U.S. patent application number 17/367909 was filed with the patent office on 2022-02-10 for cytotoxicity-inducing therapeutic agent.
The applicant listed for this patent is Chugai Seiyaku Kabushiki Kaisha. Invention is credited to Tomoyuki Igawa, Takahiro Ishiguro, Yumiko Kawai, Taichi Kuramochi, Atsushi Narita, Junichi Nezu, Akihisa Sakamoto.
Application Number | 20220041756 17/367909 |
Document ID | / |
Family ID | 1000005916338 |
Filed Date | 2022-02-10 |
United States Patent
Application |
20220041756 |
Kind Code |
A1 |
Nezu; Junichi ; et
al. |
February 10, 2022 |
CYTOTOXICITY-INDUCING THERAPEUTIC AGENT
Abstract
By replacing the antigen-binding domain, the present inventors
discovered novel polypeptide complexes that retain BiTE's strong
anti-tumor activity and excellent safety properties, as well as
have long half-life in blood and can damage various different
target cells.
Inventors: |
Nezu; Junichi; (Shizuoka,
JP) ; Ishiguro; Takahiro; (Kanagawa, JP) ;
Narita; Atsushi; (Shizuoka, JP) ; Sakamoto;
Akihisa; (Shizuoka, JP) ; Kawai; Yumiko;
(Kanagawa, JP) ; Igawa; Tomoyuki; (Shizuoka,
JP) ; Kuramochi; Taichi; (Shizuoka, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chugai Seiyaku Kabushiki Kaisha |
Tokyo |
|
JP |
|
|
Family ID: |
1000005916338 |
Appl. No.: |
17/367909 |
Filed: |
July 6, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13990088 |
Dec 19, 2013 |
11066483 |
|
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PCT/JP2011/077603 |
Nov 30, 2011 |
|
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17367909 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/94 20130101;
C07K 16/30 20130101; C07K 16/2809 20130101; C07K 16/303 20130101;
C07K 2317/60 20130101; C07K 16/2863 20130101; C07K 2317/55
20130101; C07K 2319/30 20130101; C07K 16/46 20130101; C07K 2317/56
20130101; C07K 2317/35 20130101; C07K 2317/41 20130101; C07K
2317/73 20130101; C07K 2319/00 20130101; C07K 2317/71 20130101;
C07K 2317/64 20130101; C07K 2317/622 20130101; C07K 2317/31
20130101; A61K 2039/505 20130101; C07K 2317/52 20130101 |
International
Class: |
C07K 16/46 20060101
C07K016/46; C07K 16/30 20060101 C07K016/30; C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 30, 2010 |
JP |
2010-266760 |
May 31, 2011 |
JP |
2011-121771 |
Oct 31, 2011 |
JP |
2011-238818 |
Claims
1-68. (canceled)
69. A method for treating cancer in a subject, the method
comprising administering to a subject in need thereof an antibody
that comprises: a first light chain comprising a first VL domain
and a first CL domain; a second light chain comprising a second VL
domain and a second CL domain; a first heavy chain comprising a
first heavy chain variable region and a first heavy chain constant
region, wherein the first heavy chain constant region is a
non-wild-type heavy chain constant region comprising the sequence
of one of SEQ ID NOs: 23, 24, 25 and 26, with one or more amino
acid substitutions and optionally a deletion of the amino acids at
EU numbering positions 446 and 447; and a second heavy chain
comprising a second heavy chain variable region and a second heavy
chain constant region, wherein the second heavy chain constant
region is a non-wild-type heavy chain constant region comprising
the sequence of one of SEQ ID NOs: 23, 24, 25 and 26, with one or
more amino acid substitutions and optionally a deletion of the
amino acids at EU numbering positions 446 and 447, wherein the
amino acid sequence of the second heavy chain constant region is
the same as or different from the amino acid sequence of the first
heavy chain constant region, and is of the same isotype as the
first heavy chain constant region; wherein the first light chain
and the first heavy chain associate to form a first antigen-binding
domain that binds to CD3, wherein the second light chain and the
second heavy chain associate to form a second antigen-binding
domain that does not bind to CD3, and that does bind to a cancer
antigen that is not CD3, wherein the first heavy chain constant
region associates with the second heavy chain constant region,
wherein, when assessed by a surface plasmon resonance technique,
the ability of the associated first and second heavy chain constant
regions to bind to a given human Fc.gamma. receptor is reduced,
compared to the ability of a wild-type human IgG of the same
isotype as the antibody to bind to the human Fc.gamma. receptor, as
a result of at least one of the one or more amino acid
substitutions within the first and second heavy chain constant
regions, wherein the at least one substitution in the first and
second heavy chain constant regions represents a change in amino
acid sequence compared to the amino acid sequence of the heavy
chain constant regions of the wild-type human IgG, and wherein at
least one of the amino acid substitutions that result in the
reduced ability to bind to the human Fc.gamma. receptor is at a
position selected from the following EU numbering positions in each
of the first and second heavy chain constant regions: 220, 226,
229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265,
266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328,
329, 330, 331, and 332.
70. The method of claim 69, wherein the amino acid sequence from
position 118 to position 260 (EU numbering) of the first heavy
chain constant region and of the second heavy chain constant region
is the sequence from the corresponding portion of SEQ ID NO: 24; or
the amino acid sequence from position 261 to position 447 (EU
numbering) of the first heavy chain constant region and of the
second heavy chain constant region is the sequence from the
corresponding portion of SEQ ID NO: 26.
71. The method of claim 69, wherein the first heavy chain constant
region and the second heavy chain constant region each comprises
the sequence of SEQ ID NO: 23 with one or more substitutions and
optionally a deletion of the two residues at the carboxyl terminus
of SEQ ID NO: 23.
72. The method of claim 71, wherein the one or more substitutions
include a substitution in both the first and the second heavy chain
constant regions at a position selected from positions 233, 234,
235, 236, 327, 330, and 331 (EU numbering), the substitution at the
selected position being by an amino acid from a corresponding
position in IgG2 or IgG4.
73. The method of claim 71, wherein the one or more substitutions
include a substitution in both the first and the second heavy chain
constant regions at position 234, 235, or 297 (EU numbering).
74. The method of claim 73, wherein the one or more substitutions
include a substitution by alanine.
75. The method of claim 69, wherein the amino acid sequence of the
second heavy chain constant region is different from the amino acid
sequence of the first heavy chain constant region.
76. The method of claim 75, wherein in one of the two heavy chain
constant regions of the antibody, the amino acids at positions 349
and 366 (EU numbering) are cysteine and tryptophan, respectively;
and in the other heavy chain constant region of the antibody, the
amino acids at positions 356, 366, 368, and 407 (EU numbering) are
cysteine, serine, alanine, and valine, respectively.
77. The method of claim 75, wherein: in one of the two heavy chain
constant regions of the antibody, the amino acid at position 356 is
lysine; in the other heavy chain constant region of the antibody,
the amino acid at position 439 is glutamic acid; and in one but not
both of the two heavy chain constant regions of the antibody, the
amino acid at position 435 is arginine (all positions being by EU
numbering).
