121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd
stysmsstlt 181 ltkdeyerhn sytceathkt stspivksfn rnec (17) Nucleic
Acid Sequence Encoding the Full Length 3D11 Heavy Chain Sequence
(3D11 Heavy Chain Variable Region and IgG1 Constant Region) (signal
sequence underlined) (SEQ ID NO. 138) 1 atggctgtcc cggtgctgtt
cctctgcctg gttgcatttc caagctgtgt cctgtcccag 61 gtacagctga
aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcact 121
tgcactgtct ctgggttttc attaaccagc tatagtttac actgggttcg ccagcctcca
181 ggaaagggtc tggaatggct gggagtaata tgggctggtg gaaacacaaa
ttataattcg 241 tctctcatgt ccagactgac catcaggaaa gacaactcca
agagccaagt tttcttaaaa 301 atgaacagtc tgcaaactga tgacacagcc
atgtactact gtgccagaga gaggtttgct 361 tactggggcc aagggactct
ggtcactgtc tctgcagcca aaacgacacc cccatctgtc 421 tatccactgg
cccctggatc tgctgcccaa actaactcca tggtgaccct gggatgcctg 481
gtcaagggct atttccctga gccagtgaca gtgacctgga actctggatc cctgtccagc
541 ggtgtgcaca ccttcccagc tgtcctgcag tctgacctct acactctgag
cagctcagtg 601 actgtcccct ccagcacctg gcccagcgag accgtcacct
gcaacgttgc ccacccggcc 661 agcagcacca aggtggacaa gaaaattgtg
cccagggatt gtggttgtaa gccttgcata 721 tgtacagtcc cagaagtatc
atctgtcttc atcttccccc caaagcccaa ggatgtgctc 781 accattactc
tgactcctaa ggtcacgtgt gttgtggtag acatcagcaa ggatgatccc 841
gaggtccagt tcagctggtt tgtagatgat gtggaggtgc acacagctca gacgcaaccc
901 cgggaggagc agttcaacag cactttccgc tcagtcagtg aacttcccat
catgcaccag 961 gactggctca atggcaagga gttcaaatgc agggtcaaca
gtgcagcttt ccctgccccc 1021 atcgagaaaa ccatctccaa aaccaaaggc
agaccgaagg ctccacaggt gtacaccatt 1081 ccacctccca aggagcagat
ggccaaggat aaagtcagtc tgacctgcat gataacagac
1141 ttcttccctg aagacattac tgtggagtgg cagtggaatg ggcagccagc
ggagaactac 1201 aagaacactc agcccatcat ggacacagat ggctcttact
tcgtctacag caagctcaat 1261 gtgcagaaga gcaactggga ggcaggaaat
actttcacct gctctgtgtt acatgagggc 1321 ctgcacaacc accatactga
gaagagcctc tcccactctc ctggtaaatg a (18) Protein Sequence Defining
the Full Length 3D11 Heavy Chain Sequence (3D11 Heavy Chain
Variable Region and IgG1 Constant Region) (without signal sequence)
(SEQ ID NO. 139) 1 qvqlkesgpg lvapsqslsi tctvsgfslt syslhwvrqp
pgkglewlgv iwaggntnyn 61 sslmsrltir kdnsksqvfl kmnslqtddt
amyycarerf aywgqgtivt vsaakttpps 121 vyplapgsaa qtnsmvtlgc
lvkgyfpepv tvtwnsgsls sgvhtfpavl qsdlytlsss 181 vtvpsstwps
etvtcnvahp asstkvdkki vprdcgckpc ictvpevssv fifppkpkdv 241
ltitltpkvt cvvvdiskdd pevqfswfvd dvevhtaqtq preeqinstf rsyselpimh
301 qdwlngkefk crvnsaafpa piektisktk grpkapqvyt ipppkeqmak
dkvsltcmit 361 dffpeditve wqwngqpaen ykntqpimdt dgsyfvyskl
nvqksnweag ntftcsvlhe 421 glhnhhteks lshspgk (19) Nucleic Acid
Sequence Encoding the Full Length 3D11 Light Chain Sequence (3D11
Kappa Variable Region and Constant Region) (signal sequence
underlined) (SEQ ID NO. 140) 1 atggattttc aagtgcagat tttcagcttc
ctgctaatca gtgcctcagt caaaatatcc 61 agaggacaaa ttgttctcac
ccagtctcca gcaatcatgt ctgcatatcc aggggagaag 121 gtcaccatga
cctgcagtgc cagctcaagt gtaagttaca tgcactggta ccagcagaag 181
tcaggcacct cccccaaaag atggatttat gacacatcca aactggcttc tggagtccct
241 gctcgcttca gtggcagtgg gtctgggacc tcttactccc tcacaatcag
tagtatggag 301 gctgaagatg ctgccactta ttactgccag cagtggagta
gtaacccact cacgttcggt 361 gctgggacca agctggagct gaaacgggct
gatgctgcac caactgtatc catcttccca 421 ccatccagtg agcagttaac
atctggaggt gcctcagtcg tgtgcttctt gaacaacttc 481 taccccaaag
acatcaatgt caagtggaag attgatggca gtgaacgaca aaatggcgtc 541
ctgaacagtt ggactgatca ggacagcaaa gacagcacct acagcatgag cagcaccctc
601 acgttgacca aggacgagta tgaacgacat aacagctata cctgtgaggc
cactcacaag 661 acatcaactt cacccattgt caagagcttc aacaggaatg agtgttag
(20) Protein Sequence Defining the Full Length 3D11 Light Chain
Sequence (3D11 Kappa Variable Region and Constant Region) (without
signal sequence) (SEQ ID NO. 141) 1 qivltqspai msaypgekvt
mtcsasssys ymhwyqqksg tspkrwiydt sklasgvpar 61 fsgsgsgtsy
sltissmeae daatyycqqw ssnpltfgag tklelkrada aptvsifpps 121
seqltsggas vvcflnnfyp kdinvkwkid gserqngvin swtdqdskds tysmsstltl
181 tkdeyerhns ytceathkts tspivksfnr nec (21) Nucleic Acid Sequence
Encoding the Full Length 1D3 Heavy Chain Sequence (1D3 Heavy Chain
Variable Region and IgG1 Constant Region) (signal sequence
underlined) (SEQ ID NO. 142) 1 atgaactttg ggctcagatt gattttcctt
gtccttgttt taaaaggtgt gaagtgtgaa 61 gtgcagctgg tggagtctgg
gggaggctta gtgcagcctg gagggtccct gaaactctcc 121 tgtgcagcct
ctggattcac tttcagtgac tattacatgt cttgggttcg ccagactcca 181
gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca
241 gacagtgtga agggtcgatt caccatctcc cgagacaatg ccaagaacac
cctgtacctg 301 caaatgagca gtctgaagtc tgaggacaca gccatatatt
actgtgtgag acaaggggat 361 ggttattacg gggactatgc tatggactac
tggggtcaag gaacctcagt catcgtctcc 421 tcagccaaaa cgacaccccc
atctgtctat ccactggccc ctggatctgc tgcccaaact 481 aactccatgg
tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg 541
acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct
601 gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc
cagcgagacc 661 gtcacctgca acgttgccca cccggccagc agcaccaagg
tggacaagaa aattgtgccc 721 agggattgtg gttgtaagcc ttgcatatgt
acagtcccag aagtatcatc tgtcttcatc 781 ttccccccaa agcccaagga
tgtgctcacc attactctga ctcctaaggt cacgtgtgtt 841 gtggtagaca
tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg 901
gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca
961 gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt
caaatgcagg 1021 gtcaacagtg cagctttccc tgcccccatc gagaaaacca
tctccaaaac caaaggcaga 1081 ccgaaggctc cacaggtgta caccattcca
cctcccaagg agcagatggc caaggataaa 1141 gtcagtctga cctgcatgat
aacagacttc ttccctgaag acattactgt ggagtggcag 1201 tggaatgggc
agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc 1261
tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact
1321 ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa
gagcctctcc 1381 cactctcctg gtaaatga (22) Protein Sequence Defining
the Full Length 1D3 Heavy chain sequence (1D3 Heavy Chain Variable
Region and IgG1 Constant Region) (without signal sequence) (SEQ ID
NO. 143) 1 evqlvesggg lvqpggslkl scaasgftfs dyymswvrqt pekrlewvay
issgggstyy 61 pdsvkgrfti srdnakntly lqmsslksed taiyycvrqg
dgyygdyamd ywgqgtsviv 121 ssakttppsv yplapgsaaq tnsmvtlgcl
vkgyfpepvt vtwnsgslss gvhtfpavlq 181 sdlytlsssv tvpsstwpse
tvtcnvahpa sstkvdkkiv prdcgckpci ctvpevssvf 241 ifppkpkdvl
titltpkvtc vvvdiskddp evqfswfvdd vevhtaqtqp reeqfnstfr 301
syselpimhq dwlngkefkc rvnsaafpap iektisktkg rpkapqyyti pppkeqmakd
361 kvsltcmitd ffpeditvew qwngqpaeny kntqpimdtd gsyfvyskln
vqksnweagn 421 tftcsvlheg lhnhhteksl shspgk (23) Nucleic Acid
Sequence Encoding the Full Length 1D3 Light Chain Sequence (1D3
Kappa Variable Region and Constant Region) (signal sequence
underlined) (SEQ ID NO. 144) 1 atgagtgtgc ccactcaggt cctggggttg
ctgctgctgt ggcttacaga tgtcagatgt 61 gacatccaga tgactcagtc
tccagcctcc ctatctgtat ctgtgggaga aactgtcacc 121 atcacatgtc
gaacaagtga gaatatttac agtaatttag cgtggtatca gcagaaacag 181
ggaaaatctc ctcagctcct aatctatgct gcaacaaact tagcagatgg tgtgccatca
241 aggttcagtg gcagtggatc aggcacacag ttttccctca ggatcaacag
cctgcagtct 301 gaagattttg ggaggtatta ctgtcaacat ttttggggga
ctccgtacac gttcggaggg 361 gggaccaaac tggaaataaa acgggctgat
gctgcaccaa ctgtatccat cttcccacca 421 tccagtgagc agttaacatc
tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 481 cccaaagaca
tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 541
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg
601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac
tcacaagaca 661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag
Kappa Variable Region and Constant Region) (without signal
sequence) (SEQ ID NO. 145) 1 diqmtqspas lsysvgetvt itcrtseniy
snlawyqqkq gkspqlliya atnladgvps 61 rfsgsgsgtq fslrinslqs
edfgryycqh fwgtpytfgg gtkleikrad aaptvsifpp 121 sseqltsgga
svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt 181
ltkdeyerhn sytceathkt stspivksfn rnec (25) Nucleic Acid Sequence
Encoding the Full Length 1F3 Heavy Chain Sequence (1F3 Heavy Chain
Variable Region and IgG1 Constant Region) (signal sequence
underlined) (SEQ ID NO. 146) 1 atgaactttg ggctcagatt gattttcctt
gtccttgttt taaaaggtgt gaagtgtgag 61 gtgcagctgg tggagtctgg
gggaggctta gtgcagtctg gagggtccct gaaactctcc 121 tgtgcggcct
ctggattcac tttcagtaac tatttcatgt cttgggttcg ccagactcca 181
gagaagaggc tggagtgggt cgcatatatt agtagtggtg gtggtagcac ctactatcca
241 gacagtgtga agggtcgatt caccatctct agagacaatg ccaagaacac
cctgtacctg 301 caaatgagca gtctgaagtc tgaggacaca gccatgtatt
actgtgtaag acaaggggat 361 ggttactacg gggactatgc tatggactac
tggggtcaag gaacctcagt caccgtctcc 421 tcagccaaaa cgacaccccc
atctgtctat ccactggccc ctggatctgc tgcccaaact 481 aactccatgg
tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg 541
acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct
601 gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc
cagcgagacc 661 gtcacctgca acgttgccca cccggccagc agcaccaagg
tggacaagaa aattgtgccc 721 agggattgtg gttgtaagcc ttgcatatgt
acagtcccag aagtatcatc tgtcttcatc 781 ttccccccaa agcccaagga
tgtgctcacc attactctga ctcctaaggt cacgtgtgtt 841 gtggtagaca
tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg 901
gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca
961 gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt
caaatgcagg 1021 gtcaacagtg cagctttccc tgcccccatc gagaaaacca
tctccaaaac caaaggcaga 1081 ccgaaggctc cacaggtgta caccattcca
cctcccaagg agcagatggc caaggataaa 1141 gtcagtctga cctgcatgat
aacagacttc ttccctgaag acattactgt ggagtggcag 1201 tggaatgggc
agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc 1261
tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact
1321 ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa
gagcctctcc 1381 cactctcctg gtaaatga (26) Protein Sequence Defining
the Full Length 1F3 Heavy Chain Sequence (1F3 Heavy Chain Variable
Region and IgG1 Constant Region) (without signal sequence) (SEQ ID
NO. 147) 1 evqlvesggg lvqsggslkl scaasgftfs nyfmswvrqt pekrlewvay
issgggstyy 61 pdsvkgrfti srdnakntly lqmsslksed tamyycvrqg
dgyygdyamd ywgqgtsvtv 121 ssakttppsv yplapgsaaq tnsmvtlgcl
vkgyfpepvt vtwnsgslss gvhtfpavlq 181 sdlytlsssv tvpsstwpse
tvtcnvahpa sstkvdkkiv prdcgckpci ctvpevssvf 241 ifppkpkdvl
titltpkvtc vvvdiskddp evqfswfvdd vevhtaqtqp reeqfnstfr
301 syselpimhq dwlngkefkc rvnsaafpap iektisktkg rpkapqvyti
pppkeqmakd 361 kvsltcmitd ffpeditvew qwngqpaeny kntqpimdtd
gsyfvyskln vqksnweagn 421 tftcsvlheg lhnhhteksl shspgk (27) Nucleic
Acid Sequence Encoding the Full Length 1F3 Light Chain Sequence
(1F3 Kappa Variable Region and Constant Region) (signal sequence
underlined) (SEQ ID NO. 148) 1 atgagtgtgc ccactcaggt cctggggttg
ctgctgctgt ggcttacaga tgccagatgt 61 gacatccaga tgactcagtc
tccagcctcc ctatctgtat ctgtgggaga aactgtcacc 121 atcacatgtc
gagcaagtga gaatatttac agtaatttag catggtatca gcagaaacag 181
ggaaaatctc ctcagctcct ggtctatgat gcaacacact taccagatgg tgtgccatca
241 aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag
cctgcagtct 301 gaagattttg ggagttatta ctgtcaacat ttttggggta
ctccgtacac gtttggaggg 361 gggaccagac tggaaattaa acgggctgat
gctgcaccaa ctgtatccat cttcccacca 421 tccagtgagc agttaacatc
tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 481 cccaaagaca
tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 541
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg
601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac
tcacaagaca 661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag
(28) Protein Sequence Defining the Full Length 1F3 Light Chain
Sequence (1F3 Kappa Variable Region and Constant Region) (without
signal sequence) (SEQ ID NO. 149) 1 diqmtqspas lsysvgetvt
itcraseniy snlawyqqkq gkspqllvyd athlpdgvps 61 rfsgsgsgtq
fslkinslqs edfgsyycqh fwgtpytfgg gtrleikrad aaptvsifpp 121
sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt
181 ltkdeyerhn sytceathkt stspivksfn rnec (29) Nucleic Acid
Sequence Encoding the Full Length 3Al2 Heavy Chain Sequence (3A12
Heavy Chain Variable Region and IgG1 Constant Region) (signal
sequence underlined) (SEQ ID NO. 150) 1 atgaactttg ggctcagatt
gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa 61 gtgcagctgg
tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaaatctcc 121
tgtgcagcct ctggatttac tttcagtaac tatttcatgt cttgggttcg ccagactcca
181 gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac
ctactatcca 241 gacagtgtga agggtcgatt caccatctcc agagacaatg
ccaagaacac cctgtacctg 301 caaatgaaca gtctgaagtc tgaggacaca
gccatgtatt actgtgtaag acaaggagat 361 ggttactatg gggactatgc
tatggactac tggggtcaag gaacctcagt caccgtctcc 421 tcagccaaaa
cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact 481
aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg
541 acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt
cctgcagtct 601 gacctctaca ctctgagcag ctcagtgact gtcccctcca
gcacctggcc cagcgagacc 661 gtcacctgca acgttgccca cccggccagc
agcaccaagg tggacaagaa aattgtgccc 721 agggattgtg gttgtaagcc
ttgcatatgt acagtcccag aagtatcatc tgtcttcatc 781 ttccccccaa
agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt 841
gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg
901 gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac
tttccgctca 961 gtcagtgaac ttcccatcat gcaccaggac tggctcaatg
gcaaggagtt caaatgcagg 1021 gtcaacagtg cagctttccc tgcccccatc
gagaaaacca tctccaaaac caaaggcaga 1081 ccgaaggctc cacaggtgta
caccattcca cctcccaagg agcagatggc caaggataaa 1141 gtcagtctga
cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag 1201
tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc
1261 tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc
aggaaatact 1321 ttcacctgct ctgtgttaca tgagggcctg cacaaccacc
atactgagaa gagcctctcc 1381 cactctcctg gtaaatga (30) Protein
Sequence Defining the Full Length 3Al2 Heavy Chain Sequence (3A12
Heavy Chain Variable Region and IgG1 Constant Region) (without
signal sequence) (SEQ ID NO. 151) 1 evqlvesggg lvqpggslki
scaasgftfs nyfmswvrqt pekrlewvay issgggstyy 61 pdsvkgrfti
srdnakntly lqmnslksed tamyycvrqg dgyygdyamd ywgqgtsvtv 121
ssakttppsv yplapgsaaq tnsmvtlgcl vkgyfpepvt vtwnsgslss gvhtfpavlq
181 sdlytlsssv tvpsstwpse tvtcnvahpa sstkvdkkiv prdcgckpci
ctvpevssvf 241 ifppkpkdvl titltpkvtc vvvdiskddp evqfswfvdd
vevhtaqtqp reeqfnstfr 301 syselpimhq dwlngkefkc rvnsaafpap
iektisktkg rpkapqvyti pppkeqmakd 361 kvsltcmitd ffpeditvew
qwngqpaeny kntqpimdtd gsyfvyskln vqksnweagn 421 tftcsvlheg
lhnhhteksl shspgk (31) Nucleic Acid Sequence Encoding the Full
Length 3A12 Light Chain Sequence (3A12 Kappa Variable Region and
Constant Region) (signal sequence underlined) (SEQ ID NO. 152) 1
atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt
61 gacatccaga tgactcagtc gccagcctcc ctatctgtat ctgtgggaga
aactgtcacc 121 atcacatgtc gagcaagtga gaatatttac attaatttag
catggtatca gcagaaacag 181 ggaaaatctc ctcagctcct ggtccatgct
gcaacaaagt tagcagatgg tgtgccatca 241 aggttcagtg gcagtggatc
aggcacacag tattccctca agatcaacag cctgcagtct 301 gaagattttg
ggagttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg 361
gggaccaaac tagaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca
421 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa
caacttctac 481 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg
aacgacaaaa tggcgtcctg 541 aacagttgga ctgatcagga cagcaaagac
agcacctaca gcatgagcag caccctcacg 601 ttgaccaagg acgagtatga
acgacataac agctatacct gtgaggccac tcacaagaca 661 tcaacttcac
ccattgtcaa gagcttcaac aggaatgagt gttag (32) Protein Sequence
Defining the Full Length 3A12 Light Chain Sequence (3A12 Kappa
Variable Region and Constant Region) (without signal sequence) (SEQ
ID NO. 153) 1 diqmtqspas lsysvgetvt itcraseniy inlawyqqkq gkspqnvha
atkladgvps 61 rfsgsgsgtq yslkinslqs edfgsyycqh fwgtpytfgg
gtkleikrad aaptvsifpp 121 sseqltsgga svvcflnnfy pkdinvkwki
dgserqngvl nswtdqdskd stysmsstlt 181 ltkdeyerhn sytceathkt
stspivksfn rnec
[0134] For convenience, Table 2 provides a concordance chart
showing the correspondence between the full length sequences of the
antibodies discussed in this Example with those presented in the
Sequence Listing.
TABLE-US-00004 TABLE 2 SEQ. ID NO. Protein or Nucleic Acid 122 1A3
Heavy Variable + IgG1 constant - nucleic acid 123 1A3 Heavy
Variable + IgG1 constant - protein 124 1A3 Light Variable +
constant - nucleic acid 125 1A3 Light Variable + constant - protein
126 2B8 Heavy Variable + IgG1 constant - nucleic acid 127 2B8 Heavy
Variable + IgG1 constant - protein 128 2B8 Light Variable +
constant - nucleic acid 129 2B8 Light Variable + constant - protein
130 2F8 Heavy Variable + IgG1 constant - nucleic acid 131 2F8 Heavy
Variable + IgG1 constant - protein 132 2F8 Light Variable +
constant - nucleic acid 133 2F8 Light Variable + constant - protein
134 3B6 Heavy Variable + IgG1 constant - nucleic acid 135 3B6 Heavy
Variable + IgG1 constant - protein 136 3B6 Light Variable +
constant - nucleic acid 137 3B6 Light Variable + constant - protein
138 3D11 Heavy Variable + IgG1 constant - nucleic acid 139 3D11
Heavy Variable + IgG1 constant - protein 140 3D11 Light Variable +
constant - nucleic acid 141 3D11 Light Variable + constant -
protein 142 1D3 Heavy Variable + IgG1 constant - nucleic acid 143
1D3 Heavy Variable + IgG1 constant - protein 144 1D3 Light Variable
+ constant - nucleic acid 145 1D3 Light Variable + constant -
protein 146 1F3 Heavy Variable + IgG1 constant - nucleic acid 147
1F3 Heavy Variable + IgG1 constant - protein 148 1F3 Light Variable
+ constant - nucleic acid 149 1F3 Light Variable + constant -
protein 150 3A12 Heavy Variable + IgG1 constant - nucleic acid 151
3A12 Heavy Variable + IgG1 constant - protein 152 3A12 Light
Variable + constant - nucleic acid 153 3A12 Light Variable +
constant - protein
Example 3
Production of Various Recombinant hHGF Proteins
[0135] This Example describes the cloning and expression of a
number of recombinant proteins used to characterize the antibodies
created in Example 1 and in Example 14. In particular, this Example
describes the cloning and expression of recombinant hHGF protein, a
recombinant hHGF protein containing a glycine to glutamate
substitution at position 555 (G555E), a recombinant hHGF protein
containing a cysteine to arginine substitution at position 561
(C561R), a recombinant mouse-human-mouse (mhm) chimeric HGF protein
containing the human V495-L585 HGF sequence disposed within mouse
HGF sequence, a recombinant mhm chimeric HGF protein containing the
human I499-R566 HGF sequence disposed within mouse HGF sequence,
and a recombinant mhm chimeric HGF protein containing human
W507-L585 HGF sequence disposed within mouse HGF sequence.
[0136] The following expression constructs were generated using
standard molecular techniques and the resulting cDNA sequences were
confirmed by DNA sequencing:
[0137] a. hHGF-Fc
[0138] In a first round of PCR, two overlapping PCR fragments were
generated introducing a Not I site and encoding a 6.times.His tag
between hHGF and hIgFc. The overlapping PCR fragments served as
template in a second round to amplify hHGF-his-IgFc. The resulting
fragment was digested by NheI and BamHI and cloned into pcDNA5/FRT
(Invitrogen, #35-3014). Then, hHGF was amplified from Invitrogen
clone ID: IOH29794 (human HGF cDNA). The sequence was found to
correspond to the sequence deposited at the NCBI under accession
number NM.sub.--000601.4.
TABLE-US-00005 (1) 5'hHGF NheI Primer (SEQ ID NO. 102)
ACTGGCTAGCATGTGGGTGACCAAACTCCT (2) 3' hHGF NotI His Tag Primer (SEQ
ID NO. 103) GTGATGGTGATGGTGATGGCGGCCGCATGACTGTGGTACCTTATATG (3) 5'
HisIgFc Primer (SEQ ID NO. 104) ACTGGCGGCCGCCATCACCATCACCATCAC (4)
3' IgFc BamHI Primer (SEQ ID NO. 105)
ACTGGGATCCTCACTATTTACCCGGGGACAG
[0139] b. hHGF-Fc G555E and hHGF-Fc C561R
[0140] hHGF-Fc mutants G555E and C561R were generated by site
directed mutagenesis using the QuikChange II XL site-directed
mutagenesis kit (Stratagene) according to manufacturer's
instructions.
TABLE-US-00006 (1) hHGF-Fc (G555E) Sense Primer (SEQ ID NO. 106)
CATGATGTCCACGAAAGAGGAGATGAG (2) hHGF-Fc (G555E) Anti-sense Primer
(SEQ ID NO. 107) CTCATCTCCTCTTTCGTGGACATCATG (3) hHGF-Fc (C561R)
Sense Primer (SEQ ID NO. 108) GGAAGAGGAGATGAGAAACGCAAACAGGTTCTCAATG
(4) hHGF-Fc (C561R) Anti-sense Primer (SEQ ID NO. 109)
CATTGAGAACCTGTTTGCGTTTCTCATCTCCTCTTCC
[0141] c. Mouse-Human-Mouse Chimera Fc
[0142] The mouse-human-mouse chimera IgFc construct contains mHGF
alpha chain-hHGF, .beta.-chain amino acids Val 495-Leu 585 of human
HGF, and mHGF C-terminal beta chain followed by 6.times.His tag and
IgG-Fc.
[0143] Human HGF cDNA encoding amino acids V495-L585 was amplified
from Invitrogen clone ID: IOH29794 (human HGF cDNA). The sequence
corresponds to the sequence deposited at the NCBI under accession
number NM.sub.--000601.4. Mouse HGF sequences were amplified by
RT-PCR from mouse liver total RNA (Clontech, #636603) using the
Super Script One Step RT-PCR kit from Invitrogen (#10928-034)
according to manufacturer's instructions. The mHGF cDNA sequence
corresponds to the sequence deposited at the NCBI under accession
number D10213.1.
[0144] Three fragments, referred to as Fragments 1, 2, and 3, were
generated using overlapping PCR primers and annealed in consecutive
rounds of PCR amplification. The final product was cleaved with
NheI and NotI and cloned into pcDNA5/FRT IgGFc.
TABLE-US-00007 (1) Fragment 1 Primers for mHGF alpha chain 5'NheI
(SEQ ID NO. 110) 5'ATCGGCTAGCATGATGTGGGGGACCAAAC (SEQ ID NO. 111)
3' GAATCCCATTTACAACCCGCAGTTGTTTTGTTTTGG (2) Fragment 2 Primers for
hHGF beta chain aa V495-L585 (SEQ ID NO. 112) 5'
CCAAAACAAAACAACTGCGGGTTGTAAATGGGATTC (SEQ ID NO. 113) 3'
CAGGATTGCAGGTCGAGCAAGCTTCATTAAAACCAGATCT (3) Fragment 3 Primer for
mHGF beta chain C-terminus 3'NotI (SEQ ID NO. 114) 5'
AGATCTGGTTTTAATGAAGCTTGCTCGACCTGCAATCCTG (SEQ ID NO. 115) 3'
GTAATTTTGACATACAAGTTGTGCGGCCGCCATCACCATCACCA TCAC
[0145] d. Construction of hHGF and mhm Chimera
[0146] The vectors encoding hHGF and mhm chimera (V495-L585),
pcDNA5/FRT hHGF and pcDNA5/FRT-mhm chimera (V495-L585), without
Fc-tag were generated by site directed mutagenesis. A stop codon
was introduced 3' of the 6.times.His tag using the QuikChange II XL
site-directed mutagenesis kit (Stratagene) according to
manufacturer's instructions. The mutagenesis primer included Primer
1: CATCACCATCACCATCACTAAGCGGGTCTGGTGCCACG (SEQ ID NO. 116), and
Primer 2: CGTGGCACCAGACCCGCTTAGTGATGGTGATGGTGATG (SEQ ID NO.
117).
[0147] In addition, two additional mhm chimeras were created from
the pcDNA51FRT-mhm (V495-L585) construct by site directed
mutagenesis using the QuikChange II XL site-directed mutagenesis
kit (Stratagene) according to manufacturer's instructions. One mhm
construct contained the region of I499-R556 of hHGF disposed
between murine sequences. The other mhm construct contained the
region of W507-L585 of hHGF disposed between murine sequences.
[0148] For the mhm chimera (I499-R556), the following point
mutations were made in order in the template pcDNA5/FRT-mhm chimera
(V495-L585) construct: D558E, C561R, V564I, V567I and M583L, using
the appropriate oligonucleotide sequences. For the mhm chimera
(W507-L585), the following point mutations were introduced in one
step in the template pcDNA5/FRT-mhm chimera (V495-L585) construct:
Q502R, N504T and I505V, using the appropriate oligonucleotide
sequences.
[0149] The resulting nucleotide sequence of the hHGF-Fc protein is
set forth as SEQ ID NO. 118, including signal sequence (nucleotides
1-93) and prodomain (nucleotides 94-162). The amino acid sequence
of the hHGF-Fc protein is set forth as SEQ ID NO. 119.
[0150] The resulting nucleotide sequence encoding the mhm
(V495-L585)-Fc chimeric protein is set forth in SEQ ID NO. 120,
including signal sequence (nucleotides 1-96) and prodomain
(nucleotides 97-165). The amino acid sequence of the mhm
(V495-L585)-Fc chimeric protein is set forth in SEQ ID NO. 121.
[0151] The resulting nucleotide sequence encoding, and the protein
sequence defining, the mhm (V495-L585) construct are set forth in
SEQ ID NOS. 211 and 212, respectively. The nucleic acid sequence
set forth in SEQ ID NO. 211 includes the signal sequence
(nucleotides 1-96) and the prodomain (nucleotides 97-165), and the
protein sequence set forth in SEQ ID NO. 212 includes the active
protein sequence (without the signal sequence or the prodomain).
The resulting nucleotide sequence encoding, and the protein
sequence defining, the mhm (I499-R556) construct are set forth in
SEQ ID NOS. 213 and 214, respectively. The nucleic acid sequence
set forth in SEQ ID NO. 213 includes the signal sequence
(nucleotides 1-96) and the prodomain (nucleotides 97-165), and the
protein sequence set forth in SEQ ID NO. 214 includes the active
protein sequence (without the signal sequence or the prodomain).
The resulting nucleotide sequence encoding, and the protein
sequence defining, the mhm (W507-L585) are set forth in SEQ ID NOS.
215 and 216, respectively. The nucleic acid sequence set forth in
SEQ ID NO. 215 includes the signal sequence (nucleotides 1-96) and
the prodomain (nucleotides 97-165), and the protein sequence set
forth in SEQ ID NO. 216 includes the active protein sequence
(without the signal sequence or the prodomain)
[0152] e. Protein Expression
[0153] (1) Cell Culture
[0154] CHO FlpIn cells (Invitrogen, Catalog No. R758-07)) were
grown in F12K media (ATCC, Catalog No. 30-2004), 10% FCS
(Invitrogen, Catalog No. 10438026), 1% Penicillin (10000
units/mL)/Streptomycin (10,000 .mu.g/mL) (Invitrogen, Catalog No.
15140-122) at 37.degree. C., 5% CO.sub.2, 100 .mu.g/mL Zeocin
(Invitrogen, Catalog No. R250-01).
[0155] (2) Generation of Stable CHO FlpIn Cell Lines
[0156] CHO FlpIn host cells were transfected with a 9:1 ratio of
pOG44:pcDNA5/FRT expression plasmid DNA using lipofectamine 2000
according to the manufacturer's instructions (Invitrogen, Catalog
No. 11668-027). As controls, cells were transfected with empty
pcDNA5/FRT vector/pOG44 and pOG44 plasmid (Invitrogen, Catalog No.
35-3018) alone. Twenty four hours after transfection, the cells
were split, and after forty eight hours 0.5 mg/mL Hygromycin B
(Sigma, Catalog No. H0654-SPEC) was added to the cells. Polyclonal
selection of stable cells was performed in F12K, 10% FCS, 1%
Penicillin/Streptomycin, 0.5 mg/mL Hygromycin B.
[0157] (3) Protein Expression in Stable CHO FlpIn Cell Lines
[0158] Approximately 2.times.10.sup.6 cells were seeded in 15 cm
plates and grown in F12K (ATCC, Catalog No. 30-2004)/DMEM high
glucose (Invitrogen, Catalog No. 11995065) 1:1, 5% ultra low IgG
FCS (Invitrogen, #16250-78) at 37.degree. C., 5% CO.sub.2 for 5-6
days. Supernatants were harvested and resulting proteins analyzed
by ELISA and by surface plasmon resonance.
Example 4
Binding Characteristics of Anti-hHGF Monoclonal Antibodies
[0159] The monoclonal antibodies produced in Example 1 were
characterized by their ability to bind hHGF, and certain of the
recombinant HGF proteins produced in Example 3.
[0160] The antibodies were analyzed by surface-plasmon resonance
using a BIAcore T100 instrument to assess their ability to bind HGF
and certain of the fusion proteins discussed in Example 3. Each
antibody was immobilized on a carboxymethylated dextran CM5 sensor
chip (BIAcore, Catalog No. BR-1006-68) by amine coupling (BIAcore,
Catalog No. BR-1000-50) using a standard coupling protocol
according to manufacturer's instructions.
[0161] Analyses were performed at 25.degree. C. using PBS (GIBCO,
Catalog No. 14040-133) containing 0.05% surfactant P20 (BIAcore,
Catalog No. R-1000-54), 2 mg/mL BSA (EMD, Catalog No. 2930) and 10
mg/mL CM-Dextran Sodium salt (Fluka, Catalog No. 86524) as running
buffer. Supernatant containing different HGF fusion proteins or
supernatant from cells transfected with empty vector were injected
over each antibody at a flow rate of 30 .mu.L/min for 3 minutes.
The resulting binding was determined as resonance units (RU) over
baseline 30 seconds after the end of injection. Binding was
compared to human HGF (R&D Systems, Catalog No. 294-HGN-025)
diluted in running buffer. Non-specific binding was monitored by
comparing binding to a control surface where mouse IgG (Rockland,
Catalog No. 010-0102) was immobilized using the same amine coupling
procedure.
[0162] The results are summarized in the Table 3.
TABLE-US-00008 TABLE 3 rhHGF (R&D rmHGF (R&D mhm chimera
Antibody Systems) Systems) (V495-L585) human HGF G555E C561R 1A3
Yes No No Yes Yes Yes 1D3 Yes No Yes Yes Yes Yes 1F3 Yes Yes Yes
Yes Yes Yes 2B8 Yes No Yes Yes Yes Yes 2F8 Yes Yes No Yes Yes Yes
3A12 Yes No No Yes Yes Yes 3B6 Yes No No Yes Yes Yes 3D11 Yes No No
Yes Yes Yes
[0163] The results in Table 3 demonstrate that each of the
antibodies bind rHGF and purified human HGF. Furthermore, all of
the antibodies bind hHGF containing point mutations G555E and
C561R. In general, all of the antibodies except for 1F3 and 2F8 did
not bind murine HGF demonstrating that the antibodies 1A3, 1D3,
2B8, 3A12, 3B6, and 3D11 specifically bind human HGF. Antibodies
1D3, 1F3, and 2B8 bind the mouse-human-mouse chimera whereas the
remaining antibodies did not. The results suggest that the
antibodies 1D3 and 2B8 at least in part bind to residues 495-585 of
human HGF. The antibodies 1A3, 3A12, 3B6, and 3D11 appear to bind
portions of human hHGF other than residues 495-585. At present, it
is uncertain why 2F8 does not bind the mhm chimera as it appears to
bind both hHGF and mHGF.
Example 5
Ability of Anti-hHGF Monoclonal Antibodies to Bind Reduced and
Non-Reduced HGF
[0164] In this Example, the anti-hHGF monoclonal antibodies
produced in Example 1 were analyzed for their ability to bind
reduced and non-reduced HGF.
[0165] The reactivity of the anti-HGF sera with the recombinant
hHGF was assessed by immunoblotting. Eight .mu.g of recombinant
hHGF protein in NuPAGE MOPS SDS running buffer (Invitrogen) with or
without NuPAGE sample reducing buffer (Invitrogen) was fractionated
on a 4-12% Bis-Tris 1.0 mm.times.2D well gel (Invitrogen, Carlsbad,
Calif.). The fractionated proteins then were transferred onto a
nitrocellulose membrane using standard procedures. The
nitrocellulose membranes were blocked with 5% nonfat milk powder
solution in Tris buffered Saline with 0.1% Tween-20.RTM. (TBST),
and then mounted onto a Mini Protean II Multi-Screen apparatus
(BioRad) for further blocking.
[0166] The resulting membranes were probed with the purified
antibodies on a Multi-Screen apparatus. The purified antibodies
were diluted to 5 .mu.g/mL in blocking buffer. The nitrocellulose
membrane then was removed from the apparatus, and incubated with
horseradish peroxidase-labeled anti-mouse IgG antibodies. The
results are summarized in Table 4, where the numbers reflect the
extent of binding with--representing the least (little or no
binding) and 3+ representing the most binding.
TABLE-US-00009 TABLE 4 Reduced Non-Reduced Antibody (exposure: 3-5
min) (exposure: 20 sec) 1A3 2+ 2+ 1D3 2+ 2+ 1F3 2+ 2+ 2B8 -- 1+ 2F8
2+ 2+ 3A12 -- 2+ 3B6 3+ 2+ 3D11 -- 3+
[0167] The data in Table 4 demonstrate that all the antibodies bind
non-reduced rhHGF. In contrast, monoclonal antibodies 1A3, 1D3,
1F3, 2F8, 3B6 bound reduced rhHGF but antibodies 2B8, 3A12, and
3D11 did not bind to reduced rhHGF.