78. The method of claim 76, wherein the amino acids at EU numbering
positions 446 and 447 are deleted in both heavy chain constant
regions of the antibody.
79. The method of claim 69, wherein the human Fc.gamma. receptor is
selected from Fc.gamma.I, Fc.gamma.IIA, Fc.gamma.IIB,
Fc.gamma.IIIA, and Fc.gamma.IIIB.
80. The method of claim 69, wherein the amino acid sequence of the
second heavy chain constant region is the same as the amino acid
sequence of the first heavy chain constant region.
81. The method of claim 69, wherein the amino acid sequence of the
second heavy chain constant region is different from the amino acid
sequence of the first heavy chain constant region, and wherein the
percentage of heterodimers formed in a mixture of the first and
second heavy chains is higher than the percentage of heterodimers
that would be formed if the two heavy chain constant regions were
identical and matched one of SEQ ID NO: 23-26.
82. The method of claim 81, wherein the difference between the
sequences of the first and second heavy chain constant regions
comprises a difference at one or more CH3 domain positions.
83. The method of claim 82, wherein the difference between the
sequences of the first and second heavy chain constant regions
further comprises a difference at one or more CH1 domain positions,
wherein the one or more CH1 domain positions interface with a CL
domain.
84. The method of claim 69, wherein the amino acid sequence of the
second heavy chain constant region is different from the amino acid
sequence of the first heavy chain constant region, and this
difference in sequences comprises a difference at one or more CH1
domain positions of the two heavy chain constant regions, wherein
the one or more CH1 domain positions interface with a CL
domain.
85. The method of claim 69, wherein the human Fc.gamma. receptor is
selected from Fc.gamma.I, Fc.gamma.IIIA, and Fc.gamma.IIIB.
86. The method of claim 69, wherein, when assessed by a surface
plasmon resonance technique, the antibody's ability to bind to each
of human Fc.gamma. receptors Fc.gamma.l, Fc.gamma.IIIA, and
Fc.gamma.IIIB is reduced compared to the ability of the wild-type
human IgG to bind to the same human Fc.gamma. receptor.
87. The method of claim 69, wherein, when assessed by a surface
plasmon resonance technique, the antibody's ability to bind to each
of human Fc.gamma. receptors Fc.gamma.I, Fc.gamma.IIA,
Fc.gamma.IIB, Fc.gamma.IIIA, and Fc.gamma.IIIB is reduced compared
to the ability of the wild-type human IgG to bind to the same human
Fc.gamma. receptor.
88. The method of claim 69, wherein the first and second heavy
chain constant regions are human heavy chain constant regions.
89. The method of claim 69, wherein the associated first and second
heavy chain constant regions have essentially no detectable ability
to bind to the given human Fc.gamma. receptor, when assessed by a
surface plasmon resonance technique.
90. The method of claim 69, wherein the associated first and second
heavy chain constant regions have essentially no detectable ability
to bind to human Fc.gamma. receptors Fc.gamma.I, Fc.gamma.IIA,
Fc.gamma.IIB, Fc.gamma.IIIA, and Fc.gamma.IIIB, when assessed by a
surface plasmon resonance technique.
91. The method of claim 73, wherein the one or more substitutions
include one of the following sets of substitutions in both the
first and second heavy chain constant regions (all positions by EU
numbering): (a) L234F, L235E, P331S; (b) C226S, C229S, P238S; (c)
C226S, C229S; (d) C226S, C229S, E233P, L234V, L235A.
92. The method of claim 69, wherein each of the first and second
heavy chain constant regions comprises the sequence of SEQ ID NO:
24 with one or more substitutions and optionally a deletion of the
two residues at the carboxyl terminus of SEQ ID NO: 24.
93. The method of claim 92, wherein the one or more substitutions
include one of the following sets of substitutions in both the
first and second heavy chain constant regions (all positions by EU
numbering): (a) H268Q, V309L, A330S, and P331S; (b) V234A; (c)
G237A; (d) V234A and G237A; (e) A235E and G237A; or (f) V234A,
A235E, and G237A.
94. The method of claim 69, wherein the first heavy chain constant
region and the second heavy chain constant region comprise the
sequence of SEQ ID NO: 25 with one or more substitutions and
optionally a deletion of the two residues at the carboxyl terminus
of SEQ ID NO: 25.
95. The method of claim 94, wherein the one or more substitutions
include one of the following substitutions in both the first and
second heavy chain constant regions (all positions by EU
numbering): (a) F241A; (b) D265A; or (c) V264A.
96. The method of claim 69, wherein the first heavy chain constant
region and the second heavy chain constant region comprise the
sequence of SEQ ID NO: 26 with one or more substitutions and
optionally a deletion of the two residues at the carboxyl terminus
of SEQ ID NO: 26.
97. The method of claim 96, wherein the one or more substitutions
include one of the following sets of substitutions in both the
first and second heavy chain constant regions (all positions by EU
numbering): (a) L235A, G237A, and E318A; (b) L235E; or (c) F234A
and L235A.
98. The method of claim 69, wherein the one or more substitutions
include a substitution at position 234 (EU numbering) in both the
first and second heavy chain constant regions.
99. The method of claim 69, wherein the one or more substitutions
include a substitution at position 235 (EU numbering) in both the
first and second heavy chain constant regions.
100. The method of claim 69, wherein the one or more substitutions
include a substitution at position 239 (EU numbering) in both the
first and second heavy chain constant regions.
101. The method of claim 69, wherein the one or more substitutions
include a substitution at position 297 (EU numbering) in both the
first and second heavy chain constant regions.
102. The method of claim 69, wherein the residues at positions 234
and 235 (both positions by EU numbering) are both alanine in both
the first and second heavy chain constant regions.
103. The method of claim 69, wherein the residues at positions 234,
235 and 297 (all positions by EU numbering) are all alanine in both
the first and second heavy chain constant regions.
104. The method of claim 69, wherein positions 446 and 447 (EU
numbering) are deleted in the sequence of each of the first and
second heavy chain constant regions.
105. The method of claim 69, wherein the only substitution(s) in
the amino acid sequence of one or both of the first and second
heavy chain constant regions, compared to the amino acid sequence
of SEQ ID NO: 23, is/are at a position or positions independently
selected from the following EU numbering positions: 220, 226, 229,
231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266,
267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329,
330, 331, and 332.
106. The method of claim 69, wherein the only substitution(s) in
the amino acid sequence of one or both of the first and second
heavy chain constant regions, compared to the amino acid sequence
of SEQ ID NO: 24, is/are at a position or positions independently
selected from the following EU numbering positions: 220, 226, 229,
231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266,
267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329,
330, 331, and 332.
107. The method of claim 69, wherein the only substitution(s) in
the amino acid sequence of one or both of the first and second
heavy chain constant regions, compared to the amino acid sequence
of SEQ ID NO: 25, is/are at a position or positions independently
selected from the following EU numbering positions: 220, 226, 229,
231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266,
267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329,
330, 331, and 332.