Example 6
Binding Affinities
[0168] The binding affinities and kinetics of interaction of each
of the antibodies produced in Example 1 against hHGF were measured
by surface plasmon resonance.
[0169] Rabbit anti-mouse immunoglobulins (BIAcore, Catalog No.
BR-1005-14) were immobilized on carboxymethylated dextran CM5
sensor chips (BIAcore, Catalog No. BR-1006-68) by amine coupling
(BIAcore, Catalog No. BR-1000-50) using a standard coupling
protocol according to manufacturer's instructions. The analyses
were performed at 25.degree. C. using PBS (GIBCO, Catalog No.
14040-133) containing 0.05% surfactant P20 (BIAcore, Catalog No.
BR-1000-54), 2 mg/mL BSA (EMD, Catalog No. 2930), and 10 mg/mL
CM-Dextran Sodium salt (Fluka, Catalog No. 86524) as running
buffer.
[0170] The antibodies were captured in an individual flow cell at a
flow rate of 10 .mu.L/min. Injection time was variable for each
antibody to yield approximately 20 RU of antibody captured for each
cycle. Buffer or HGF (R&D Systems, Catalog No. 294-HGN-025)
diluted in running buffer was injected sequentially over a
reference surface (no antibody captured) and the active surface
(antibody to be tested) for 2 minutes at 60 .mu.L/min. The
dissociation phase was monitored for 15 or 90 minutes, depending on
concentration. The surface then was regenerated with 10 mM
Glycine-HCl, pH 1.7 (BIAcore, Catalog No. BR-1003-54) injected for
3 minutes at a flow rate of 60 .mu.L/min before another cycle was
initiated. HGF concentrations tested were 0.46 nM to 7.5 nM.
[0171] Kinetic parameters were determined using the kinetic
function of the BIAevalutation software with reference subtraction.
Kinetic parameters for each antibody, k.sub.a (association rate
constant), k.sub.d (dissociation rate constant) and K.sub.D
(equilibrium dissociation constant) are summarized in Table 5.
TABLE-US-00010 TABLE 5 (pM) Antibody ka (1/Ms) SE (ka) kd (1/s) SE
(kd) K.sub.D SD 1A3 1.7 .times. 10.sup.6 7.3 .times. 10.sup.4 5.2
.times. 10.sup.-5 8.4 .times. 10.sup.-7 30.1 5.6 1D3 1.7 .times.
10.sup.6 3.1 .times. 10.sup.4 8.2 .times. 10.sup.-5 1.7 .times.
10.sup.-6 54.2 27.4 1F3 1.5 .times. 10.sup.6 5.0 .times. 10.sup.4
2.6 .times. 10.sup.-5 6.6 .times. 10.sup.-7 18.1 8.2 2B8 1.6
.times. 10.sup.6 2.9 .times. 10.sup.4 2.1 .times. 10.sup.-5 1.4
.times. 10.sup.-7 13.5 4.4 3A12 1.6 .times. 10.sup.6 3.7 .times.
10.sup.4 1.6 .times. 10.sup.-4 1.6 .times. 10.sup.-6 103.0 10.4 3B6
2.0 .times. 10.sup.6 6.5 .times. 10.sup.4 3.9 .times. 10.sup.-5 3.2
.times. 10.sup.-7 17.0 3.4
[0172] The data in Table 5 demonstrate that the antibodies bind
hHGF with a K.sub.D of about 100 pM or less, about 50 pM or less,
or 20 pM or less.
Example 7
Neutralization Activity of Anti-hHGF Antibodies
[0173] In this Example, the antibodies produced in Example 1 were
characterized for their ability to (a) inhibit the binding of hHGF
to c-Met, and (b) inhibit HGF stimulated BrdU incorporation in 4
MBr-5 cells.
[0174] a. HGF-Met Binding Inhibition Assay (Neutralization
Assay)
[0175] The antibodies were tested by ELISA for their ability to
inhibit hHGF binding to c-Met.
[0176] Specifically, Wallac 96-well DELFIA assay plates (Wallac
Inc., Catalog No. AAAND-0001) were coated with 100 .mu.L of 6.25
.mu.g/mL HGF (R&D Systems, Catalog No. 294-HGN-025) in
carbonate coating buffer (15 mM Na.sub.2CO.sub.3 and 34 mM
NaHCO.sub.3, pH 9.0) for 16 hours at 4.degree. C. The plates then
were blocked with 200 .mu.L of 5% non-fat dry milk in PBS for 1
hour at room temperature. The antibodies were prepared in a
separate plate by adding increasing concentrations of the
antibodies under investigation (0.033-667 nM, 3-fold-serial
dilution) to 2 nM c-Met (R&D Systems, Catalog No. 358-MT/CF) in
5% non-fat dry milk in PBS. 100 .mu.L of sample per well was
transferred to the assay plate and incubated overnight at 4.degree.
C. The assay plates then were washed 3 times with PBS-0.1% Tween
20, and incubated for 2 hours at room temperature with 100
.mu.L/well of 2 .mu.g/mL biotinylated anti-human c-Met antibody
(R&D Systems, Catalog No. BAF358) prepared in 5% non-fat dry
milk in PBS.
[0177] The resulting plates then were washed three times with
PBS-0.1% Tween 20, and incubated for 1 hour at room temperature
with Eu-labeled Streptavidin (Wallac, Catalog No. 1244-360) diluted
1:1000 in DELFIA assay buffer (Wallac, Catalog No. 4002-0010). The
resulting plates were washed 3 times with DELFIA wash solution
(Wallac, Catalog No. 4010-0010) and incubated with 100 .mu.L/well
DELFIA enhancement solution (Wallac #4001-0010) for 15 minutes at
room temperature with agitation.
[0178] The plates were read on Victor.sup.3V instrument (Perkin
Elmer) using the Europium method. The IC.sub.50 values were
calculated and are summarized in Table 6.
TABLE-US-00011 TABLE 6 Antibody IC.sub.50 (nM) SD 1A3 5.65 0.91 1D3
4.43 2.27 1F3 6.57 0.28 2B8 5.57 1.19 2F8 5.36 0.88 3A12 5.26 2.11
3B6 -- -- 3D11 5.66 2.75
[0179] The results demonstrate that all the antibodies (i.e., 1D3,
1A3, 2B8, 3A12, 1F3, 3D11, and 2F8) other than 3B6 efficiently
neutralize HGF binding to c-Met.
[0180] b. Neutralization of HGF Stimulated BrdU Incorporation in 4
MBr-5 Cells
[0181] Ten .mu.L of 12.5 nM of hHGF was dispensed into individual
wells of a 96-well tissue culture microtiter plate (Costar Catalog
No. 3903). Ten .mu.L of serially diluted antibodies at
concentrations of 6667, 2222, 740, 247, 82, 27, 9.1, 3.0, 1.0, 0.33
nM were added to each well. The HGF antibody mixture then was
incubated at room temperature for 30 minutes. Monkey bronchial
epithelial cells 4 MBr-5 (ATCC, CCL208) cultured in F-12K (ATCC,
30-2004), 15% FBS (Gibco 10438-026), 30 ng/mL EGF (Sigma E9644), 1%
penicillin/streptomycin (PS, Gibco Catalog No. 15140-122) were
dissociated with Trypsin (Gibco Catalog No. 25200-056), resuspended
in assay media (F-12K, 2.5% FBS, 1% PS) at 75,000 cells/mL, and 80
.mu.L of the cell suspension was dispensed to the HGF antibody
mixture.
[0182] The resulting cells were incubated at 37.degree. C., 5%
CO.sub.2. Forty eight hours later, 10 .mu.L of 100 .mu.M BrdU
(Roche Catalog No. 1669915) was added. Seventy two hours later, the
media was removed, the plates were dried with a hair dryer and were
processed with the BrdU ELISA in accordance with manufacturer's
instructions (Roche Catalog No. 1669915).
[0183] The luminescent signal was quantified by a Synergy HT plate
reader (Bio-Tek). The data were fit to a sigmoidal dose response
with variable slope with the equation y=bottom+(top-bottom)/(1+10
(log(EC50-x)*hill slope)) in GraphPad Prism (GraphPad Software).
Each experiment was repeated at least 3 times in duplicates, and
average EC.sub.50 values are presented in Table 7.
TABLE-US-00012 TABLE 7 Antibody IC.sub.50 (nM) 1A3 4.69 1D3 4.99
1F3 1.94 2B8 1.41 2F8 19.24 3A12 30.30 3B6 36.08 3D11 51.12
[0184] The results in Table 7 demonstrate that all of the
antibodies, 1A3, 1D3, 1F3, 2B8, 2F8, 3A12, 3B6, and 3D11 inhibit
HGF induced proliferation in 4 MBr-5 cells.
Example 8
Anti-Scatter Activity of Anti-hHGF Antibodies
[0185] This Example describes a characterization of the antibodies
produced in Example 1 for their ability to inhibit HGF induced
scatter activity. HGF induces "scattering" (motility) of clusters
in MDCK cells (ATCC, Manassas, Va., Catalog No. CCL-34).
[0186] MDCK cells were seeded in 96-well Costar tissue culture
plates (Corning Incorporated, Corning, N.Y., Catalog No. 3595) at a
density of 4.times.10.sup.3 cells per well in 80 .mu.L MEM (ATCC,
Manassas, Va., Catalog No. 30-2003) containing 10% Fetal Bovine
Serum (Invitrogen Catalog No. 10438026), and 1%
penicillin-streptomycin (Invitrogen Catalog No. 15140122). Each of
the antibodies to be investigated was diluted to 6,667 nM in MEM
containing 10% Fetal Bovine Serum and 1% penicillin-streptomycin.
Each of the different antibody dilutions, as well as MEM containing
10% Fetal Bovine Serum and 1% penicillin-streptomycin without
antibody, then was separately combined with an equal volume of MEM
containing 10% Fetal Bovine Serum and 1% penicillin-streptomycin,
and 100 ng/ml HGF (R&D Systems Catalog No. 294-HGN-025). The
antibody/HGF dilutions were incubated for 30 minutes at 25.degree.
C. Twenty .mu.L of each antibody/HGF dilution was added separately
to individual wells, yielding a final antibody concentration of
666.7 nM, and a final HGF concentration of 10 ng/ml. The MDCK cells
then were incubated for 24 hours at 37.degree. C. with 5%
CO.sub.2.
[0187] After 24 hours incubation, the MDCK cells were carefully
washed once with 100 .mu.L per well of ice-cold PBS (Invitrogen
Catalog No. 14190144), and fixed with 100 .mu.L per well of
ice-cold methanol while rocking for 10 minutes at 25.degree. C. The
plates then were washed carefully once with distilled water. A
volume of 100 .mu.L crystal violet solution, consisting of 0.5%
crystal violet (Sigma, St. Louis, Mo., Catalog No. C3886) and 50%
ethanol in distilled water, was added to each well, and the cells
were incubated for 20 minutes at 25.degree. C. while rocking.
[0188] Following staining with crystal violet solution, the cells
were washed carefully three times with distilled water. Then, PBS
was added to each well to prevent drying of samples. The cells were
imaged using the Leica DMIRB microscope (Leica Microsystems GmbH,
Wetzler, Germany), DC500 camera (Leica Microsystems GmbH, Wetzler,
Germany), and MagnaFire 2.1C software (Optronics, Goleta, Calif.),
and samples were rated for level of scattering. The results are
summarized in Table 8.
TABLE-US-00013 TABLE 8 Inhibition of HGF-induced MDCK Cell
Scattering Antibody Trial 1 Trial 2 1A3 ++ + 1D3 ++ ++ 1F3 + + 2B8
+++ +++ 2F8 + + 3A12 - -/+ 3B6 ++ ++ 3D11 - - - No Inhibition +++
Very strong, nearly complete inhibition ++ Strong inhibition +
Detectable inhibition
[0189] The results in Table 8 demonstrate that antibody 2B8
inhibited HGF-induced scattering more than the other antibodies.
Antibodies 1D3 and 3B6 displayed an intermediate level of
inhibition; antibody 1A3 displayed a low to intermediate level of
inhibition: antibodies 1F3 and 2F8 displayed a low level of
inhibition; and antibodies 3A12 and 3D11 gave little or no
detectable inhibition.
Example 9
Inhibition of HGF-Stimulated c-Met Phosphorylation
[0190] This Example describes a characterization of the antibodies
produced in Example 1 for their ability to inhibit the
HGF-stimulated c-Met phosphorylation in PC-3 cells. HGF induces
phosphorylation of Met in PC-3 cells (ATCC No. CRL-1435).
[0191] PC-3 cells were seeded into individual wells of 96-well
Costar tissue culture plates (Corning Catalog No. 3595) at a
density of 4.5.times.10.sup.4 cells per well in 100 .mu.L F-12K
(ATCC, Manassas, Va., Catalog No. 30-2004) containing 10% Fetal
Bovine Serum (Invitrogen Catalog No. 10438026) and 1%
penicillin-streptomycin (Invitrogen Catalog No. 15140122). After 24
hours at 37.degree. C. with 5% CO.sub.2, the media was removed, and
cells were rinsed once with serum-free F-12K containing 1%
penicillin-streptomycin. Cells then were incubated for 24 hours in
100 .mu.L serum-free F-12K containing 1%
penicillin-streptomycin.
[0192] The following 10 different dilutions of each of the
antibodies being investigated were prepared in serum-free F-12K
containing 1% penicillin-streptomycin: 6667 nM, 2222 nM, 741 nM,
247 nM, 82.3 nM, 27.4 nM, 9.1 nM, 3.0 nM, 1.0 nM, and 0.3 nM. Each
antibody dilution, and, serum-free F-12K containing 1%
penicillin-streptomycin without antibody, were separately combined
with an equal volume of serum-free F-12K containing 1%
penicillin-streptomycin and 500 ng/mL HGF (R&D Systems Catalog
No. 294-HGN-025). These antibody/HGF dilutions were incubated for
30 minutes at 25.degree. C. This resulted in a final concentration
of 1.25 nM HGF.
[0193] The PC-3 cells then were rinsed once with serum-free F-12K
containing 1% penicillin-streptomycin. Next, 70 .mu.L of serum-free
F-12K containing 1% penicillin-streptomycin was added to the cells,
followed by 10 .mu.L of 10 mM Na.sub.3VO.sub.4 (Sigma Catalog No.
S6508) in serum-free F-12K containing 1% penicillin-streptomycin.
The cells then were incubated for 60 minutes at 37.degree. C. with
5% CO.sub.2. Following this incubation, 20 .mu.L of each
antibody/HGF dilution was added separately to separate wells,
yielding a final HGF concentration of 50 ng/mL, and the following
final concentrations of each antibody: 666.7 nM, 222.2 nM, 74.1 nM,
24.7 nM, 8.23 nM, 2.74 nM, 0.91 nM, 0.30 nM, 0.10 nM, 0.03 nM. The
cells then were incubated for 10 minutes at 37.degree. C. with 5%
CO.sub.2, after which point the media/antibody/HGF mixture was
removed, the plates were placed on ice. The cells then were rinsed
once with 100 .mu.L per well of ice-cold PBS (Invitrogen Catalog
No. 14190144) containing 1 mM Na.sub.3VO.sub.4. The cells then were
incubated for 30 minutes at 4.degree. C. in 100 .mu.L per well
ice-cold lysis buffer consisting of 1% OmniPur Triton X-100 (MERCK
KGaA, Darmstadt, Germany, Catalog No. 9410), 50 mM Tris-HCl pH 8.0,
100 mM NaCl, 0.3 mM Na.sub.3VO.sub.4, 1.times. protease inhibitor
cocktail (Sigma Catalog No. P8340), and 1.times. phosphatase
inhibitor cocktail 2 (Sigma Catalog No. 5726).
[0194] Biotinylated anti-human HGF-R (c-met) antibody (R&D
Systems Catalog No. BAF358) was diluted to a concentration of 2
.mu.g/mL in DELFIA Assay Buffer (PerkinElmer, Turku, Finland,
Catalog No. 4002-0010) containing 1% bovine serum albumin (Sigma
Catalog No. A2153), and 50 .mu.L of this dilution was added per
well of yellow streptavidin microtitration plates (PerkinElmer
Catalog No. AAAND-0005). The plates then were incubated with
antibody for 30 minutes at 25.degree. C. with rocking. Following
incubation, the plates were washed with DELFIA wash solution
(PerkinElmer Catalog No. 4010-0010), and 80 .mu.L of each of the
different PC-3 cell lysates was added separately to individual
wells of the washed streptavidin microtitration plates.
[0195] The streptavidin microtitration plates containing PC-3 cell
lysates were incubated for 60 minutes at 25.degree. C. with
shaking, and then washed with DELFIA wash solution. 100 .mu.L of
600 ng/mL DELFIA Eu-N1 P-Tyr-100 antibody (PerkinElmer Catalog No.
AD0159) diluted in DELFIA Assay Buffer containing 1% bovine serum
albumin was added to each well of the washed streptavidin
microtitration plates previously incubated with PC-3 cell lysates.
The plates were incubated for 60 minutes at 25.degree. C., with
rocking. The plates were washed a final time with DELFIA wash
solution. Then 200 .mu.L of DELFIA Enhancement Solution
(PerkinElmer Catalog No. 4001-0010) was added to each well of the
washed streptavidin microtitration plates, and the plates were
incubated in the dark for 5 minutes at 25.degree. C., with
shaking.
[0196] Signal then was measured using the Europium protocol on the
Victor3V reader (PerkinElmer). EC.sub.50 values were calculated
using Prism 4 for Windows (GraphPad Software, Inc., San Diego,
Calif.) and the sigmoidal dose-response equation.
[0197] The results summarized as EC50s in nM are tabulated in Table
9.
TABLE-US-00014 TABLE 9 Average of Standard Antibody Two Trials
Deviation 1A3 0.684 0.242 1D3 0.984 0.129 1F3 1.19 1.01 2B8 0.287
0.104 2F8 1.39 2.12 3A12 2.00 0.553 3B6 1.01 1.11 3D11 2.28 N/A
[0198] The data in Table 9 demonstrate that all eight antibodies
are potent inhibitors of HGF-induced c-Met phosphorylation in PC-3
cells.
Example 10
Tumor Inhibition in U87MG Xenograft Model
[0199] The ability of murine monoclonal antibodies of the invention
to inhibit tumor growth was tested in an U87MG xenograft model.
U87MG cells (ATCC) were expanded in culture at 37.degree. C. in an
atmosphere containing 5% CO2 and 95% air, using a medium comprising
Dulbecco's Modified Eagle medium (DMEM) with 10% fetal bovine
serum, 100 units/mL penicillin and 100 .mu.g/mL streptomycin. The
cells were subcultured and maintained by detaching the cells from
the wall of the culture dish using trypsin-EDTA.
[0200] Near-confluent cells were collected by trypsinization and
then 5.times.10.sup.6 cells in 50% Matrigel (BD Biosciences;
catalog no. 356237) were injected subcutaneously into the upper
dorsal area between the shoulder blades of 7-week old female ICR
SCID mice (Taconic Labs). The long (L) and short (W) diameters (mm)
of tumors were measured with a caliper. Tumor volume (vol.) was
calculated as: volume (mm.sup.3)=L.times.W.sup.2/2. When the tumors
grew to approximately 200 mm.sup.3, the tumor-bearing mice were
randomized into 5 groups of 10 mice each. One group received PBS.
Each of the other 4 groups received one of the antibody 1A3, 1D3,
1F3 or 2B8. All antibodies were dosed at 1 mg/kg body weight, twice
per week, by intra-peritoneal injections of 5 doses. Tumor volumes
and mouse body weights were recorded twice per week. Tumor growth
inhibition was analyzed using Student's t-test. The results are
summarized in FIG. 6 and Table 10.
TABLE-US-00015 TABLE 10 Percent Inhibition 2B8 vs PBS 93% p = 0.001
1A3 vs PBS 73% p = 0.0075 1D3 vs PBS 51% p = 0.075 1F3 vs PBS 60% p
= 0.027
[0201] Partial regression was achieved in 2B8 treated group (FIG.
6). Statistically significant growth inhibition was observed in the
1A3-treated and 1F3-treated groups (Table 10). There was 51% tumor
growth inhibition for 1D3 with a p value of 0.075. No significant
body weight loss was observed.
Example 11
Tumor Inhibition in U118 Xenograft Model
[0202] The ability of the antibodies 1A3, 1D3, 1F3 and 2B8 to
inhibit tumor growth was tested in an U118 xenograft model. U118
cells (ATCC) were expanded as described in Example 10 (above) with
respect to the U87MG cells.
[0203] Subcutaneous tumors were established as described in Example
10 above, except that the mice used were 7 weeks old female NCr
nude mice (Taconic), and treatment was started when the tumors grew
to approximately 80 mm.sup.3. As in the U87MG model, all the
antibodies were dosed at 1 mg/kg body weight twice a week by
intra-peritoneal injections for 4 doses. Tumor volumes and body
weights of the mice were recorded twice per week. Tumor growth
inhibition was analyzed using Student's t-test. The results are
summarized in FIG. 7 and Table 11.
TABLE-US-00016 TABLE 11 Percent Inhibition 2B8 vs IgG 75% p = 0.007
1A3 vs IgG 57% p = 0.01 1D3 vs IgG 47% p = 0.12 1F3 vs IgG 30% p =
0.39
[0204] Statistically significant tumor growth inhibition was
observed in 2B8 and 1A3 treated groups (FIG. 7). There was modest
tumor growth inhibition in 1F3 and 1D3 groups with p values less
than 0.05, which was defined as statistical significance in this
study (Table 11). No significant body weight loss was observed.
Example 12
Humanization of Murine Monoclonal Antibodies
[0205] This Example describes the humanization of the murine 2B8
antibody, together with a characterization of the resulting
humanized antibodies. The murine 2B8 Heavy and Light Variable
Regions were "humanized" by two methods.
A. Humanization Procedure 1
[0206] In the first method, three humanized heavy chain variable
regions and two humanized kappa light chain variable regions were
designed based on the "superhumanization" method described in Hwang
et al. (2005) METHODS 36:35-42; Tan et al. (2002) J. IMMUNOL.
169:1119-1125; U.S. Pat. No. 6,881,557.
[0207] The Chothia canonical structural class was determined for
each mouse 2B8 CDR based on CDR length and amino acid composition.
Human germline variable regions consisting of the same Chothia
canonical structural class light and heavy variable regions were
identified based on known human germline variable region reference
alleles described at the International Immunogentics Information
System (IMGT) website (available on the world wide web at
imgt.cines.fr and biochem.unizh.ch/antibody/Sequences/index.html).
These human germline variable regions of the same structural class
were compared to murine 2B8 variable regions by calculating the
percent identity or similarity between CDR amino acid residues.
Those human germline variable regions with the highest identity
and/or similarity with mouse 2B8 CDR residues were chosen for CDR
grafting. The framework residues of the human germline variable
regions were preserved while the mouse 2B8 CDR residues were used
to replace the corresponding human germline variable region
residues that were different between mouse 2B8 CDR and human
germline CDRs. The human J region that was most similar to the 2B8
mouse J region was then added to the carboxyl terminus of the
"superhumanized" variable region. A signal sequence was then added
to the amino terminus of the "superhumanized" variable regions and
these amino acid sequences were converted into nucleic acid
sequences.
[0208] The complete variable region nucleic acid sequence was
constructed using gene synthesis PCR methods (Young et al. (2004)
NUCL. ACIDSRES. 32:e59) and cloned into a mammalian expression
vector (based on pcDNA3.2 DEST (Invitrogen)) containing human
constant IgG1 (G1m(17,1) allotype) or Kappa (Km(3) allotype (allele
2)) regions (downstream of the variable regions) using standard
molecular biology techniques. All four heavy chain IgG1 antibodies
(chimeric 2B8 and 3 humanized heavy chains (Hu2B8 Hv1-f.1, Hu2B8
Hv5-a.1, Hu2B8 Hv5-51.1) were expressed in the possible
combinations with all 3 kappa chain antibodies (chimera 2B8 and 2
humanized light chains (Hu2B8 Kv1-39.1 and Hu2B8 Kv3-15.1) creating
12 different antibody proteins. Binding of the chimeric,
chimeric/humanized, and humanized antibodies to human HGF was then
measured as described below and the results are summarized in FIG.
8. Each of the possible combinations of immunoglobulin heavy chain
and immunoglobulin light chain variable regions are set forth below
in Table 12A.
TABLE-US-00017 TABLE 12A Heavy Chain Variable Region Light Chain
Variable Region Chimeric 2B8 (SEQ ID NO: 12) Chimeric 2B8 (SEQ ID
NO: 14) Chimeric 2B8 (SEQ ID NO: 12) Hu2B8 Kv1-39.1 (SEQ ID NO:
173) Chimeric 2B8 (SEQ ID NO: 12) Hu2B8 Kv3-15.1 (SEQ ID NO: 179)
Hu2B8 Hv1-f.1 (SEQ ID NO: 159) Chimeric 2B8 (SEQ ID NO: 14) Hu2B8
Hv1-f.1 (SEQ ID NO: 159) Hu2B8 Kv1-39.1 (SEQ ID NO: 173) Hu2B8
Hv1-f.1 (SEQ ID NO: 159) Hu2B8 Kv3-15.1 (SEQ ID NO: 179) Hu2B8
Hv5-a.1 (SEQ ID NO: 165) Chimeric 2B8 (SEQ ID NO: 14) Hu2B8 Hv5-a.1
(SEQ ID NO: 165) Hu2B8 Kv1-39.1 (SEQ ID NO: 173) Hu2B8 Hv5-a.1 (SEQ
ID NO: 165) Hu2B8 Kv3-15.1 (SEQ ID NO: 179) Hu2B8 Hv5-51.1 (SEQ ID
NO: 169) Chimeric 2B8 (SEQ ID NO: 14) Hu2B8 Hv5-51.1 (SEQ ID NO:
169) Hu2B8 Kv1-39.1 (SEQ ID NO: 173) Hu2B8 Hv5-51.1 (SEQ ID NO:
169) Hu2B8 Kv3-15.1 (SEQ ID NO: 179)
[0209] Each of the possible combinations of immunoglobulin heavy
chains and immunoglobulin light chains are set forth below in Table
12B.
TABLE-US-00018 TABLE 12B Immunoglobulin Heavy Chain Immunoglobulin
Light Chain Chimeric 2B8 IgG1 (SEQ ID NO: 155) Chimeric 2B8 Kappa
(Km(3)) (SEQ ID NO: 157) Chimeric 2B8 IgG1 (SEQ ID NO: 155) Hu2B8
Kv1-39.1 + Kappa Constant (Km(3) allotype) (allele 2) (SEQ ID NO:
177) Chimeric 2B8 IgG1 (SEQ ID NO: 155) Hu2B8 Kv3-15.1 + Kappa
Constant (Km(3) allotype) (allele 2) (SEQ ID NO: 181) Hu2B8 Hv1-f.1
+ IgG1 Constant Chimeric 2B8 Kappa (Km(3)) (G1M(17,1)) allotype
(SEQ ID NO: 163) (SEQ ID NO: 157) Hu2B8 Hv1-f.1 + IgG1 Constant
Hu2B8 Kv1-39.1 + Kappa Constant (Km(3) (G1M(17,1)) allotype (SEQ ID
NO: 163) allotype) (allele 2) (SEQ ID NO: 177) Hu2B8 Hv1-f.1 + IgG1
Constant Hu2B8 Kv3-15.1 + Kappa Constant (Km(3) (G1M(17,1))
allotype (SEQ ID NO: 163) allotype) (allele 2) (SEQ ID NO: 181)
Hu2B8 Hv5-a.1 + IgG1 Constant Chimeric 2B8 Kappa (Km(3))
(G1M(17,1)) allotype (SEQ ID NO: 167) (SEQ ID NO: 157) Hu2B8
Hv5-a.1 + IgG1 Constant Hu2B8 Kv1-39.1 + Kappa Constant (Km(3)
(G1M(17,1)) allotype (SEQ ID NO: 167) allotype) (allele 2) (SEQ ID
NO: 177) Hu2B8 Hv5-a.1 + IgG1 Constant Hu2B8 Kv3-15.1 + Kappa
Constant (Km(3) (G1M(17,1)) allotype (SEQ ID NO: 167) allotype)
(allele 2) (SEQ ID NO: 181) Hu2B8 Hv5-51.1 + IgG1 Constant Chimeric
2B8 Kappa (Km(3)) (G1M(17,1)) allotype (SEQ ID NO: 171) (SEQ ID NO:
157) Hu2B8 Hv5-51.1 + IgG1 Constant Hu2B8 Kv1-39.1 + Kappa Constant
(Km(3) (G1M(17,1)) allotype (SEQ ID NO: 171) allotype) (allele 2)
(SEQ ID NO: 177) Hu2B8 Hv5-51.1 + IgG1 Constant Hu2B8 Kv3-15.1 +
Kappa Constant (Km(3) (G1M(17,1)) allotype (SEQ ID NO: 171)
allotype) (allele 2) (SEQ ID NO: 181)
[0210] Two of the possible antibody constructs containing the full
length immunoglobulin heavy and light chains containing humanized
variable regions are designated below: [0211] sh2B8-9
(G1m(17,1))=hu2B8 Hv5-51.1 (+IgG1 constant region (G1m(17,1)
allotype) (SEQ ID NO. 171) plus hu2B8 Kv 1-39.1 (+Kappa constant
region (Km(3) allotype (allele 2))) (SEQ ID NO. 177) [0212]
sh2B8-12 (G1m(17,1))=hu2B8 Hv5-51.1 (+IgG1 constant region
(G1m(17,1) allotype)) (SEQ ID NO. 171) plus hu2B8 Kv 3-15.1 (+Kappa
constant region (Km(3) allotype (allele 2))) (SEQ ID NO. 181).
[0213] The nucleic acid sequences encoding and the protein
sequences defining each of the humanized antibodies are summarized
below. In this section, the last nucleotide of each variable region
is the first base of the next codon generated by the
variable/constant region junction. This nucleotide is included in
the Variable Region because it is part of that exon. Amino acid
sequences of Constant Regions listed below include the translation
of this junction codon.
TABLE-US-00019 (1) Nucleic Acid Sequence Encoding the Full Length
Chimeric 2B8 Heavy Chain (Mouse Variable Region and Human IgG1
Constant Region) (allotype G1m(17,1)) (signal sequence underlined)
(SEQ ID NO. 154) 1 atgggatgga gctatatcat cctctttttg gtagcaacag
ctacagatgt ccactcccag 61 gtccaactgc agcagcctgg ggctgaactg
gtgaagcctg ggacttcagt gaagctgtcc 121 tgcaaggctt ctggctacac
cttcaccacc tactggatgc actgggtgaa tcagaggcct 181 ggacaaggcc
ttgagtggat tggagagatt aatcctacca acggtcatac taactacaat 241
gagaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg
301 caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag
aaactatgtt 361 ggtagcatct ttgactactg gggccaaggc accactctca
ccgtctcctc agcctccacc 421 aagggcccat cggtcttccc cctggcaccc
tcctccaaga gcacctctgg gggcacagcg 481 gccctgggct gcctggtcaa
ggactacttc cccgaaccgg tgacggtgtc gtggaactca 541 ggcgccctga
ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 601
tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
661 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc
caaatcttgt 721 gacaaaactc acacatgccc accgtgccca gcacctgaac
tcctgggggg accgtcagtc 781 ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca 841 tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 901 ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 961
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag
1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc
caaagccaaa 1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggatga gctgaccaag 1141 aaccaggtca gcctgacctg cctggtcaaa
ggcttctatc ccagcgacat cgccgtggag 1201 tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1261 gacggctcct
tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1321
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc
1381 ctctccctgt ctccgggtaa atga (2) Protein Sequence Defining the
Full Length Chimeric 2B8 Heavy Chain (Chimeric 2B8 IgG1 (G1m(17,1)
allotype) (without signal sequence) (SEQ ID NO. 155) 1 qvqlqqpgae
lvkpgtsvkl sckasgytft tywmhwvnqr pgqglewige inptnghtny 61
nekfkskatl tvdkssstay mqlssltsed savyycarny vgsifdywgq gttltvssas
121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht
fpavlqssgl 181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks
cdkthtcppc papellggps 241 vflfppkpkd tlmisrtpev tcvvvdvshe
dpevkfnwyv dgvevhnakt kpreeqynst 301 yrvvsvltvl hqdwlngkey
kckvsnkalp apiektiska kgqprepqvy tlppsrdelt 361 knqvsltclv
kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq 421
gnvfscsvmh ealhnhytqk slslspgk (3) Nucleic Acid Sequence Encoding
the Full Length Chimeric 2B8 Light Chain (Mouse Variable Region and
Human Constant Region) (Chimeric 2B8 Kappa (Km(3))) (signal
sequence underlined) (SEQ ID NO. 156) 1 atggaatcac agactctggt
cttcatatcc atactgctct ggttatatgg tgctgatggg 61 aacattgtaa
tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc 121
ttgagctgca aggccagtga gaatgtggtt tcttatgtat cctggtatca acagaaacca
181 gcgcagtctc ctaaactgct gatatacggg gcatccaacc ggaacactgg
ggtccccgat 241 cgcttcacag gcagtggatc tgcaacagat ttcactctga
ccatcagcag tgtgcgggct 301 gaagaccttg cagattatca ctgtgggcag
agttacaact atccgtacac gttcggaggg 361 gggaccaggc tggaaataaa
acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 421 tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 481
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag
541 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag
caccctgacg 601 ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 661 ctgagctcgc ccgtcacaaa gagcttcaac
aggggagagt gttga (4) Protein Sequence Defining the Full Length
Chimeric 2B8 Light Chain (Chimeric 2B8 Kappa (Km(3))) (without
signal sequence) (SEQ ID NO. 157) 1 nivmtqspks msmsvgervt
lsckasenvv syvswyqqkp aqspklliyg asnrntgvpd 61 rftgsgsatd
ftltissvra edladyhcgq synypytfgg gtrleikrtv aapsvfifpp 121
sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt
181 lskadyekhk vyacevthqg lsspvtksfn rgec (5) Nucleic Acid Sequence
Encoding Humanized Hu2B8 Hv1-f.1 Heavy Chain Variable Region
(signal sequence underlined) (SEQ ID NO. 158) 1 atggactgca
cctggaggat cctcctcttg gtggcagcag ctacaggcac ccacgccgag 61
gtccagctgg tacagtctgg ggctgaggtg aagaagcctg gggctacagt gaaaatctcc
121 tgcaaggttt ctggatacac cttcaccacc tactggatgc actgggtgca
acaggcccct 181 ggaaaagggc ttgagtggat gggagagatt aatcctacca
acggtcatac taactacaat 241 gagaagttcc agggcagagt caccataacc
gcggacacgt ctacagacac agcctacatg 301 gagctgagca gcctgagatc
tgaggacacg gccgtgtatt actgtgcaac aaactatgtt 361 ggtagcatct
ttgactactg gggccaagga accctggtca ccgtctcctc ag (6) Protein Sequence
Defining Humanized Hu2B8 Hv1-f.1 Heavy Chain Variable Region
(without signal sequence) (SEQ ID NO. 159) 1 evqlvqsgae vkkpgatvki
sckvsgytft tywmhwvqqa pgkglewmge inptnghtny 61 nekfqgrvti
tadtstdtay melsslrsed tavyycatny vgsifdywgq gtlvtvss (7) Nucleic
Acid Sequence Encoding Human IgG1 Heavy Chain Constant Region
(G1m(17,1) allotype) (SEQ ID NO. 160) 1 cctccaccaa gggcccatcg
gtcttccccc tggcaccctc ctccaagagc acctctgggg 61 gcacagcggc
cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt 121
ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag
181 gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc
acccagacct 241 acatctgcaa cgtgaatcac aagcccagca acaccaaggt
ggacaagaaa gttgagccca 301 aatcttgtga caaaactcac acatgcccac
cgtgcccagc acctgaactc ctggggggac 361 cgtcagtctt cctcttcccc
ccaaaaccca aggacaccct catgatctcc cggacccctg 421 aggtcacatg
cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt 481
acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca
541 gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg
aatggcaagg 601 agtacaagtg caaggtctcc aacaaagccc tcccagcccc
catcgagaaa accatctcca 661 aagccaaagg gcagccccga gaaccacagg
tgtacaccct gcccccatcc cgggatgagc 721 tgaccaagaa ccaggtcagc
ctgacctgcc tggtcaaagg cttctatccc agcgacatcg 781 ccgtggagtg
ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc 841
tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc
901 agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac
cactacacgc 961 agaagagcct ctccctgtct ccgggtaaat ga (8) Protein
Sequence Defining Human IgG1 Heavy Chain Constant Region (G1m(17,1)
allotype) (SEQ ID NO. 161). The first amino acid is derived from
translation of the last nucleotide of variable region and beginning
two nucleotides of the IgG1 Heavy Chain sequence. 1 astkgpsvfp
lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss 61
glyslssvvt vpssslgtqt yicnvnhkps ntkvdkkvep kscdkthtcp pcpapellgg
121 psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna
ktkpreeqyn 181 styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis
kakgqprepq vytlppsrde 241 ltknqvsltc lvkgfypsdi avewesngqp
ennykttppv ldsdgsffly skltvdksrw 301 qqgnvfscsv mhealhnhyt
qkslslspgk (9) Nucleic Acid Sequence Encoding the Full Length Heavy
Chain Humanized Hu2B8 Hv1f.1 Variable Region and Human IgG1
(G1m(17,1) allotype) Heavy Chain Constant Region (signal sequence
underlined) (SEQ ID NO. 162) 1 atggactgca cctggaggat cctcctcttg
gtggcagcag ctacaggcac ccacgccgag 61 gtccagctgg tacagtctgg
ggctgaggtg aagaagcctg gggctacagt gaaaatctcc 121 tgcaaggttt
ctggatacac cttcaccacc tactggatgc actgggtgca acaggcccct 181
ggaaaagggc ttgagtggat gggagagatt aatcctacca acggtcatac taactacaat
241 gagaagttcc agggcagagt caccataacc gcggacacgt ctacagacac
agcctacatg 301 gagctgagca gcctgagatc tgaggacacg gccgtgtatt
actgtgcaac aaactatgtt 361 ggtagcatct ttgactactg gggccaagga
accctggtca ccgtctcctc agcctccacc 421 aagggcccat cggtcttccc
cctggcaccc tcctccaaga gcacctctgg gggcacagcg 481 gccctgggct
gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 541
ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac
601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac
ctacatctgc 661 aacgtgaatc acaagcccag caacaccaag gtggacaaga
aagttgagcc caaatcttgt 721 gacaaaactc acacatgccc accgtgccca
gcacctgaac tcctgggggg accgtcagtc 781 ttcctcttcc ccccaaaacc
caaggacacc ctcatgatct cccggacccc tgaggtcaca 841 tgcgtggtgg
tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 901
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac
961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa
ggagtacaag 1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga
aaaccatctc caaagccaaa 1081 gggcagcccc gagaaccaca ggtgtacacc
ctgcccccat cccgggatga gctgaccaag 1141 aaccaggtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1201 tgggagagca
atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1261
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg
1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac
gcagaagagc 1381 ctctccctgt ctccgggtaa atga (10) Protein Sequence
Defining the Full Length Heavy Chain Humanized Hu2B8 Hv1f.1
Variable Region and Human IgG1 Heavy Chain Constant Region
(G1m(17,1) allotype) (without signal sequence) (SEQ ID NO. 163) 1
evqlvqsgae vkkpgatvki sckvsgytft tywmhwvqqa pgkglewmge inptnghtny
61 nekfqgrvti tadtstdtay melsslrsed tavyycatny vgsifdywgq
gtlvtvssas 121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn
sgaltsgvht fpavlqssgl 181 yslsavvtvp ssslgtqtyi cnvnhkpsnt
kvdkkvepks cdkthtcppc papellggps 241 vflfppkpkd tlmisrtpev
tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst 301 yrvvsvltvl
hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsrdelt 361
knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq
421 gnvfscsvmh ealhnhytqk slslspgk (11) Nucleic Acid Sequence
Encoding Humanized Hu2B8 Hy5a.1 Heavy Chain Variable Region (signal
sequence underlined) (SEQ ID NO. 164) 1 atggggtcaa ccgccatcct
cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa 61 gtgcagctgg
tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaggatctcc 121
tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc
181 gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac
taactacaat 241 ccgtccttcc aaggccacgt caccatctca gctgacaagt
ccatcagcac tgcctacctg 301 cagtggagca gcctgaaggc ctcggacacc
gccatgtatt actgtgcgag aaactatgtt 361 ggtagcatct ttgactactg
gggccaagga accctggtca ccgtctcctc ag (12) Protein Sequence Defining
Humanized Hu2B8 Hv5a.1 Heavy Chain Variable Region (without signal
sequence) (SEQ ID NO. 165) 1 evqlvqsgae vkkpgeslri sckgsgysft
tywmhwvrqm pgkglewmge inptnghtny 61 npsfqghvti sadksistay
lqwsslkasd tamyycarny vgsifdywgq gtlvtvss (13) Nucleic Acid
Sequence Encoding the Full Length Humanized Hu2B8 Hv5a.1 Heavy
Chain Variable Region and Human IgG1 (G1m(17,1) allotype) Heavy
Chain Constant Region (signal sequence underlined) (SEQ ID NO. 166)
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa
61 gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct
gaggatctcc 121 tgtaagggtt ctggatacag ctttaccacc tactggatgc
actgggtgcg ccagatgccc 181 gggaaaggcc tggagtggat gggggagatt
aatcctacca acggtcatac taactacaat 241 ccgtccttcc aaggccacgt
caccatctca gctgacaagt ccatcagcac tgcctacctg 301 cagtggagca
gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt 361
ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc
421 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg
gggcacagcg 481 gccctgggct gcctggtcaa ggactacttc cccgaaccgg
tgacggtgtc gtggaactca 541 ggcgccctga ccagcggcgt gcacaccttc
ccggctgtcc tacagtcctc aggactctac 601 tccctcagca gcgtggtgac
cgtgccctcc agcagcttgg gcacccagac ctacatctgc 661 aacgtgaatc
acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 721
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc
781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc
tgaggtcaca 841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca
agttcaactg gtacgtggac 901 ggcgtggagg tgcataatgc caagacaaag
ccgcgggagg agcagtacaa cagcacgtac 961 cgtgtggtca gcgtcctcac
cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1021 tgcaaggtct
ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1081
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag
1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat
cgccgtggag 1201 tgggagagca atgggcagcc ggagaacaac tacaagacca
cgcctcccgt gctggactcc 1261 gacggctcct tcttcctcta cagcaagctc
accgtggaca agagcaggtg gcagcagggg 1321 aacgtcttct catgctccgt
gatgcatgag gctctgcaca accactacac gcagaagagc 1381 ctctccctgt
ctccgggtaa atga (14) Protein Sequence Defining the Full Length
Humanized Hu2B8 Hv5a.1 Heavy Chain Variable Region and Human IgG1
(G1m(17,1) allotype) Heavy Chain Constant Region (without signal
sequence) (SEQ ID NO. 167) 1 evqlvqsgae vkkpgeslri sckgsgysft
tywmhwvrqm pgkglewmge inptnghtny 61 npsfqghvti sadksistay
lqwsslkasd tamyycarny vgsifdywgq gtivtvssas 121 tkgpsvfpla
psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl 181
yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks cdkthtcppc papellggps
241 vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt
kpreeqynst 301 yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska
kgqprepqvy tlppsrdelt 361 knqvsltclv kgfypsdiav ewesngqpen
nykttppvld sdgsfflysk ltvdksrwqq 421 gnvfscsvmh ealhnhytqk slslspgk
(15) Nucleic Acid Sequence Encoding Humanized Hu2B8 Hv5-51.1 Heavy
Chain Variable Region (signal sequence underlined) (SEQ ID NO. 168)
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa
61 gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct
gaagatctcc 121 tgtaagggtt ctggatacag ctttaccacc tactggatgc
actgggtgcg ccagatgccc 181 gggaaaggcc tggagtggat gggggagatt
aatcctacca acggtcatac taactacaat 241 ccgtccttcc aaggccaggt
caccatctca gctgacaagt ccatcagcac tgcctacctg 301 cagtggagca
gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt 361
ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc ag (16)
Protein Sequence Defining Humanized Hu2B8 Hv5-51.1 Heavy Chain
Variable Sequence (without signal sequence) (SEQ ID NO. 169) 1
evqlvqsgae vkkpgeslki sckgsgysft tywmhwvrqm pgkglewmge inptnghtny
61 npsfqgqvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvss
(17) Nucleic Acid Sequence Encoding the Full Length Humanized Hu2B8
Hv5-51.1 Heavy Chain Variable Region and Human IgG1 (G1m(17,1)
allotype) Heavy Chain Constant Region (signal sequence underlined)
(SEQ ID NO. 170) 1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc
tccaaggagt ctgtgccgaa 61 gtgcagctgg tgcagtctgg agcagaggtg
aaaaagcccg gggagtctct gaagatctcc 121 tgtaagggtt ctggatacag
ctttaccacc tactggatgc actgggtgcg ccagatgccc 181 gggaaaggcc
tggagtggat gggggagatt aatcctacca acggtcatac taactacaat 241
ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac tgcctacctg
301 cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag
aaactatgtt 361 ggtagcatct ttgactactg gggccaagga accctggtca
ccgtctcctc agcctccacc 421 aagggcccat cggtcttccc cctggcaccc
tcctccaaga gcacctctgg gggcacagcg 481 gccctgggct gcctggtcaa
ggactacttc cccgaaccgg tgacggtgtc gtggaactca 541 ggcgccctga
ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 601
tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
661 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc
caaatcttgt 721 gacaaaactc acacatgccc accgtgccca gcacctgaac
tcctgggggg accgtcagtc
781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc
tgaggtcaca 841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca
agttcaactg gtacgtggac 901 ggcgtggagg tgcataatgc caagacaaag
ccgcgggagg agcagtacaa cagcacgtac 961 cgtgtggtca gcgtcctcac
cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1021 tgcaaggtct
ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1081
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag
1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat
cgccgtggag 1201 tgggagagca atgggcagcc ggagaacaac tacaagacca
cgcctcccgt gctggactcc 1261 gacggctcct tcttcctcta cagcaagctc
accgtggaca agagcaggtg gcagcagggg 1321 aacgtcttct catgctccgt
gatgcatgag gctctgcaca accactacac gcagaagagc 1381 ctctccctgt
ctccgggtaa atga (18) Protein Sequence Defining the Full Length
Humanized Hu2B8 Hv5-51.1 Heavy Chain Variable Region and Human IgG1
(G1m(17,1) allotype) Heavy Chain Constant Region (without signal
sequence) (SEQ ID NO. 171) 1 evqlvqsgae vkkpgeslki sckgsgysft
tywmhwvrqm pgkglewmge inptnghtny 61 npsfqgqvti sadksistay
lqwsslkasd tamyycarny vgsifdywgq gtivtvssas 121 tkgpsvfpla
psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl 181
yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks cdkthtcppc papellggps
241 vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt
kpreeqynst 301 yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska
kgqprepqvy tlppsrdelt 361 knqvsltclv kgfypsdiav ewesngqpen
nykttppvld sdgsfflysk ltvdksrwqq 421 gnvfscsvmh ealhnhytqk slslspgk
(19) Nucleic Acid Sequence Encoding Humanized Hu2B8 Kv1-39.1 Kappa
Chain Variable Region (signal sequence underlined) (SEQ ID NO.
172). Two possible start ATGs are shown in uppercase. 1 ATGgacATGa
gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc 61
agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga
121 gtcaccatca cttgcaaggc cagtgagaat gtggtttctt atgtatcctg
gtatcagcag 181 aaaccaggga aagcccctaa gctcctgatc tatggggcat
ccaaccggaa cactggggtc 241 ccatcaaggt tcagtggcag tggatctggg
acagatttca ctctcaccat cagcagtctg 301 caacctgaag attttgcaac
ttactactgt gggcagagtt acaactatcc gtacacgttt 361 ggccagggga
ccaagctgga gatcaaac (20) Protein Sequence Defining Humanized Hu2B8
Kv1-39.1 Kappa Chain Variable Region (without signal sequence) (SEQ
ID NO. 173) 1 diqmtqspss lsasvgdrvt itckasenvv syvswyqqkp
gkapklliyg asnrntgvps 61 rfsgsgsgtd ftltisslqp edfatyycgq
synypytfgq gtkleik (21) Nucleic Acid Sequence Encoding Human Kappa
Chain Constant Region (Km(3) allotype) (allele 2) (SEQ ID NO. 174)
1 gaactgtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg
61 gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc
aaagtacagt 121 ggaaggtgga taacgccctc caatcgggta actcccagga
gagtgtcaca gagcaggaca 181 gcaaggacag cacctacagc ctcagcagca
ccctgacgct gagcaaagca gactacgaga 241 aacacaaagt ctacgcctgc
gaagtcaccc atcagggcct gagctcgccc gtcacaaaga 301 gcttcaacag
gggagagtgt tga (22) Protein Sequence Defining Human Kappa Chain
Constant Region (Km(3) allotype) (allele 2) (SEQ ID NO. 175). The
first amino acid is derived from translation of the last nucleotide
of variable region and beginning two nucleotides of the Kappa Light
Chain sequence. 1 rtvaapsvfi fppsdeqlks gtasvvclln nfypreakvq
wkvdnalqsg nsqesvteqd 61 skdstyslss tltlskadye khkvyacevt
hqglsspvtk sfnrgec (23) Nucleic Acid Sequence Encoding the Full
Length Humanized Hu2B8 Kv1-39.1 Light Chain Variable Region and
Human Kappa Chain Constant Region (Km(3) allotype) (allele 2)
(signal sequence underlined) (SEQ ID NO. 176) 1 atggacatga
gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc 61
agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga
121 gtcaccatca cttgcaaggc cagtgagaat gtggtttctt atgtatcctg
gtatcagcag 181 aaaccaggga aagcccctaa gctcctgatc tatggggcat
ccaaccggaa cactggggtc 241 ccatcaaggt tcagtggcag tggatctggg
acagatttca ctctcaccat cagcagtctg 301 caacctgaag attttgcaac
ttactactgt gggcagagtt acaactatcc gtacacgttt 361 ggccagggga
ccaagctgga gatcaaacga actgtggctg caccatctgt cttcatcttc 421
ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac
481 ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca
atcgggtaac 541 tcccaggaga gtgtcacaga gcaggacagc aaggacagca
cctacagcct cagcagcacc 601 ctgacgctga gcaaagcaga ctacgagaaa
cacaaagtct acgcctgcga agtcacccat 661 cagggcctga gctcgcccgt
cacaaagagc ttcaacaggg gagagtgttg a (24) Protein Sequence Defining
the Full Length Humanized Hu2B8 Kv1-39.1 Light Chain Variable
Region and Human Kappa Chain Constant Region (Km(3) allotype)
(allele 1) (SEQ ID NO. 177) 1 diqmtqspss lsasvgdrvt itckasenvy
syvswyqqkp gkapklliyg asnrntgvps 61 rfsgsgsgtd ftltisslqp
edfatyycgq synypytfgq gtkleikrtv aapsvfifpp 121 sdeqlksgta
svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt 181
lskadyekhk vyacevthqg lsspvtksfn rgec (25) Nucleic Acid Sequence
Encoding Humanized Hu2B8 Kv3-15.1 Light Chain Variable Region
(signal sequence underlined) (SEQ ID NO. 178) 1 atggaagccc
cagcgcagct tctcttcctc ctgctactct ggctcccaga taccactgga 61
gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc
121 ctctcctgca aggccagtga gaatgtggtt tcttatgtat cctggtacca
gcagaaacct 181 ggccaggctc ccaggctcct catctatggg gcatccaacc
ggaacactgg tatcccagcc 241 aggttcagtg gcagtgggtc tgggacagag
ttcactctca ccatcagcag cctgcagtct 301 gaagattttg cagtttatta
ctgtgggcag agttacaact atccgtacac gtttggccag 361 gggaccaagc
tggagatcaa ac (26) Protein Sequence Defining Humanized Hu2B8
Kv3-15.1 Light Chain Variable Region (without signal sequence) (SEQ
ID NO. 179) 1 eivmtqspat lsyspgerat lsckasenvv syvswyqqkp
gqaprlliyg asnrntgipa 61 rfsgsgsgte ftltisslqs edfavyycgq
synypytfgq gtkleik (27) Nucleic Acid Encoding the Full Length
Humanized Hu2B8 Ky3-15.1 Light Chain Variable Region and Human
Kappa Chain Constant Region (Km(3) allotype) (allele 2) (signal
sequence underlined) (SEQ ID NO. 180) 1 atggaagccc cagcgcagct
tctcttcctc ctgctactct ggctcccaga taccactgga 61 gaaatagtga
tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 121
ctctcctgca aggccagtga gaatgtggtt tcttatgtat cctggtacca gcagaaacct
181 ggccaggctc ccaggctcct catctatggg gcatccaacc ggaacactgg
tatcccagcc 241 aggttcagtg gcagtgggtc tgggacagag ttcactctca
ccatcagcag cctgcagtct 301 gaagattttg cagtttatta ctgtgggcag
agttacaact atccgtacac gtttggccag 361 gggaccaagc tggagatcaa
acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 421 tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 481
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag
541 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag
caccctgacg 601 ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 661 ctgagctcgc ccgtcacaaa gagcttcaac
aggggagagt gttga (28) Protein Sequence Defining Humanized Hu2B8
Kv3-15.1 Light Chain Variable Region and Human Kappa Chain Constant
Region (Km(3) allotype) (allele 2) (without signal sequence) (SEQ
ID NO. 181) 1 eivmtqspat lsyspgerat lsckasenvv syvswyqqkp
gqaprlliyg asnrntgipa 61 rfsgsgsgte ftltisslqs edfavyycgq
synypytfgq gtkleikrtv aapsvfifpp 121 sdeqlksgta svvcllnnfy
preakvqwkv dnalqsgnsq esvteqdskd styslsstlt 181 lskadyekhk
vyacevthgg lsspvtksfn rgec
[0214] For convenience, Table 13 provides a concordance chart
showing the correspondence between the full length sequences and of
the antibodies discussed in this section with those presented in
the Sequence Listing.
TABLE-US-00020 TABLE 13 SEQ. ID NO. Protein or Nucleic Acid 154
Chimeric 2B8 IgG1 (G1m(17,1)) - nucleic acid 155 Chimeric 2B8 IgG1
(G1m(17,1)) - protein 156 Chimeric 2B8 Kappa (Km(3)) - nucleic acid
157 Chimeric 2B8 Kappa (Km(3)) - protein 158 Hu2B8 Hv1f.1 Heavy
Chain Variable Region - nucleic acid 159 Hu2B8 Hv1f.1 Heavy Chain
Variable Region - protein 160 Human IgG1 Heavy Chain Constant
Region (G1m(17,1)) allotype - nucleic acid 161 Human IgG1 Heavy
Chain Constant Region (G1m(17,1)) allotype - protein 162 Hu2B8
Hv1f.1 + IgG1 Constant (G1m(17,1) allotype) - nucleic acid 163
Hu2B8 Hv1f.1 + IgG1 Constant (G1m(17,1) allotype) - protein 164
Hu2B8 Hv5a.1 Heavy Chain Variable Region - nucleic acid 165 Hu2B8
Hv5a.1 Heavy Chain Variable Region - protein 166 Hu2B8 Hv5a.1 +
IgG1 Constant (G1m(17,1) allotype) - nucleic acid 167 Hu2B8 Hv5a.1
+ IgG1 Constant (G1m(17,1) allotype) - protein 168 Hu2B8 Hv5-51.1
Heavy Chain Variable Region - nucleic acid 169 Hu2B8 Hv5-51.1 Heavy
Chain Variable Region - protein 170 Hu2B8 Hv5-51.1 + IgG1 Constant
(G1m(17,1 allotype) - nucleic acid 171 Hu2B8 Hv5-51.1 + IgG1
Constant (G1m(17,1 allotype) - protein 172 Hu2B8 Kv1-39.1 Kappa
Chain Variable Region - nucleic acid 173 Hu2B8 Kv1-39.1 Kappa Chain
Variable Region - protein 174 Human Kappa Chain Constant Region
(Km(3) allotype) (allele 2) - nucleic acid 175 Human Kappa Chain
Constant Region (Km(3) allotype) (allele 2) - protein 176 Hu2B8
Kv1-39.1 + Kappa Constant (Km(3) allotype) (allele 2) - nucleic
acid 177 Hu2B8 Kv1-39.1 + Kappa Constant (Km(3) allotype) (allele
2) - protein 178 Hu2B8 Kv3-15.1 Kappa Chain Variable Region -
nucleic acid 179 Hu2B8 Kv3-15.1 Kappa Chain Variable Region -
protein 180 Hu2B8 Kv3-15.1 + Kappa Constant (Km(3) allotype)
(allele 2) - nucleic acid 181 Hu2B8 Kv3-15.1 + Kappa Constant
(Km(3) allotype) (allele 2) - protein
B. Humanization Procedure 2
[0215] The second humanization method employed for reducing
immunogenicity of the mouse 2B8 antibody is based on the method
described in Studnicka et al. (1994) PROTEIN ENG. 7:805-814. The
heavy and kappa human germline variable regions most identical (at
the amino acid level) to those of mouse 2B8 were identified.
Residues that differed between mouse and human were converted into
the human sequence depending on the likely risk that such a change
would affect binding or immunogenicity. Low risk residues (i.e.,
residues that when changed would likely not affect antigen binding
and would also reduce potential immunogenicity) were changed to the
human amino acid in the heavy variable region (creating LR2B8HC)
and the kappa variable region (creating LR2B8LC). Additionally, low
risk and medium risk (i.e., residues that when changed are somewhat
likely to have an effect on antigen binding residues and would also
reduce potential immunogenicity) were changed to the human amino
acid in the heavy variable region (creating LRMR2B8HC) and the
kappa variable region (creating LRMR2B8LC). The human IgG1 heavy
chain constant region (G1m(3) allotype (allele 1)) was added to the
carboxyl terminus of the two human engineered heavy variable
regions and the human Kappa constant region (Km(3) allotype (allele
1)) was added to the carboxyl terminus of two human engineered
light variable regions, thus creating four human engineered
antibody chains. Variable region nucleic acid sequences were first
synthesized by gene synthesis methods and then added to human
constant region sequences. These human engineered antibodies were
cloned into mammalian protein expression vectors, and protein was
expressed in the four possible combinations of heavy chain plus
light chain. Binding of the chimeric, chimeric/humanized, or
humanized antibodies to human HGF was measured using conventional
techniques, as described below.
[0216] The nucleic acid sequences encoding and the protein
sequences defining each of the humanized antibodies are summarized
below. In this section, the last nucleotide of each variable region
is the first base of the next codon generated by the
variable/constant region junction. This nucleotide is included in
the Variable Region because it is part of that exon. Amino acid
sequences of Constant Regions listed below include the translation
of this junction codon.
TABLE-US-00021 (1) Nucleic Acid Sequence Encoding the Humanized
LR2B8HC Heavy Chain Variable Region (signal sequence underlined)
(SEQ ID NO. 182) 1 atgggctggt catatattat tctctttctt gttgctaccg
ctaccgatgt gcactctcaa 61 gtccaactcg tacaaccagg cgctgaagtc
gtaaaacccg gaacatctgt taaactctca 121 tgcaaagcct caggatacac
tttcacaact tactggatgc attgggtcaa tcaagccccc 181 ggacaaggcc
tcgaatggat tggcgaaatt aacccaacta acggacatac taattataat 241
gaaaaattta agggcaaagc tacactcacc gtcgataaat caacctctac agcttatatg
301 gaactttcat ccctgagatc agaagataca gccgtctact attgcgccag
aaactacgta 361 ggatcaatat tcgattactg gggtcaaggc actctcctca
cagtcagctc ag c2) Protein Sequence Defining Humanized LR2B8HC Heavy
Chain Variable Region (without signal sequence) (SEQ ID NO. 183) 1
qvqlvqpgae vvkpgtsvkl sckasgytft tywmhwvnqa pgqglewige inptnghtny
61 nekfkgkatl tvdkststay melsslrsed tavyycarny vgsifdywgq gtlltvss
(3) Nucleic Acid Sequence Encoding the Human IgG1 Heavy Chain
Constant Region (G1m(3) allotype) (allele 1) (SEQ ID NO. 184) 1
ccagcacaaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg
61 gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg
acggtgtcgt 121 ggaactcagg cgccctgacc agcggcgtgc acaccttccc
ggctgtccta cagtcctcag 181 gactctactc cctcagcagc gtggtgaccg
tgccctccag cagcttgggc acccagacct 241 acatctgcaa cgtgaatcac
aagcccagca acaccaaggt ggacaagaga gttgagccca 301 aatcttgtga
caaaactcac acatgtccac cgtgcccagc acctgaactc ctggggggac 361
cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg
421 aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag
ttcaactggt 481 acgtggacgg cgtggaggtg cataatgcca agacaaagcc
gcgggaggag cagtacaaca 541 gcacgtaccg tgtggtcagc gtcctcaccg
tcctgcacca ggactggctg aatggcaagg 601 agtacaagtg caaggtctcc
aacaaagccc tcccagcccc catcgagaaa accatctcca 661 aagccaaagg
gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggaggaga 721
tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg
781 ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg
cctcccgtgc 841 tggactccga cggctccttc ttcctctata gcaagctcac
cgtggacaag ageaggtggc 901 agcaggggaa cgtcttctca tgctccgtga
tgcatgaggc tctgcacaac cactacacgc 961 agaagagcct ctccctgtcc
ccgggtaaat ga (4) Protein Sequence Defining Human IgG1 Heavy Chain
Constant Region (G1m(3) allotype) (allele 1 or 2) (SEQ ID NO. 185).
The first amino acid is derived from translation of the last
nucleotide of variable region and the beginning two nucleotides of
the IgG1 Heavy Chain sequence. 1 astkgpsvfp lapsskstsg gtaalgclvk
dyfpepvtvs wnsgaltsgv htfpavlqss 61 glyslssvvt vpssslgtqt
yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg 121 psvflfppkp
kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn 181
styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree
241 mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly
skltvdksrw 301 qqgnvfscsv mhealhnhyt qks1s1spgk (5) Nucleic Acid
Sequence Encoding the Full Length Heavy Chain Humanized LR2B8HC
Heavy Chain Variable Region and Human IgG1 Heavy Chain Constant
Region (G1m(3) allotype) (allele 1) (signal sequence underlined)
(SEQ ID NO. 186) 1 atgggctggt catatattat tctctttctt gttgctaccg
ctaccgatgt gcactctcaa 61 gtccaactcg tacaaccagg cgctgaagtc
gtaaaacccg gaacatctgt taaactctca 121 tgcaaagcct caggatacac
tttcacaact tactggatgc attgggtcaa tcaagccccc 181 ggacaaggcc
tcgaatggat tggcgaaatt aacccaacta acggacatac taattataat 241
gaaaaattta agggcaaagc tacactcacc gtcgataaat caacctctac agcttatatg
301 gaactttcat ccctgagatc agaagataca gccgtctact attgcgccag
aaactacgta 361 ggatcaatat tcgattactg gggtcaaggc actctcctca
cagtcagctc agccagcaca 421 aagggcccat cggtcttccc cctggcaccc
tcctccaaga gcacctctgg gggcacagcg 481 gccctgggct gcctggtcaa
ggactacttc cccgaaccgg tgacggtgtc gtggaactca 541 ggcgccctga
ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 601
tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
661 aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc
caaatcttgt 721 gacaaaactc acacatgtcc accgtgccca gcacctgaac
tcctgggggg accgtcagtc 781 ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca 841 tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 901 ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 961
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag
1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc
caaagccaaa 1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggagga gatgaccaag 1141 aaccaggtca gcctgacctg cctggtcaaa
ggcttctatc ccagcgacat cgccgtggag 1201 tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1261 gacggctcct
tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1321
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc
1381 ctctccctgt ccccgggtaa atga (6) Protein Sequence Defining the
Full Length Heavy Chain Humanized LR2B8HC Heavy Chain Variable
Region and Human IgG1 Heavy Chain Constant Region (G1m(3) allotype)
(allele 1) (without signal sequence) (SEQ ID NO. 187) 1 qvqlvqpgae
vvkpgtsvkl sckasgytft tywmhwvnqa pgqglewige inptnghtny 61
nekfkgkatl tvdkststay melsslrsed tavyycarny vgsifdywgq gtlltvssas
121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht
fpavlqssgl 181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkrvepks
cdkthtcppc papellggps 241 vflfppkpkd tlmisrtpev tcvvvdvshe
dpevkfnwyv dgvevhnakt kpreeqynst 301 yrvvsvltvl hqdwlngkey
kckvsnkalp apiektiska kgqprepqvy tlppsreemt 361 knqvsltclv
kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq 421
gnvfscsvmh ealhnhytqk slslspgk (7) Nucleic Acid Sequence Encoding
the Humanized LRMR2B8HC Heavy Chain Variable Region (signal
sequence underlined) (SEQ ID NO. 188) 1 atgggttggt catatattat
actctttctc gtagccaccg ccaccgacgt acactctcag 61 gttcaactcg
tacaacccgg cgccgaagtc aagaaaccag gaacatcagt caaactctca 121
tgtaaagcaa gcggatacac ctttactact tattggatgc attgggtaag acaagccccc
181 ggacaaggac tcgaatggat aggcgaaata aatcccacta atggacatac
aaattataat 241 caaaaatttc aaggacgcgc tacactcacc gtcgataaat
caacctcaac cgcatacatg 301 gaactcagct ccctccgatc cgaagacact
gccgtttatt attgtgccag aaactatgta 361 ggatctattt tcgattactg
gggacaagga acacttctca ccgtaagctc ag (8) Protein Sequence Defining
Humanized LRMR2B8HC Heavy Chain Variable Region (without signal
sequence) (SEQ ID NO. 189) 1 qvqlvqpgae vkkpgtsvkl sckasgytft
tywmhwvrqa pgqglewige inptnghtny 61 nqkfqgratl tvdkststay
melsslrsed tavyycarny vgsifdywgq gtlltvss (9) Nucleic Acid Sequence
Encoding the Full Length Heavy Chain Humanized LRMR2B8HC Heavy
Chain Variable Region and Human IgG1 Heavy Chain Constant Region
(G1m(3) allotype) (allele 1) (signal sequence underlined) (SEQ ID
NO. 190) 1 atgggttggt catatattat actctttctc gtagccaccg ccaccgacgt
acactctcag 61 gttcaactcg tacaacccgg cgccgaagtc aagaaaccag
gaacatcagt caaactctca 121 tgtaaagcaa gcggatacac ctttactact
tattggatgc attgggtaag acaagccccc 181 ggacaaggac tcgaatggat
aggcgaaata aatcccacta atggacatac aaattataat 241 caaaaatttc
aaggacgcgc tacactcacc gtcgataaat caacctcaac cgcatacatg 301
gaactcagct ccctccgatc cgaagacact gccgtttatt attgtgccag aaactatgta
361 ggatctattt tcgattactg gggacaagga acacttctca ccgtaagctc
agccagcaca 421 aagggcccat cggtcttccc cctggcaccc tcctccaaga
gcacctctgg gggcacagcg 481 gccctgggct gcctggtcaa ggactacttc
cccgaaccgg tgacggtgtc gtggaactca 541 ggcgccctga ccagcggcgt
gcacaccttc ccggctgtcc tacagtcctc aggactctac 601 tccctcagca
gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 661
aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt
721 gacaaaactc acacatgtcc accgtgccca gcacctgaac tcctgggggg
accgtcagtc 781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct
cccggacccc tgaggtcaca 841 tgcgtggtgg tggacgtgag ccacgaagac
cctgaggtca agttcaactg gtacgtggac 901 ggcgtggagg tgcataatgc
caagacaaag ccgcgggagg agcagtacaa cagcacgtac 961 cgtgtggtca
gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1021
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa
1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga
gatgaccaag 1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc
ccagcgacat cgccgtggag 1201 tgggagagca atgggcagcc ggagaacaac
tacaagacca cgcctcccgt gctggactcc 1261 gacggctcct tcttcctcta
tagcaagctc accgtggaca agagcaggtg gcagcagggg 1321 aacgtcttct
catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1381
ctctccctgt ccccgggtaa atga (10) Protein Sequence Defining the Full
Length Heavy Chain Humanized LRMR2B8HC Heavy Chain Variable Region
and Human IgG1 Heavy Chain Constant Region (G1m(3) allotype)
(allele 1) (without signal sequence) (SEQ ID NO. 191) 1 qvqlvqpgae
vkkpgtsvkl sckasgytft tywmhwvrqa pgqglewige inptnghtny 61
nqkfqgratl tvdkststay melsslrsed tavyycarny vgsifdywgq gtlltvssas
121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht
fpavlqssgl 181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkrvepks
cdkthtcppc papellggps 241 vflfppkpkd tlmisrtpev tcvvvdvshe
dpevkfnwyv dgvevhnakt kpreeqynst 301 yrvvsvltvl hqdwlngkey
kckvsnkalp apiektiska kgqprepqvy tlppsreemt 361 knqvsltclv
kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq 421
gnvfscsvmh ealhnhytqk slslspgk (11) Nucleic Acid Sequence Encoding
the Humanized LR2B8LC Light Chain Variable Region (signal sequence
underlined) (SEQ ID NO. 192) 1 atggaaagtc agacccttgt attcatctct
attcttcttt ggttgtatgg agcagacggc 61 gacattgtga tgacccaatc
ccccgatagt atggccatga gtgtaggaga aagagtcacc 121 cttaattgca
aagcctccga aaatgtcgtt tcatatgtgt cttggtatca acaaaaaccc 181
ggccaatcac ccaaacttct catatacggc gcttcaaaca gaaacacagg cgttcccgac
241 agatttagtg gatccggatc agctacagat ttcaccctta ccatcagttc
agttcaagca 301 gaagacgttg cagactatca ttgcggacaa tcttataact
acccttacac attcggacaa (12) Protein Sequence Defining Humanized
LR2B8LC Light Chain Variable Region (without signal sequence) (SEQ
ID NO. 193) 1 divmtqspds mamsvgervt lnckasenvv syvswyqqkp
gqspklliyg asnrntgvpd 61 rfsgsgsatd ftltissvqa edvadyhcgq
synypytfgq gtkleik (13) Nucleic Acid Sequence Encoding the Human
Kappa Chain Constant Region (Km(3) allotype) (allele 1) (SEQ ID NO.
194) 1 gtacggtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag
ttgaaatctg 61 gaactgcctc tgttgtgtgc ctgctgaata acttctatcc
cagagaggcc aaagtacagt 121 ggaaggtgga taacgccctc caatcgggta
actcccagga gagtgtcaca gagcaggaca 181 gcaaggacag cacctacagc
ctcagcagca ccctgacgct gagcaaagca gactacgaga 241 aacacaaagt
ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga 301
gcttcaacag gggagagtgt tag (14) Protein Sequence Defining the Human
Kappa Chain Constant Region (Km(3) allotype) (allele 1) (SEQ ID NO.
195). The first amino acid derived from translation of the last
nucleotide of variable region and beginning two nucleotides of the
Kappa Light Chain sequence. 1 rtvaapsvfi fppsdeqlks gtasvvclln
nfypreakvq wkvdnalqsg nsqesvteqd 61 skdstyslss tltlskadye
khkvyacevt hqglsspvtk sfnrgec (15) Nucleic Acid Sequence Encoding
the Full Length Humanized LR2B8LC Light Chain Variable Region and
the Human Kappa Chain Constant Region (Km(3) allotype) (allele 1)
(SEQ ID NO. 196) 1 atggaaagtc agacccttgt attcatctct attcttcttt
ggttgtatgg agcagacggc 61 gacattgtga tgacccaatc ccccgatagt
atggccatga gtgtaggaga aagagtcacc 121 cttaattgca aagcctccga
aaatgtcgtt tcatatgtgt cttggtatca acaaaaaccc 181 ggccaatcac
ccaaacttct catatacggc gcttcaaaca gaaacacagg cgttcccgac 241
agatttagtg gatccggatc agctacagat ttcaccctta ccatcagttc agttcaagca
301 gaagacgttg cagactatca ttgcggacaa tcttataact acccttacac
attcggacaa 361 ggaaccaaac tcgaaattaa acgtacggtg gctgcaccat
ctgtcttcat cttcccgcca 421 tctgatgagc agttgaaatc tggaactgcc
tctgttgtgt gcctgctgaa taacttctat 481 cccagagagg ccaaagtaca
gtggaaggtg gataacgccc tccaatcggg taactcccag 541 gagagtgtca
cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 601
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc
661 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag (16) Protein
Sequence Encoding the Full Length Humanized LR2B8LC Light Chain
Variable Region and the Human Kappa Chain Constant Region (Km(3)
allotype) (allele 1) (SEQ ID NO. 197) 1 divmtqspds mamsvgervt
lnckasenvv syvswyqqkp gqspklliyg asnrntgvpd 61 rfsgsgsatd
ftltissvqa edvadyhcgq synypytfgq gtkleikrtv aapsvfifpp 121
sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt
181 lskadyekhk vyacevthqg lsspvtksfn rgec (17) Nucleic Acid
Sequence Encoding the Humanized LRMR2B8LC Light Chain Variable
Region (signal sequence underlined) (SEQ ID NO. 198) 1 atggaatccc
aaacccttgt tttcatctct atccttctct ggctttatgg cgccgacgga 61
gacatcgtaa tgacacaatc ccctgactct cttgctatga gcttgggcga acgagtaaca
121 cttaactgca aagcatccga aaatgtcgta tcttacgtat cctggtatca
gcaaaaacct 181 ggtcaaagtc ctaaacttct tatatatggt gcaagtaatc
gtgaaagtgg cgtcccagac 241 agatttagcg gttcaggttc agcaactgac
tttacactta caatttctag cgttcaggcc 301 gaagacgttg cagactatca
ttgtggacaa tcttataact atccttatac tttcggacaa 361 ggcactaaac
ttgaaattaa ac (18) Protein Sequence Defining the Humanized
LRMR2B8LC Light Chain Variable Region (without signal sequence)
(SEQ ID NO. 199) 1 divmtqspds lamslgervt lnckasenyv syvswyqqkp
gqspklliyg asnresgvpd 61 rfsgsgsatd ftltissvqa edvadyhcgq
synypytfgq gtkleik (19) Nucleic Acid Sequence Encoding the Full
Length Humanized LRMR2B8LC Light Chain Variable Region and the
Human Kappa Chain Constant Region (Km(3) allotype) (allele 1)
(signal sequence underlined) (SEQ ID NO. 200) 1 atggaatccc
aaacccttgt tttcatctct atccttctct ggctttatgg cgccgacgga 61
gacatcgtaa tgacacaatc ccctgactct cttgctatga gcttgggcga acgagtaaca
121 cttaactgca aagcatccga aaatgtcgta tcttacgtat cctggtatca
gcaaaaacct 181 ggtcaaagtc ctaaacttct tatatatggt gcaagtaatc
gtgaaagtgg cgtcccagac 241 agatttagcg gttcaggttc agcaactgac
tttacactta caatttctag cgttcaggcc 301 gaagacgttg cagactatca
ttgtggacaa tcttataact atccttatac tttcggacaa 361 ggcactaaac
ttgaaattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 421
tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat
481 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 541 gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 601 ctgagcaaag cagactacga gaaacacaaa
gtctacgcct gcgaagtcac ccatcagggc 661 ctgagctcgc ccgtcacaaa
gagcttcaac aggggagagt gttag (20) Protein Sequence Defining the Full
Length Humanized LRMR2B8LC Light Chain Variable Region and the
Human Kappa Chain Constant Region (Km(3) allotype) (allele 1) (SEQ
ID NO. 201) 1 divmtqspds lamslgervt lnckasenvv syvswyqqkp
gqspklliyg asnresgvpd 61 rfsgsgsatd ftltissvqa edvadyhcgq
synypytfgq gtkleikrtv aapsvfifpp
121 sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd
styslsstlt 181 lskadyekhk vyacevthqg lsspvtksfn rgec
[0217] For convenience, Table 14 provides a concordance chart
showing the correspondence between the full length sequences and of
the antibodies discussed in this section with those presented in
the Sequence Listing.