108. The method of claim 69, wherein the only substitution(s) in
the amino acid sequence of one or both of the first and second
heavy chain constant regions, compared to the amino acid sequence
of SEQ ID NO: 26, is/are at a position or positions independently
selected from the following EU numbering positions: 220, 226, 229,
231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266,
267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329,
330, 331, and 332.
109. The method of claim 69, wherein the amino acid sequence of one
or both of the first and second heavy chain constant regions has a
substitution, compared to the amino acid sequence of SEQ ID NO: 23,
at a position or positions independently selected from the
following EU numbering positions: 229, 239, 240, 264, 265, 267,
297, 298, 299, 329, 331, and 332.
110. The method of claim 69, wherein the amino acid sequence of one
or both of the first and second heavy chain constant regions has a
substitution, compared to the amino acid sequence of SEQ ID NO: 24,
at a position or positions independently selected from the
following EU numbering positions: 229, 239, 240, 264, 265, 267,
297, 298, 299, 329, 331, and 332.
111. The method of claim 69, wherein the amino acid sequence of one
or both of the first and second heavy chain constant regions has a
substitution, compared to the amino acid sequence of SEQ ID NO: 25,
at a position or positions independently selected from the
following EU numbering positions: 229, 239, 240, 264, 265, 267,
297, 298, 299, 329, 331, and 332.
112. The method of claim 69, wherein the amino acid sequence of one
or both of the first and second heavy chain constant regions has a
substitution, compared to the amino acid sequence of SEQ ID NO: 26,
at a position or positions independently selected from the
following EU numbering positions: 229, 239, 240, 264, 265, 267,
297, 298, 299, 329, and 332.
113. The method of claim 69, wherein the subject is a human and the
antibody is a human or humanized antibody.
114. The method of claim 69, wherein the subject is a human and the
isotype of the antibody is human IgG1, human IgG2, human IgG3, or
human IgG4.
115. The method of claim 69, wherein the subject is a human and the
isotype of each of the first and second heavy chain constant
regions is independently selected from human IgG1, human IgG2,
human IgG3, and human IgG4.
116. The method of claim 69, wherein the cancer is liver cancer or
lung cancer.
117. The method of claim 69, wherein the subject is a human.
118. The method of claim 117, wherein the cancer antigen is
selected from a group consisting of human glypican-3 (GPC3), human
epidermal growth factor receptor (EGFR), and human epithelial cell
adhesion molecule (EpCAM).
119. The method of claim 69, wherein the first antigen-binding
domain is in the form of a first Fab, and the second
antigen-binding domain is in the form of a second Fab.
120. The method of claim 84, wherein one or more of the following
(i) to (iv) is true: (i) a charged amino acid residue at one of the
one or more CH1 domain positions in the first heavy chain constant
region interfaces with, and has a charge opposite to that of, a
charged amino acid residue in the first CL domain; (ii) a charged
amino acid residue at one of the one or more CH1 domain positions
in the second heavy chain constant region interfaces with, and has
a charge opposite to that of, a charged amino acid residue in the
second CL domain; (iii) a charged amino acid residue at one of the
one or more CH1 domain positions in the first heavy chain constant
region interfaces with, and has the same charge as, a charged amino
acid residue in the second CL domain; (iv) a charged amino acid
residue at one of the one or more CH1 domain positions in the
second heavy chain constant region interfaces with, and has the
same charge as, a charged amino acid residue in the first CL
domain.
121. The method of claim 120, wherein the two charged amino acid
residues of any of (i) to (iv) are at a pair of positions
independently selected from the following pairs (all positions by
EU numbering): (a) CH1 domain position 147 and CL domain position
180; (b) CH1 domain position 147 and CL domain position 131; (c)
CH1 domain position 147 and CL domain position 164; (d) CH1 domain
position 147 and CL domain position 138; (e) CH1 domain position
147 and CL domain position 123; (f) CH1 domain position 175 and CL
domain position 160; and (g) CH1 domain position 213 and CL domain
position 123.
122. The method of claim 120, wherein if (i) is true, one of the
charged amino acids of (i) is selected from glutamic acid and
aspartic acid, and the other is selected from lysine, arginine and
histidine; if (ii) is true, one of the charged amino acids of (ii)
is selected from glutamic acid and aspartic acid, and the other is
selected from lysine, arginine and histidine; if (iii) is true,
either both the charged amino acids of (iii) are independently
selected from glutamic acid and aspartic acid, or both are
independently selected from lysine, arginine and histidine; if (iv)
is true, either both of the charged amino acids of (iv) are
independently selected from glutamic acid and aspartic acid, or
both are independently selected from lysine, arginine and
histidine.
123. The method of claim 121, wherein either (1) the first heavy
chain constant region CH1 domain comprises a glutamic acid at
position 213 (EU numbering), and the first CL domain comprises a
lysine at position 123 (EU numbering); or (2) the second heavy
chain constant region CH1 domain comprises a glutamic acid at
position 213 (EU numbering), and the second CL domain comprises a
lysine at position 123 (EU numbering).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 13/990,088, filed on Dec. 19, 2013, which is the National Stage
of International Patent Application No. PCT/JP2011/077603, filed on
Nov. 30, 2011, which claims the benefit of Japanese Patent
Application No. 2010-266760, filed on Nov. 30, 2010, Japanese
Patent Application No. 2011-121771, filed on May 31, 2011, and
Japanese Patent Application No. 2011-238818, filed on Oct. 31,
2011.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing that has been
submitted electronically as an ASCII text file named
SequenceListing.txt. The ASCII text file, created on Apr. 2, 2021,
is 272 kilobytes in size. The material in the ASCII text file is
hereby incorporated by reference in its entirety.
TECHNICAL FIELD
[0003] The present invention relates to polypeptide complexes that
enable cancer treatment by having T cells close to target cancer
cells and using the cytotoxic activity of T cells against the
target cancer cells, methods for producing the polypeptide
complexes, and therapeutic agents that contain such a polypeptide
complex as an active ingredient for inducing cellular cytotoxicity.
The present invention also relates to pharmaceutical compositions
for treating or preventing various cancers, which contain an
above-mentioned therapeutic agent for inducing cellular
cytotoxicity as an active ingredient, and therapeutic methods using
such pharmaceutical compositions.
BACKGROUND ART
[0004] To date, multiple therapeutic antibodies having an excellent
anti-tumor effect have been developed as pharmaceuticals for
treating cancer (Non-patent Document 1). These therapeutic
antibodies are known to exert their anti-tumor effect on cancer
cells through inhibition of signals essential for cancer cell
growth, induction of cell death signals, antibody dependent
cell-mediated cytotoxicity (ADCC), or complement dependent
cytotoxicity (CDC) (Non-patent Document 2). ADCC is a cytotoxicity
exerted by effector cells such as NK cells and macrophages against
antibody-bound target cancer cells when the Fc region of an
antibody binds to an Fc receptor on the effector cells. Meanwhile,
a complement complex binds to the complement-binding site in an
antibody structure. CDC is cytotoxicity that occurs when a
complement component in the complex forms a pore through the cell
membrane of an antibody-bound cell, enhancing water or ion influx
into the cell. Although conventional therapeutic antibodies show
excellent activities, to date administration of such antibodies led
to only unsatisfactory therapeutic outcomes. Thus, it is desirable
to develop therapeutic antibodies that exert a greater cell-killing
activity against cancer.