TABLE-US-00022 TABLE 14 SEQ. ID NO. Protein or Nucleic Acid 182
LR2B8HC Heavy Chain Variable Region - nucleic acid 183 LR2B8HC
Heavy Chain Variable Region - protein 184 Human IgG1 Heavy Chain
Constant Region (G1m(3) allotype) (allele 1) - nucleic acid 185
Human IgG1 Heavy Chain Constant Region (G1m(3) allotype) (allele 1)
- protein 186 LR2B8HC + IgG1 Constant (G1m(3) allotype) (allele 1)
- nucleic acid 187 LR2B8HC + IgG1 Constant (G1m(3) allotype)
(allele 1) - protein 188 LRMR2B8HC Heavy Chain Variable Region -
nucleic acid 189 LRMR2B8HC Heavy Chain Variable Region - protein
190 LRMR2B8HC + IgG1 Constant (G1m(3) allotype) (allele 1) -
nucleic acid 191 LRMR2B8HC + IgG1 Constant (G1m(3) allotype)
(allele 1) - protein 192 LR2B8LC Light Chain Variable Region -
nucleic acid 193 LR2B8LC Light Chain Variable Region - protein 194
Human Kappa Chain Constant Region (Km(3) allotype) (allele 1) -
nucleic acid 195 Human Kappa Chain Constant Region (Km(3) allotype)
(allele 1) - protein 196 LR2B8LC + Kappa Constant (Km(3) allotype)
(allele 1) - nucleic acid 197 LR2B8LC + Kappa Constant (Km(3)
allotype) (allele 1) - protein 198 LRMR2B8LC Light Chain Variable
Region - nucleic acid 199 LRMR2B8LC Light Chain Variable Region -
protein 200 LRMR2B8LC + Kappa Constant (Km(3) allotype) (allele 1)
- nucleic acid 201 LRMR2B8LC + Kappa Constant (Km(3) allotype)
(allele 1) - protein
[0218] Table 15 summarizes the heavy chain CDR sequences (Kabat
Definition) of the humanized 2B8 antibodies prepared by
humanization procedure 1 and by humanization procedure 2 described
herein above in this Example.
TABLE-US-00023 TABLE 15 Full Length Heavy Chain Antibody CDR1 CDR2
CDR3 Variable Region Murine 2B8 TYWMH EINPTNGHTNYNEKFKS NYVGSIFDY
SEQ ID NO: 12 Heavy (SEQ ID NO: 15) (SEQ ID NO: 16) (SEQ ID NO: 17)
Hu2B8 Hv1f.1 TYWMH EINPTNGHTNYNEKFQG NYVGSIFDY SEQ ID NO: 159 (SEQ
ID NO: 15) (SEQ ID NO: 202) (SEQ ID NO: 17) Hu2B8 Hv5a.1 TYWMH
EINPTNGHTNYNPSFQG NYVGSIFDY SEQ ID NO: 165 (SEQ ID NO: 15) (SEQ ID
NO: 203) (SEQ ID NO: 17) Hu2B8 Hv5- TYWMH EINPTNGHTNYNPSFQG
NYVGSIFDY SEQ ID NO: 169 51.1 (SEQ ID NO: 15) (SEQ ID NO: 203) (SEQ
ID NO: 17) LR2B8HC TYWMH EINPTNGHTNYNEKFKG NYVGSIFDY SEQ ID NO: 183
(SEQ ID NO: 15) (SEQ ID NO: 204) (SEQ ID NO: 17) LRMR2B8HC TYWMH
EINPTNGHTNYNQKFQG NYVGSIFDY SEQ ID NO: 189 (SEQ ID NO: 15) (SEQ ID
NO: 205) (SEQ ID NO: 17)
[0219] Table 16 summarizes the light chain CDR sequences (Kabat
Definition) of the humanized 2B8 antibodies prepared by
humanization procedure 1 and by humanization procedure 2 described
herein above in this Example.
TABLE-US-00024 TABLE 16 Full Length Light Chain Antibody CDR1 CDR2
CDR3 Variable Region Murine 2B8 Light KASENVVSYVS GASNRNT GQSYNYPYT
SEQ ID NO: 14 (SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 20) Hu2B8
Kv1-39.1 KASENVVSYVS GASNRNT GQSYNYPYT SEQ ID NO: 173 (SEQ ID NO:
18) (SEQ ID NO: 19) (SEQ ID NO: 20) Hu2B8 Kv3-15.1 KASENVVSYVS
GASNRNT GQSYNYPYT SEQ ID NO: 179 (SEQ ID NO: 18) (SEQ ID NO: 19)
(SEQ ID NO: 20) LR2B8LC KASENVVSYVS GASNRNT GQSYNYPYT SEQ ID NO:
193 (SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 20) LRMR2B8LC
KASENVVSYVS GASNRES GQSYNYPYT SEQ ID NO: 199 (SEQ ID NO: 18) (SEQ
ID NO: 206) (SEQ ID NO: 20)
C. Binding Affinity of Humanized 2B8 Antibodies
[0220] Antigen-binding affinity and kinetics of interaction were
assessed by surface plasmon resonance technology using a BIAcore
T100 instrument. Mouse anti-human immunoglobulins (Jackson
ImmunoResearch Labs, 209-005-098) were immobilized on
carboxymethylated dextran CM4 sensor chips (BIAcore, Catalog No.
BR-1005-34) by amine coupling (BIAcore, Catalog No. BR-1000-50)
using a standard coupling protocol according to manufacturer's
recommendations. The analyses were performed at 25.degree. C. using
PBS (GIBCO, Catalog No. 14040-133) containing 0.05% surfactant P20
(BIAcore, Catalog No. BR-1000-54), 2 mg/mL BSA (EMD, Catalog No.
2930) and 10 mg/mL CM-Dextran Sodium salt (Fluka, Catalog No.
86524) as running buffer.
[0221] The antibodies were captured on individual flow cell at a
flow rate of 10 .mu.L/min. Injection time was variable for each
antibody to yield approximately 20 RU of antibody captured for each
cycle. Buffer or HGF (R&D Systems, Catalog No. 294-HGN-025)
diluted in running buffer was injected sequentially over a
reference surface (no antibody captured) and the active surface
(antibody to be tested) for 2 minutes at 60 .mu.L/min. The
dissociation phase was monitored for 15 or 90 minutes, depending on
concentration. The surface then was regenerated with 10 mM
Glycine-HCl, pH 2.0 (BIAcore, Catalog No. BR-1003-55) injected for
3 minutes at a flow rate of 60 .mu.L/min before another cycle was
initiated. HGF concentrations tested were 1.88, 3.75 and 7.5 nM.
Determination of kinetic parameters was achieved using the kinetic
function of the BIAevalutation software with reference subtraction.
Kinetic parameters for each antibody, k.sub.a (association rate
constant), k.sub.d (dissociation rate constant) and K.sub.D
(equilibrium dissociation constant) are summarized in FIG. 8.
[0222] The results summarized in FIG. 8 show that certain
combinations of superhumanized heavy chains (Hu2B8 Hv5a.1, Hu2B8
Hv5-51.1 or Hu2B8 Hv1-f.1) and light chains (Hu2B8 Kv1-39.1 or
Hu2B8 Kv3-15.1) retain similar binding affinity (K.sub.D) to HGF as
chimeric 2B8 (mouse variable regions with human constant regions)
and 2B8 (Table 5).
D. Mutually Exclusive Binding Assay
[0223] Mutually exclusive binding to HGF was assessed by surface
plasmon resonance technology using a BIAcore T100 instrument. Mouse
anti-human immunoglobulins (Jackson ImmunoResearch Labs,
209-005-098) were immobilized on carboxymethylated dextran CM5
sensor chips (BIAcore, Catalog No. BR-1006-68) by amine coupling
(BIAcore, Catalog No. BR-1000-50) using a standard coupling
protocol according to manufacturer's recommendations. The analyses
were performed at 25.degree. C. using PBS (GIBCO, Catalog No.
14040-133) containing 0.05% surfactant P20 (BIAcore, #BR-1000-54),
2 mg/mL BSA (EMD, Catalog No. 2930) and 10 mg/ml CM-Dextran Sodium
salt (Fluka, Catalog No. 86524) as running buffer.
[0224] The humanized antibodies were captured on an individual flow
cell at a flow rate of 30 .mu.L/min. Injection time was variable
for each antibody to yield approximately 150 RU of antibody
captured for each cycle. HGF (R&D Systems, Catalog No.
294-HGN-025) diluted in running buffer at a final concentration of
7.5 .mu.g/mL was injected for 90 sec at 30 .mu.L/min over the
captured humanized antibodies. Binding of HGF was monitored before
subsequent injection of mouse 2B8 antibody or polyclonal goat
anti-HGF antibody (R & D Systems, AF294) for 3 min at 30
.mu.L/min. The surface then was regenerated with 10 mM Glycine-HCl,
pH 2.0 (BIAcore, Catalog No. BR-1003-55) injected for 3 min at a
flow rate of 60 .mu.L/min before another antibody was tested. The
results are summarized in FIG. 9.
[0225] Results summarized in FIG. 9 show that both humanized 2B8
antibodies and chimeric 2B8 antibodies prevent murine 2B8 from
binding HGF. These results demonstrate that the humanized
antibodies still bind the same HGF epitope as the original 2B8
antibody.
Example 13
Production of Humanized 2B8 Variants
a. HUMAN ENGINEERED.TM. Antibodies
[0226] Codon- and expression-optimized low risk and
low-plus-moderate risk Human Engineered light chain (LR2B8LC and
LRMR2B8LC, respectively) and heavy chains (LR2B8HC and LRMR2B8HC,
respectively) were cloned in-phase into XOMA's transient antibody
expression vectors, which contain human Kappa and Gamma-1 constant
regions modules. The four Human Engineered 2B8 variants were
produced by transient transfection in HEK293E cells. The following
four antibodies were produced: [0227] HE2B8-1=LR2B8HC (+IgG1
constant region (G1m(3) allotype (allele 1)) (SEQ ID NO. 187) plus
LR2B8LC (+Kappa constant region (Km(3) allotype (allele 1))) (SEQ
ID NO. 197) [0228] HE2B8-2=LR2B8HC (+IgG1 constant region (G1m(3)
allotype (allele 1)) (SEQ ID NO. 187) plus LRMR2B8LC (+Kappa
constant region (Km(3) allotype (allele 1))) (SEQ ID NO. 201)
[0229] HE2B8-3=LRMR2B8HC (+IgG1 constant region (G1m(3) allotype
(allele 1)) (SEQ ID NO. 191) plus LR2B8LC (+Kappa constant region
(Km(3) allotype (allele 1))) (SEQ ID NO. 197) [0230]
HE2B8-4=LRMR2B8HC (+IgG1 constant region (G1m(3) allotype (allele
1)) (SEQ ID NO. 191) plus LRMR2B8LC (+Kappa constant region (Km(3)
allotype (allele 1))) (SEQ ID NO. 201)
[0231] The light and heavy chains were co-transfected into XOMA's
suspension adapted HEK293E cells grown in IS293 media (Irvine
Scientific, Irvine, Calif.) using 2 liter shake flasks. After 24
hours in the shake flasks, 200 mL of transfected cells were
centrifuged, resuspended in 40 mL of fresh medium and transferred
to Integra flasks (Wilson Wolf Manufacturing Inc., MN) for
production. After incubation for seven days, the cell suspensions
were removed from the Integra flasks, centrifuged and the culture
supernatants retained. Antibodies in the culture supernatants were
purified on protein A spin columns (Pro-Chem), dialyzed against
PBS, concentrated and sterile filtered.
b. SUPERHUMANIZED.TM. Antibodies
[0232] Full length Hu2B8_Hv5-51.1+human IgG1 constant domain
(G1m(3) allotype) cDNA was cloned into pEE6.4 (Lonza Biologics,
Berkshire, UK) using HindIII and EcoRI restriction sites. Full
length Hu2B8-Kv1-39.1 variable region+human Kappa constant domain
cDNA and full length Hu2B8_Kv3-15.1 variable region+human Kappa
constant domain cDNA were each cloned into pEE14.4 (Lonza
Biologics) using HindIII and EcoRI restriction sites. The hCMV-MIE
promoter+full length Hu2B8_Hv5-51.1+human IgG1 constant domain
(G1m(3) allotype) cDNA+SV40 poly A fragment (in pEE6.4) was removed
by NotI/SalI digestion and inserted into either Kappa chain pEE14.4
vector through NotI/SalI sites, thus creating 2 different
expression vectors that each simultaneously express heavy and light
chain to make the following antibodies: [0233] sh2B8-9
(G1m(3))=hu2B8 Hv5-51.1 (+IgG1 constant region (G1m(3) allotype)
(allele 2)) (SEQ ID NO. 210) plus hu2B8 Kv 1-39.1 (+Kappa constant
region (Km(3) allotype (allele 2))) (SEQ ID NO: 177) [0234]
sh2B8-12 (G1m(3))=hu2B8 Hv5-51.1 (+IgG1 constant region (G1m(3)
allotype) (allele 2)) (SEQ ID NO. 210) plus hu2B8 Kv 3-15.1 (+Kappa
constant region (Km(3) allotype (allele 2))) (SEQ ID No. 181)
[0235] The nucleic acid sequences encoding and the protein
sequences defining the human IgG1 Heavy Constant Region G1m(3)
allotype (allele 2) and each of the full length heavy chain
sequences are set forth below. The light chain sequences were the
same as described in Example 12.
TABLE-US-00025 (1) Nucleic Acid Sequence Encoding Human IgG1 Heavy
Chain Constant Region (G1m(3) allotype) (allele 2) (SEQ ID NO. 207)
1 cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg
61 gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg
acggtgtcgt 121 ggaactcagg cgccctgacc agcggcgtgc acaccttccc
ggctgtccta cagtcctcag 181 gactctactc cctcagcagc gtggtgaccg
tgccctccag cagcttgggc acccagacct 241 acatctgcaa cgtgaatcac
aagcccagca acaccaaggt ggacaagaga gttgagccca 301 aatcttgtga
caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac 361
cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg
421 aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag
ttcaactggt 481 acgtggacgg cgtggaggtg cataatgcca agacaaagcc
gcgggaggag cagtacaaca 541 gcacgtaccg tgtggtcagc gtcctcaccg
tcctgcacca ggactggctg aatggcaagg 601 agtacaagtg caaggtctcc
aacaaagccc tcccagcccc catcgagaag accatctcca 661 aagccaaagg
gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggaggaga 721
tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg
781 ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg
cctcccgtgc 841 tggactccga cggctccttc ttcctctaca gcaagctcac
cgtggacaag agcaggtggc 901 agcaggggaa cgtcttctca tgctccgtga
tgcatgaggc tctgcacaac cactacacgc 961 agaagagcct ctccctgtct
ccgggtaaat ga (2) Protein Sequence Defining Human IgG1 Heavy Chain
Constant Region (G1m(3) allotype) (allele 1 or 2) (SEQ ID NO. 208).
The first amino acid is derived from translation of the last
nucleotide of variable region and the beginning two nucleotides of
the IgG1 Heavy Chain sequence. 1 astkgpsvfp lapsskstsg gtaalgclvk
dyfpepvtvs wnsgaltsgv htfpavlqss 61 glyslssvvt vpssslgtqt
yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg 121 psvflfppkp
kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn 181
styrvvsvlt vlhqdwlngh eykckvsnka lpapieltis kakgqprepq vytlppsree
241 mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly
skltvdksrw 301 qqgnvfscsv mhealhnhyt qkslslspgk (3) Nucleic Acid
Sequence Encoding the Full Length Chain Containing Humanized Hu2B8
Hv5-51.1 Heavy Chain Variable Region and the Human IgG1 Heavy Chain
Constant Region G1m(3) allotype (allele 2) (signal sequence
underlined) (SEQ ID NO. 209) 1 atggggtcaa ccgccatcct cgccctcctc
ctggctgttc tccaaggagt ctgtgccgaa 61 gtgcagctgg tgcagtctgg
agcagaggtg aaaaagcccg gggagtctct gaagatctcc 121 tgtaagggtt
ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc 181
gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat
241 ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac
tgcctacctg 301 cagtggagca gcctgaaggc ctcggacacc gccatgtatt
actgtgcgag aaactatgtt 361 ggtagcatct ttgactactg gggccaagga
accctggtca ccgtctcctc agcctccacc 421 aagggcccat cggtcttccc
cctggcaccc tcctccaaga gcacctctgg gggcacagcg 481 gccctgggct
gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 541
ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac
601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac
ctacatctgc 661 aacgtgaatc acaagcccag caacaccaag gtggacaaga
gagttgagcc caaatcttgt 721 gacaaaactc acacatgccc accgtgccca
gcacctgaac tcctgggggg accgtcagtc 781 ttcctcttcc ccccaaaacc
caaggacacc ctcatgatct cccggacccc tgaggtcaca 841 tgcgtggtgg
tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 901
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac
961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa
ggagtacaag 1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga
agaccatctc caaagccaaa 1081 gggcagcccc gagaaccaca ggtgtacacc
ctgcccccat cccgggagga gatgaccaag 1141 aaccaggtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1201 tgggagagca
atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1261
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg
1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac
gcagaagagc 1381 ctctccctgt ctccgggtaa atga (4) Protein Sequence
Defining the Full Length Heavy Chain Containing Humanized Hu2B8
Hv5-51.1 and the Human IgG1 Heavy Chain Constant Region G1m(3)
allotype (allele 2) (without signal sequence) (SEQ ID NO. 210) 1
evqlvqsgae vkkpgeslki sckgsgysft tywmhwvrqm pgkglewmge inptnghtny
61 npsfqgqvti sadksistay lqwsslkasd tamyycarny vgsifdywgq
gtlvtvssas 121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn
sgaltsgvht fpavlqssgl 181 yslssvvtvp ssslgtqtyi cnvnhkpsnt
kvdkrvepks cdkthtcppc papellggps 241 vflfppkpkd tlmisrtpev
tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst 301 yrvvsvltvl
hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsreemt 361
knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq
421 gnvfscsvmh ealhnhytqk slslspgk
[0236] Each dual expression vector was transfected into 293T cells
for transient expression using DMEM 10% fetal bovine serum.
Forty-eight hours after transfection, cells were washed with and
then replaced with serum free medium, IS GRO.TM. (Irvine
Scientific, Santa Ana, Calif.) containing 4 mM L-Glutamine.
Supernatant was harvested daily and replaced with fresh media for
10 days. The culture supernatants were centrifuged, filtered (0.45
.mu.m) and concentrated 10-100 fold. Antibodies were purified on
ProSep vA resin (Millipore), dialyzed against PBS, concentrated and
sterile filtered.
Example 14
Binding Characteristics of Humanized 2B8 Variants
[0237] The humanized antibodies produced in Example 13 were
characterized by their ability to bind hHGF and the recombinant HGF
proteins produced in Example 3.
[0238] The antibodies were analyzed by surface-plasmon resonance
using a BIAcore T100 instrument to assess their ability to bind
hHGF and the fusion proteins discussed in Example 3. Each antibody
was immobilized on a carboxymethylated dextran CM5 sensor chip
(BIAcore, Catalog No. BR-1006-68) by amine coupling (BIAcore,
Catalog No. BR-1000-50) using a standard coupling protocol
according to manufacturer's instructions.
[0239] Analyses were performed at 25.degree. C. using PBS (GIBCO,
Catalog No. 14040-133) containing 0.05% surfactant P20 (BIAcore,
Catalog No. R-1000-54), 2 mg/mL BSA (EMD, Catalog No. 2930) and 10
mg/mL CM-Dextran Sodium salt (Fluka, Catalog No. 86524) as running
buffer. Supernatant containing different HGF fusion proteins or
supernatant from cells transfected with empty vector were injected
over each antibody at a flow rate of 30 .mu.L/min for 3 minutes.
The resulting binding was determined as resonance units (RU) over
baseline 30 seconds after the end of injection. Binding was
compared to human HGF (R&D Systems, Catalog No. 294-HGN-025)
diluted in running buffer. Non-specific binding was monitored by
comparing binding to a control surface. The results are summarized
in the Table 17.
TABLE-US-00026 TABLE 17 rhHGF (R&D rmHGF (R&D MHM chimera
MHM chimera MHM chimera Antibody Systems) Systems) (495-585)
(507-585) (499-556) 2B8 Yes No Yes Yes Yes HE2B8-1 Yes No Yes Yes
Yes HE2B8-2 Yes No Yes Yes Yes HE2B8-3 Yes No Yes Yes Yes HE2B8-4
Yes No Yes Yes Yes sh2B8-9 Yes No Yes Yes Yes (G1m(3)) sh2B8-12 Yes
No Yes Yes Yes (G1m(3))
[0240] The results in Table 17 demonstrate that each of the
humanized 2B8-based antibodies bind rhHGF and all three
mouse-human-mouse chimeras.
Example 15
Binding Affinities of Humanized 2B8 Variants
[0241] The binding affinities and kinetics of interaction of the
antibodies listed in Table 15 were measured by surface plasmon
resonance.
[0242] Mouse anti-human immunoglobulins (Jackson Labs, Catalog No.
209-005) were immobilized on carboxymethylated dextran CM4 sensor
chips (BIAcore, Catalog No. BR-1006-68) by amine coupling (BIAcore,
Catalog No. BR-1000-50) using a standard coupling protocol
according to manufacturer's instructions. The analyses were
performed at 25.degree. C. using PBS (GIBCO, Catalog No. 14040-133)
containing 0.05% surfactant P20 (BIAcore, Catalog No. BR-1000-54),
and 2 mg/mL BSA (EMD, Catalog No. 2930).
[0243] The antibodies were captured in an individual flow cell at a
flow rate of 10 .mu.L/min. Injection time was variable for each
antibody to yield approximately 20 RU of antibody captured for each
cycle. Buffer or HGF (R&D Systems, Catalog No. 294-HGN-025)
diluted in running buffer was injected sequentially over a
reference surface (no antibody captured) and the active surface
(antibody to be tested) for 2 minutes at 60 .mu.L/min. The
dissociation phase was monitored for 15 or 90 minutes, depending on
concentration. The surface then was regenerated with 10 mM
Glycine-HCl, pH 2.2 (BIAcore, Catalog No. BR-1003-54) injected for
3 minutes at a flow rate of 60 .mu.L/min before another cycle was
initiated. HGF concentrations tested were 0.46 nM to 7.5 nM.
[0244] Kinetic parameters were determined using the kinetic
function of the BIAevalutation.TM. software with reference
subtraction. Kinetic parameters for each antibody, k.sub.a
(association rate constant), k.sub.d (dissociation rate constant)
and K.sub.D (equilibrium dissociation constant) are summarized in
Table 18.
TABLE-US-00027 TABLE 18 Antibody k.sub.a (1/Ms) k.sub.d (1/s)
K.sub.D (pM) SD 2B8 1.4 .times. 10.sup.6 1.0 .times. 10.sup.-5 7.3
-- HE2B8-1 2.2 .times. 10.sup.6 1.4 .times. 10.sup.-5 7.1 5.2
HE2B8-2 1.8 .times. 10.sup.6 9.6 .times. 10.sup.-6 5.2 2.7 HE2B8-3
2.0 .times. 10.sup.6 4.1 .times. 10.sup.-6 2.0 1.1 HE2B8-4 1.7
.times. 10.sup.6 1.1 .times. 10.sup.-5 6.5 1.3 sh2B8-9 (G1m(17,1)
2.0 .times. 10.sup.6 1.7 .times. 10.sup.-5 8.1 5.3 sh2B8-12
(G1m(17,1) 1.9 .times. 10.sup.6 2.3 .times. 10.sup.-5 12 0.4
[0245] These data show that the humanized antibodies have fast
association rates (k.sub.a), very slow dissociation rates
(k.sub.d), and very high affinities (K.sub.D). In particular, the
antibodies have affinities ranging from 2.0-12 pM.
Example 16
Comparison of Binding Affinities at 25.degree. C. and 37.degree.
C.
[0246] The binding affinities and kinetics of interaction of
antibody HE2B8-4, sh2B8-9, sh2B8-12, and murine 2B8 were measured
by surface plasmon resonance under different conditions.
[0247] Mouse anti-human immunoglobulins (Jackson Labs, Catalog No.
209-005) or rabbit anti-mouse immunoglobulins (BIAcore, Catalog No.
BR-1005-14) were immobilized on carboxymethylated dextran CM4
sensor chips (BIAcore, Catalog No. BR-1006-68) by amine coupling
(BIAcore, Catalog No. BR-1000-50) using a standard coupling
protocol according to manufacturer's instructions. In the case of
25.degree. C. measurements for sh2b8-9 and sh2B8-12, a CM5 sensor
chip (BIAcore, Catalog No. BR-1006-68) was used. The analyses were
performed at 25.degree. C. and 37.degree. C. using PBS (GIBCO,
Catalog No. 14040-133) containing 0.05% surfactant P20 (BIAcore,
Catalog No. BR-1000-54), and 2 mg/mL BSA (EMD, Catalog No. 2930) as
running buffer.
[0248] The antibodies were captured in an individual flow cell at a
flow rate of 10 .mu.L/min. Injection time was variable for each
antibody to yield approximately 20 RU of antibody captured for each
cycle. Buffer or HGF (R&D Systems, Catalog No. 294-HGN-025)
diluted in running buffer was injected sequentially over a
reference surface (no antibody captured) and the active surface
(antibody to be tested) for 2 minutes at 60 .mu.L/min. The
dissociation phase was monitored for 15 or 90 minutes, depending on
concentration. The surface of mouse anti-human immunoglobulins
sensor chips was then regenerated with 10 mM Glycine-HCl, pH 2.2
(BIAcore, Catalog No. BR-1003-54) injected for 3 minutes at a flow
rate of 60 .mu.L/min before another cycle was initiated. The
surface of rabbit anti-mouse immunoglobulins sensor chips was
regenerated with 10 mM Glycine-HCl, pH 1.7 (BIAcore, Catalog No.
BR-1003-54) injected for 3 minutes at a flow rate of 60 .mu.L/min
before another cycle was initiated. HGF concentrations tested were
0.46 nM to 7.5 nM.
[0249] Kinetic parameters were determined using the kinetic
function of the BIAevaluation software with reference subtraction.
Kinetic parameters for each antibody, k.sub.a (association rate
constant), k.sub.d (dissociation rate constant) and K.sub.D
(equilibrium dissociation constant) are summarized below in Table
19.
TABLE-US-00028 TABLE 19 Antibody Temp. (.degree. C.) k.sub.a (1/Ms)
k.sub.d (1/s) K.sub.D (pM) 2B8 25 1.6 .times. 10.sup.6 2.1 .times.
10.sup.-5 13.5 2B8 37 2.8 .times. 10.sup.6 1.3 .times. 10.sup.-5
4.5 HE2B8-4 25 2.0 .times. 10.sup.6 1.2 .times. 10.sup.-5 5.6
HE2B8-4 37 3.1 .times. 10.sup.6 1.0 .times. 10.sup.-5 3.3 sh2B8-9
25 2.0 .times. 10.sup.6 1.7 .times. 10.sup.-5 8.1 (G1m(17,1))
sh2B8-9 37 2.5 .times. 10.sup.6 1.4 .times. 10.sup.-5 5.8 (G1m(3))
sh2B8-12 25 1.9 .times. 10.sup.6 2.3 .times. 10.sup.-5 12.0
(G1m(17,1)) sh2B8-12 37 2.4 .times. 10.sup.6 1.1 .times. 10.sup.-5
4.8 (G1m(3))
[0250] As expected, the association rate constants increased with
an increase in the temperature. Surprisingly, the dissociation
constants did not change significantly with a corresponding
increase in temperature. Consequently, the overall equilibrium
dissociation constants (K.sub.D) were approximately 1.4 to 3 times
smaller (higher affinity) at physiological temperature (37.degree.
C.).
Example 17
Neutralization Activity of Humanized 2B8 Variants
[0251] The antibodies described in Example 14 were characterized
for their ability to (a) inhibit the binding of hHGF to c-Met, and
(b) inhibit HGF stimulated BrdU incorporation in 4MBr-5 cells.
[0252] HGF-Met Binding Inhibition Assay (Neutralization Assay) was
performed as described in as follows. The antibodies were tested by
ELISA for their ability to inhibit hHGF binding to c-Met.
Specifically, Wallac 96-well DELFIA assay plates (Wallac Inc.,
Catalog No. AAAND-0001) were coated with 100 .mu.L of 6.25 .mu.g/mL
HGF (R&D Systems, Catalog No. 294-HGN-025) in carbonate coating
buffer (15 mM Na.sub.2CO.sub.3 and 34 mM NaHCO.sub.3, pH 9.0) for
16 hours at 4.degree. C. The plates then were blocked with 200
.mu.L of 5% non-fat dry milk in PBS for 1 hour at room temperature.
The antibodies were prepared in a separate plate by adding
increasing concentrations of the antibodies under investigation
(0.033-250 nM, 2-fold-serial dilution) to 2 nM biotinylated c-Met
in 5% non-fat dry milk in PBS. c-Met (R&D Systems, Catalog No.
358-MT/CF) is biotinylated according to manufacturer's instruction
at 10:1 biotin to c-Met ratio (Pierce, Catalog No. 21335). 100
.mu.L of sample per well was transferred to the assay plate and
incubated for 2 hours at room temperature. The resulting plates
were washed three times with PBS-0.1% Tween 20, and incubated for 1
hour at room temperature with Eu-labeled Streptavidin (Wallac,
Catalog No. 1244-360) diluted 1:1000 in DELFIA assay buffer
(Wallac, Catalog No. 4002-0010). The resulting plates were washed 3
times with DELFIA wash solution (Wallac, Catalog No. 4010-0010) and
incubated with 100 .mu.L/well DELFIA enhancement solution (Wallac
#4001-0010) for 15 minutes at room temperature with agitation. The
plates were read on Victor.sup.3V instrument (Perkin Elmer) using
the Europium method. The IC.sub.50 values were calculated using
Prism.
[0253] The IC.sub.50 values obtained are shown in Table 20.
TABLE-US-00029 TABLE 20 Antibody IC.sub.50 (nM) SD 2B8 9.2 1.2
HE2B8-1 6.0 1.2 HE2B8-2 5.7 1.1 HE2B8-3 5.9 1.1 HE2B8-4 6.5 1.2
sh2B8-9 (G1m(3)) 4.2 -- sh2B8-12 (G1m(3) 6.8 --
[0254] These results from Table 20 demonstrate that the humanized
antibodies tested efficiently neutralize HGF binding to c-Met.
[0255] The antibodies in Table 17 were also tested in the cell
proliferation assay described in Example 7(b). The results are
summarized below in Table 21.
TABLE-US-00030 TABLE 21 Antibody IC.sub.50 (nM) SD 2B8 0.86 0.35
HE2B8-1 0.47 0.15 HE2B8-2 0.66 0.13 HE2B8-3 0.55 0.28 HE2B8-4 0.58
0.26 sh2B8-9 (G1m(3)) 0.52 0.11 sh2B8-12 (G1m(3)) 0.81 0.22
[0256] The results from Table 21 demonstrate that all the humanized
antibodies tested inhibit HGF-induced proliferation of 4 MBr-5
cells.
Example 18
Anti-Scatter Activity of Humanized 2B8 Variants
[0257] The antibodies in Table 17 were tested in the anti-scatter
assay described in Example 8. The results are summarized below in
Table 22.
TABLE-US-00031 TABLE 22 Inhibition of HGF-induced MDCK Cell
Scattering Antibody Trial 1 Trial 2 2B8 ++ ++ HE2B8-1 ++ ++ HE2B8-2
++ ++ HE2B8-3 ++ ++ HE2B8-4 ++ ++ sh2B8-9 (G1m(3)) ++ ++ sh2B8-12
(G1m(3)) ++ ++ - No Inhibition +++ Very strong, nearly complete
inhibition ++ Strong inhibition + Detectable inhibition
[0258] The results in Table 22 demonstrate that all the humanized
antibodies tested inhibited HGF-induced scattering to the same
extent as the murine monoclonal antibody 2B8.
Example 19
Inhibition of HGF-Stimulated c-Met Phosphorylation
[0259] The antibodies in Table 17 were tested in the c-Met
phosphorylation assay described in Example 9. The results are
summarized below in Table 23.
TABLE-US-00032 TABLE 23 Average of Standard Antibody Two Trials
Deviation 2B8 0.91 0.02 he2B8-1 0.80 0.04 he2B8-2 0.88 0.15 he2B8-3
0.79 0.05 he2B8-4 0.75 0.14 sh2B8-9 (G1m(3)) 0.93 0.03 sh2B8-12
(G1m(3)) 0.81 0.07
[0260] The results in Table 23 demonstrate that all the humanized
antibodies tested are potent inhibitors of HGF-induced c-Met
phosphorylation in PC-3 cells.
Example 20
Tumor Inhibition in U87MG Xenograft Model
[0261] The ability of the humanized monoclonal antibodies of the
invention to inhibit tumor growth was tested in an U87MG xenograft
model. U87MG cells (ATCC) were expanded in culture at 37.degree. C.
in an atmosphere containing 5% CO.sub.2 and 95% air, using a medium
comprising Dulbecco's Modified Eagle medium (DMEM) with 10% fetal
bovine serum, 100 units/mL penicillin and 100 .mu.g/mL
streptomycin. The cells were subcultured and maintained by
detaching the cells from the wall of the culture dish using
trypsin-EDTA.
[0262] Near-confluent cells were collected by trypsinization and
then 5.times.10.sup.6 cells in 50% Matrigel (BD Biosciences;
catalog no. 356237) were injected subcutaneously into the upper
dorsal area between the shoulder blades of 7-week old female ICR
SCID mice (Taconic Labs). The long (L) and short (W) diameters (mm)
of tumors were measured with a caliper. Tumor volume (vol.) was
calculated as: volume (mm.sup.3)=L.times.W.sup.2/2. When the tumors
grew to approximately 200 mm.sup.3, the tumor-bearing mice were
randomized into 5 groups of 10 mice each. One group received PBS
and one group received human IgG control. Each of the other 4
groups received one of the humanized antibodies (HE2B8-1, HE2B8-2,
HE2B8-3, and HE2B8-4). All the antibodies were dosed at 0.25 mg/kg
body weight, twice per week, by intra-peritoneal injections of 5
doses. Tumor volumes and mouse body weights were recorded twice per
week. Tumor growth inhibition was analyzed using Student's
t-test.
[0263] The humanized antibodies tested were active in vivo. There
was 57% tumor growth inhibition for HE2B8-1 with a p value of 0.02,
61% tumor growth inhibition for HE2B8-2 with a p value of 0.02, 85%
tumor growth inhibition for HE2B8-3, with a p value of 0.0004, and
74% tumor growth inhibition for HE2B8-4 with a p value of 0.001. No
significant body weight loss was observed.
[0264] A subsequent study was performed as described above in
female NCR nude mice (Taconic Labs) bearing subcutaneous U87MG
tumors inoculated in the flank. Each group (10 mice each) received
one of the following treatments at 0.5 mg/kg: PBS vehicle control,
huIgG control, HE2B8-4, or sh2B8-9. Treatment was given
intra-peritoneal twice weekly for a minimum of 5 weeks. Each
treatment group demonstrated similar tumor regression with tumor
growth inhibition of 113% for sh2B8-9 and 115% for HE2B8-4, and a
minimum tumor growth delay of 30 days. Both treatments were
well-tolerated with no significant body weight loss.
INCORPORATION BY REFERENCE
[0265] The entire disclosure of each of the patent documents and
scientific articles referred to herein is incorporated by reference
for all purposes.
EQUIVALENTS
[0266] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are intended to be embraced therein.
Sequence CWU 1
1
2161424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 1atgaactttg ggctcagatt gattttcctt
gtccttgttt taaaaggtgt gaagtgtgaa 60gtgcagctgg tggagtctgg gggaggctta
gtgcagcctg gagggtccct gaaactctcc 120tgtgcagcct ctgaattcac
tttcagtaac tattacatgt cttgggttcg ccagactcca 180gagaagaggc
tgcagtgggt cgcatacatt agtcctggtg gtggtagctc ctactatcca
240gccagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac
cctgtacctg 300caaatgagca gtctgaagtc tgaggacaca gccatgtatt
actgtgcaag acaaggggat 360ggttactacg gggactatgc tatggactac
tggggtcaag gaacctcagt caccgtctcc 420tcag 4242141PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1
5 10 15 Val Lys Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln 20 25 30 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Glu
Phe Thr Phe 35 40 45 Ser Asn Tyr Tyr Met Ser Trp Val Arg Gln Thr
Pro Glu Lys Arg Leu 50 55 60 Gln Trp Val Ala Tyr Ile Ser Pro Gly
Gly Gly Ser Ser Tyr Tyr Pro 65 70 75 80 Ala Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln
Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met 100 105 110 Tyr Tyr Cys
Ala Arg Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met 115 120 125 Asp
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 130 135 140
3382DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 3atgagtgtgc ccactcaggt cctggggttg
ctgctgctgt ggcttacaga tgccagatgt 60gacatccaga tgactcagtc tccagcctcc
ctatctgttt ctgtgggaga aactgtcacc 120atcacatgtc gagcaagtga
gaatatttat agtaatttag catggtatca gcagaaacag 180ggaaaatctc
ctcagctcct ggtctatgct gcaacaaact tagcagatgg tgtgccatca
240aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag
cctgcagtct 300gaagattttg ggacttatta ctgtcaacat ttttggggta
ctccgtacac gttcggaggg 360gggaccaagc tggaaataaa ac
3824127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 4Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu
Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys Asp Ile Gln Met Thr
Gln Ser Pro Ala Ser Leu Ser 20 25 30 Val Ser Val Gly Glu Thr Val
Thr Ile Thr Cys Arg Ala Ser Glu Asn 35 40 45 Ile Tyr Ser Asn Leu
Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro 50 55 60 Gln Leu Leu
Val Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser 65 70 75 80 Arg
Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn 85 90
95 Ser Leu Gln Ser Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp
100 105 110 Gly Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 115 120 125 55PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Asn Tyr Tyr Met Ser 1 5
617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Tyr Ile Ser Pro Gly Gly Gly Ser Ser Tyr Tyr Pro
Ala Ser Val Lys 1 5 10 15 Gly 713PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 7Gln Gly Asp Gly Tyr Tyr
Gly Asp Tyr Ala Met Asp Tyr 1 5 10 811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Arg
Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala 1 5 10 97PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Ala
Ala Thr Asn Leu Ala Asp 1 5 109PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 10Gln His Phe Trp Gly Thr Pro
Tyr Thr 1 5 11412DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 11atgggatgga gctatatcat
cctctttttg gtagcaacag ctacagatgt ccactcccag 60gtccaactgc agcagcctgg
ggctgaactg gtgaagcctg ggacttcagt gaagctgtcc 120tgcaaggctt
ctggctacac cttcaccacc tactggatgc actgggtgaa tcagaggcct
180ggacaaggcc ttgagtggat tggagagatt aatcctacca acggtcatac
taactacaat 240gagaagttca agagcaaggc cacactgact gtagacaaat
cctccagcac agcctacatg 300caactcagca gcctgacatc tgaggactct
gcggtctatt actgtgcaag aaactatgtt 360ggtagcatct ttgactactg
gggccaaggc accactctca cagtctcctc ag 41212137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
12Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Asp 1
5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val
Lys 20 25 30 Pro Gly Thr Ser Val Lys Leu Ser Cys Lys Ala Ser Gly
Tyr Thr Phe 35 40 45 Thr Thr Tyr Trp Met His Trp Val Asn Gln Arg
Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Asn Pro Thr
Asn Gly His Thr Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Ser Lys Ala
Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys
Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly 115 120 125 Gln
Gly Thr Thr Leu Thr Val Ser Ser 130 135 13382DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
13atggaatcac agactctggt cttcatatcc atactgctct ggttatatgg tgctgatggg
60aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc
120ttgagctgca aggccagtga gaatgtggtt tcttatgtat cctggtatca
acagaaacca 180gcgcagtctc ctaaactgct gatatacggg gcatccaacc
ggaacactgg ggtccccgat 240cgcttcacag gcagtggatc tgcaacagat
ttcactctga ccatcagcag tgtgcgggct 300gaagaccttg cagattatca
ctgtgggcag agttacaact atccgtacac gttcggaggg 360gggaccaggc
tggaaataaa ac 38214127PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 14Met Glu Ser Gln Thr Leu
Val Phe Ile Ser Ile Leu Leu Trp Leu Tyr 1 5 10 15 Gly Ala Asp Gly
Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser 20 25 30 Met Ser
Val Gly Glu Arg Val Thr Leu Ser Cys Lys Ala Ser Glu Asn 35 40 45
Val Val Ser Tyr Val Ser Trp Tyr Gln Gln Lys Pro Ala Gln Ser Pro 50
55 60 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Asn Thr Gly Val Pro
Asp 65 70 75 80 Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu
Thr Ile Ser 85 90 95 Ser Val Arg Ala Glu Asp Leu Ala Asp Tyr His
Cys Gly Gln Ser Tyr 100 105 110 Asn Tyr Pro Tyr Thr Phe Gly Gly Gly
Thr Arg Leu Glu Ile Lys 115 120 125 155PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Thr
Tyr Trp Met His 1 5 1617PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 16Glu Ile Asn Pro Thr Asn Gly
His Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser 179PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Asn
Tyr Val Gly Ser Ile Phe Asp Tyr 1 5 1811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Lys
Ala Ser Glu Asn Val Val Ser Tyr Val Ser 1 5 10 197PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Gly
Ala Ser Asn Arg Asn Thr 1 5 209PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 20Gly Gln Ser Tyr Asn Tyr Pro
Tyr Thr 1 5 21412DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 21atggaatgga gctgggtctt
tctcttcctc ctgtcagtaa ctgcaggtgt ccactgccag 60gtccagctga agcagtctgg
agctgagctg gtgaggcctg ggacttcagt gaagatgtcc 120tgcaaggctt
ctggctacac cttcactacc tactatatac actgggtgaa tcagaggcct
180ggacagggcc ttgagtggat tggaaagatt ggtcctggaa gtggtagtac
ttactacaat 240gagatgttca aagacaaggc cacattgact gtagacacat
cctccagcac agcctacatg 300cagctcagca gcctgacatc tgacgactct
gcggtctatt tctgtgcaag aaggggactg 360ggacgtggct ttgactactg
gggccaaggc accactctca cagtctcctc ag 41222137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
22Met Glu Trp Ser Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Gly 1
5 10 15 Val His Cys Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val
Arg 20 25 30 Pro Gly Thr Ser Val Lys Met Ser Cys Lys Ala Ser Gly
Tyr Thr Phe 35 40 45 Thr Thr Tyr Tyr Ile His Trp Val Asn Gln Arg
Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Lys Ile Gly Pro Gly
Ser Gly Ser Thr Tyr Tyr Asn 65 70 75 80 Glu Met Phe Lys Asp Lys Ala
Thr Leu Thr Val Asp Thr Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln
Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val 100 105 110 Tyr Phe Cys
Ala Arg Arg Gly Leu Gly Arg Gly Phe Asp Tyr Trp Gly 115 120 125 Gln
Gly Thr Thr Leu Thr Val Ser Ser 130 135 23394DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
23atggagacag acacaatcct gctatgggtg ctgctgctct gggttccagg ctccactggt
60gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc
120atctcctgca aggccagcca aagtgttgat tatgatggta atagttatat
caactggtac 180caacagaaac caggacagcc acccaaagtc ctcatctatg
ttgcatccaa tctagaatct 240gggatcccag ccaggtttag tggcagtggg
tctgggacag acttcaccct caacatccat 300cctgtggagg aggaggatgc
tgcaacctat tactgtcagc aaagtattga ggatcctccc 360acgttcggtg
ctgggaccaa gctggagctg aaac 39424131PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
24Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1
5 10 15 Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala 20 25 30 Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala
Ser Gln Ser 35 40 45 Val Asp Tyr Asp Gly Asn Ser Tyr Ile Asn Trp
Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Val Leu Ile Tyr
Val Ala Ser Asn Leu Glu Ser 65 70 75 80 Gly Ile Pro Ala Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Asn Ile His Pro
Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Gln Gln Ser
Ile Glu Asp Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu 115 120 125 Glu
Leu Lys 130 255PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 25Thr Tyr Tyr Ile His 1 5
2617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 26Lys Ile Gly Pro Gly Ser Gly Ser Thr Tyr Tyr Asn
Glu Met Phe Lys 1 5 10 15 Asp 279PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 27Arg Gly Leu Gly Arg Gly
Phe Asp Tyr 1 5 2815PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 28Lys Ala Ser Gln Ser Val Asp Tyr Asp
Gly Asn Ser Tyr Ile Asn 1 5 10 15 297PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 29Val
Ala Ser Asn Leu Glu Ser 1 5 309PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 30Gln Gln Ser Ile Glu Asp Pro
Pro Thr 1 5 31418DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 31atggaatggc cttgtatctt
tctcttcctc ctgtcagtaa ctgaaggtgt ccactcccag 60gttcagctgc agcagtctgg
ggctgaactg gtgaggcctg ggtcctcagt gaagatttcc 120tgcaaggctt
ctggctatgt attcagtagc tactggatga actgggtgaa gcagaggcct
180ggacagggtc ttgagtggat tggacagatt tatcctggag atggtgatag
taactacaat 240ggaaacttca agggtaaagc cacactgact gcagacaaat
cctccagtac agcctacatg 300cagctcagca gcctaacatc tgaggactct
gcggtctatt tctgtgcatc ccagctcggg 360ctacgtgaga actactttga
ctactggggc caaggcacca ctctcacagt ctcctcag 41832139PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
32Met Glu Trp Pro Cys Ile Phe Leu Phe Leu Leu Ser Val Thr Glu Gly 1
5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val
Arg 20 25 30 Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Val Phe 35 40 45 Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg
Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Gln Ile Tyr Pro Gly
Asp Gly Asp Ser Asn Tyr Asn 65 70 75 80 Gly Asn Phe Lys Gly Lys Ala
Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Phe Cys
Ala Ser Gln Leu Gly Leu Arg Glu Asn Tyr Phe Asp Tyr 115 120 125 Trp
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 130 135 33388DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
33atggacatga ggacccctgc tcagtttctt ggaatcttgt tgctctggtt tccaggtatc
60aaatgtgaca tcaagatgac ccagtctcca tcttccatgt atgcatctct aggagagaga
120gtcacaatca cttgcaaggc gagtcaggac attaaaagct atttaagctg
gttccagcag 180aaaccaggga aatctcctaa gaccctgatc tatcgtgtaa
acagattggt agatggggtc 240ccatcaaggt tcagtggcag tggatctggg
caagattctt ctctcaccat caccagcctg 300gagaatgaag atatgggaat
ttattattgt ctacagtatg atgagtttcc gttcacgttc 360ggagggggga
ccaagctgga aataaagc 38834129PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 34Met Asp Met Arg Thr Pro
Ala Gln Phe Leu Gly Ile Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ile
Lys Cys Asp Ile Lys Met Thr Gln Ser Pro Ser Ser 20 25 30 Met Tyr
Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser 35 40 45
Gln Asp Ile Lys Ser Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys 50
55 60 Ser Pro Lys Thr Leu Ile Tyr Arg Val Asn Arg Leu Val Asp Gly
Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Ser
Ser Leu Thr 85 90 95 Ile Thr Ser Leu Glu Asn Glu Asp Met Gly Ile
Tyr Tyr Cys Leu Gln 100 105 110 Tyr Asp Glu Phe Pro Phe Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile 115 120 125 Lys 355PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 35Ser
Tyr Trp Met Asn 1 5 3617PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 36Gln Ile Tyr Pro Gly Asp Gly
Asp Ser Asn Tyr Asn Gly Asn Phe Lys 1 5 10 15 Gly 3711PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Gln
Leu Gly Leu Arg Glu Asn Tyr Phe Asp Tyr 1 5 10 3811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Lys
Ala Ser Gln Asp Ile Lys Ser Tyr Leu Ser 1 5 10 397PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 39Arg
Val Asn Arg Leu Val Asp 1 5 409PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 40Leu Gln Tyr Asp Glu Phe Pro
Phe Thr 1 5
41397DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 41atggctgtcc cggtgctgtt cctctgcctg
gttgcatttc caagctgtgt cctgtcccag 60gtacagctga aggagtcagg acctggcctg
gtggcgccct cacagagcct gtccatcact 120tgcactgtct ctgggttttc
attaaccagc tatagtttac actgggttcg ccagcctcca 180ggaaagggtc
tggaatggct gggagtaata tgggctggtg gaaacacaaa ttataattcg
240tctctcatgt ccagactgac catcaggaaa gacaactcca agagccaagt
tttcttaaaa 300atgaacagtc tgcaaactga tgacacagcc atgtactact
gtgccagaga gaggtttgct 360tactggggcc aagggactct ggtcactgtc tctgcag
39742132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 42Met Ala Val Pro Val Leu Phe Leu Cys Leu Val
Ala Phe Pro Ser Cys 1 5 10 15 Val Leu Ser Gln Val Gln Leu Lys Glu
Ser Gly Pro Gly Leu Val Ala 20 25 30 Pro Ser Gln Ser Leu Ser Ile
Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40 45 Thr Ser Tyr Ser Leu
His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60 Glu Trp Leu
Gly Val Ile Trp Ala Gly Gly Asn Thr Asn Tyr Asn Ser 65 70 75 80 Ser
Leu Met Ser Arg Leu Thr Ile Arg Lys Asp Asn Ser Lys Ser Gln 85 90
95 Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr
100 105 110 Tyr Cys Ala Arg Glu Arg Phe Ala Tyr Trp Gly Gln Gly Thr
Leu Val 115 120 125 Thr Val Ser Ala 130 43385DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
43atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt caaaatatcc
60agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatatcc aggggagaag
120gtcaccatga cctgcagtgc cagctcaagt gtaagttaca tgcactggta
ccagcagaag 180tcaggcacct cccccaaaag atggatttat gacacatcca
aactggcttc tggagtccct 240gctcgcttca gtggcagtgg gtctgggacc
tcttactccc tcacaatcag tagtatggag 300gctgaagatg ctgccactta
ttactgccag cagtggagta gtaacccact cacgttcggt 360gctgggacca
agctggagct gaaac 38544128PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 44Met Asp Phe Gln Val Gln
Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Lys Ile Ser
Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser
Ala Tyr Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser 35 40 45
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser 50
55 60 Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val
Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
Leu Thr Ile 85 90 95 Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Leu Thr Phe Gly Ala
Gly Thr Lys Leu Glu Leu Lys 115 120 125 455PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 45Ser
Tyr Ser Leu His 1 5 4616PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 46Val Ile Trp Ala Gly Gly Asn
Thr Asn Tyr Asn Ser Ser Leu Met Ser 1 5 10 15 475PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 47Glu
Arg Phe Ala Tyr 1 5 4810PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 48Ser Ala Ser Ser Ser Val Ser
Tyr Met His 1 5 10 497PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 49Asp Thr Ser Lys Leu Ala Ser
1 5 509PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5
51424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 51atgaactttg ggctcagatt gattttcctt
gtccttgttt taaaaggtgt gaagtgtgaa 60gtgcagctgg tggagtctgg gggaggctta
gtgcagcctg gagggtccct gaaactctcc 120tgtgcagcct ctggattcac
tttcagtgac tattacatgt cttgggttcg ccagactcca 180gagaagaggc
tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca
240gacagtgtga agggtcgatt caccatctcc cgagacaatg ccaagaacac
cctgtacctg 300caaatgagca gtctgaagtc tgaggacaca gccatatatt
actgtgtgag acaaggggat 360ggttattacg gggactatgc tatggactac
tggggtcaag gaacctcagt catcgtctcc 420tcag 42452141PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
52Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1
5 10 15 Val Lys Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln 20 25 30 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe 35 40 45 Ser Asp Tyr Tyr Met Ser Trp Val Arg Gln Thr
Pro Glu Lys Arg Leu 50 55 60 Glu Trp Val Ala Tyr Ile Ser Ser Gly
Gly Gly Ser Thr Tyr Tyr Pro 65 70 75 80 Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln
Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Ile 100 105 110 Tyr Tyr Cys
Val Arg Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met 115 120 125 Asp
Tyr Trp Gly Gln Gly Thr Ser Val Ile Val Ser Ser 130 135 140
53382DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 53atgagtgtgc ccactcaggt cctggggttg
ctgctgctgt ggcttacaga tgtcagatgt 60gacatccaga tgactcagtc tccagcctcc
ctatctgtat ctgtgggaga aactgtcacc 120atcacatgtc gaacaagtga
gaatatttac agtaatttag cgtggtatca gcagaaacag 180ggaaaatctc
ctcagctcct aatctatgct gcaacaaact tagcagatgg tgtgccatca
240aggttcagtg gcagtggatc aggcacacag ttttccctca ggatcaacag
cctgcagtct 300gaagattttg ggaggtatta ctgtcaacat ttttggggga
ctccgtacac gttcggaggg 360gggaccaaac tggaaataaa ac
38254127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 54Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu
Leu Leu Trp Leu Thr 1 5 10 15 Asp Val Arg Cys Asp Ile Gln Met Thr
Gln Ser Pro Ala Ser Leu Ser 20 25 30 Val Ser Val Gly Glu Thr Val
Thr Ile Thr Cys Arg Thr Ser Glu Asn 35 40 45 Ile Tyr Ser Asn Leu
Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro 50 55 60 Gln Leu Leu
Ile Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser 65 70 75 80 Arg
Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Arg Ile Asn 85 90
95 Ser Leu Gln Ser Glu Asp Phe Gly Arg Tyr Tyr Cys Gln His Phe Trp
100 105 110 Gly Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 115 120 125 555PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 55Asp Tyr Tyr Met Ser 1 5
5617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Tyr Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro
Asp Ser Val Lys 1 5 10 15 Gly 5713PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 57Gln Gly Asp Gly Tyr Tyr
Gly Asp Tyr Ala Met Asp Tyr 1 5 10 5811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 58Arg
Thr Ser Glu Asn Ile Tyr Ser Asn Leu Ala 1 5 10 597PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 59Ala
Ala Thr Asn Leu Ala Asp 1 5 609PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 60Gln His Phe Trp Gly Thr Pro
Tyr Thr 1 5 61424DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 61atgaactttg ggctcagatt
gattttcctt gtccttgttt taaaaggtgt gaagtgtgag 60gtgcagctgg tggagtctgg
gggaggctta gtgcagtctg gagggtccct gaaactctcc 120tgtgcggcct
ctggattcac tttcagtaac tatttcatgt cttgggttcg ccagactcca
180gagaagaggc tggagtgggt cgcatatatt agtagtggtg gtggtagcac
ctactatcca 240gacagtgtga agggtcgatt caccatctct agagacaatg
ccaagaacac cctgtacctg 300caaatgagca gtctgaagtc tgaggacaca
gccatgtatt actgtgtaag acaaggggat 360ggttactacg gggactatgc
tatggactac tggggtcaag gaacctcagt caccgtctcc 420tcag
42462141PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 62Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val
Leu Val Leu Lys Gly 1 5 10 15 Val Lys Cys Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln 20 25 30 Ser Gly Gly Ser Leu Lys Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Asn Tyr Phe Met
Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu Trp Val
Ala Tyr Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro 65 70 75 80 Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90
95 Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met
100 105 110 Tyr Tyr Cys Val Arg Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr
Ala Met 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
Ser 130 135 140 63382DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 63atgagtgtgc
ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt 60gacatccaga
tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc
120atcacatgtc gagcaagtga gaatatttac agtaatttag catggtatca
gcagaaacag 180ggaaaatctc ctcagctcct ggtctatgat gcaacacact
taccagatgg tgtgccatca 240aggttcagtg gcagtggatc aggcacacag
ttttccctca agatcaacag cctgcagtct 300gaagattttg ggagttatta
ctgtcaacat ttttggggta ctccgtacac gtttggaggg 360gggaccagac
tggaaattaa ac 38264127PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 64Met Ser Val Pro Thr Gln
Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser 20 25 30 Val Ser
Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn 35 40 45
Ile Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro 50
55 60 Gln Leu Leu Val Tyr Asp Ala Thr His Leu Pro Asp Gly Val Pro
Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu
Lys Ile Asn 85 90 95 Ser Leu Gln Ser Glu Asp Phe Gly Ser Tyr Tyr
Cys Gln His Phe Trp 100 105 110 Gly Thr Pro Tyr Thr Phe Gly Gly Gly
Thr Arg Leu Glu Ile Lys 115 120 125 655PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 65Asn
Tyr Phe Met Ser 1 5 6617PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 66Tyr Ile Ser Ser Gly Gly Gly
Ser Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly 6713PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 67Gln
Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met Asp Tyr 1 5 10
6811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 68Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala 1 5
10 697PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 69Asp Ala Thr His Leu Pro Asp 1 5
709PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 70Gln His Phe Trp Gly Thr Pro Tyr Thr 1 5
71424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 71atgaactttg ggctcagatt gattttcctt
gtccttgttt taaaaggtgt gaagtgtgaa 60gtgcagctgg tggagtctgg gggaggctta
gtgcagcctg gagggtccct gaaaatctcc 120tgtgcagcct ctggatttac
tttcagtaac tatttcatgt cttgggttcg ccagactcca 180gagaagaggc
tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca
240gacagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac
cctgtacctg 300caaatgaaca gtctgaagtc tgaggacaca gccatgtatt
actgtgtaag acaaggagat 360ggttactatg gggactatgc tatggactac
tggggtcaag gaacctcagt caccgtctcc 420tcag 42472141PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
72Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1
5 10 15 Val Lys Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln 20 25 30 Pro Gly Gly Ser Leu Lys Ile Ser Cys Ala Ala Ser Gly
Phe Thr Phe 35 40 45 Ser Asn Tyr Phe Met Ser Trp Val Arg Gln Thr
Pro Glu Lys Arg Leu 50 55 60 Glu Trp Val Ala Tyr Ile Ser Ser Gly
Gly Gly Ser Thr Tyr Tyr Pro 65 70 75 80 Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln
Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met 100 105 110 Tyr Tyr Cys
Val Arg Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met 115 120 125 Asp
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 130 135 140
73382DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 73atgagtgtgc ccactcaggt cctggggttg
ctgctgctgt ggcttacaga tgccagatgt 60gacatccaga tgactcagtc gccagcctcc
ctatctgtat ctgtgggaga aactgtcacc 120atcacatgtc gagcaagtga
gaatatttac attaatttag catggtatca gcagaaacag 180ggaaaatctc
ctcagctcct ggtccatgct gcaacaaagt tagcagatgg tgtgccatca
240aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag
cctgcagtct 300gaagattttg ggagttatta ctgtcaacat ttttggggta
ctccgtacac gttcggaggg 360gggaccaaac tagaaataaa ac
38274127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 74Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu
Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys Asp Ile Gln Met Thr
Gln Ser Pro Ala Ser Leu Ser 20 25 30 Val Ser Val Gly Glu Thr Val
Thr Ile Thr Cys Arg Ala Ser Glu Asn 35 40 45 Ile Tyr Ile Asn Leu
Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro 50 55 60 Gln Leu Leu
Val His Ala Ala Thr Lys Leu Ala Asp Gly Val Pro Ser 65 70 75 80 Arg
Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn 85 90
95 Ser Leu Gln Ser Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp
100 105 110 Gly Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 115 120 125 755PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 75Asn Tyr Phe Met Ser 1 5
7617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 76Tyr Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro
Asp Ser Val Lys 1 5 10 15 Gly 7713PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 77Gln Gly Asp Gly Tyr Tyr
Gly Asp Tyr Ala Met Asp Tyr 1 5 10 7811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 78Arg
Ala Ser Glu Asn Ile Tyr Ile Asn Leu Ala 1 5 10 797PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 79Ala
Ala Thr Lys Leu Ala Asp 1 5 809PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 80Gln
His Phe Trp Gly Thr Pro Tyr Thr 1 5 81974DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
81ccaaaacgac acccccatct gtctatccac tggcccctgg atctgctgcc caaactaact
60ccatggtgac cctgggatgc ctggtcaagg gctatttccc tgagccagtg acagtgacct
120ggaactctgg atccctgtcc agcggtgtgc acaccttccc agctgtcctg
gagtctgacc 180tctacactct gagcagctca gtgactgtcc cctccagccc
tcggcccagc gagaccgtca 240cctgcaacgt tgcccacccg gccagcagca
ccaaggtgga caagaaaatt gtgcccaggg 300attgtggttg taagccttgc
atatgtacag tcccagaagt atcatctgtc ttcatcttcc 360ccccaaagcc
caaggatgtg ctcaccatta ctctgactcc taaggtcacg tgtgttgtgg
420tagacatcag caaggatgat cccgaggtcc agttcagctg gtttgtagat
gatgtggagg 480tgcacacagc tcagacgcaa ccccgggagg agcagttcaa
cagcactttc cgctcagtca 540gtgaacttcc catcatgcac caggactggc
tcaatggcaa ggagttcaaa tgcagggtca 600acagtgcagc tttccctgcc
cccatcgaga aaaccatctc caaaaccaaa ggcagaccga 660aggctccaca
ggtgtacacc attccacctc ccaaggagca gatggccaag gataaagtca
720gtctgacctg catgataaca gacttcttcc ctgaagacat tactgtggag
tggcagtgga 780atgggcagcc agcggagaac tacaagaaca ctcagcccat
catgaacacg aatggctctt 840acttcgtcta cagcaagctc aatgtgcaga
agagcaactg ggaggcagga aatactttca 900cctgctctgt gttacatgag
ggcctgcaca accaccatac tgagaagagc ctctcccact 960ctcctggtaa atga
97482974DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 82ccaaaacgac acccccatct gtctatccac
tggcccctgg atctgctgcc caaactaact 60ccatggtgac cctgggatgc ctggtcaagg
gctatttccc tgagccagtg acagtgacct 120ggaactctgg atccctgtcc
agcggtgtgc acaccttccc agctgtcctg cagtctgacc 180tctacactct
gagcagctca gtgactgtcc cctccagcac ctggcccagc gagaccgtca
240cctgcaacgt tgcccacccg gccagcagca ccaaggtgga caagaaaatt
gtgcccaggg 300attgtggttg taagccttgc atatgtacag tcccagaagt
atcatctgtc ttcatcttcc 360ccccaaagcc caaggatgtg ctcaccatta
ctctgactcc taaggtcacg tgtgttgtgg 420tagacatcag caaggatgat
cccgaggtcc agttcagctg gtttgtagat gatgtggagg 480tgcacacagc
tcagacgcaa ccccgggagg agcagttcaa cagcactttc cgctcagtca
540gtgaacttcc catcatgcac caggactggc tcaatggcaa ggagttcaaa
tgcagggtca 600acagtgcagc tttccctgcc cccatcgaga aaaccatctc
caaaaccaaa ggcagaccga 660aggctccaca ggtgtacacc attccacctc
ccaaggagca gatggccaag gataaagtca 720gtctgacctg catgataaca
gacttcttcc ctgaagacat tactgtggag tggcagtgga 780atgggcagcc
agcggagaac tacaagaaca ctcagcccat catggacaca gatggctctt
840acttcgtcta cagcaagctc aatgtgcaga agagcaactg ggaggcagga
aatactttca 900cctgctctgt gttacatgag ggcctgcaca accaccatac
tgagaagagc ctctcccact 960ctcctggtaa atga 97483323DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
83gggctgatgc tgcaccaact gtatccatct tcccaccatc cagtgagcag ttaacatctg
60gaggtgcctc agtcgtgtgc ttcttgaaca acttctaccc caaagacatc aatgtcaagt
120ggaagattga tggcagtgaa cgacaaaatg gcgtcctgaa cagttggact
gatcaggaca 180gcaaagacag cacctacagc atgagcagca ccctcacgtt
gaccaaggac gagtatgaac 240gacataacag ctatacctgt gaggccactc
acaagacatc aacttcaccc attgtcaaga 300gcttcaacag gaatgagtgt tag
32384323DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 84gggctgatgc tgcaccaact gtatccatct
tcccaccatc cagtgagcag ttaacatctg 60gaggtgcctc agtcgtgtgc ttcttgaaca
acttctaccc caaagacatc aatgtcaagt 120ggaagattga tggcagtgaa
cgacaaaatg gcgtcctgaa cagttggact gatcaggaca 180gcaaagacag
cacctacagc atgagcagca ccctcatgtt gaccaaggac gagtatgaac
240gacataacag ctatacctgt gaggccactc acaagacatc aacttcaccc
attgtcaaga 300gcttcaacag gaatgagtgt tag 3238530DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
85aagcagtggt atcaacgcag agtacgcggg 308627DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
86tttttttttt tttttttttt tttttvn 278745DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
87ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt
458822DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 88ctaatacgac tcactatagg gc 228921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
89tatgcaaggc ttacaaccac a 219028DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 90gccagtggat agacagatgg
gggtgtcg 289127DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 91ctcattcctg ttgaagctct tgacaat
279223DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 92cgactgaggc acctccagat gtt 239320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
93taatacgact cactataggg 209417DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 94gtaaaacgac ggccagt
179518DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 95caggaaacag ctatgacc 189663DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
96ggggacaagt ttgtacaaaa aagcaggctg ccaccatgaa ctttgggctc agattgattt
60tcc 639754DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 97ggggaccact ttgtacaaga aagctgggtt
catttaccag gagagtggga gagg 549862DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 98ggggacaagt ttgtacaaaa
aagcaggctg ccaccatggg atggagctat atcatcctct 60tt
629953DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 99ggggaccact ttgtacaaga aagctgggtt catttaccag
gagagtggga gag 5310062DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 100ggggacaagt ttgtacaaaa
aagcaggctg ccaccatgga atcacagact ctggtcttca 60ta
6210154DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 101ggggaccact ttgtacaaga aagctgggtc taacactcat
tcctgttgaa gctc 5410230DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 102actggctagc atgtgggtga
ccaaactcct 3010346DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 103gtgatggtga tggtgatggc ggccgcatga
ctgtggtacc ttatat 4610430DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 104actggcggcc gccatcacca
tcaccatcac 3010531DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 105actgggatcc tcactattta cccggggaca g
3110627DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 106catgatgtcc acgaaagagg agatgag
2710727DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 107ctcatctcct ctttcgtgga catcatg
2710837DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 108ggaagaggag atgagaaacg caaacaggtt ctcaatg
3710937DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 109cattgagaac ctgtttgcgt ttctcatctc ctcttcc
3711029DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 110atcggctagc atgatgtggg ggaccaaac
2911136DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 111ggttttgttt tgttgacgcc caacatttac cctaag
3611236DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 112ccaaaacaaa acaactgcgg gttgtaaatg ggattc
3611340DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 113tctagaccaa aattacttcg aacgagctgg acgttaggac
4011440DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 114agatctggtt ttaatgaagc ttgctcgacc tgcaatcctg
4011548DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 115cactaccact accactaccg ccggcgtgtt gaacatacag
ttttaatg 4811638DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 116catcaccatc accatcacta agcgggtctg
gtgccacg 3811738DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 117cgtggcacca gacccgctta gtgatggtga
tggtgatg 381182922DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 118atgtgggtga ccaaactcct
gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60ctgctcccca tcgccatccc
ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120gaattcaaaa
aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa
180accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa
taaaggactt 240ccattcactt gcaaggcttt tgtttttgat aaagcaagaa
aacaatgcct ctggttcccc 300ttcaatagca tgtcaagtgg agtgaaaaaa
gaatttggcc atgaatttga cctctatgaa 360aacaaagact acattagaaa
ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420tctatcacta
agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac
480agctttttgc cttcgagcta tcggggtaaa gacctacagg aaaactactg
tcgaaatcct 540cgaggggaag aagggggacc ctggtgtttc acaagcaatc
cagaggtacg ctacgaagtc 600tgtgacattc ctcagtgttc agaagttgaa
tgcatgacct gcaatgggga gagttatcga 660ggtctcatgg atcatacaga
atcaggcaag atttgtcagc gctgggatca tcagacacca 720caccggcaca
aattcttgcc tgaaagatat cccgacaagg gctttgatga taattattgc
780cgcaatcccg atggccagcc gaggccatgg tgctatactc ttgaccctca
cacccgctgg 840gagtactgtg caattaaaac atgcgctgac aatactatga
atgacactga tgttcctttg 900gaaacaactg aatgcatcca aggtcaagga
gaaggctaca ggggcactgt caataccatt 960tggaatggaa ttccatgtca
gcgttgggat tctcagtatc ctcacgagca tgacatgact 1020cctgaaaatt
tcaagtgcaa ggacctacga gaaaattact gccgaaatcc agatgggtct
1080gaatcaccct ggtgttttac cactgatcca aacatccgag ttggctactg
ctcccaaatt 1140ccaaactgtg atatgtcaca tggacaagat tgttatcgtg
ggaatggcaa aaattatatg 1200ggcaacttat cccaaacaag atctggacta
acatgttcaa tgtgggacaa gaacatggaa 1260gacttacatc gtcatatctt
ctgggaacca gatgcaagta agctgaatga gaattactgc 1320cgaaatccag
atgatgatgc tcatggaccc tggtgctaca cgggaaatcc actcattcct
1380tgggattatt gccctatttc tcgttgtgaa ggtgatacca cacctacaat
agtcaattta 1440gaccatcccg taatatcttg tgccaaaacg aaacaattgc
gagttgtaaa tgggattcca 1500acacgaacaa acataggatg gatggttagt
ttgagataca gaaataaaca tatctgcgga 1560ggatcattga taaaggagag
ttgggttctt actgcacgac agtgtttccc ttctcgagac 1620ttgaaagatt
atgaagcttg gcttggaatt catgatgtcc acggaagagg agatgagaaa
1680tgcaaacagg ttctcaatgt ttcccagctg gtatatggcc ctgaaggatc
agatctggtt 1740ttaatgaagc ttgccaggcc tgctgtcctg gatgattttg
ttagtacgat tgatttacct 1800aattatggat gcacaattcc tgaaaagacc
agttgcagtg tttatggctg gggctacact 1860ggattgatca actatgatgg
cctattacga gtggcacatc tctatataat gggaaatgag 1920aaatgcagcc
agcatcatcg agggaaggtg actctgaatg agtctgaaat atgtgctggg
1980gctgaaaaga ttggatcagg accatgtgag ggggattatg gtggcccact
tgtttgtgag 2040caacataaaa tgagaatggt tcttggtgtc attgttcctg
gtcgtggatg tgccattcca 2100aatcgtcctg gtatttttgt ccgagtagca
tattatgcaa aatggataca caaaattatt 2160ttaacatata aggtaccaca
gtcatgcggc cgccatcacc atcaccatca ctccgcgggt 2220ctggtgccac
gcggtagtga caaaactcac acatgcccac cgtgcccagc acctgaactc
2280ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct
catgatctcc 2340cggacccctg aggtcacatg cgtggtggtg gacgtgagcc
acgaagaccc tgaggtcaag 2400ttcaactggt acgtggacgg cgtggaggtg
cataatgcca agacaaagcc gcgggaggag 2460cagtacaaca gcacgtaccg
tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 2520aatggcaagg
agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa
2580accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct
gcccccatcc 2640cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc
tggtcaaagg cttctatccc 2700agcgacatcg ccgtggagtg ggagagcaat
gggcagccgg agaacaacta caagaccacg 2760cctcccgtgc tggactccga
cggctccttc ttcctctata gcaagctcac cgtggacaag 2820agcaggtggc
agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac
2880cactacacgc agaagagcct ctccctgtcc ccgggtaaat ag
2922119919PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 119Pro Ala Leu Lys Ile Lys Thr Lys Lys Val
Asn Thr Ala Asp Gln Cys 1 5 10 15 Ala Asn Arg Cys Thr Arg Asn Lys
Gly Leu Pro Phe Thr Cys Lys Ala 20 25 30 Phe Val Phe Asp Lys Ala
Arg Lys Gln Cys Leu Trp Phe Pro Phe Asn 35 40 45 Ser Met Ser Ser
Gly Val Lys Lys Glu Phe Gly His Glu Phe Asp Leu 50 55 60 Tyr Glu
Asn Lys Asp Tyr Ile Arg Asn Cys Ile Ile Gly Lys Gly Arg 65 70 75 80
Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln 85
90 95 Pro Trp Ser Ser Met Ile Pro His Glu His Ser Phe Leu Pro Ser
Ser 100 105 110 Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn
Pro Arg Gly 115 120 125 Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn
Pro Glu Val Arg Tyr 130 135 140 Glu Val Cys Asp Ile Pro Gln Cys Ser
Glu Val Glu Cys Met Thr Cys 145 150 155 160 Asn Gly Glu Ser Tyr Arg
Gly Leu Met Asp His Thr Glu Ser Gly Lys 165 170 175 Ile Cys Gln Arg
Trp Asp His Gln Thr Pro His Arg His Lys Phe Leu 180 185 190 Pro Glu
Arg Tyr Pro Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg Asn 195 200 205
Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr Thr Leu Asp Pro His Thr 210
215 220 Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys Ala Asp Asn Thr Met
Asn 225 230 235 240 Asp Thr Asp Val Pro Leu Glu Thr Thr Glu Cys Ile
Gln Gly Gln Gly 245 250 255 Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile
Trp Asn Gly Ile Pro Cys 260 265 270 Gln Arg Trp Asp Ser Gln Tyr Pro
His Glu His Asp Met Thr Pro Glu 275 280 285 Asn Phe Lys Cys Lys Asp
Leu Arg Glu Asn Tyr Cys Arg Asn Pro Asp 290 295 300 Gly Ser Glu Ser
Pro Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg Val 305 310 315 320 Gly
Tyr Cys Ser Gln Ile Pro Asn Cys Asp Met Ser His Gly Gln Asp 325 330
335 Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met Gly Asn Leu Ser Gln Thr
340 345 350 Arg Ser Gly Leu Thr Cys Ser Met Trp Asp Lys Asn Met Glu