[0005] In addition to the above-mentioned antibodies which adopt
ADCC as their anti-tumor mechanism by recruiting NK cells or
macrophages as effector cells, T cell-recruiting antibodies (TR
antibodies) which adopt cytotoxicity as their anti-tumor mechanism
by recruiting T cells as effector cells have been known since the
1980s (Non-patent Documents 3 to 5). A TR antibody is a bispecific
antibody that contains an antibody against any one of the subunits
forming a T-cell receptor (TCR) complex on T cells, in particular
an antibody that binds to the CD3 epsilon chain, and an antibody
that binds to an antigen on target cancer cells. A T cell gets near
a cancer cell when the TR antibody binds to both the CD3 epsilon
chain and cancer antigen at the same time, and this causes an
anti-tumor effect against the cancer cell due to the cytotoxic
activity of the T cell.
[0006] An antibody called "trifunctional antibody" is also known as
a TR antibody (Non-patent Documents 6 and 7). A trifunctional
antibody is a whole IgG-type bispecific antibody in which one arm
contains an Fab that binds to a cancer antigen and the other arm
contains an Fab that binds to the CD3 epsilon chain. Therapeutic
effect against malignant ascites has been demonstrated by
administering catumaxomab, which is a trifunctional antibody
against EpCAM, into the peritoneal cavities of malignant ascites
patients having EpCAM expression-positive cancer cells. The use of
catumaxomab has been approved in the EU for the above
treatment.
[0007] Furthermore, a TR antibody called "bispecific T-cell engager
(BiTE)" has been recently found to exhibit a strong anti-tumor
effect (Non-patent Documents 8 and 9). BiTE is a TR antibody with a
molecular form in which the scFv of an antibody against a cancer
antigen is linked to the scFv of an antibody against the CD3
epsilon chain via a short polypeptide linker. BiTE has been
reported to have an anti-tumor activity superior to those of the
various known TR antibodies (Non-patent Documents 9 and 10).
Specifically, as compared to other TR antibodies, BiTE exerts an
anti-tumor effect even at a significantly lower concentration and
lower effector cell/cancer cell ratio (ET ratio). It has been also
demonstrated that the effect can be exerted without the need to
activate effector cells using IL-2, a CD28 agonistic antibody or
such in advance. Blinatumomab (MT103), which is a BiTE against
CD19, exhibits a much stronger cytotoxic activity against cancer
cells in vitro than that of Rituxan which is known to produce an
excellent clinical effect. Furthermore, blinatumomab has been
reported to show an extremely superior anti-tumor effect in phase I
and II clinical trials conducted recently (Non-patent Document
11).
[0008] The fact that catumaxomab has been approved as a therapeutic
agent that demonstrates clinical drug effect, and that multiple
BiTEs including blinatumomab exert a strong anti-tumor effect,
suggests that TR antibodies that recruit T cells as effector cells
have a significantly higher potential as an anti-tumor agent as
compared to conventional antibodies that use ADCC as their
mechanism of action.
[0009] However, it is known that a trifunctional antibody binds to
both a T cell and a cell such as an NK cell or macrophage at the
same time in a cancer antigen-independent manner, and as a result
receptors expressed on the cells are cross-linked, and expression
of various cytokines is induced in a cancer antigen-independent
manner. Systemic administration of a trifunctional antibody is
thought to cause cytokine storm-like side effects as a result of
such induction of cytokine expression. In fact, it has been
reported that, in the phase I clinical trial, a very low dose of 5
.mu.g/body was the maximum tolerance dose for systemic
administration of catumaxomab to patients with non-small cell lung
cancer, and that administration of a higher dose causes various
severe side effects (Non-patent Document 12). When administered at
such a low dose, catumaxomab can never reach the effective blood
level. That is, the expected anti-tumor effect cannot be achieved
by administrating catumaxomab at such a low dose.
[0010] Meanwhile, unlike catumaxomab, BiTE has no Fc.gamma.
receptor-binding site, and therefore it does not cross-link the
receptors expressed on T cells and cells such as NK cells and
macrophages in a cancer antigen-dependent manner. Thus, it has been
demonstrated that BiTE does not cause cancer antigen-independent
cytokine induction which is observed when catumaxomab is
administered. However, since BiTE is a modified
low-molecular-weight antibody molecule without an Fc region, the
problem is that its blood half-life after administration to a
patient is significantly shorter than IgG-type antibodies
conventionally used as therapeutic antibodies. In fact, the blood
half-life of BiTE administered in vivo has been reported to be
about several hours (Non-patent Documents 13 and 14). In the
clinical trials of blinatumomab, it is administered by continuous
intravenous infusion using a minipump. This administration method
is not only extremely inconvenient for patients but also has the
potential risk of medical accidents due to device malfunction or
the like. Thus, it cannot be said that such an administration
method is desirable.
PRIOR ART DOCUMENTS
Non-Patent Documents
[Non-patent Document 1] Clin Cancer Res. (2010) 16 (1), 11-20
[Non-patent Document 2] Drug Des Devel Ther (2009) 3, 7-16
[Non-patent Document 3] Nature (1985) 314 (6012), 628-31
[Non-patent Document 4] Int J Cancer (1988) 41 (4), 609-15
[Non-patent Document 5] Proc Natl Acad Sci USA (1986) 83 (5),
1453-7
[Non-patent Document 6] Cancer Treat Rev. (2010) 36 (6), 458-67
[Non-patent Document 7] Expert Opin Biol Ther (2010) 10 (8),
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[Non-patent Document 8] Proc Natl Acad Sci USA. (1995) 92 (15),
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[Non-patent Document 9] Drug Discov Today (2005), 10 (18),
1237-44
[Non-patent Document 10] Trends Biotechnol (2004) 22 (5),
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[Non-patent Document 11] Science (2008), 321 (5891), 974-7
[Non-patent Document 12] Cancer Immunol Immunother (2007) 56 (10),
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[Non-patent Document 13] Cancer Immunol Immunother. (2006) 55 (5),
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[Non-patent Document 14] Cancer Immunol Immunother. (2009) 58 (1),
95-109
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0011] The present invention was achieved in view of the above
circumstances. An objective of the present invention is to provide
polypeptide complexes that enable cancer treatment by having T
cells close to target cancer cells and using the cytotoxicity of T
cells against the target cancer cells, methods for producing the
polypeptide complexes, and therapeutic agents containing such a
polypeptide complex as an active ingredient for inducing cellular
cytotoxicity. Another objective of the present invention is to
provide pharmaceutical compositions for treating or preventing
various cancers, which comprise an above-mentioned therapeutic
agent for inducing cellular cytotoxicity as an active ingredient,
and therapeutic methods using the pharmaceutical compositions.