Asp Leu 355 360 365 His Arg His Ile Phe Trp Glu Pro Asp Ala Ser Lys
Leu Asn Glu Asn 370 375 380 Tyr Cys Arg Asn Pro Asp Asp Asp Ala His
Gly Pro Trp Cys Tyr Thr 385 390 395 400 Gly Asn Pro Leu Ile Pro Trp
Asp Tyr Cys Pro Ile Ser Arg Cys Glu 405 410 415 Gly Asp Thr Thr Pro
Thr Ile Val Asn Leu Asp His Pro Val Ile Ser 420 425 430 Cys Ala Lys
Thr Lys Gln Leu Arg Val Val Asn Gly Ile Pro Thr Arg 435 440 445 Thr
Asn Ile Gly Trp Met Val Ser Leu Arg Tyr Arg Asn Lys His Ile 450 455
460 Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp Val Leu Thr Ala Arg Gln
465 470 475 480 Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr Glu Ala Trp
Leu Gly Ile 485 490 495 His Asp Val His Gly Arg Gly Asp Glu Lys Cys
Lys Gln Val Leu Asn 500 505 510 Val Ser Gln Leu Val Tyr Gly Pro Glu
Gly Ser Asp Leu Val Leu Met 515 520 525 Lys Leu Ala Arg Pro Ala Val
Leu Asp Asp Phe Val Ser Thr Ile Asp 530 535 540 Leu Pro Asn Tyr Gly
Cys Thr Ile Pro Glu Lys Thr Ser Cys Ser Val 545 550 555 560 Tyr Gly
Trp Gly Tyr Thr Gly Leu Ile Asn Tyr Asp Gly Leu Leu Arg 565 570 575
Val Ala His Leu Tyr Ile Met Gly Asn Glu Lys Cys Ser Gln His His 580
585 590 Arg Gly Lys Val Thr Leu Asn Glu Ser Glu Ile Cys Ala Gly Ala
Glu 595 600 605 Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp Tyr Gly Gly
Pro Leu Val 610 615 620 Cys Glu Gln His
Lys Met Arg Met Val Leu Gly Val Ile Val Pro Gly 625 630 635 640 Arg
Gly Cys Ala Ile Pro Asn Arg Pro Gly Ile Phe Val Arg Val Ala 645 650
655 Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile Leu Thr Tyr Lys Val Pro
660 665 670 Gln Ser Cys Gly Arg His His His His His His Ser Ala Gly
Leu Val 675 680 685 Pro Arg Gly Ser Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro 690 695 700 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 705 710 715 720 Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val 725 730 735 Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 740 745 750 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 755 760 765 Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 770 775
780 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
785 790 795 800 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg 805 810 815 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys 820 825 830 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 835 840 845 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 850 855 860 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 865 870 875 880 Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 885 890 895
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 900
905 910 Leu Ser Leu Ser Pro Gly Lys 915 1202901DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
120atgatgtggg ggaccaaact tctgccggtc ctgttgctgc agcatgtcct
cctgcacctc 60ctcctgcttc atgtcgccat cccctatgca gaaggacaga agaaaagaag
aaatacactt 120catgaattta aaaagtcagc aaaaactact cttaccaagg
aagacccatt actgaagatt 180aaaaccaaaa aagtgaactc tgcagatgag
tgtgccaaca ggtgtatcag gaacaggggc 240tttacgttca cttgcaaggc
cttcgttttt gataagtcaa gaaaacgatg ctactggtat 300cctttcaata
gtatgtcaag tggagtgaaa aaagggtttg gccatgaatt tgacctctat
360gaaaacaaag actatattag aaactgcatc attggtaaag gaggcagcta
taaagggacg 420gtatccatca ctaagagtgg catcaaatgc cagccttgga
attccatgat cccccatgaa 480cacagctatc gcggtaaaga cctacaggaa
aactactgtc gaaatcctcg aggggaagaa 540gggggaccct ggtgtttcac
aagcaatcca gaggtacgct acgaagtctg tgacattcct 600cagtgttcag
aagttgaatg catgacctgc aatggtgaaa gctacagagg tcccatggat
660cacacagaat caggcaagac ttgtcagcgc tgggaccagc agacaccaca
ccggcacaag 720ttcttgccag aaagatatcc cgacaagggc tttgatgata
attattgccg caatcctgat 780ggcaagccga ggccatggtg ctacactctt
gaccctgaca ccccttggga gtattgtgca 840attaaaacgt gcgctcacag
tgctgtgaat gagactgatg tccctatgga aacaactgaa 900tgcattcaag
gccaaggaga aggttacagg ggaaccagca ataccatttg gaatggaatt
960ccctgtcagc gttgggattc gcagtaccct cacaagcatg atatcactcc
cgagaacttc 1020aaatgcaagg accttagaga aaattattgc cgcaatccag
atggggctga atcaccatgg 1080tgttttacca ctgacccaaa catccgagtt
ggctactgct ctcaaattcc caagtgtgac 1140gtgtcaagtg gacaagattg
ttatcgtggc aatgggaaaa attacatggg caacttatcc 1200aaaacaaggt
ctggacttac atgttccatg tgggacaaga atatggagga tttacaccgt
1260catatcttct gggagccaga tgctagcaaa ttgaataaga attactgccg
gaatcctgat 1320gatgatgccc atggaccttg gtgctacacg gggaatcctc
ttattccttg ggattattgc 1380cctatttccc gttgtgaagg agatactaca
cctacaattg tcaatttgga ccatcctgta 1440atatcctgtg ccaaaacaaa
acaactgcgg gttgtaaatg ggattccaac acgaacaaac 1500ataggatgga
tggttagttt gagatacaga aataaacata tctgcggagg atcattgata
1560aaggagagtt gggttcttac tgcacgacag tgtttccctt ctcgagactt
gaaagattat 1620gaagcttggc ttggaattca tgatgtccac ggaagaggag
atgagaaatg caaacaggtt 1680ctcaatgttt cccagctggt atatggccct
gaaggatcag atctggtttt aatgaagctt 1740gctcgacctg caatcctgga
taactttgtc agtacaattg atttacctag ttatggttgt 1800acaatccctg
aaaagaccac ttgcagtatt tacggctggg gctacactgg attgatcaac
1860gcggatggtt tattacgagt agctcatctg tatattatgg ggaatgagaa
atgcagtcag 1920caccatcaag gcaaggtgac tttgaatgag tctgagttat
gtgctggggc tgaaaagatt 1980ggatcaggac catgtgaggg agattatggt
ggcccactca tttgtgaaca acacaaaatg 2040agaatggttc ttggtgtcat
tgttcctggt cgtggatgtg ccatcccaaa tcgtcctggt 2100atttttgttc
gagtagcata ttatgcaaaa tggatacaca aagtaatttt gacatacaag
2160ttgtgcggcc gccatcacca tcaccatcac tccgcgggtc tggtgccacg
cggtagtgac 2220aaaactcaca catgcccacc gtgcccagca cctgaactcc
tggggggacc gtcagtcttc 2280ctcttccccc caaaacccaa ggacaccctc
atgatctccc ggacccctga ggtcacatgc 2340gtggtggtgg acgtgagcca
cgaagaccct gaggtcaagt tcaactggta cgtggacggc 2400gtggaggtgc
ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt
2460gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga
gtacaagtgc 2520aaggtctcca acaaagccct cccagccccc atcgagaaaa
ccatctccaa agccaaaggg 2580cagccccgag aaccacaggt gtacaccctg
cccccatccc gggaggagat gaccaagaac 2640caggtcagcc tgacctgcct
ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 2700gagagcaatg
ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac
2760ggctccttct tcctctatag caagctcacc gtggacaaga gcaggtggca
gcaggggaac 2820gtcttctcat gctccgtgat gcatgaggct ctgcacaacc
actacacgca gaagagcctc 2880tccctgtccc cgggtaaata g
2901121911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 121Pro Leu Leu Lys Ile Lys Thr Lys Lys Val
Asn Ser Ala Asp Glu Cys 1 5 10 15 Ala Asn Arg Cys Ile Arg Asn Arg
Gly Phe Thr Phe Thr Cys Lys Ala 20 25 30 Phe Val Phe Asp Lys Ser
Arg Lys Arg Cys Tyr Trp Tyr Pro Phe Asn 35 40 45 Ser Met Ser Ser
Gly Val Lys Lys Gly Phe Gly His Glu Phe Asp Leu 50 55 60 Tyr Glu
Asn Lys Asp Tyr Ile Arg Asn Cys Ile Ile Gly Lys Gly Gly 65 70 75 80
Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln 85
90 95 Pro Trp Asn Ser Met Ile Pro His Glu His Ser Tyr Arg Gly Lys
Asp 100 105 110 Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu
Gly Gly Pro 115 120 125 Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr
Glu Val Cys Asp Ile 130 135 140 Pro Gln Cys Ser Glu Val Glu Cys Met
Thr Cys Asn Gly Glu Ser Tyr 145 150 155 160 Arg Gly Pro Met Asp His
Thr Glu Ser Gly Lys Thr Cys Gln Arg Trp 165 170 175 Asp Gln Gln Thr
Pro His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro 180 185 190 Asp Lys
Gly Phe Asp Asp Asn Tyr Cys Arg Asn Pro Asp Gly Lys Pro 195 200 205
Arg Pro Trp Cys Tyr Thr Leu Asp Pro Asp Thr Pro Trp Glu Tyr Cys 210
215 220 Ala Ile Lys Thr Cys Ala His Ser Ala Val Asn Glu Thr Asp Val
Pro 225 230 235 240 Met Glu Thr Thr Glu Cys Ile Gln Gly Gln Gly Glu
Gly Tyr Arg Gly 245 250 255 Thr Ser Asn Thr Ile Trp Asn Gly Ile Pro
Cys Gln Arg Trp Asp Ser 260 265 270 Gln Tyr Pro His Lys His Asp Ile
Thr Pro Glu Asn Phe Lys Cys Lys 275 280 285 Asp Leu Arg Glu Asn Tyr
Cys Arg Asn Pro Asp Gly Ala Glu Ser Pro 290 295 300 Trp Cys Phe Thr
Thr Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln 305 310 315 320 Ile
Pro Lys Cys Asp Val Ser Ser Gly Gln Asp Cys Tyr Arg Gly Asn 325 330
335 Gly Lys Asn Tyr Met Gly Asn Leu Ser Lys Thr Arg Ser Gly Leu Thr
340 345 350 Cys Ser Met Trp Asp Lys Asn Met Glu Asp Leu His Arg His
Ile Phe 355 360 365 Trp Glu Pro Asp Ala Ser Lys Leu Asn Lys Asn Tyr
Cys Arg Asn Pro 370 375 380 Asp Asp Asp Ala His Gly Pro Trp Cys Tyr
Thr Gly Asn Pro Leu Ile 385 390 395 400 Pro Trp Asp Tyr Cys Pro Ile
Ser Arg Cys Glu Gly Asp Thr Thr Pro 405 410 415 Thr Ile Val Asn Leu
Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys 420 425 430 Gln Leu Arg
Val Val Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp 435 440 445 Met
Val Ser Leu Arg Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu 450 455
460 Ile Lys Glu Ser Trp Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg
465 470 475 480 Asp Leu Lys Asp Tyr Glu Ala Trp Leu Gly Ile His Asp
Val His Gly 485 490 495 Arg Gly Asp Glu Lys Cys Lys Gln Val Leu Asn
Val Ser Gln Leu Val 500 505 510 Tyr Gly Pro Glu Gly Ser Asp Leu Val
Leu Met Lys Leu Ala Arg Pro 515 520 525 Ala Ile Leu Asp Asn Phe Val
Ser Thr Ile Asp Leu Pro Ser Tyr Gly 530 535 540 Cys Thr Ile Pro Glu
Lys Thr Thr Cys Ser Ile Tyr Gly Trp Gly Tyr 545 550 555 560 Thr Gly
Leu Ile Asn Ala Asp Gly Leu Leu Arg Val Ala His Leu Tyr 565 570 575
Ile Met Gly Asn Glu Lys Cys Ser Gln His His Gln Gly Lys Val Thr 580
585 590 Leu Asn Glu Ser Glu Leu Cys Ala Gly Ala Glu Lys Ile Gly Ser
Gly 595 600 605 Pro Cys Glu Gly Asp Tyr Gly Gly Pro Leu Ile Cys Glu
Gln His Lys 610 615 620 Met Arg Met Val Leu Gly Val Ile Val Pro Gly
Arg Gly Cys Ala Ile 625 630 635 640 Pro Asn Arg Pro Gly Ile Phe Val
Arg Val Ala Tyr Tyr Ala Lys Trp 645 650 655 Ile His Lys Val Ile Leu
Thr Tyr Lys Leu Cys Gly Arg His His His 660 665 670 His His His Ser
Ala Gly Leu Val Pro Arg Gly Ser Asp Lys Thr His 675 680 685 Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 690 695 700
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 705
710 715 720 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu 725 730 735 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys 740 745 750 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser 755 760 765 Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys 770 775 780 Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 785 790 795 800 Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 805 810 815 Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 820 825
830 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
835 840 845 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser 850 855 860 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 865 870 875 880 Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu 885 890 895 His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 900 905 910 1221398DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
122atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt
gaagtgtgaa 60gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct
gaaactctcc 120tgtgcagcct ctgaattcac tttcagtaac tattacatgt
cttgggttcg ccagactcca 180gagaagaggc tgcagtgggt cgcatacatt
agtcctggtg gtggtagctc ctactatcca 240gccagtgtga agggtcgatt
caccatctcc agagacaatg ccaagaacac cctgtacctg 300caaatgagca
gtctgaagtc tgaggacaca gccatgtatt actgtgcaag acaaggggat
360ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt
caccgtctcc 420tcagccaaaa cgacaccccc atctgtctat ccactggccc
ctggatctgc tgcccaaact 480aactccatgg tgaccctggg atgcctggtc
aagggctatt tccctgagcc agtgacagtg 540acctggaact ctggatccct
gtccagcggt gtgcacacct tcccagctgt cctgcagtct 600gacctctaca
ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc
660gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa
aattgtgccc 720agggattgtg gttgtaagcc ttgcatatgt acagtcccag
aagtatcatc tgtcttcatc 780ttccccccaa agcccaagga tgtgctcacc
attactctga ctcctaaggt cacgtgtgtt 840gtggtagaca tcagcaagga
tgatcccgag gtccagttca gctggtttgt agatgatgtg 900gaggtgcaca
cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca
960gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt
caaatgcagg 1020gtcaacagtg cagctttccc tgcccccatc gagaaaacca
tctccaaaac caaaggcaga 1080ccgaaggctc cacaggtgta caccattcca
cctcccaagg agcagatggc caaggataaa 1140gtcagtctga cctgcatgat
aacagacttc ttccctgaag acattactgt ggagtggcag 1200tggaatgggc
agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc
1260tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc
aggaaatact 1320ttcacctgct ctgtgttaca tgagggcctg cacaaccacc
atactgagaa gagcctctcc 1380cactctcctg gtaaatga
1398123446PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 123Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala
Ser Glu Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ser Trp Val Arg
Gln Thr Pro Glu Lys Arg Leu Gln Trp Val 35 40 45 Ala Tyr Ile Ser
Pro Gly Gly Gly Ser Ser Tyr Tyr Pro Ala Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85
90 95 Ala Arg Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr
Thr Pro Pro 115 120 125 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala
Gln Thr Asn Ser Met 130 135 140 Val Thr Leu Gly Cys Leu Val Lys Gly
Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Thr Trp Asn Ser Gly
Ser Leu Ser Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 180 185 190 Pro Ser
Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His 195 200 205
Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys 210
215 220 Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val
Phe 225 230 235 240 Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile
Thr Leu Thr Pro 245 250 255 Lys Val Thr Cys Val Val Val Asp Ile Ser
Lys Asp Asp Pro Glu Val 260 265 270 Gln Phe Ser Trp Phe Val Asp Asp
Val Glu Val His Thr Ala Gln Thr 275 280 285 Gln Pro Arg Glu Glu Gln
Phe Asn Ser Thr Phe Arg Ser Val Ser Glu 290 295 300 Leu Pro Ile Met
His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys 305 310 315 320 Arg
Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330
335 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro
340 345 350 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys
Met Ile 355 360 365 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp
Gln Trp Asn Gly 370 375
380 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp
385 390 395 400 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys
Ser Asn Trp 405 410 415 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu
His Glu Gly Leu His 420 425 430 Asn His His Thr Glu Lys Ser Leu Ser
His Ser Pro Gly Lys 435 440 445 124705DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
124atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga
tgccagatgt 60gacatccaga tgactcagtc tccagcctcc ctatctgttt ctgtgggaga
aactgtcacc 120atcacatgtc gagcaagtga gaatatttat agtaatttag
catggtatca gcagaaacag 180ggaaaatctc ctcagctcct ggtctatgct
gcaacaaact tagcagatgg tgtgccatca 240aggttcagtg gcagtggatc
aggcacacag ttttccctca agatcaacag cctgcagtct 300gaagattttg
ggacttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg
360gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatccat
cttcccacca 420tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt
gcttcttgaa caacttctac 480cccaaagaca tcaatgtcaa gtggaagatt
gatggcagtg aacgacaaaa tggcgtcctg 540aacagttgga ctgatcagga
cagcaaagac agcacctaca gcatgagcag caccctcatg 600ttgaccaagg
acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca
660tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag
705125214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 125Asp Ile Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Val Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg
Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Ala Ala Thr
Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Ser 65 70 75 80
Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr 85
90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala
Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu
Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser
Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Met
Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys
Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205
Phe Asn Arg Asn Glu Cys 210 1261386DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
126atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt
ccactcccag 60gtccaactgc agcagcctgg ggctgaactg gtgaagcctg ggacttcagt
gaagctgtcc 120tgcaaggctt ctggctacac cttcaccacc tactggatgc
actgggtgaa tcagaggcct 180ggacaaggcc ttgagtggat tggagagatt
aatcctacca acggtcatac taactacaat 240gagaagttca agagcaaggc
cacactgact gtagacaaat cctccagcac agcctacatg 300caactcagca
gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactatgtt
360ggtagcatct ttgactactg gggccaaggc accactctca cagtctcctc
agccaaaacg 420acacccccat ctgtctatcc actggcccct ggatctgctg
cccaaactaa ctccatggtg 480accctgggat gcctggtcaa gggctatttc
cctgagccag tgacagtgac ctggaactct 540ggatccctgt ccagcggtgt
gcacaccttc ccagctgtcc tgcagtctga cctctacact 600ctgagcagct
cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac
660gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag
ggattgtggt 720tgtaagcctt gcatatgtac agtcccagaa gtatcatctg
tcttcatctt ccccccaaag 780cccaaggatg tgctcaccat tactctgact
cctaaggtca cgtgtgttgt ggtagacatc 840agcaaggatg atcccgaggt
ccagttcagc tggtttgtag atgatgtgga ggtgcacaca 900gctcagacgc
aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt
960cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt
caacagtgca 1020gctttccctg cccccatcga gaaaaccatc tccaaaacca
aaggcagacc gaaggctcca 1080caggtgtaca ccattccacc tcccaaggag
cagatggcca aggataaagt cagtctgacc 1140tgcatgataa cagacttctt
ccctgaagac attactgtgg agtggcagtg gaatgggcag 1200ccagcggaga
actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc
1260tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt
cacctgctct 1320gtgttacatg agggcctgca caaccaccat actgagaaga
gcctctccca ctctcctggt 1380aaatga 1386127442PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
127Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Thr
1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Thr Tyr 20 25 30 Trp Met His Trp Val Asn Gln Arg Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Thr Asn Gly His Thr
Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val
Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Tyr
Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu
Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 115 120 125
Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly 130
135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp
Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val
Thr Val Pro Ser Ser Thr 180 185 190 Trp Pro Ser Glu Thr Val Thr Cys
Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys
Ile Val Pro Arg Asp Cys Gly Cys Lys Pro 210 215 220 Cys Ile Cys Thr
Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro 225 230 235 240 Lys
Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys 245 250
255 Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp
260 265 270 Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro
Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu
Leu Pro Ile Met 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Phe
Lys Cys Arg Val Asn Ser 305 310 315 320 Ala Ala Phe Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Thr Lys Gly 325 330 335 Arg Pro Lys Ala Pro
Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln 340 345 350 Met Ala Lys
Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe 355 360 365 Pro
Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu 370 375
380 Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
385 390 395 400 Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu
Ala Gly Asn 405 410 415 Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu
His Asn His His Thr 420 425 430 Glu Lys Ser Leu Ser His Ser Pro Gly
Lys 435 440 128705DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 128atggaatcac agactctggt
cttcatatcc atactgctct ggttatatgg tgctgatggg 60aacattgtaa tgacccaatc
tcccaaatcc atgtccatgt cagtaggaga gagggtcacc 120ttgagctgca
aggccagtga gaatgtggtt tcttatgtat cctggtatca acagaaacca
180gcgcagtctc ctaaactgct gatatacggg gcatccaacc ggaacactgg
ggtccccgat 240cgcttcacag gcagtggatc tgcaacagat ttcactctga
ccatcagcag tgtgcgggct 300gaagaccttg cagattatca ctgtgggcag
agttacaact atccgtacac gttcggaggg 360gggaccaggc tggaaataaa
acgggctgat gctgcaccaa ctgtatccat cttcccacca 420tccagtgagc
agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac
480cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa
tggcgtcctg 540aacagttgga ctgatcagga cagcaaagac agcacctaca
gcatgagcag caccctcacg 600ttgaccaagg acgagtatga acgacataac
agctatacct gtgaggccac tcacaagaca 660tcaacttcac ccattgtcaa
gagcttcaac aggaatgagt gttag 705129214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
129Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly
1 5 10 15 Glu Arg Val Thr Leu Ser Cys Lys Ala Ser Glu Asn Val Val
Ser Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Ala Gln Ser Pro
Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Asn Thr Gly Val
Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr
Leu Thr Ile Ser Ser Val Arg Ala 65 70 75 80 Glu Asp Leu Ala Asp Tyr
His Cys Gly Gln Ser Tyr Asn Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly
Gly Thr Arg Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr
Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130
135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val
Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr
Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr
Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys
210 1301386DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 130atggaatgga gctgggtctt tctcttcctc
ctgtcagtaa ctgcaggtgt ccactgccag 60gtccagctga agcagtctgg agctgagctg
gtgaggcctg ggacttcagt gaagatgtcc 120tgcaaggctt ctggctacac
cttcactacc tactatatac actgggtgaa tcagaggcct 180ggacagggcc
ttgagtggat tggaaagatt ggtcctggaa gtggtagtac ttactacaat
240gagatgttca aagacaaggc cacattgact gtagacacat cctccagcac
agcctacatg 300cagctcagca gcctgacatc tgacgactct gcggtctatt
tctgtgcaag aaggggactg 360ggacgtggct ttgactactg gggccaaggc
accactctca cagtctcctc agccaaaacg 420acacccccat ctgtctatcc
actggcccct ggatctgctg cccaaactaa ctccatggtg 480accctgggat
gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct
540ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga
cctctacact 600ctgagcagct cagtgactgt cccctccagc acctggccca
gcgagaccgt cacctgcaac 660gttgcccacc cggccagcag caccaaggtg
gacaagaaaa ttgtgcccag ggattgtggt 720tgtaagcctt gcatatgtac
agtcccagaa gtatcatctg tcttcatctt ccccccaaag 780cccaaggatg
tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc
840agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga
ggtgcacaca 900gctcagacgc aaccccggga ggagcagttc aacagcactt
tccgctcagt cagtgaactt 960cccatcatgc accaggactg gctcaatggc
aaggagttca aatgcagggt caacagtgca 1020gctttccctg cccccatcga
gaaaaccatc tccaaaacca aaggcagacc gaaggctcca 1080caggtgtaca
ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc
1140tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg
gaatgggcag 1200ccagcggaga actacaagaa cactcagccc atcatggaca
cagatggctc ttacttcgtc 1260tacagcaagc tcaatgtgca gaagagcaac
tgggaggcag gaaatacttt cacctgctct 1320gtgttacatg agggcctgca
caaccaccat actgagaaga gcctctccca ctctcctggt 1380aaatga
1386131442PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 131Gln Val Gln Leu Lys Gln Ser Gly Ala Glu
Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Tyr Ile His Trp Val Asn
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Lys Ile Gly
Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Met Phe 50 55 60 Lys Asp
Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85
90 95 Ala Arg Arg Gly Leu Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110 Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser
Val Tyr Pro 115 120 125 Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser
Met Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu
Pro Val Thr Val Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr
Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr 180 185 190 Trp Pro
Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205
Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro 210
215 220 Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro
Pro 225 230 235 240 Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro
Lys Val Thr Cys 245 250 255 Val Val Val Asp Ile Ser Lys Asp Asp Pro
Glu Val Gln Phe Ser Trp 260 265 270 Phe Val Asp Asp Val Glu Val His
Thr Ala Gln Thr Gln Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr
Phe Arg Ser Val Ser Glu Leu Pro Ile Met 290 295 300 His Gln Asp Trp
Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser 305 310 315 320 Ala
Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly 325 330
335 Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln
340 345 350 Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp
Phe Phe 355 360 365 Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly
Gln Pro Ala Glu 370 375 380 Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp
Thr Asp Gly Ser Tyr Phe 385 390 395 400 Val Tyr Ser Lys Leu Asn Val
Gln Lys Ser Asn Trp Glu Ala Gly Asn 405 410 415 Thr Phe Thr Cys Ser
Val Leu His Glu Gly Leu His Asn His His Thr 420 425 430 Glu Lys Ser
Leu Ser His Ser Pro Gly Lys 435 440 132717DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
132atggagacag acacaatcct gctatgggtg ctgctgctct gggttccagg
ctccactggt 60gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca
gagggccacc 120atctcctgca aggccagcca aagtgttgat tatgatggta
atagttatat caactggtac 180caacagaaac caggacagcc acccaaagtc
ctcatctatg ttgcatccaa tctagaatct 240gggatcccag ccaggtttag
tggcagtggg tctgggacag acttcaccct caacatccat 300cctgtggagg
aggaggatgc tgcaacctat tactgtcagc aaagtattga ggatcctccc
360acgttcggtg ctgggaccaa gctggagctg aaacgggctg atgctgcacc
aactgtatcc 420atcttcccac catccagtga gcagttaaca tctggaggtg
cctcagtcgt gtgcttcttg 480aacaacttct accccaaaga catcaatgtc
aagtggaaga ttgatggcag tgaacgacaa 540aatggcgtcc tgaacagttg
gactgatcag gacagcaaag acagcaccta cagcatgagc 600agcaccctca
cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc
660actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgttag
717133218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 133Asp Ile Val Leu Thr Gln Ser Pro Ala Ser
Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys
Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30 Gly Asn Ser Tyr Ile Asn Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro 35 40 45 Lys Val Leu Ile Tyr Val Ala Ser Asn Leu Glu Ser
Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln Ser Ile 85 90 95 Glu Asp Pro Pro Thr Phe
Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 110 Ala Asp Ala Ala
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln 115 120 125 Leu Thr
Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 130 135 140
Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 145
150 155 160 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp
Ser Thr 165 170 175 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp
Glu Tyr Glu Arg 180 185 190 His Asn Ser Tyr Thr Cys Glu Ala Thr His
Lys Thr Ser Thr Ser Pro 195 200 205 Ile Val Lys Ser Phe Asn Arg Asn
Glu Cys 210 215 1341392DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 134atggaatggc
cttgtatctt tctcttcctc ctgtcagtaa ctgaaggtgt ccactcccag 60gttcagctgc
agcagtctgg ggctgaactg gtgaggcctg ggtcctcagt gaagatttcc
120tgcaaggctt ctggctatgt attcagtagc tactggatga actgggtgaa
gcagaggcct 180ggacagggtc ttgagtggat tggacagatt tatcctggag
atggtgatag taactacaat 240ggaaacttca agggtaaagc cacactgact
gcagacaaat cctccagtac agcctacatg 300cagctcagca gcctaacatc
tgaggactct gcggtctatt tctgtgcatc ccagctcggg 360ctacgtgaga
actactttga ctactggggc caaggcacca ctctcacagt ctcctcagcc
420aaaacgacac ccccatctgt ctatccactg gcccctggat ctgctgccca
aactaactcc 480atggtgaccc tgggatgcct ggtcaagggc tatttccctg
agccagtgac agtgacctgg 540aactctggat ccctgtccag cggtgtgcac
accttcccag ctgtcctgca gtctgacctc 600tacactctga gcagctcagt
gactgtcccc tccagcacct ggcccagcga gaccgtcacc 660tgcaacgttg
cccacccggc cagcagcacc aaggtggaca agaaaattgt gcccagggat
720tgtggttgta agccttgcat atgtacagtc ccagaagtat catctgtctt
catcttcccc 780ccaaagccca aggatgtgct caccattact ctgactccta
aggtcacgtg tgttgtggta 840gacatcagca aggatgatcc cgaggtccag
ttcagctggt ttgtagatga tgtggaggtg 900cacacagctc agacgcaacc
ccgggaggag cagttcaaca gcactttccg ctcagtcagt 960gaacttccca
tcatgcacca ggactggctc aatggcaagg agttcaaatg cagggtcaac
1020agtgcagctt tccctgcccc catcgagaaa accatctcca aaaccaaagg
cagaccgaag 1080gctccacagg tgtacaccat tccacctccc aaggagcaga
tggccaagga taaagtcagt 1140ctgacctgca tgataacaga cttcttccct
gaagacatta ctgtggagtg gcagtggaat 1200gggcagccag cggagaacta
caagaacact cagcccatca tggacacaga tggctcttac 1260ttcgtctaca
gcaagctcaa tgtgcagaag agcaactggg aggcaggaaa tactttcacc
1320tgctctgtgt tacatgaggg cctgcacaac caccatactg agaagagcct
ctcccactct 1380cctggtaaat ga 1392135444PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
135Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser
1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Val Phe Ser
Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45 Gly Gln Ile Tyr Pro Gly Asp Gly Asp Ser
Asn Tyr Asn Gly Asn Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala
Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Ser Gln Leu
Gly Leu Arg Glu Asn Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr
Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val 115 120 125
Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr 130
135 140 Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val
Thr 145 150 155 160 Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr
Phe Pro Ala Val 165 170 175 Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser
Ser Val Thr Val Pro Ser 180 185 190 Ser Thr Trp Pro Ser Glu Thr Val
Thr Cys Asn Val Ala His Pro Ala 195 200 205 Ser Ser Thr Lys Val Asp
Lys Lys Ile Val Pro Arg Asp Cys Gly Cys 210 215 220 Lys Pro Cys Ile
Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe 225 230 235 240 Pro
Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val 245 250
255 Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe
260 265 270 Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr
Gln Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val
Ser Glu Leu Pro 290 295 300 Ile Met His Gln Asp Trp Leu Asn Gly Lys
Glu Phe Lys Cys Arg Val 305 310 315 320 Asn Ser Ala Ala Phe Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr 325 330 335 Lys Gly Arg Pro Lys
Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys 340 345 350 Glu Gln Met
Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp 355 360 365 Phe
Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro 370 375
380 Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser
385 390 395 400 Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn
Trp Glu Ala 405 410 415 Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu
Gly Leu His Asn His 420 425 430 His Thr Glu Lys Ser Leu Ser His Ser
Pro Gly Lys 435 440 136711DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 136atggacatga
ggacccctgc tcagtttctt ggaatcttgt tgctctggtt tccaggtatc 60aaatgtgaca
tcaagatgac ccagtctcca tcttccatgt atgcatctct aggagagaga
120gtcacaatca cttgcaaggc gagtcaggac attaaaagct atttaagctg
gttccagcag 180aaaccaggga aatctcctaa gaccctgatc tatcgtgtaa
acagattggt agatggggtc 240ccatcaaggt tcagtggcag tggatctggg
caagattctt ctctcaccat caccagcctg 300gagaatgaag atatgggaat
ttattattgt ctacagtatg atgagtttcc gttcacgttc 360ggagggggga
ccaagctgga aataaagcgg gctgatgctg caccaactgt atccatcttc
420ccaccatcca gtgagcagtt aacatctgga ggtgcctcag tcgtgtgctt
cttgaacaac 480ttctacccca aagacatcaa tgtcaagtgg aagattgatg
gcagtgaacg acaaaatggc 540gtcctgaaca gttggactga tcaggacagc
aaagacagca cctacagcat gagcagcacc 600ctcacgttga ccaaggacga
gtatgaacga cataacagct atacctgtga ggccactcac 660aagacatcaa
cttcacccat tgtcaagagc ttcaacagga atgagtgtta g
711137214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 137Asp Ile Lys Met Thr Gln Ser Pro Ser Ser
Met Tyr Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Asp Ile Lys Ser Tyr 20 25 30 Leu Ser Trp Phe Gln Gln
Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Val Asn
Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Gln Asp Ser Ser Leu Thr Ile Thr Ser Leu Glu Asn 65 70 75 80
Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Phe 85
90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala
Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu
Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser
Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr
Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys
Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205
Phe Asn Arg Asn Glu Cys 210 1381361DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
138atggctgtcc cggtgctgtt cctctgcctg gttgcatttc caagctgtgt
cctgtcccag 60gtacagctga aggagtcagg acctggcctg gtggcgccct cacagagcct
gtccatcact 120tgcactgtct ctgggttttc attaaccagc tatagtttac
actgggttcg ccagcctcca 180ggaaagggtc tggaatggct gggagtaata
tgggctggtg gaaacacaaa ttataattcg 240tctctcatgt ccagactgac
catcaggaaa gacaactcca agagccaagt tttcttaaaa 300atgaacagtc
tgcaaactga tgacacagcc atgtactact gtgccagaga gaggtttgct
360tactggggcc aagggactct ggtcactgtc tctgcagcca aaacgacacc
cccatctgtc 420tatccactgg cccctggatc tgctgcccaa actaactcca
tggtgaccct gggatgcctg 480gtcaagggct atttccctga gccagtgaca
gtgacctgga actctggatc cctgtccagc 540ggtgtgcaca ccttcccagc
tgtcctgcag tctgacctct acactctgag cagctcagtg 600actgtcccct
ccagcacctg gcccagcgag accgtcacct gcaacgttgc ccacccggcc
660agcagcacca aggtggacaa gaaaattgtg cccagggatt gtggttgtaa
gccttgcata 720tgtacagtcc cagaagtatc atctgtcttc atcttccccc
caaagcccaa ggatgtgctc 780accattactc tgactcctaa ggtcacgtgt
gttgtggtag acatcagcaa ggatgatccc 840gaggtccagt tcagctggtt
tgtagatgat gtggaggtgc acacagctca gacgcaaccc 900cgggaggagc
agttcaacag cactttccgc tcagtcagtg aacttcccat catgcaccag
960gactggctca atggcaagga gttcaaatgc agggtcaaca gtgcagcttt
ccctgccccc 1020atcgagaaaa ccatctccaa aaccaaaggc agaccgaagg
ctccacaggt gtacaccatt 1080ccacctccca aggagcagat ggccaaggat
aaagtcagtc tgacctgcat gataacagac 1140ttcttccctg aagacattac
tgtggagtgg cagtggaatg ggcagccagc ggagaactac 1200aagaacactc
agcccatcat ggacacagat ggctcttact tcgtctacag caagctcaat
1260gtgcagaaga gcaactggga ggcaggaaat actttcacct gctctgtgtt
acatgagggc 1320ctgcacaacc accatactga tcccactctc ctggtaaatg a
1361139437PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 139Gln Val Gln Leu Lys Glu Ser Gly Pro Gly
Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Ser Leu His Trp Val Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp
Ala Gly Gly Asn Thr Asn Tyr Asn Ser Ser Leu Met 50 55 60 Ser Arg
Leu Thr Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85
90 95 Arg Glu Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser 100 105 110 Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala
Pro Gly Ser 115 120 125 Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly
Cys Leu Val Lys Gly 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Thr
Trp Asn Ser Gly Ser Leu Ser 145 150 155 160 Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr 165 170 175 Leu Ser Ser Ser
Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr 180 185 190 Val Thr
Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys 195 200 205
Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val 210
215 220 Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp
Val 225 230 235 240 Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val
Val Val Asp Ile 245 250 255 Ser Lys Asp Asp Pro Glu Val Gln Phe Ser
Trp Phe Val Asp Asp Val 260 265 270 Glu Val His Thr Ala Gln Thr Gln
Pro Arg Glu Glu Gln Phe Asn Ser 275 280 285 Thr Phe Arg Ser Val Ser
Glu Leu Pro Ile Met His Gln Asp Trp Leu 290 295 300 Asn Gly Lys Glu
Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala 305 310 315 320 Pro
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro 325 330
335 Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys
340 345 350 Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp
Ile Thr 355 360 365 Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn
Tyr Lys Asn Thr 370 375 380 Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr
Phe Val Tyr Ser Lys Leu 385 390 395 400 Asn Val Gln Lys Ser Asn Trp
Glu Ala Gly Asn Thr Phe Thr Cys Ser 405 410 415 Val Leu His Glu Gly
Leu His Asn His His Thr Glu Lys Ser Leu Ser 420 425 430 His Ser Pro
Gly Lys 435 140708DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 140atggattttc aagtgcagat
tttcagcttc ctgctaatca gtgcctcagt caaaatatcc 60agaggacaaa ttgttctcac
ccagtctcca gcaatcatgt ctgcatatcc aggggagaag 120gtcaccatga
cctgcagtgc cagctcaagt gtaagttaca tgcactggta ccagcagaag
180tcaggcacct cccccaaaag atggatttat gacacatcca aactggcttc
tggagtccct 240gctcgcttca gtggcagtgg gtctgggacc tcttactccc
tcacaatcag tagtatggag 300gctgaagatg ctgccactta ttactgccag
cagtggagta gtaacccact cacgttcggt 360gctgggacca agctggagct
gaaacgggct gatgctgcac caactgtatc catcttccca 420ccatccagtg
agcagttaac atctggaggt gcctcagtcg tgtgcttctt gaacaacttc
480taccccaaag acatcaatgt caagtggaag attgatggca gtgaacgaca
aaatggcgtc 540ctgaacagtt ggactgatca ggacagcaaa gacagcacct
acagcatgag cagcaccctc 600acgttgacca aggacgagta tgaacgacat
aacagctata cctgtgaggc cactcacaag 660acatcaactt cacccattgt
caagagcttc aacaggaatg agtgttag 708141213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
141Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Tyr Pro Gly
1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser
Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys
Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu
Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro 100 105 110 Thr Val
Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120 125
Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 130
135 140 Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
Asn 145 150 155 160 Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Met Ser Ser 165 170 175 Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
Arg His Asn Ser Tyr Thr 180 185 190 Cys Glu Ala Thr His Lys Thr Ser
Thr Ser Pro Ile Val Lys Ser Phe 195 200 205 Asn Arg Asn Glu Cys 210
1421398DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 142atgaactttg ggctcagatt gattttcctt
gtccttgttt taaaaggtgt gaagtgtgaa 60gtgcagctgg tggagtctgg gggaggctta
gtgcagcctg gagggtccct gaaactctcc 120tgtgcagcct ctggattcac
tttcagtgac tattacatgt cttgggttcg ccagactcca 180gagaagaggc
tggagtgggt cgcatacatt agtagtggtg
gtggtagcac ctactatcca 240gacagtgtga agggtcgatt caccatctcc
cgagacaatg ccaagaacac cctgtacctg 300caaatgagca gtctgaagtc
tgaggacaca gccatatatt actgtgtgag acaaggggat 360ggttattacg
gggactatgc tatggactac tggggtcaag gaacctcagt catcgtctcc
420tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc
tgcccaaact 480aactccatgg tgaccctggg atgcctggtc aagggctatt
tccctgagcc agtgacagtg 540acctggaact ctggatccct gtccagcggt
gtgcacacct tcccagctgt cctgcagtct 600gacctctaca ctctgagcag
ctcagtgact gtcccctcca gcacctggcc cagcgagacc 660gtcacctgca
acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc
720agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc
tgtcttcatc 780ttccccccaa agcccaagga tgtgctcacc attactctga
ctcctaaggt cacgtgtgtt 840gtggtagaca tcagcaagga tgatcccgag
gtccagttca gctggtttgt agatgatgtg 900gaggtgcaca cagctcagac
gcaaccccgg gaggagcagt tcaacagcac tttccgctca 960gtcagtgaac
ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg
1020gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac
caaaggcaga 1080ccgaaggctc cacaggtgta caccattcca cctcccaagg
agcagatggc caaggataaa 1140gtcagtctga cctgcatgat aacagacttc
ttccctgaag acattactgt ggagtggcag 1200tggaatgggc agccagcgga
gaactacaag aacactcagc ccatcatgga cacagatggc 1260tcttacttcg
tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact
1320ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa
gagcctctcc 1380cactctcctg gtaaatga 1398143446PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
143Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg
Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Gly Gly Ser Thr
Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu
Lys Ser Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Val Arg Gln Gly
Asp Gly Tyr Tyr Gly Asp Tyr Ala Met Asp Tyr Trp 100 105 110 Gly Gln
Gly Thr Ser Val Ile Val Ser Ser Ala Lys Thr Thr Pro Pro 115 120 125
Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met 130
135 140 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val
Thr 145 150 155 160 Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val
His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
Ser Ser Ser Val Thr Val 180 185 190 Pro Ser Ser Thr Trp Pro Ser Glu
Thr Val Thr Cys Asn Val Ala His 195 200 205 Pro Ala Ser Ser Thr Lys
Val Asp Lys Lys Ile Val Pro Arg Asp Cys 210 215 220 Gly Cys Lys Pro
Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe 225 230 235 240 Ile
Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro 245 250
255 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val
260 265 270 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala
Gln Thr 275 280 285 Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg
Ser Val Ser Glu 290 295 300 Leu Pro Ile Met His Gln Asp Trp Leu Asn
Gly Lys Glu Phe Lys Cys 305 310 315 320 Arg Val Asn Ser Ala Ala Phe
Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Thr Lys Gly Arg
Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro 340 345 350 Pro Lys Glu
Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile 355 360 365 Thr
Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly 370 375
380 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp
385 390 395 400 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys
Ser Asn Trp 405 410 415 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu
His Glu Gly Leu His 420 425 430 Asn His His Thr Glu Lys Ser Leu Ser
His Ser Pro Gly Lys 435 440 445 144705DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
144atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga
tgtcagatgt 60gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga
aactgtcacc 120atcacatgtc gaacaagtga gaatatttac agtaatttag
cgtggtatca gcagaaacag 180ggaaaatctc ctcagctcct aatctatgct
gcaacaaact tagcagatgg tgtgccatca 240aggttcagtg gcagtggatc
aggcacacag ttttccctca ggatcaacag cctgcagtct 300gaagattttg
ggaggtatta ctgtcaacat ttttggggga ctccgtacac gttcggaggg
360gggaccaaac tggaaataaa acgggctgat gctgcaccaa ctgtatccat
cttcccacca 420tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt
gcttcttgaa caacttctac 480cccaaagaca tcaatgtcaa gtggaagatt
gatggcagtg aacgacaaaa tggcgtcctg 540aacagttgga ctgatcagga
cagcaaagac agcacctaca gcatgagcag caccctcacg 600ttgaccaagg
acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca
660tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag
705145214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 145Asp Ile Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Val Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg
Thr Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ala Ala Thr
Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Gln Phe Ser Leu Arg Ile Asn Ser Leu Gln Ser 65 70 75 80
Glu Asp Phe Gly Arg Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr 85
90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala
Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu
Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser
Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr
Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys
Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205
Phe Asn Arg Asn Glu Cys 210 1461398DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
146atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt
gaagtgtgag 60gtgcagctgg tggagtctgg gggaggctta gtgcagtctg gagggtccct
gaaactctcc 120tgtgcggcct ctggattcac tttcagtaac tatttcatgt
cttgggttcg ccagactcca 180gagaagaggc tggagtgggt cgcatatatt
agtagtggtg gtggtagcac ctactatcca 240gacagtgtga agggtcgatt
caccatctct agagacaatg ccaagaacac cctgtacctg 300caaatgagca
gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggggat
360ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt
caccgtctcc 420tcagccaaaa cgacaccccc atctgtctat ccactggccc
ctggatctgc tgcccaaact 480aactccatgg tgaccctggg atgcctggtc
aagggctatt tccctgagcc agtgacagtg 540acctggaact ctggatccct
gtccagcggt gtgcacacct tcccagctgt cctgcagtct 600gacctctaca
ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc
660gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa
aattgtgccc 720agggattgtg gttgtaagcc ttgcatatgt acagtcccag
aagtatcatc tgtcttcatc 780ttccccccaa agcccaagga tgtgctcacc
attactctga ctcctaaggt cacgtgtgtt 840gtggtagaca tcagcaagga
tgatcccgag gtccagttca gctggtttgt agatgatgtg 900gaggtgcaca
cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca
960gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt
caaatgcagg 1020gtcaacagtg cagctttccc tgcccccatc gagaaaacca
tctccaaaac caaaggcaga 1080ccgaaggctc cacaggtgta caccattcca
cctcccaagg agcagatggc caaggataaa 1140gtcagtctga cctgcatgat
aacagacttc ttccctgaag acattactgt ggagtggcag 1200tggaatgggc
agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc
1260tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc
aggaaatact 1320ttcacctgct ctgtgttaca tgagggcctg cacaaccacc
atactgagaa gagcctctcc 1380cactctcctg gtaaatga
1398147446PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 147Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Ser Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Phe Met Ser Trp Val Arg
Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser
Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85
90 95 Val Arg Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr
Thr Pro Pro 115 120 125 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala
Gln Thr Asn Ser Met 130 135 140 Val Thr Leu Gly Cys Leu Val Lys Gly
Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Thr Trp Asn Ser Gly
Ser Leu Ser Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 180 185 190 Pro Ser
Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His 195 200 205
Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys 210
215 220 Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val
Phe 225 230 235 240 Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile
Thr Leu Thr Pro 245 250 255 Lys Val Thr Cys Val Val Val Asp Ile Ser
Lys Asp Asp Pro Glu Val 260 265 270 Gln Phe Ser Trp Phe Val Asp Asp
Val Glu Val His Thr Ala Gln Thr 275 280 285 Gln Pro Arg Glu Glu Gln
Phe Asn Ser Thr Phe Arg Ser Val Ser Glu 290 295 300 Leu Pro Ile Met
His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys 305 310 315 320 Arg
Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330
335 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro
340 345 350 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys
Met Ile 355 360 365 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp
Gln Trp Asn Gly 370 375 380 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln
Pro Ile Met Asp Thr Asp 385 390 395 400 Gly Ser Tyr Phe Val Tyr Ser
Lys Leu Asn Val Gln Lys Ser Asn Trp 405 410 415 Glu Ala Gly Asn Thr
Phe Thr Cys Ser Val Leu His Glu Gly Leu His 420 425 430 Asn His His
Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 445
148705DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 148atgagtgtgc ccactcaggt cctggggttg
ctgctgctgt ggcttacaga tgccagatgt 60gacatccaga tgactcagtc tccagcctcc
ctatctgtat ctgtgggaga aactgtcacc 120atcacatgtc gagcaagtga
gaatatttac agtaatttag catggtatca gcagaaacag 180ggaaaatctc
ctcagctcct ggtctatgat gcaacacact taccagatgg tgtgccatca
240aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag
cctgcagtct 300gaagattttg ggagttatta ctgtcaacat ttttggggta
ctccgtacac gtttggaggg 360gggaccagac tggaaattaa acgggctgat
gctgcaccaa ctgtatccat cttcccacca 420tccagtgagc agttaacatc
tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 480cccaaagaca
tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg
540aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag
caccctcacg 600ttgaccaagg acgagtatga acgacataac agctatacct
gtgaggccac tcacaagaca 660tcaacttcac ccattgtcaa gagcttcaac
aggaatgagt gttag 705149214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 149Asp Ile Gln Met Thr
Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Glu Thr Val
Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40
45 Tyr Asp Ala Thr His Leu Pro Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu
Gln Ser 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp
Gly Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile
Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro
Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys
Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp
Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn
Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170
175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val
Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210
1501398DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 150atgaactttg ggctcagatt gattttcctt
gtccttgttt taaaaggtgt gaagtgtgaa 60gtgcagctgg tggagtctgg gggaggctta
gtgcagcctg gagggtccct gaaaatctcc 120tgtgcagcct ctggatttac
tttcagtaac tatttcatgt cttgggttcg ccagactcca 180gagaagaggc
tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca
240gacagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac
cctgtacctg 300caaatgaaca gtctgaagtc tgaggacaca gccatgtatt
actgtgtaag acaaggagat 360ggttactatg gggactatgc tatggactac
tggggtcaag gaacctcagt caccgtctcc 420tcagccaaaa cgacaccccc
atctgtctat ccactggccc ctggatctgc tgcccaaact 480aactccatgg
tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg
540acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt
cctgcagtct 600gacctctaca ctctgagcag ctcagtgact gtcccctcca
gcacctggcc cagcgagacc 660gtcacctgca acgttgccca cccggccagc
agcaccaagg tggacaagaa aattgtgccc 720agggattgtg gttgtaagcc
ttgcatatgt acagtcccag aagtatcatc tgtcttcatc 780ttccccccaa
agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt
840gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt
agatgatgtg 900gaggtgcaca cagctcagac gcaaccccgg gaggagcagt
tcaacagcac tttccgctca 960gtcagtgaac ttcccatcat gcaccaggac
tggctcaatg gcaaggagtt caaatgcagg 1020gtcaacagtg cagctttccc
tgcccccatc gagaaaacca tctccaaaac caaaggcaga 1080ccgaaggctc
cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa
1140gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt
ggagtggcag 1200tggaatgggc agccagcgga gaactacaag aacactcagc
ccatcatgga cacagatggc 1260tcttacttcg tctacagcaa gctcaatgtg
cagaagagca actgggaggc aggaaatact 1320ttcacctgct ctgtgttaca
tgagggcctg cacaaccacc atactgagaa gagcctctcc 1380cactctcctg gtaaatga
1398151446PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 151Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Ile Ser Cys Ala Ala
Ser Gly Phe
Thr Phe Ser Asn Tyr 20 25 30 Phe Met Ser Trp Val Arg Gln Thr Pro
Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Gly
Gly Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Val
Arg Gln Gly Asp Gly Tyr Tyr Gly Asp Tyr Ala Met Asp Tyr Trp 100 105
110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro
115 120 125 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn
Ser Met 130 135 140 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro
Glu Pro Val Thr 145 150 155 160 Val Thr Trp Asn Ser Gly Ser Leu Ser
Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Asp Leu
Tyr Thr Leu Ser Ser Ser Val Thr Val 180 185 190 Pro Ser Ser Thr Trp
Pro Ser Glu Thr Val Thr Cys Asn Val Ala His 195 200 205 Pro Ala Ser
Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys 210 215 220 Gly
Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe 225 230
235 240 Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr
Pro 245 250 255 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp
Pro Glu Val 260 265 270 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val
His Thr Ala Gln Thr 275 280 285 Gln Pro Arg Glu Glu Gln Phe Asn Ser
Thr Phe Arg Ser Val Ser Glu 290 295 300 Leu Pro Ile Met His Gln Asp
Trp Leu Asn Gly Lys Glu Phe Lys Cys 305 310 315 320 Arg Val Asn Ser
Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Thr
Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro 340 345 350
Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile 355
360 365 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn
Gly 370 375 380 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met
Asp Thr Asp 385 390 395 400 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
Val Gln Lys Ser Asn Trp 405 410 415 Glu Ala Gly Asn Thr Phe Thr Cys
Ser Val Leu His Glu Gly Leu His 420 425 430 Asn His His Thr Glu Lys
Ser Leu Ser His Ser Pro Gly Lys 435 440 445 152705DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
152atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga
tgccagatgt 60gacatccaga tgactcagtc gccagcctcc ctatctgtat ctgtgggaga
aactgtcacc 120atcacatgtc gagcaagtga gaatatttac attaatttag
catggtatca gcagaaacag 180ggaaaatctc ctcagctcct ggtccatgct
gcaacaaagt tagcagatgg tgtgccatca 240aggttcagtg gcagtggatc
aggcacacag tattccctca agatcaacag cctgcagtct 300gaagattttg
ggagttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg
360gggaccaaac tagaaataaa acgggctgat gctgcaccaa ctgtatccat
cttcccacca 420tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt
gcttcttgaa caacttctac 480cccaaagaca tcaatgtcaa gtggaagatt
gatggcagtg aacgacaaaa tggcgtcctg 540aacagttgga ctgatcagga
cagcaaagac agcacctaca gcatgagcag caccctcacg 600ttgaccaagg
acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca
660tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag
705153214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 153Asp Ile Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Val Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg
Ala Ser Glu Asn Ile Tyr Ile Asn 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 His Ala Ala Thr
Lys Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser 65 70 75 80
Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr 85
90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala
Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu
Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser
Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr
Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys
Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205
Phe Asn Arg Asn Glu Cys 210 1541404DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
154atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt
ccactcccag 60gtccaactgc agcagcctgg ggctgaactg gtgaagcctg ggacttcagt
gaagctgtcc 120tgcaaggctt ctggctacac cttcaccacc tactggatgc
actgggtgaa tcagaggcct 180ggacaaggcc ttgagtggat tggagagatt
aatcctacca acggtcatac taactacaat 240gagaagttca agagcaaggc
cacactgact gtagacaaat cctccagcac agcctacatg 300caactcagca
gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactatgtt
360ggtagcatct ttgactactg gggccaaggc accactctca ccgtctcctc
agcctccacc 420aagggcccat cggtcttccc cctggcaccc tcctccaaga
gcacctctgg gggcacagcg 480gccctgggct gcctggtcaa ggactacttc
cccgaaccgg tgacggtgtc gtggaactca 540ggcgccctga ccagcggcgt
gcacaccttc ccggctgtcc tacagtcctc aggactctac 600tccctcagca
gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
660aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc
caaatcttgt 720gacaaaactc acacatgccc accgtgccca gcacctgaac
tcctgggggg accgtcagtc 780ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca 840tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 900ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac
960cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa
ggagtacaag 1020tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga
aaaccatctc caaagccaaa 1080gggcagcccc gagaaccaca ggtgtacacc
ctgcccccat cccgggatga gctgaccaag 1140aaccaggtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200tgggagagca
atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc
1260gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg
gcagcagggg 1320aacgtcttct catgctccgt gatgcatgag gctctgcaca
accactacac gcagaagagc 1380ctctccctgt ctccgggtaa atga
1404155448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 155Gln Val Gln Leu Gln Gln Pro Gly Ala Glu
Leu Val Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Val Asn
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn
Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser
Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110 Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330
335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445
156705DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 156atggaatcac agactctggt cttcatatcc
atactgctct ggttatatgg tgctgatggg 60aacattgtaa tgacccaatc tcccaaatcc
atgtccatgt cagtaggaga gagggtcacc 120ttgagctgca aggccagtga
gaatgtggtt tcttatgtat cctggtatca acagaaacca 180gcgcagtctc
ctaaactgct gatatacggg gcatccaacc ggaacactgg ggtccccgat
240cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag
tgtgcgggct 300gaagaccttg cagattatca ctgtgggcag agttacaact
atccgtacac gttcggaggg 360gggaccaggc tggaaataaa acgaactgtg
gctgcaccat ctgtcttcat cttcccgcca 420tctgatgagc agttgaaatc
tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480cccagagagg
ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag
540gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag
caccctgacg 600ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 660ctgagctcgc ccgtcacaaa gagcttcaac
aggggagagt gttga 705157214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 157Asn Ile Val Met Thr
Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val
Thr Leu Ser Cys Lys Ala Ser Glu Asn Val Val Ser Tyr 20 25 30 Val
Ser Trp Tyr Gln Gln Lys Pro Ala Gln Ser Pro Lys Leu Leu Ile 35 40
45 Tyr Gly Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val
Arg Ala 65 70 75 80 Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Ser Tyr
Asn Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 158412DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
158atggactgca cctggaggat cctcctcttg gtggcagcag ctacaggcac
ccacgccgag 60gtccagctgg tacagtctgg ggctgaggtg aagaagcctg gggctacagt
gaaaatctcc 120tgcaaggttt ctggatacac cttcaccacc tactggatgc
actgggtgca acaggcccct 180ggaaaagggc ttgagtggat gggagagatt
aatcctacca acggtcatac taactacaat 240gagaagttcc agggcagagt
caccataacc gcggacacgt ctacagacac agcctacatg 300gagctgagca
gcctgagatc tgaggacacg gccgtgtatt actgtgcaac aaactatgtt
360ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc ag
412159118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 159Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val
Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Val Gln
Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Asn
Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Gln Gly
Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Thr Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110 Leu Val Thr Val Ser Ser 115 160992DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
160cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc
acctctgggg 60gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg
acggtgtcgt 120ggaactcagg cgccctgacc agcggcgtgc acaccttccc
ggctgtccta cagtcctcag 180gactctactc cctcagcagc gtggtgaccg
tgccctccag cagcttgggc acccagacct 240acatctgcaa cgtgaatcac
aagcccagca acaccaaggt ggacaagaaa gttgagccca 300aatcttgtga
caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac
360cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc
cggacccctg 420aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc
tgaggtcaag ttcaactggt 480acgtggacgg cgtggaggtg cataatgcca
agacaaagcc gcgggaggag cagtacaaca 540gcacgtaccg tgtggtcagc
gtcctcaccg tcctgcacca ggactggctg aatggcaagg 600agtacaagtg
caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca
660aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc
cgggatgagc 720tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg
cttctatccc agcgacatcg 780ccgtggagtg ggagagcaat gggcagccgg
agaacaacta caagaccacg cctcccgtgc 840tggactccga cggctccttc
ttcctctaca gcaagctcac cgtggacaag agcaggtggc 900agcaggggaa
cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc
960agaagagcct ctccctgtct ccgggtaaat ga 992161330PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
161Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 325 330 1621404DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
162atggactgca cctggaggat cctcctcttg gtggcagcag ctacaggcac
ccacgccgag 60gtccagctgg tacagtctgg ggctgaggtg aagaagcctg gggctacagt
gaaaatctcc 120tgcaaggttt ctggatacac cttcaccacc tactggatgc
actgggtgca acaggcccct 180ggaaaagggc ttgagtggat gggagagatt
aatcctacca acggtcatac taactacaat 240gagaagttcc agggcagagt
caccataacc gcggacacgt ctacagacac agcctacatg 300gagctgagca
gcctgagatc tgaggacacg gccgtgtatt actgtgcaac aaactatgtt
360ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc
agcctccacc 420aagggcccat cggtcttccc cctggcaccc tcctccaaga
gcacctctgg gggcacagcg 480gccctgggct gcctggtcaa ggactacttc
cccgaaccgg tgacggtgtc gtggaactca 540ggcgccctga ccagcggcgt
gcacaccttc ccggctgtcc tacagtcctc aggactctac 600tccctcagca
gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
660aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc
caaatcttgt 720gacaaaactc acacatgccc accgtgccca gcacctgaac
tcctgggggg accgtcagtc 780ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca 840tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 900ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac
960cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa
ggagtacaag 1020tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga
aaaccatctc caaagccaaa 1080gggcagcccc gagaaccaca ggtgtacacc
ctgcccccat cccgggatga gctgaccaag 1140aaccaggtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200tgggagagca
atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc
1260gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg
gcagcagggg 1320aacgtcttct catgctccgt gatgcatgag gctctgcaca
accactacac gcagaagagc 1380ctctccctgt ctccgggtaa atga
1404163448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 163Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val
Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Val Gln
Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Asn
Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Gln Gly
Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Thr Asn Tyr Val Gly Ser Ile Phe Asp 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 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330
335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445
164412DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 164atggggtcaa ccgccatcct cgccctcctc
ctggctgttc tccaaggagt ctgtgccgaa 60gtgcagctgg tgcagtctgg agcagaggtg
aaaaagcccg gggagtctct gaggatctcc 120tgtaagggtt ctggatacag
ctttaccacc tactggatgc actgggtgcg ccagatgccc 180gggaaaggcc
tggagtggat gggggagatt aatcctacca acggtcatac taactacaat
240ccgtccttcc aaggccacgt caccatctca gctgacaagt ccatcagcac
tgcctacctg 300cagtggagca gcctgaaggc ctcggacacc gccatgtatt
actgtgcgag aaactatgtt 360ggtagcatct ttgactactg gggccaagga
accctggtca ccgtctcctc ag 412165118PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 165Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Arg
Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr 20 25 30 Trp
Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40
45 Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Pro Ser Phe
50 55 60 Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr
Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala
Met Tyr Tyr Cys 85 90 95 Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
1661404DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 166atggggtcaa ccgccatcct cgccctcctc
ctggctgttc tccaaggagt ctgtgccgaa 60gtgcagctgg tgcagtctgg agcagaggtg
aaaaagcccg gggagtctct gaggatctcc 120tgtaagggtt ctggatacag
ctttaccacc tactggatgc actgggtgcg ccagatgccc 180gggaaaggcc
tggagtggat gggggagatt aatcctacca acggtcatac taactacaat
240ccgtccttcc aaggccacgt caccatctca gctgacaagt ccatcagcac
tgcctacctg 300cagtggagca gcctgaaggc ctcggacacc gccatgtatt
actgtgcgag aaactatgtt 360ggtagcatct ttgactactg gggccaagga
accctggtca ccgtctcctc agcctccacc 420aagggcccat cggtcttccc
cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480gccctgggct
gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca
540ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc
aggactctac 600tccctcagca gcgtggtgac cgtgccctcc agcagcttgg
gcacccagac ctacatctgc 660aacgtgaatc acaagcccag caacaccaag
gtggacaaga aagttgagcc caaatcttgt 720gacaaaactc acacatgccc
accgtgccca gcacctgaac tcctgggggg accgtcagtc 780ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca
840tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg
gtacgtggac 900ggcgtggagg tgcataatgc caagacaaag ccgcgggagg
agcagtacaa cagcacgtac 960cgtgtggtca gcgtcctcac cgtcctgcac
caggactggc tgaatggcaa ggagtacaag 1020tgcaaggtct ccaacaaagc
cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080gggcagcccc
gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag
1140aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat
cgccgtggag 1200tgggagagca atgggcagcc ggagaacaac tacaagacca
cgcctcccgt gctggactcc 1260gacggctcct tcttcctcta cagcaagctc
accgtggaca agagcaggtg gcagcagggg 1320aacgtcttct catgctccgt
gatgcatgag gctctgcaca accactacac gcagaagagc 1380ctctccctgt
ctccgggtaa atga 1404167448PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 167Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Arg
Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr 20 25 30 Trp
Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40
45 Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Pro Ser Phe
50 55 60 Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr
Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala
Met Tyr Tyr Cys 85 90 95 Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp
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 Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295
300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420
425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 435 440 445 168412DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 168atggggtcaa
ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa 60gtgcagctgg
tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc
120tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg
ccagatgccc 180gggaaaggcc tggagtggat gggggagatt aatcctacca
acggtcatac taactacaat 240ccgtccttcc aaggccaggt caccatctca
gctgacaagt ccatcagcac tgcctacctg 300cagtggagca gcctgaaggc
ctcggacacc gccatgtatt actgtgcgag aaactatgtt 360ggtagcatct
ttgactactg gggccaagga accctggtca ccgtctcctc ag
412169118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 169Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly
Ser Gly Tyr Ser Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Val Arg
Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Asn
Pro Thr Asn Gly His Thr Asn Tyr Asn Pro Ser Phe 50 55 60 Gln Gly
Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85
90 95 Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110 Leu Val Thr Val Ser Ser 115 1701404DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
170atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt
ctgtgccgaa 60gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct
gaagatctcc 120tgtaagggtt ctggatacag ctttaccacc tactggatgc
actgggtgcg ccagatgccc 180gggaaaggcc tggagtggat gggggagatt
aatcctacca acggtcatac taactacaat 240ccgtccttcc aaggccaggt
caccatctca gctgacaagt ccatcagcac tgcctacctg 300cagtggagca
gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt
360ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc
agcctccacc 420aagggcccat cggtcttccc cctggcaccc tcctccaaga
gcacctctgg gggcacagcg 480gccctgggct gcctggtcaa ggactacttc
cccgaaccgg tgacggtgtc gtggaactca 540ggcgccctga ccagcggcgt
gcacaccttc ccggctgtcc tacagtcctc aggactctac 600tccctcagca
gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
660aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc
caaatcttgt 720gacaaaactc acacatgccc accgtgccca gcacctgaac
tcctgggggg accgtcagtc 780ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca 840tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 900ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac
960cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa
ggagtacaag 1020tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga
aaaccatctc caaagccaaa 1080gggcagcccc gagaaccaca ggtgtacacc
ctgcccccat cccgggatga gctgaccaag 1140aaccaggtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200tgggagagca
atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc
1260gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg
gcagcagggg 1320aacgtcttct catgctccgt gatgcatgag gctctgcaca
accactacac gcagaagagc 1380ctctccctgt ctccgggtaa atga
1404171448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 171Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile
Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr 20 25 30 Trp Met
His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45
Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Pro Ser Phe 50
55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala
Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met
Tyr Tyr Cys 85 90 95 Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp 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 Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305
310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425
430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445 172388DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 172atggacatga gggtccccgc
tcagctcctg gggctcctgc tactctggct ccgaggtgcc 60agatgtgaca tccagatgac
ccagtctcca tcctccctgt ctgcatctgt aggagacaga 120gtcaccatca
cttgcaaggc cagtgagaat gtggtttctt atgtatcctg gtatcagcag
180aaaccaggga aagcccctaa gctcctgatc tatggggcat ccaaccggaa
cactggggtc 240ccatcaaggt tcagtggcag tggatctggg acagatttca
ctctcaccat cagcagtctg 300caacctgaag attttgcaac ttactactgt
gggcagagtt acaactatcc gtacacgttt 360ggccagggga ccaagctgga gatcaaac
388173107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 173Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys
Ala Ser Glu Asn Val Val Ser Tyr 20 25 30 Val Ser Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser
Asn Arg Asn Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gln Ser Tyr Asn Tyr Pro Tyr 85
90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
174323DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 174gaactgtggc tgcaccatct gtcttcatct
tcccgccatc tgatgagcag ttgaaatctg 60gaactgcctc tgttgtgtgc ctgctgaata
acttctatcc cagagaggcc aaagtacagt 120ggaaggtgga taacgccctc
caatcgggta actcccagga gagtgtcaca gagcaggaca 180gcaaggacag
cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga
240aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc
gtcacaaaga 300gcttcaacag gggagagtgt tga 323175107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
175Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 100 105 176711DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
176atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct
ccgaggtgcc 60agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt
aggagacaga 120gtcaccatca cttgcaaggc cagtgagaat gtggtttctt
atgtatcctg gtatcagcag 180aaaccaggga aagcccctaa gctcctgatc
tatggggcat ccaaccggaa cactggggtc 240ccatcaaggt tcagtggcag
tggatctggg acagatttca ctctcaccat cagcagtctg 300caacctgaag
attttgcaac ttactactgt gggcagagtt acaactatcc gtacacgttt
360ggccagggga ccaagctgga gatcaaacga actgtggctg caccatctgt
cttcatcttc 420ccgccatctg atgagcagtt gaaatctgga actgcctctg
ttgtgtgcct gctgaataac 480ttctatccca gagaggccaa agtacagtgg
aaggtggata acgccctcca atcgggtaac 540tcccaggaga gtgtcacaga
gcaggacagc aaggacagca cctacagcct cagcagcacc 600ctgacgctga
gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat
660cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgttg a
711177214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 177Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys
Ala Ser Glu Asn Val Val Ser Tyr 20 25 30 Val Ser Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser
Asn Arg Asn Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gln Ser Tyr Asn Tyr Pro Tyr 85
90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205
Phe Asn Arg Gly Glu Cys 210 178382DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 178atggaagccc
cagcgcagct tctcttcctc ctgctactct ggctcccaga taccactgga 60gaaatagtga
tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc
120ctctcctgca aggccagtga gaatgtggtt tcttatgtat cctggtacca
gcagaaacct 180ggccaggctc ccaggctcct catctatggg gcatccaacc
ggaacactgg tatcccagcc 240aggttcagtg gcagtgggtc tgggacagag
ttcactctca ccatcagcag cctgcagtct 300gaagattttg cagtttatta
ctgtgggcag agttacaact atccgtacac gtttggccag 360gggaccaagc
tggagatcaa ac 382179107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 179Glu Ile Val Met Thr
Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Lys Ala Ser Glu Asn Val Val Ser Tyr 20 25 30 Val
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Gly Ala Ser Asn Arg Asn Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gly Gln Ser Tyr
Asn Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 105 180705DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 180atggaagccc cagcgcagct
tctcttcctc ctgctactct ggctcccaga taccactgga 60gaaatagtga tgacgcagtc
tccagccacc ctgtctgtgt ctccagggga aagagccacc 120ctctcctgca
aggccagtga gaatgtggtt tcttatgtat cctggtacca gcagaaacct
180ggccaggctc ccaggctcct catctatggg gcatccaacc ggaacactgg