Means for Solving the Problems
[0012] The present inventors discovered novel polypeptide complexes
that retain the strong anti-tumor activity possessed by BiTE and
have a long half-life in blood, as well as excellent safety
properties that result in no induction of cancer
antigen-independent cytokine storm or such. The present inventors
also found that the polypeptide complexes can damage various target
cells when antigen-binding domains of the polypeptide complexes are
substituted. Based on the above findings, the present inventors
demonstrated that the polypeptide complexes of the present
invention damage cancer cells. The present inventors also revealed
that more efficient cellular cytotoxicity is achieved by regulating
the CH1/CL interface association and introducing Knobs-into-Holes
(KiH) modifications into the polypeptide complexes. In addition,
the present inventors demonstrated that various cancers can be
treated or prevented using therapeutic agents for inducing cellular
cytotoxicity comprising a polypeptide complex of the present
invention as an active ingredient.
[0013] More specifically, the present invention provides the
following:
[1] a polypeptide complex which comprises:
[0014] (1) an antigen-binding domain;
[0015] (2) a domain comprising an Fc region that has reduced
Fc.gamma. receptor-binding activity; and
[0016] (3) a T cell receptor complex-binding domain;
[2] the polypeptide complex of [1], wherein the T cell receptor
complex-binding domain is a T cell receptor-binding domain; [3] the
polypeptide complex of [1], wherein the T cell receptor
complex-binding domain is a CD3-binding domain; [4] the polypeptide
complex of any one of [1] to [3], wherein the antigen-binding
domain is a bivalent antigen-binding domain; [5] the polypeptide
complex of [4], wherein the bivalent antigen-binding domain is a
domain having an F(ab')2 structure; [6] the polypeptide complex of
[5], in which two polypeptides forming a heavy chain constant
region of the domain having the F(ab')2 structure are individually
linked to either of the two polypeptides forming an Fc domain; [7]
the polypeptide complex of [6], in which a CD3-binding domain is
linked to either or both of the two CH3s forming the Fc domain; [8]
the polypeptide complex of [7], in which a heavy chain Fv fragment
forming the CD3-binding domain is linked to one of the CH3s forming
the Fc domain and a light chain Fv fragment forming the CD3-binding
domain is linked to the other CH3 forming the Fc domain; [9] the
polypeptide complex of [8], in which an antibody CH1 domain is
linked to the heavy chain Fv fragment forming the CD3-binding
domain and an antibody CL domain is linked to the light chain Fv
fragment; [10] the polypeptide complex of [6], in which the
CD3-binding domain is linked to either or both of the two CLs
forming the F(ab')2; [11] the polypeptide complex of [6], in which
the CD3-binding domain is linked to either or both of the two VHs
forming the F(ab')2; [12] the polypeptide complex of [6], in which
the CD3-binding domain is linked to either or both of the two VLs
forming the F(ab')2; [13] the polypeptide complex of any one of [1]
to [12], wherein the CD3-binding domain is Fv; [14] the polypeptide
complex of any one of [1] to [7] and [10] to [12], wherein the
CD3-binding domain is Fab; [15] the polypeptide complex of any one
of [1] to [7] and [10] to [12], wherein the CD3-binding domain is
scFv; [16] the polypeptide complex of any one of [1] to [15],
wherein the CD3-binding domain is monovalent; [17] the polypeptide
complex of any one of [1] to [3], wherein the antigen-binding
domain is a monovalent scFv and a monovalent Fab; [18] the
polypeptide complex of [17], in which the monovalent scFv is linked
to one of the polypeptides forming the Fc domain via the scFv that
forms the CD3-binding domain; the heavy chain Fv fragment of the
monovalent Fab is linked to one of the polypeptides forming the Fc
domain via a CH1 domain; and the light chain Fv fragment of the Fab
is linked to the CL domain; [19] the polypeptide complex of any one
of [1] to [3], wherein the antigen-binding domain is a bivalent
scFv; [20] the polypeptide complex of [19], in which one monovalent
scFv is linked to one of the polypeptides forming the Fc domain via
the heavy chain Fv fragment forming the CD3-binding domain, and the
other monovalent scFv is linked to the other polypeptide forming
the Fc domain via the light chain Fv fragment forming the
CD3-binding domain; [21] the polypeptide complex of [19], wherein
one monovalent scFv is linked to one of the polypeptides forming
the Fc domain via the scFv forming the CD3-binding domain, and the
other monovalent scFv is linked to the other polypeptide forming
the Fc domain; [22] the polypeptide complex of any one of [1] to
[3], wherein the antigen-binding domain and T cell receptor
complex-binding domain are each a monovalent Fab; [23] the
polypeptide complex of [22], in which the heavy chain Fv fragment
of a monovalent Fab forming the antigen-binding domain is linked to
one of the polypeptides forming the Fc domain via a CH1 domain and
the light chain Fv fragment of the Fab is linked to a CL domain;
and the heavy chain Fv fragment of Fab forming the T cell
receptor-binding domain is linked to the other polypeptide forming
the Fc domain via a CH1 domain and the light chain Fv fragment of
the Fab is linked to a CL domain; [24] the polypeptide complex of
[22], in which the heavy chain Fv fragment of a monovalent Fab
forming the antigen-binding domain is linked to one of the
polypeptides forming the Fc domain via a CH1 domain and the light
chain Fv fragment of the Fab is linked to a CL domain; and the
light chain Fv fragment of Fab forming the T cell receptor-binding
domain is linked to the other polypeptide forming the Fc domain via
a CH1 domain and the heavy chain Fv fragment of the Fab is linked
to a CL domain; [25] the polypeptide complex of [22], in which the
heavy chain Fv fragment of a monovalent Fab forming the
antigen-binding domain is linked to one of the polypeptides forming
the Fc domain via a CH1 domain and the light chain Fv fragment of
the Fab is linked to a CL domain; and the heavy chain Fv fragment
of the Fab forming the T cell receptor-binding domain is linked to
the other polypeptide forming the Fc domain via a CL domain and the
light chain Fv fragment of the Fab is linked to a CH1 domain; [26]
the polypeptide complex of [22], in which the heavy chain Fv
fragment of a monovalent Fab forming the T cell receptor-binding
domain is linked to one of the polypeptides forming the Fc domain
via a CH1 domain and the light chain Fv fragment of the Fab is
linked to a CL domain; and the light chain Fv fragment of Fab
forming the antigen-binding domain is linked to the other
polypeptide forming the Fc domain via a CH1 domain and the heavy
chain Fv fragment of the Fab is linked to a CL domain; [27] the
polypeptide complex of [22], in which the heavy chain Fv fragment
of a monovalent Fab forming the T cell receptor-binding domain is
linked to one of the polypeptide forming the Fc domain via a CH1
domain and the light chain Fv fragment of the Fab is linked to a CL
domain; and the heavy chain Fv fragment of the Fab forming the
antigen-binding domain to the other polypeptide forming the Fc
domain via a CL domain and the light chain Fv fragment of the Fab
is linked to a CH1 domain; [28] the polypeptide complex of [22]
comprising:
[0017] (1) an antigen-binding domain in which the heavy chain Fv
fragment of a monovalent Fab structure that binds to an antigen is
linked via a CH1 domain to one of the polypeptides forming the Fc
domain, and the light chain Fv fragment of the Fab structure is
linked to a CL domain; and
[0018] (2) a T cell receptor complex-binding domain in which the
heavy chain Fv fragment of a monovalent Fab structure that binds to
a T cell receptor complex is linked via a CH1 domain to the other
polypeptide forming the Fc domain, and the light chain Fv fragment
of the Fab structure is linked to a CL domain;
wherein electric charges of the CH1 and CL domains are controlled
so that the heavy chain Fv fragment of the antigen-binding domain
assembles with the light chain Fv fragment of the antigen-binding
domain, or the heavy chain Fv fragment of the T cell
receptor-binding domain assembles with the light chain Fv fragment
of the T cell receptor-binding domain; [29] the polypeptide complex
of [28], wherein an amino acid residue in the CH1 domain linked to
the heavy chain Fv fragment of the T cell receptor complex-binding
domain has the same type of electric charge as an amino acid
residue in the CL domain linked to the light chain Fv fragment of
the antigen-binding domain; [30] the polypeptide complex of [28],
wherein an amino acid residue in the CH1 domain linked to the heavy
chain Fv fragment of the antigen-binding domain has the same type
of electric charge as an amino acid residue in the CL domain linked
to the light chain Fv fragment of the T cell receptor
complex-binding domain; [31] the polypeptide complex of [28],
wherein an amino acid residue in the CH1 domain linked to the heavy
chain Fv fragment of the T cell receptor complex-binding domain has
the same type of electric charge as an amino acid in the CL domain
linked to the light chain Fv fragment of the antigen-binding
domain, and an amino acid residue in the CH1 domain linked to the
heavy chain Fv fragment of the antigen-binding domain has the same
type of electric charge as an amino acid in the CL domain linked to
the light chain Fv fragment of the T cell receptor complex-binding
domain; [32] the polypeptide complex of [29] or [31], wherein an
amino acid residue in the CH1 domain linked to the heavy chain Fv
fragment of the T cell receptor complex-binding domain has an
electric charge opposite to that of an amino acid residue in the CL
domain linked to the light chain Fv fragment of the T cell
receptor-binding domain; [33] the polypeptide complex of [30] or
[31], wherein an amino acid residue in the CH1 domain linked to the
heavy chain Fv fragment of the antigen-binding domain has an
electric charge opposite to that of an amino acid residue in the CL
domain linked to the light chain Fv fragment of the antigen-binding
domain; [34] the polypeptide complex of any one of [22] to [33],
wherein the T cell receptor complex-binding domain is a T cell
receptor-binding domain; [35] the polypeptide complex of [34],
wherein the T cell receptor-binding domain is a CD3-binding domain;
[36] the polypeptide complex of [32] or [33], wherein the amino
acid residues in the CH1 and CL domains are one, two, or more
combinations of amino acid residues selected from the group
consisting of the combinations of:
[0019] (a) the amino acid residue at position 147 (EU numbering) in
the CH1 domain, and the amino acid residue at position 180 (EU
numbering) in the CL domain;
[0020] (b) the amino acid residue at position 147 (EU numbering) in
the CH1 domain, and the amino acid residue at position 131 (EU
numbering) in the CL domain;
[0021] (c) the amino acid residue at position 147 (EU numbering) in
the CH1 domain, and the amino acid residue at position 164 (EU
numbering) in the CL domain;
[0022] (d) the amino acid residue at position 147 (EU numbering) in
the CH1 domain, and the amino acid residue at position 138 (EU
numbering) in the CL domain;
[0023] (e) the amino acid residue at position 147 (EU numbering) in
the CH1 domain, and the amino acid residue at position 123 (EU
numbering) in the CL domain; and
[0024] (f) the amino acid residue at position 175 (EU numbering) in
the CH1 domain, and the amino acid residue at position 160 (EU
numbering) in the CL domain;
and in which the amino acid residue in the CH1 domain has an
electric charge opposite to that of the amino acid residue in the
CL domain; [37] the polypeptide complex of [36], wherein the amino
acid residues are selected from the group additionally comprising
the combination of amino acid residues:
[0025] (g) the amino acid residue at position 213 (EU numbering) in
the CH1 domain, and the amino acid residue at position 123 (EU
numbering) in the CL domain;
[38] the polypeptide complex of [36] or [37], wherein the amino
acid residue having an opposite electric charge is selected from an
amino acid residue in either group:
[0026] (X) glutamic acid (E) and aspartic acid (D); or
[0027] (Y) lysine (K), arginine (R), and histidine (H);
[39] the polypeptide complex of any one of [36] to [38], wherein
the amino acid residues having an opposite electric charge are: Lys
at position 175 (EU numbering) in the CH1 domain, and Glu at
positions 131, 160, and 180 (EU numbering) in the CL domain; [40]
the polypeptide complex of any one of [36] to [38], wherein the
amino acid residues having an opposite electric charge are: Glu at
positions 147 and 175 (EU numbering) in the CH1 domain, and Lys at
positions 131, 160, and 180 (EU numbering) in the CL domain; [41]
the polypeptide complex of [40], wherein the amino acid residue at
position 213 (EU numbering) in the CH1 domain is Glu, and the amino
acid residue of position 123 (EU numbering) in the CL domain is
Lys; [42] the polypeptide complex of any one of [1] to [41],
wherein the Fc domain exhibits impaired Fc.gamma. receptor-binding
activity to Fc.gamma.I, Fc.gamma.IIA, Fc.gamma.IIB, Fc.gamma.IIIA,
and/or Fc.gamma.IIIB; [43] the polypeptide complex of any one of
[1] to [42], wherein the Fc domain is any one of the Fc domains of
SEQ ID NOs: 23, 24, 25, and 26 in which an amino acid(s) forming
the Fc domain is mutated; [44] the polypeptide complex of [43],
wherein the Fc domain comprises any one of the amino acids below:
the amino acid sequence of positions 118 to 260 (EU numbering) is
the sequence of SEQ ID NO: 24; or the amino acid sequence at
positions 261 to 447 (EU numbering) is the sequence of SEQ ID NO:
26; [45] the polypeptide complex of [43], wherein the amino acids
forming the Fc domain comprises a mutation at any one of the
following positions: 220, 226, 229, 231, 232, 233, 234, 235, 236,
237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297,
298, 299, 300, 325, 327, 328, 329, 330, 331, and 332 (EU
numbering); [46] the polypeptide complex of [45], wherein the Fc
domain comprises a mutation in the amino acids of SEQ ID NO: 23
forming the Fc domain; [47] the polypeptide complex of [46],