tatcccagcc 240aggttcagtg gcagtgggtc tgggacagag ttcactctca
ccatcagcag cctgcagtct 300gaagattttg cagtttatta ctgtgggcag
agttacaact atccgtacac gtttggccag 360gggaccaagc tggagatcaa
acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat
480cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 540gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 600ctgagcaaag cagactacga gaaacacaaa
gtctacgcct gcgaagtcac ccatcagggc 660ctgagctcgc ccgtcacaaa
gagcttcaac aggggagagt gttga 705181214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
181Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Glu Asn Val Val
Ser Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Asn Thr Gly Ile
Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr
Tyr Cys Gly Gln Ser Tyr Asn Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130
135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys
210 182412DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 182atgggctggt catatattat tctctttctt
gttgctaccg ctaccgatgt gcactctcaa 60gtccaactcg tacaaccagg cgctgaagtc
gtaaaacccg gaacatctgt taaactctca 120tgcaaagcct caggatacac
tttcacaact tactggatgc attgggtcaa tcaagccccc 180ggacaaggcc
tcgaatggat tggcgaaatt aacccaacta acggacatac taattataat
240gaaaaattta agggcaaagc tacactcacc gtcgataaat caacctctac
agcttatatg 300gaactttcat ccctgagatc agaagataca gccgtctact
attgcgccag aaactacgta 360ggatcaatat tcgattactg gggtcaaggc
actctcctca cagtcagctc ag 412183118PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 183Gln Val Gln Leu Val
Gln Pro Gly Ala Glu Val Val Lys Pro Gly Thr 1 5 10 15 Ser Val Lys
Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp
Met His Trp Val Asn Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe
50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Leu Thr Val Ser Ser 115
184992DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 184ccagcacaaa gggcccatcg gtcttccccc
tggcaccctc ctccaagagc acctctgggg 60gcacagcggc cctgggctgc ctggtcaagg
actacttccc cgaaccggtg acggtgtcgt 120ggaactcagg cgccctgacc
agcggcgtgc acaccttccc ggctgtccta cagtcctcag 180gactctactc
cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct
240acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga
gttgagccca 300aatcttgtga caaaactcac acatgtccac cgtgcccagc
acctgaactc ctggggggac 360cgtcagtctt cctcttcccc ccaaaaccca
aggacaccct catgatctcc cggacccctg 420aggtcacatg cgtggtggtg
gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt 480acgtggacgg
cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca
540gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg
aatggcaagg 600agtacaagtg caaggtctcc aacaaagccc tcccagcccc
catcgagaaa accatctcca 660aagccaaagg gcagccccga gaaccacagg
tgtacaccct gcccccatcc cgggaggaga 720tgaccaagaa ccaggtcagc
ctgacctgcc tggtcaaagg cttctatccc agcgacatcg 780ccgtggagtg
ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc
840tggactccga cggctccttc ttcctctata gcaagctcac cgtggacaag
agcaggtggc 900agcaggggaa cgtcttctca tgctccgtga tgcatgaggc
tctgcacaac cactacacgc 960agaagagcct ctccctgtcc ccgggtaaat ga
992185330PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 185Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
1861404DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 186atgggctggt catatattat tctctttctt
gttgctaccg ctaccgatgt gcactctcaa 60gtccaactcg tacaaccagg cgctgaagtc
gtaaaacccg gaacatctgt taaactctca 120tgcaaagcct caggatacac
tttcacaact tactggatgc attgggtcaa tcaagccccc 180ggacaaggcc
tcgaatggat tggcgaaatt aacccaacta acggacatac taattataat
240gaaaaattta agggcaaagc tacactcacc gtcgataaat caacctctac
agcttatatg 300gaactttcat ccctgagatc agaagataca gccgtctact
attgcgccag aaactacgta 360ggatcaatat tcgattactg gggtcaaggc
actctcctca cagtcagctc agccagcaca 420aagggcccat cggtcttccc
cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480gccctgggct
gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca
540ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc
aggactctac 600tccctcagca gcgtggtgac cgtgccctcc agcagcttgg
gcacccagac ctacatctgc 660aacgtgaatc acaagcccag caacaccaag
gtggacaaga gagttgagcc caaatcttgt 720gacaaaactc acacatgtcc
accgtgccca gcacctgaac tcctgggggg accgtcagtc 780ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca
840tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg
gtacgtggac 900ggcgtggagg tgcataatgc caagacaaag ccgcgggagg
agcagtacaa cagcacgtac 960cgtgtggtca gcgtcctcac cgtcctgcac
caggactggc tgaatggcaa ggagtacaag 1020tgcaaggtct ccaacaaagc
cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080gggcagcccc
gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag
1140aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat
cgccgtggag 1200tgggagagca atgggcagcc ggagaacaac tacaagacca
cgcctcccgt gctggactcc 1260gacggctcct tcttcctcta tagcaagctc
accgtggaca agagcaggtg gcagcagggg 1320aacgtcttct catgctccgt
gatgcatgag gctctgcaca accactacac gcagaagagc 1380ctctccctgt
ccccgggtaa atga 1404187448PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 187Gln Val Gln Leu Val
Gln Pro Gly Ala Glu Val Val Lys Pro Gly Thr 1 5 10 15 Ser Val Lys
Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp
Met His Trp Val Asn Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe
50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Leu Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295
300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420
425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 435 440 445 188412DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 188atgggttggt
catatattat actctttctc gtagccaccg ccaccgacgt acactctcag 60gttcaactcg
tacaacccgg cgccgaagtc aagaaaccag gaacatcagt caaactctca
120tgtaaagcaa gcggatacac ctttactact tattggatgc attgggtaag
acaagccccc 180ggacaaggac tcgaatggat aggcgaaata aatcccacta
atggacatac aaattataat 240caaaaatttc aaggacgcgc tacactcacc
gtcgataaat caacctcaac cgcatacatg 300gaactcagct ccctccgatc
cgaagacact gccgtttatt attgtgccag aaactatgta 360ggatctattt
tcgattactg gggacaagga acacttctca ccgtaagctc ag
412189118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 189Gln Val Gln Leu Val Gln Pro Gly Ala Glu
Val Lys Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn
Pro Thr Asn Gly His Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Gln Gly
Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110 Leu Leu Thr Val Ser Ser 115 1901404DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
190atgggttggt catatattat actctttctc gtagccaccg ccaccgacgt
acactctcag 60gttcaactcg tacaacccgg cgccgaagtc aagaaaccag gaacatcagt
caaactctca 120tgtaaagcaa gcggatacac ctttactact tattggatgc
attgggtaag acaagccccc 180ggacaaggac tcgaatggat aggcgaaata
aatcccacta atggacatac aaattataat 240caaaaatttc aaggacgcgc
tacactcacc gtcgataaat caacctcaac cgcatacatg 300gaactcagct
ccctccgatc cgaagacact gccgtttatt attgtgccag aaactatgta
360ggatctattt tcgattactg gggacaagga acacttctca ccgtaagctc
agccagcaca 420aagggcccat cggtcttccc cctggcaccc tcctccaaga
gcacctctgg gggcacagcg 480gccctgggct gcctggtcaa ggactacttc
cccgaaccgg tgacggtgtc gtggaactca 540ggcgccctga ccagcggcgt
gcacaccttc ccggctgtcc tacagtcctc aggactctac 600tccctcagca
gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
660aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc
caaatcttgt 720gacaaaactc acacatgtcc accgtgccca gcacctgaac
tcctgggggg accgtcagtc 780ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca 840tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 900ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac
960cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa
ggagtacaag 1020tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga
aaaccatctc caaagccaaa 1080gggcagcccc gagaaccaca ggtgtacacc
ctgcccccat cccgggagga gatgaccaag 1140aaccaggtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200tgggagagca
atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc
1260gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg
gcagcagggg 1320aacgtcttct catgctccgt gatgcatgag gctctgcaca
accactacac gcagaagagc 1380ctctccctgt ccccgggtaa atga
1404191448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 191Gln Val Gln Leu Val Gln Pro Gly Ala Glu
Val Lys Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn
Pro Thr Asn Gly His Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Gln Gly
Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110 Leu Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330
335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445
192360DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 192atggaaagtc agacccttgt attcatctct
attcttcttt ggttgtatgg agcagacggc 60gacattgtga tgacccaatc ccccgatagt
atggccatga gtgtaggaga aagagtcacc 120cttaattgca aagcctccga
aaatgtcgtt tcatatgtgt cttggtatca acaaaaaccc 180ggccaatcac
ccaaacttct catatacggc gcttcaaaca gaaacacagg cgttcccgac
240agatttagtg gatccggatc agctacagat ttcaccctta ccatcagttc
agttcaagca 300gaagacgttg cagactatca ttgcggacaa tcttataact
acccttacac attcggacaa 360193107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 193Asp Ile Val Met Thr
Gln Ser Pro Asp Ser Met Ala Met Ser Val Gly 1 5 10 15 Glu Arg Val
Thr Leu Asn Cys Lys Ala Ser Glu Asn Val Val Ser Tyr 20 25 30 Val
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40
45 Tyr Gly Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Ser Gly
50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val
Gln Ala 65 70 75 80 Glu Asp Val Ala Asp Tyr His Cys Gly Gln Ser Tyr
Asn Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 105 194323DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 194gtacggtggc tgcaccatct
gtcttcatct tcccgccatc tgatgagcag ttgaaatctg 60gaactgcctc tgttgtgtgc
ctgctgaata acttctatcc cagagaggcc aaagtacagt 120ggaaggtgga
taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca
180gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca
gactacgaga 240aacacaaagt ctacgcctgc gaagtcaccc atcagggcct
gagctcgccc gtcacaaaga 300gcttcaacag gggagagtgt tag
323195107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 195Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85
90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
196705DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 196atggaaagtc agacccttgt attcatctct
attcttcttt ggttgtatgg agcagacggc 60gacattgtga tgacccaatc ccccgatagt
atggccatga gtgtaggaga aagagtcacc 120cttaattgca aagcctccga
aaatgtcgtt tcatatgtgt cttggtatca acaaaaaccc 180ggccaatcac
ccaaacttct catatacggc gcttcaaaca gaaacacagg cgttcccgac
240agatttagtg gatccggatc agctacagat ttcaccctta ccatcagttc
agttcaagca 300gaagacgttg cagactatca ttgcggacaa tcttataact
acccttacac attcggacaa 360ggaaccaaac tcgaaattaa acgtacggtg
gctgcaccat ctgtcttcat cttcccgcca 420tctgatgagc agttgaaatc
tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480cccagagagg
ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag
540gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag
caccctgacg 600ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 660ctgagctcgc ccgtcacaaa gagcttcaac
aggggagagt gttag 705197214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 197Asp Ile Val Met Thr
Gln Ser Pro Asp Ser Met Ala Met Ser Val Gly 1 5 10 15 Glu Arg Val
Thr Leu Asn Cys Lys Ala Ser Glu Asn Val Val Ser Tyr 20 25 30 Val
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40
45 Tyr Gly Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Ser Gly
50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser
Ser Val Gln Ala 65 70 75 80 Glu Asp Val Ala Asp Tyr His Cys Gly Gln
Ser Tyr Asn Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155
160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
198382DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 198atggaatccc aaacccttgt tttcatctct
atccttctct ggctttatgg cgccgacgga 60gacatcgtaa tgacacaatc ccctgactct
cttgctatga gcttgggcga acgagtaaca 120cttaactgca aagcatccga
aaatgtcgta tcttacgtat cctggtatca gcaaaaacct 180ggtcaaagtc
ctaaacttct tatatatggt gcaagtaatc gtgaaagtgg cgtcccagac
240agatttagcg gttcaggttc agcaactgac tttacactta caatttctag
cgttcaggcc 300gaagacgttg cagactatca ttgtggacaa tcttataact
atccttatac tttcggacaa 360ggcactaaac ttgaaattaa ac
382199107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 199Asp Ile Val Met Thr Gln Ser Pro Asp Ser
Leu Ala Met Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Leu Asn Cys Lys
Ala Ser Glu Asn Val Val Ser Tyr 20 25 30 Val Ser Trp Tyr Gln Gln
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser
Asn Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80
Glu Asp Val Ala Asp Tyr His Cys Gly Gln Ser Tyr Asn Tyr Pro Tyr 85
90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
200705DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 200atggaatccc aaacccttgt tttcatctct
atccttctct ggctttatgg cgccgacgga 60gacatcgtaa tgacacaatc ccctgactct
cttgctatga gcttgggcga acgagtaaca 120cttaactgca aagcatccga
aaatgtcgta tcttacgtat cctggtatca gcaaaaacct 180ggtcaaagtc
ctaaacttct tatatatggt gcaagtaatc gtgaaagtgg cgtcccagac
240agatttagcg gttcaggttc agcaactgac tttacactta caatttctag
cgttcaggcc 300gaagacgttg cagactatca ttgtggacaa tcttataact
atccttatac tttcggacaa 360ggcactaaac ttgaaattaa acgtacggtg
gctgcaccat ctgtcttcat cttcccgcca 420tctgatgagc agttgaaatc
tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480cccagagagg
ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag
540gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag
caccctgacg 600ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 660ctgagctcgc ccgtcacaaa gagcttcaac
aggggagagt gttag 705201214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 201Asp Ile Val Met Thr
Gln Ser Pro Asp Ser Leu Ala Met Ser Leu Gly 1 5 10 15 Glu Arg Val
Thr Leu Asn Cys Lys Ala Ser Glu Asn Val Val Ser Tyr 20 25 30 Val
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40
45 Tyr Gly Ala Ser Asn Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly
50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val
Gln Ala 65 70 75 80 Glu Asp Val Ala Asp Tyr His Cys Gly Gln Ser Tyr
Asn Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 20217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 202Glu
Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe Gln 1 5 10
15 Gly 20317PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 203Glu Ile Asn Pro Thr Asn Gly His Thr
Asn Tyr Asn Pro Ser Phe Gln 1 5 10 15 Gly 20417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 204Glu
Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10
15 Gly 20517PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 205Glu Ile Asn Pro Thr Asn Gly His Thr
Asn Tyr Asn Gln Lys Phe Gln 1 5 10 15 Gly 2067PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 206Gly
Ala Ser Asn Arg Glu Ser 1 5 207992DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 207cctccaccaa
gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg 60gcacagcggc
cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt
120ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta
cagtcctcag 180gactctactc cctcagcagc gtggtgaccg tgccctccag
cagcttgggc acccagacct 240acatctgcaa cgtgaatcac aagcccagca
acaccaaggt ggacaagaga gttgagccca 300aatcttgtga caaaactcac
acatgcccac cgtgcccagc acctgaactc ctggggggac 360cgtcagtctt
cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg
420aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag
ttcaactggt 480acgtggacgg cgtggaggtg cataatgcca agacaaagcc
gcgggaggag cagtacaaca 540gcacgtaccg tgtggtcagc gtcctcaccg
tcctgcacca ggactggctg aatggcaagg 600agtacaagtg caaggtctcc
aacaaagccc tcccagcccc catcgagaag accatctcca 660aagccaaagg
gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggaggaga
720tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc
agcgacatcg 780ccgtggagtg ggagagcaat gggcagccgg agaacaacta
caagaccacg cctcccgtgc 840tggactccga cggctccttc ttcctctaca
gcaagctcac cgtggacaag agcaggtggc 900agcaggggaa cgtcttctca
tgctccgtga tgcatgaggc tctgcacaac cactacacgc 960agaagagcct
ctccctgtct ccgggtaaat ga 992208330PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 208Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
2091404DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 209atggggtcaa ccgccatcct cgccctcctc
ctggctgttc tccaaggagt ctgtgccgaa 60gtgcagctgg tgcagtctgg agcagaggtg
aaaaagcccg gggagtctct gaagatctcc 120tgtaagggtt ctggatacag
ctttaccacc tactggatgc actgggtgcg ccagatgccc 180gggaaaggcc
tggagtggat gggggagatt aatcctacca acggtcatac taactacaat
240ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac
tgcctacctg 300cagtggagca gcctgaaggc ctcggacacc gccatgtatt
actgtgcgag aaactatgtt 360ggtagcatct ttgactactg gggccaagga
accctggtca ccgtctcctc agcctccacc 420aagggcccat cggtcttccc
cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480gccctgggct
gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca
540ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc
aggactctac 600tccctcagca gcgtggtgac cgtgccctcc agcagcttgg
gcacccagac ctacatctgc 660aacgtgaatc acaagcccag caacaccaag
gtggacaaga gagttgagcc caaatcttgt 720gacaaaactc acacatgccc
accgtgccca gcacctgaac tcctgggggg accgtcagtc 780ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca
840tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg
gtacgtggac 900ggcgtggagg tgcataatgc caagacaaag ccgcgggagg
agcagtacaa cagcacgtac 960cgtgtggtca gcgtcctcac cgtcctgcac
caggactggc tgaatggcaa ggagtacaag 1020tgcaaggtct ccaacaaagc
cctcccagcc cccatcgaga agaccatctc caaagccaaa 1080gggcagcccc
gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag
1140aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat
cgccgtggag 1200tgggagagca atgggcagcc ggagaacaac tacaagacca
cgcctcccgt gctggactcc 1260gacggctcct tcttcctcta cagcaagctc
accgtggaca agagcaggtg gcagcagggg 1320aacgtcttct catgctccgt
gatgcatgag gctctgcaca accactacac gcagaagagc 1380ctctccctgt
ctccgggtaa atga 1404210448PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 210Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys
Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr 20 25 30 Trp
Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40
45 Gly Glu Ile Asn Pro Thr Asn Gly His Thr Asn Tyr Asn Pro Ser Phe
50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr
Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala
Met Tyr Tyr Cys 85 90 95 Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp
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 Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295
300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420
425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 435 440 445 2112209DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 211atgatgtggg
ggaccaaact tctgccggtc ctgttgctgc agcatgtcct cctgcacctc 60ctcctgcttc
atgtcgccat cccctatgca gaaggacaga agaaaagaag aaatacactt
120catgaattta aaaagtcagc aaaaactact cttaccaagg aagacccatt
actgaagatt 180aaaaccaaaa aagtgaactc tgcagatgag tgtgccaaca
ggtgtatcag gaacaggggc 240tttacgttca cttgcaaggc cttcgttttt
gataagtcaa gaaaacgatg ctactggtat 300cctttcaata gtatgtcaag
tggagtgaaa aaagggtttg gccatgaatt tgacctctat 360gaaaacaaag
actatattag aaactgcatc attggtaaag gaggcagcta taaagggacg
420gtatccatca ctaagagtgg catcaaatgc cagccttgga attccatgat
cccccatgaa 480cacagctttt tgccttcgag ctatcgcggt aaagacctac
aggaaaacta ctgtcgaaat 540cctcgagggg aagaaggggg accctggtgt
ttcacaagca atccagaggt acgctacgaa 600gtctgtgaca ttcctcagtg
ttcagaagtt gaatgcatga cctgcaatgg tgaaagctac 660agaggtccca
tggatcacac agaatcaggc aagacttgtc agcgctggga ccagcagaca
720ccacaccggc acaagttctt gccagaaaga tatcccgaca agggctttga
tgataattat 780tgccgcaatc ctgatggcaa gccgaggcca tggtgctaca
ctcttgaccc tgacaccact 840tgggagtatt gtgcaattaa aacgtgcgct
cacagtgctg tgaatgagac tgatgtccct 900atggaaacaa ctgaatgcat
tcaaggccaa ggagaaggtt acaggggaac cagcaatacc 960atttggaatg
gaattccctg tcagcgttgg gattcgcagt accctcacaa gcatgatatc
1020actcccgaga acttcaaatg caaggacctt agagaaaatt attgccgcaa
tccagatggg 1080gctgaatcac catggtgttt taccactgac ccaaacatcc
gagttggcta ctgctctcag 1140attcccaagt gtgacgtgtc aagtggacaa
gattgttatc gtggcaatgg gaaaaattac 1200atgggcaact tatccaaaac
aaggtctgga cttacatgtt ccatgtggga caagaatatg 1260gaggatttac
accgtcatat cttctgggag ccagatgcta gcaaattgaa taagaattac
1320tgccggaatc ctgatgatga tgcccatgga ccttggtgct acacggggaa
tcctcttatt 1380ccttgggatt attgccctat ttcccgttgt gaaggagata
ctacacctac aattgtcaat 1440ttggaccatc ctgtaatatc ctgtgccaaa
acaaaacaac tgcgggttgt aaatgggatt 1500ccaacacgaa caaacatagg
atggatggtt agtttgagat acagaaataa acatatctgc 1560ggaggatcat
tgataaagga gagttgggtt cttactgcac gacagtgttt cccttctcga
1620gacttgaaag attatgaagc ttggcttgga attcatgatg tccacggaag
aggagatgag 1680aaatgcaaac aggttctcaa tgtttcccag ctggtatatg
gccctgaagg atcagatctg
1740gttttaatga agcttgctcg acctgcaatc ctggataact ttgtcagtac
aattgattta 1800cctagttatg gttgtacaat ccctgaaaag accacttgca
gtatttacgg ctggggctac 1860actggattga tcaacgcgga tggtttatta
cgagtagctc atctgtatat tatggggaat 1920gagaaatgca gtcagcacca
tcaaggcaag gtgactttga atgagtctga gttatgtgct 1980ggggctgaaa
agattggatc aggaccatgt gagggagatt atggtggccc actcatttgt
2040gaacaacaca aaatgagaat ggttcttggt gtcattgttc ctggtcgtgg
atgtgccatc 2100ccaaatcgtc ctgttatttt tgttcgagta gcatattatg
caaaatggat acacaaagta 2160attttgacat acaagttgtg cggccgccat
caccatcacc atcactaag 2209212680PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 212Pro Leu Leu Lys Ile
Lys Thr Lys Lys Val Asn Ser Ala Asp Glu Cys 1 5 10 15 Ala Asn Arg
Cys Ile Arg Asn Arg Gly Phe Thr Phe Thr Cys Lys Ala 20 25 30 Phe
Val Phe Asp Lys Ser Arg Lys Arg Cys Tyr Trp Tyr Pro Phe Asn 35 40
45 Ser Met Ser Ser Gly Val Lys Lys Gly Phe Gly His Glu Phe Asp Leu
50 55 60 Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys Ile Ile Gly Lys
Gly Gly 65 70 75 80 Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly
Ile Lys Cys Gln 85 90 95 Pro Trp Asn Ser Met Ile Pro His Glu His
Ser Phe Leu Pro Ser Ser 100 105 110 Tyr Arg Gly Lys Asp Leu Gln Glu
Asn Tyr Cys Arg Asn Pro Arg Gly 115 120 125 Glu Glu Gly Gly Pro Trp
Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr 130 135 140 Glu Val Cys Asp
Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr Cys 145 150 155 160 Asn
Gly Glu Ser Tyr Arg Gly Pro Met Asp His Thr Glu Ser Gly Lys 165 170
175 Thr Cys Gln Arg Trp Asp Gln Gln Thr Pro His Arg His Lys Phe Leu
180 185 190 Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp Asn Tyr Cys
Arg Asn 195 200 205 Pro Asp Gly Lys Pro Arg Pro Trp Cys Tyr Thr Leu
Asp Pro Asp Thr 210 215 220 Thr Trp Glu Tyr Cys Ala Ile Lys Thr Cys
Ala His Ser Ala Val Asn 225 230 235 240 Glu Thr Asp Val Pro Met Glu
Thr Thr Glu Cys Ile Gln Gly Gln Gly 245 250 255 Glu Gly Tyr Arg Gly
Thr Ser Asn Thr Ile Trp Asn Gly Ile Pro Cys 260 265 270 Gln Arg Trp
Asp Ser Gln Tyr Pro His Lys His Asp Ile Thr Pro Glu 275 280 285 Asn
Phe Lys Cys Lys Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro Asp 290 295
300 Gly Ala Glu Ser Pro Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg Val
305 310 315 320 Gly Tyr Cys Ser Gln Ile Pro Lys Cys Asp Val Ser Ser
Gly Gln Asp 325 330 335 Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met Gly
Asn Leu Ser Lys Thr 340 345 350 Arg Ser Gly Leu Thr Cys Ser Met Trp
Asp Lys Asn Met Glu Asp Leu 355 360 365 His Arg His Ile Phe Trp Glu
Pro Asp Ala Ser Lys Leu Asn Lys Asn 370 375 380 Tyr Cys Arg Asn Pro
Asp Asp Asp Ala His Gly Pro Trp Cys Tyr Thr 385 390 395 400 Gly Asn
Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys Glu 405 410 415
Gly Asp Thr Thr Pro Thr Ile Val Asn Leu Asp His Pro Val Ile Ser 420
425 430 Cys Ala Lys Thr Lys Gln Leu Arg Val Val Asn Gly Ile Pro Thr
Arg 435 440 445 Thr Asn Ile Gly Trp Met Val Ser Leu Arg Tyr Arg Asn
Lys His Ile 450 455 460 Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp Val
Leu Thr Ala Arg Gln 465 470 475 480 Cys Phe Pro Ser Arg Asp Leu Lys
Asp Tyr Glu Ala Trp Leu Gly Ile 485 490 495 His Asp Val His Gly Arg
Gly Asp Glu Lys Cys Lys Gln Val Leu Asn 500 505 510 Val Ser Gln Leu
Val Tyr Gly Pro Glu Gly Ser Asp Leu Val Leu Met 515 520 525 Lys Leu
Ala Arg Pro Ala Ile Leu Asp Asn Phe Val Ser Thr Ile Asp 530 535 540
Leu Pro Ser Tyr Gly Cys Thr Ile Pro Glu Lys Thr Thr Cys Ser Ile 545
550 555 560 Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn Ala Asp Gly Leu
Leu Arg 565 570 575 Val Ala His Leu Tyr Ile Met Gly Asn Glu Lys Cys
Ser Gln His His 580 585 590 Gln Gly Lys Val Thr Leu Asn Glu Ser Glu
Leu Cys Ala Gly Ala Glu 595 600 605 Lys Ile Gly Ser Gly Pro Cys Glu
Gly Asp Tyr Gly Gly Pro Leu Ile 610 615 620 Cys Glu Gln His Lys Met
Arg Met Val Leu Gly Val Ile Val Pro Gly 625 630 635 640 Arg Gly Cys
Ala Ile Pro Asn Arg Pro Val Ile Phe Val Arg Val Ala 645 650 655 Tyr
Tyr Ala Lys Trp Ile His Lys Val Ile Leu Thr Tyr Lys Leu Cys 660 665
670 Gly Arg His His His His His His 675 680 2132194DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
213atgatgtggg ggaccaaact tctgccggtc ctgttgctgc agcatgtcct
cctgcacctc 60ctcctgcttc atgtcgccat cccctatgca gaaggacaga agaaaagaag
aaatacactt 120catgaattta aaaagtcagc aaaaactact cttaccaagg
aagacccatt actgaagatt 180aaaaccaaaa aagtgaactc tgcagatgag
tgtgccaaca ggtgtatcag gaacaggggc 240tttacgttca cttgcaaggc
cttcgttttt gataagtcaa gaaaacgatg ctactggtat 300cctttcaata
gtatgtcaag tggagtgaaa aaagggtttg gccatgaatt tgacctctat
360gaaaacaaag actatattag aaactgcatc attggtaaag gaggcagcta
taaagggacg 420gtatccatca ctaagagtgg catcaaatgc cagccttgga
attccatgat cccccatgaa 480cacagctatc gcggtaaaga cctacaggaa
aactactgtc gaaatcctcg aggggaagaa 540gggggaccct ggtgtttcac
aagcaatcca gaggtacgct acgaagtctg tgacattcct 600cagtgttcag
aagttgaatg catgacctgc aatggtgaaa gctacagagg tcccatggat
660cacacagaat caggcaagac ttgtcagcgc tgggaccagc agacaccaca
ccggcacaag 720ttcttgccag aaagatatcc cgacaagggc tttgatgata
attattgccg caatcctgat 780ggcaagccga ggccatggtg ctacactctt
gaccctgaca ccccttggga gtattgtgca 840attaaaacgt gcgctcacag
tgctgtgaat gagactgatg tccctatgga aacaactgaa 900tgcattcaag
gccaaggaga aggttacagg ggaaccagca ataccatttg gaatggaatt
960ccctgtcagc gttgggattc gcagtaccct cacaagcatg atatcactcc
cgagaacttc 1020aaatgcaagg accttagaga aaattattgc cgcaatccag
atggggctga atcaccatgg 1080tgttttacca ctgacccaaa catccgagtt
ggctactgct ctcagattcc caagtgtgac 1140gtgtcaagtg gacaagattg
ttatcgtggc aatgggaaaa attacatggg caacttatcc 1200aaaacaaggt
ctggacttac atgttccatg tgggacaaga atatggagga tttacaccgt
1260catatcttct gggagccaga tgctagcaaa ttgaataaga attactgccg
gaatcctgat 1320gatgatgccc atggaccttg gtgctacacg gggaatcctc
ttattccttg ggattattgc 1380cctatttccc gttgtgaagg agatactaca
cctacaattg tcaatttgga ccatcctgta 1440atatcctgtg ccaaaacaaa
acaactgcgg gttgtaaatg ggattccaac acgaacaaac 1500ataggatgga
tggttagttt gagatacaga aataaacata tctgcggagg atcattgata
1560aaggagagtt gggttcttac tgcacgacag tgtttccctt ctcgagactt
gaaagattat 1620gaagcttggc ttggaattca tgatgtccac ggaagaggag
aggagaaaag aaaacagatt 1680ctcaatattt cccagctggt atatggccct
gaaggatcag atctggtttt actgaagctt 1740gctcgacctg caatcctgga
taactttgtc agtacaattg atttacctag ttatggttgt 1800acaatccctg
aaaagaccac ttgcagtatt tacggctggg gctacactgg attgatcaac
1860gcggatggtt tattacgagt agctcatctg tatattatgg ggaatgagaa
atgcagtcag 1920caccatcaag gcaaggtgac tttgaatgag tctgagttat
gtgctggggc tgaaaagatt 1980ggatcaggac catgtgaggg agattatggt
ggcccactca tttgtgaaca acacaaaatg 2040agaatggttc ttggtgtcat
tgttcctggt cgtggatgtg ccatcccaaa tcgtcctggt 2100atttttgttc
gagtagcata ttatgcaaaa tggatacaca aagtaatttt gacatacaag
2160ttgtgcggcc gccatcacca tcaccatcac taag 2194214675PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
214Pro Leu Leu Lys Ile Lys Thr Lys Lys Val Asn Ser Ala Asp Glu Cys
1 5 10 15 Ala Asn Arg Cys Ile Arg Asn Arg Gly Phe Thr Phe Thr Cys
Lys Ala 20 25 30 Phe Val Phe Asp Lys Ser Arg Lys Arg Cys Tyr Trp
Tyr Pro Phe Asn 35 40 45 Ser Met Ser Ser Gly Val Lys Lys Gly Phe
Gly His Glu Phe Asp Leu 50 55 60 Tyr Glu Asn Lys Asp Tyr Ile Arg
Asn Cys Ile Ile Gly Lys Gly Gly 65 70 75 80 Ser Tyr Lys Gly Thr Val
Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln 85 90 95 Pro Trp Asn Ser
Met Ile Pro His Glu His Ser Tyr Arg Gly Lys Asp 100 105 110 Leu Gln
Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro 115 120 125
Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile 130
135 140 Pro Gln Cys Ser Glu Val Glu Cys Met Thr Cys Asn Gly Glu Ser
Tyr 145 150 155 160 Arg Gly Pro Met Asp His Thr Glu Ser Gly Lys Thr
Cys Gln Arg Trp 165 170 175 Asp Gln Gln Thr Pro His Arg His Lys Phe
Leu Pro Glu Arg Tyr Pro 180 185 190 Asp Lys Gly Phe Asp Asp Asn Tyr
Cys Arg Asn Pro Asp Gly Lys Pro 195 200 205 Arg Pro Trp Cys Tyr Thr
Leu Asp Pro Asp Thr Pro Trp Glu Tyr Cys 210 215 220 Ala Ile Lys Thr
Cys Ala His Ser Ala Val Asn Glu Thr Asp Val Pro 225 230 235 240 Met
Glu Thr Thr Glu Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly 245 250
255 Thr Ser Asn Thr Ile Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser
260 265 270 Gln Tyr Pro His Lys His Asp Ile Thr Pro Glu Asn Phe Lys
Cys Lys 275 280 285 Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro Asp Gly
Ala Glu Ser Pro 290 295 300 Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg
Val Gly Tyr Cys Ser Gln 305 310 315 320 Ile Pro Lys Cys Asp Val Ser
Ser Gly Gln Asp Cys Tyr Arg Gly Asn 325 330 335 Gly Lys Asn Tyr Met
Gly Asn Leu Ser Lys Thr Arg Ser Gly Leu Thr 340 345 350 Cys Ser Met
Trp Asp Lys Asn Met Glu Asp Leu His Arg His Ile Phe 355 360 365 Trp
Glu Pro Asp Ala Ser Lys Leu Asn Lys Asn Tyr Cys Arg Asn Pro 370 375
380 Asp Asp Asp Ala His Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile
385 390 395 400 Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys Glu Gly Asp
Thr Thr Pro 405 410 415 Thr Ile Val Asn Leu Asp His Pro Val Ile Ser
Cys Ala Lys Thr Lys 420 425 430 Gln Leu Arg Val Val Asn Gly Ile Pro
Thr Arg Thr Asn Ile Gly Trp 435 440 445 Met Val Ser Leu Arg Tyr Arg
Asn Lys His Ile Cys Gly Gly Ser Leu 450 455 460 Ile Lys Glu Ser Trp
Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg 465 470 475 480 Asp Leu
Lys Asp Tyr Glu Ala Trp Leu Gly Ile His Asp Val His Gly 485 490 495
Arg Gly Glu Glu Lys Arg Lys Gln Ile Leu Asn Ile Ser Gln Leu Val 500
505 510 Tyr Gly Pro Glu Gly Ser Asp Leu Val Leu Leu Lys Leu Ala Arg
Pro 515 520 525 Ala Ile Leu Asp Asn Phe Val Ser Thr Ile Asp Leu Pro
Ser Tyr Gly 530 535 540 Cys Thr Ile Pro Glu Lys Thr Thr Cys Ser Ile
Tyr Gly Trp Gly Tyr 545 550 555 560 Thr Gly Leu Ile Asn Ala Asp Gly
Leu Leu Arg Val Ala His Leu Tyr 565 570 575 Ile Met Gly Asn Glu Lys
Cys Ser Gln His His Gln Gly Lys Val Thr 580 585 590 Leu Asn Glu Ser
Glu Leu Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly 595 600 605 Pro Cys
Glu Gly Asp Tyr Gly Gly Pro Leu Ile Cys Glu Gln His Lys 610 615 620
Met Arg Met Val Leu Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile 625
630 635 640 Pro Asn Arg Pro Gly Ile Phe Val Arg Val Ala Tyr Tyr Ala
Lys Trp 645 650 655 Ile His Lys Val Ile Leu Thr Tyr Lys Leu Cys Gly
Arg His His His 660 665 670 His His His 675 2152194DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
215atgatgtggg ggaccaaact tctgccggtc ctgttgctgc agcatgtcct
cctgcacctc 60ctcctgcttc atgtcgccat cccctatgca gaaggacaga agaaaagaag
aaatacactt 120catgaattta aaaagtcagc aaaaactact cttaccaagg
aagacccatt actgaagatt 180aaaaccaaaa aagtgaactc tgcagatgag
tgtgccaaca ggtgtatcag gaacaggggc 240tttacgttca cttgcaaggc
cttcgttttt gataagtcaa gaaaacgatg ctactggtat 300cctttcaata
gtatgtcaag tggagtgaaa aaagggtttg gccatgaatt tgacctctat
360gaaaacaaag actatattag aaactgcatc attggtaaag gaggcagcta
taaagggacg 420gtatccatca ctaagagtgg catcaaatgc cagccttgga
attccatgat cccccatgaa 480cacagctatc gcggtaaaga cctacaggaa
aactactgtc gaaatcctcg aggggaagaa 540gggggaccct ggtgtttcac
aagcaatcca gaggtacgct acgaagtctg tgacattcct 600cagtgttcag
aagttgaatg catgacctgc aatggtgaaa gctacagagg tcccatggat
660cacacagaat caggcaagac ttgtcagcgc tgggaccagc agacaccaca
ccggcacaag 720ttcttgccag aaagatatcc cgacaagggc tttgatgata
attattgccg caatcctgat 780ggcaagccga ggccatggtg ctacactctt
gaccctgaca ccccttggga gtattgtgca 840attaaaacgt gcgctcacag
tgctgtgaat gagactgatg tccctatgga aacaactgaa 900tgcattcaag
gccaaggaga aggttacagg ggaaccagca ataccatttg gaatggaatt
960ccctgtcagc gttgggattc gcagtaccct cacaagcatg atatcactcc
cgagaacttc 1020aaatgcaagg accttagaga aaattattgc cgcaatccag
atggggctga atcaccatgg 1080tgttttacca ctgacccaaa catccgagtt
ggctactgct ctcagattcc caagtgtgac 1140gtgtcaagtg gacaagattg
ttatcgtggc aatgggaaaa attacatggg caacttatcc 1200aaaacaaggt
ctggacttac atgttccatg tgggacaaga atatggagga tttacaccgt
1260catatcttct gggagccaga tgctagcaaa ttgaataaga attactgccg
gaatcctgat 1320gatgatgccc atggaccttg gtgctacacg gggaatcctc
ttattccttg ggattattgc 1380cctatttccc gttgtgaagg agatactaca
cctacaattg tcaatttgga ccatcctgta 1440atatcctgtg ccaaaacaaa
acaactgcgg gttgtaaatg ggattccaac acaaacaaca 1500gtaggatgga
tggttagttt gagatacaga aataaacata tctgcggagg atcattgata
1560aaggagagtt gggttcttac tgcacgacag tgtttccctt ctcgagactt
gaaagattat 1620gaagcttggc ttggaattca tgatgtccac ggaagaggag
atgagaaatg caaacaggtt 1680ctcaatgttt cccagctggt atatggccct
gaaggatcag atctggtttt aatgaagctt 1740gctcgacctg caatcctgga
taactttgtc agtacaattg atttacctag ttatggttgt 1800acaatccctg
aaaagaccac ttgcagtatt tacggctggg gctacactgg attgatcaac
1860gcggatggtt tattacgagt agctcatctg tatattatgg ggaatgagaa
atgcagtcag 1920caccatcaag gcaaggtgac tttgaatgag tctgagttat
gtgctggggc tgaaaagatt 1980ggatcaggac catgtgaggg agattatggt
ggcccactca tttgtgaaca acacaaaatg 2040agaatggttc ttggtgtcat
tgttcctggt cgtggatgtg ccatcccaaa tcgtcctggt 2100atttttgttc
gagtagcata ttatgcaaaa tggatacaca aagtaatttt gacatacaag
2160ttgtgcggcc gccatcacca tcaccatcac taag 2194216675PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
216Pro Leu Leu Lys Ile Lys Thr Lys Lys Val Asn Ser Ala Asp Glu Cys
1 5 10 15 Ala Asn Arg Cys Ile Arg Asn Arg Gly Phe Thr Phe Thr Cys
Lys Ala 20 25 30 Phe Val Phe Asp Lys Ser Arg Lys Arg Cys Tyr Trp
Tyr Pro Phe Asn 35 40 45 Ser Met Ser Ser Gly Val Lys Lys Gly Phe
Gly His Glu Phe Asp Leu 50 55 60 Tyr Glu Asn Lys Asp Tyr Ile Arg
Asn Cys Ile Ile Gly Lys Gly Gly 65 70 75 80 Ser Tyr Lys Gly Thr Val
Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln 85 90 95 Pro Trp Asn Ser
Met Ile Pro His Glu His Ser Tyr Arg Gly Lys Asp 100 105 110 Leu Gln
Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro 115 120 125
Trp Cys Phe Thr Ser Asn Pro
Glu Val Arg Tyr Glu Val Cys Asp Ile 130 135 140 Pro Gln Cys Ser Glu
Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr 145 150 155 160 Arg Gly
Pro Met Asp His Thr Glu Ser Gly Lys Thr Cys Gln Arg Trp 165 170 175
Asp Gln Gln Thr Pro His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro 180
185 190 Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg Asn Pro Asp Gly Lys
Pro 195 200 205 Arg Pro Trp Cys Tyr Thr Leu Asp Pro Asp Thr Pro Trp
Glu Tyr Cys 210 215 220 Ala Ile Lys Thr Cys Ala His Ser Ala Val Asn
Glu Thr Asp Val Pro 225 230 235 240 Met Glu Thr Thr Glu Cys Ile Gln
Gly Gln Gly Glu Gly Tyr Arg Gly 245 250 255 Thr Ser Asn Thr Ile Trp
Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser 260 265 270 Gln Tyr Pro His
Lys His Asp Ile Thr Pro Glu Asn Phe Lys Cys Lys 275 280 285 Asp Leu
Arg Glu Asn Tyr Cys Arg Asn Pro Asp Gly Ala Glu Ser Pro 290 295 300
Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln 305
310 315 320 Ile Pro Lys Cys Asp Val Ser Ser Gly Gln Asp Cys Tyr Arg
Gly Asn 325 330 335 Gly Lys Asn Tyr Met Gly Asn Leu Ser Lys Thr Arg
Ser Gly Leu Thr 340 345 350 Cys Ser Met Trp Asp Lys Asn Met Glu Asp
Leu His Arg His Ile Phe 355 360 365 Trp Glu Pro Asp Ala Ser Lys Leu
Asn Lys Asn Tyr Cys Arg Asn Pro 370 375 380 Asp Asp Asp Ala His Gly
Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile 385 390 395 400 Pro Trp Asp
Tyr Cys Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro 405 410 415 Thr
Ile Val Asn Leu Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys 420 425
430 Gln Leu Arg Val Val Asn Gly Ile Pro Thr Gln Thr Thr Val Gly Trp
435 440 445 Met Val Ser Leu Arg Tyr Arg Asn Lys His Ile Cys Gly Gly
Ser Leu 450 455 460 Ile Lys Glu Ser Trp Val Leu Thr Ala Arg Gln Cys
Phe Pro Ser Arg 465 470 475 480 Asp Leu Lys Asp Tyr Glu Ala Trp Leu
Gly Ile His Asp Val His Gly 485 490 495 Arg Gly Asp Glu Lys Cys Lys
Gln Val Leu Asn Val Ser Gln Leu Val 500 505 510 Tyr Gly Pro Glu Gly
Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro 515 520 525 Ala Ile Leu
Asp Asn Phe Val Ser Thr Ile Asp Leu Pro Ser Tyr Gly 530 535 540 Cys
Thr Ile Pro Glu Lys Thr Thr Cys Ser Ile Tyr Gly Trp Gly Tyr 545 550
555 560 Thr Gly Leu Ile Asn Ala Asp Gly Leu Leu Arg Val Ala His Leu
Tyr 565 570 575 Ile Met Gly Asn Glu Lys Cys Ser Gln His His Gln Gly
Lys Val Thr 580 585 590 Leu Asn Glu Ser Glu Leu Cys Ala Gly Ala Glu
Lys Ile Gly Ser Gly 595 600 605 Pro Cys Glu Gly Asp Tyr Gly Gly Pro
Leu Ile Cys Glu Gln His Lys 610 615 620 Met Arg Met Val Leu Gly Val
Ile Val Pro Gly Arg Gly Cys Ala Ile 625 630 635 640 Pro Asn Arg Pro
Gly Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp 645 650 655 Ile His
Lys Val Ile Leu Thr Tyr Lys Leu Cys Gly Arg His His His 660 665 670
His His His 675
* * * * *
References