wherein the Fc domain is an Fc domain comprising a substitution of
the amino acid at position 233, 234, 235, 236, 237, 327, 330, or
331 (EU numbering) by the amino acid at a corresponding position
(EU numbering) in the corresponding IgG2 or IgG4; [48] the
polypeptide complex of [46], wherein the Fc domain comprises an
amino acid mutation at position 234, 235, or 297 (EU numbering);
[49] the polypeptide complex of [48], in which the amino acid(s) at
position 234, 235, and/or 297 is substituted with alanine; [50] the
polypeptide complex of any one of [43] to [49], wherein the
sequences of two polypeptides forming the Fc domain are different
from each other; [51] the polypeptide complex of any one of [1] to
[50], wherein the amino acid at position 349 is substituted with
cysteine and the amino acid at position 366 (EU numbering) is
substituted with tryptophan among the amino acid residues of one of
the two polypeptides forming the Fc domain; and wherein the amino
acid at position 356 is substituted with cysteine, the amino acid
at position 366 is substituted with serine, the amino acid at
position 368 is substituted with alanine, and the amino acid at
position 407 (EU numbering) is substituted with valine among the
amino acid residues of the other polypeptide; [52] the polypeptide
complex of any one of [1] to [50], wherein the amino acid at
position 356 (EU numbering) is substituted with lysine among the
amino acid residues of one of the two polypeptides forming the Fc
domain; the amino acid at position 439 (EU numbering) is
substituted with glutamic acid in the other polypeptide; and the
amino acid at position 435 (EU numbering) is substituted with
arginine among the amino acid residues of either of the two
polypeptides; [53] the polypeptide complex of [51] or [52], wherein
the sequence GK is deleted from the carboxyl termini of two
polypeptides forming the Fc domain; [54] the polypeptide complex of
any one of [1] to [53], wherein the antigen-binding domains bind to
the same epitope; [55] the polypeptide complex of [54], wherein the
same epitope is present in a protein comprising the amino acid
sequence of SEQ ID NO: 2; [56] the polypeptide complex of [54],
wherein the same epitope is present in a protein comprising the
amino acid sequence of SEQ ID NO: 4; [57] the polypeptide complex
of any one of [1] to [53], wherein the antigen-binding domains each
bind to a different epitope; [58] the polypeptide complex of [57],
wherein the different epitope is present in a protein comprising
the amino acid sequence of SEQ ID NO: 2; [59] the polypeptide
complex of [57], wherein the different epitope is present in a
protein comprising the amino acid sequence of SEQ ID NO: 4; [60] a
polynucleotide encoding the polypeptide complex of any one of [1]
to [59]; [61] a vector comprising the polynucleotide of [60]; [62]
a cell comprising the vector of [61]; [63] a method for producing a
polypeptide complex, which comprises culturing the cell of [62] and
isolating the polypeptide complex from the culture supernatant;
[64] a therapeutic agent for inducing cellular cytotoxicity, which
comprises as an active ingredient the polypeptide complex of any
one of [1] to [59]; [65] the therapeutic agent of [64], wherein the
therapeutic agent for inducing cellular cytotoxicity is a
therapeutic agent for cancer; [66] the therapeutic agent of [65],
wherein the cancer is liver cancer or lung cancer; [67] a method
for treating or preventing cancer, in which the polypeptide complex
of any one of [1] to [59] is administered to a patient in need
thereof; and [68] the therapeutic or preventive method of [67],
wherein the cancer is liver cancer or lung cancer.
[0028] The present invention also relates to kits for use in a
method of the present invention, which contain a polypeptide
complex of the present invention or a polypeptide complex produced
by a method of the present invention. The present invention also
relates to use of a polypeptide complex of the present invention or
a polypeptide complex produced by a method of the present invention
in producing a therapeutic agent for inducing cellular
cytotoxicity. The present invention also relates to polypeptide
complexes of the present invention or polypeptide complexes
produced by methods of the present invention for use in a method of
the present invention.
Effects of the Invention
[0029] The present invention provides novel polypeptide complexes
that retain the strong anti-tumor activity of BiTE and have a long
half-life in blood, as well as excellent safety properties that
result in no induction of cancer antigen-independent cytokine storm
or such. When the antigen-binding domain of a polypeptide complex
of the present invention is substituted, therapeutic agents that
comprise the polypeptide complex as an active ingredient for
inducing cellular cytotoxicity can target and damage various cells
including cancer cells. Thus, various cancers can be treated or
prevented. This allows desirable treatments that are highly safe
and convenient, and reduce the physical burden for patients.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 is a graph showing comparison of the cytotoxic
activities of GPC3 ERY1 (GPC3 BiTE), GPC3 ERY2, and an IgG-type
anti-GPC3 antibody. Closed square (.box-solid.), closed triangle
(.tangle-solidup.), and open square (.quadrature.) represent the
cytotoxic activities of GPC3 ERY1 (GPC3 BiTE), GPC3 ERY2, the
IgG-type anti-GPC3 antibody, respectively.
[0031] FIG. 2 is a graph showing comparison of the cytotoxic
activities of GPC3 BiTE and GPC3 ERY5. Closed square (.box-solid.)
and open circle (.smallcircle.) represent the cytotoxic activities
of GPC3 BiTE and GPC3 ERY5, respectively.
[0032] FIG. 3 is a graph showing comparison of the cytotoxic
activities of GPC3 BiTE and GPC3 ERY6. Closed square (.box-solid.)
and closed triangle (.tangle-solidup.) represent the cytotoxic
activities of GPC3 BiTE and GPC3 ERY6, respectively.
[0033] FIG. 4 is a graph showing comparison of the cytotoxic
activities of GPC3 BiTE and GPC3 ERY7. Closed square (.box-solid.)
and closed diamond (.diamond-solid.) represent the cytotoxic
activities of GPC3 BiTE and GPC3 ERY7, respectively.
[0034] FIG. 5 is a graph showing comparison of the cytotoxic
activities of GPC3 BiTE, GPC3 ERY8-2, GPC3 ERY9-1, and GPC3
ERY10-1. Closed square (.box-solid.), closed triangle
(.tangle-solidup.), open circle (.smallcircle.), and open square
(.quadrature.) represent the cytotoxic activities of GPC3 BiTE,
GPC3 ERY8-2, GPC3 ERY9-1, and GPC3 ERY10-1, respectively.
[0035] FIG. 6 is a graph showing the in vivo anti-tumor effect of
GPC3 ERY8-2 in the PC-10 pre-mix model. Open square (.quadrature.)
and closed diamond (.diamond-solid.) indicate changes in the tumor
volume of the GPC3 ERY7 administration group and control (PBS
administration) group, respectively.
[0036] FIG. 7 is a graph showing the in vivo anti-tumor effect of
GPC3 ERY10-1 in the PC-10 pre-mix model. Open square (.quadrature.)
and closed diamond (.diamond-solid.) indicate changes in the tumor
volume of the GPC3 ERY10-1 administration group and control (PBS
administration) group, respectively.
[0037] FIG. 8 is a graph showing the in vivo anti-tumor effect of
GPC3 ERY10-1 in the PC-10 T cell transfer model. Open square
(.quadrature.) and closed diamond (.diamond-solid.) indicate
changes in the tumor volume of the GPC3 ERY10-1 administration
group and control (PBS administration) group, respectively.
[0038] FIG. 9 is a graph showing a time course of the plasma
concentrations of GPC3 ERY9-1 and GPC3 ERY10-1 determined using
GPC3-expressing Ba/F3 cells. Closed diamond (.diamond-solid.) and
open square (.quadrature.) indicate the plasma concentration time
course for GPC3 ERY9-1 and GPC3 ERY10-1, respectively.
[0039] FIG. 10 is a graph showing time course of the plasma
concentrations of GPC3 ERY9-1 and GPC3 ERY10-1 determined using
CD3-expressing Ba/F3 cells. Closed diamond (.diamond-solid.) and
open square (.quadrature.) indicate the plasma concentration time
course for GPC3 ERY9-1 and GPC3 ERY10-1, respectively.
[0040] FIG. 11 is a graph showing assessment of GPC3 BiTE, GPC3
ERY9-1, GPC3 ERY10-1, GPC3 ERY15-1, and catumaxomab for their
ability to induce cytokines in a cancer antigen-independent
manner.
[0041] FIG. 12 is a graph showing the in vitro cytotoxicities of
GPC3 ERY18 L1, GPC3 ERY18L2, GPC3 ERY18L3, GPC3 ERY18L4, and GPC3
ERY18S1. Closed triangle (.tangle-solidup.), closed circle
(.circle-solid.), closed square (.box-solid.), open square
(.quadrature.), and open diamond (.diamond.) represent the
cytotoxic activities of GPC3 ERY18 L1, GPC3 ERY18 L2, GPC3 ERY18
L3, GPC3 ERY18 L4, and GPC3 ERY18 S1, respectively.
[0042] FIG. 13 is a graph showing comparison of the in vitro
cytotoxic activities of GPC3 ERY18 L3 and GPC3 ERY10-1. Closed
square (.box-solid.) and open square (.quadrature.) represent the
cytotoxic activities of GPC3 ERY18 L3 and GPC3 ERY10-1,
respectively.
[0043] FIG. 14 is a graph showing comparison of the in vitro
cytotoxic activities of GPC3 ERY19-3 and GPC3 BiTE. Open square
(.quadrature.) and closed square (.box-solid.) represent the
cytotoxic activities of GPC3 ERY19-3 and GPC3 BiTE,
respectively.
[0044] FIG. 15 shows a set of chromatograms. Diagram A is a
chromatogram showing results of the size exclusion chromatography
analysis of CM in which NTA1L/NTA1R/GC33-k0 was expressed. Diagram
B is a chromatogram showing results of the size exclusion
chromatography analysis of CM in which NTA2L/NTA2R/GC33-k0 was
expressed.
[0045] FIG. 16 shows diagrams that represent domains forming the
following polypeptide complexes described in the Examples herein:
GPC3 BiTE, GPC3 ERY2, GPC3 ERY5, GPC3 ERY6, GPC3 ERY7, GPC3 ERY8-2,
GPC3 ERY9-1, GPC3 ERY 10-1, GPC3 ERY15, GPC3 ERY18, and GPC3
ERY19-3. The domain with cross-hatched lines represents the H-chain
variable region of the anti-cancer antigen (GPC3, EpCAM, EGFR)
antibody; the domain with diagonal lines represents the L-chain
variable region of the anti-cancer antigen (GPC3, EpCAM, EGFR)
antibody; the domain with dotted lines represents the H-chain
variable region of the anti-CD3 antibody; the closed domain
represents the L-chain variable region of the anti-CD3 antibody;
the open domain represents the antibody constant region; the cross
represents a silent Fc mutation; and the star represents a mutation
promoting heteromeric Fc association.
[0046] FIG. 17 shows diagrams of GPC3 BiTE (A); GPC3 ERY 10 (B);
GPC3 ERY2 (C); GPC3 ERY5 (D); GPC3 ERY6 (E); GPC3 ERY7 (F); GPC3
ERY8-2 (G); GPC3 ERY9-1 (H); GPC3 ERY10-1 (I); GPC3 ERY15 (J); GPC3
ERY18 (K); and GPC3 ERY19-3 (L).
[0047] FIG. 18 shows the amino acid residues forming the Fc domains
of IgG1, IgG2, IgG3, and IgG4 and their Kabat EU numbering (herein,
also called "EU INDEX").
[0048] FIG. 19 shows diagrams A and B that represent domains
forming the following polypeptide complexes described in the
Examples herein: GPC3 ERY17-2, GPC3 ERY17-3, EpCAM ERY17-2, and
EpCAM ERY17-3. The domain with cross-hatched lines represents the
H-chain variable region of the anti-cancer antigen (GPC3, EpCAM,
EGFR) antibody; the domain with diagonal lines represents the
L-chain variable region of the anti-cancer antigen (GPC3, EpCAM,
EGFR) antibody; the domain with dotted lines represents the H-chain
variable region of the anti-CD3 antibody; the closed domain
represents the L-chain variable region of the anti-CD3 antibody;
the open domain represents the antibody constant region; the cross
represents a silent Fc mutation; and the star represents a mutation
promoting heteromeric Fc association.
[0049] FIG. 20 is a graph showing comparison of the cytotoxic
activities of GPC3 BiTE, GPC3 ERY17-2, GPC3 ERY17-3, and GPC3
ERY10-1. Closed square (.box-solid.), closed triangle
(.tangle-solidup.), open circle (.smallcircle.), and open square
(.quadrature.) represent the cytotoxic activities of GPC3 BiTE,
GPC3 ERY17-2, GPC3 ERY17-3, and GPC3 ERY10-1, respectively.
[0050] FIG. 21 is a graph showing the in vivo anti-tumor effect of
GPC3 ERY17-2 in the PC-10 T cell transfer model. Open square
(.quadrature.) and closed diamond (.diamond-solid.) indicate
changes in the tumor volume of the GPC3 ERY17-2 administration
group and control (PBS administration) group, respectively.
[0051] FIG. 22 is a graph showing comparison of the cytotoxic
activities of GPC3 ERY17-2 and GPC3 ERY17-2-M20. Closed triangle
(.tangle-solidup.) and open circle (.smallcircle.) represent the
cytotoxic activities of GPC3 ERY17-2 and GPC3 ERY17-2-M20,
respectively.
[0052] FIG. 23 is a graph showing comparison of the cytotoxic
activities of EpCAM ERY17-2 and EpCAM ERY17-3. Closed triangle
(.tangle-solidup.) and open square (.quadrature.) represent the
cytotoxic activities of EpCAM ERY17-2 and EpCAM ERY17-3,
respectively.
[0053] FIG. 24 shows diagrams that represent domains forming the
following polypeptide complexes described in the Examples herein:
GM1, GM2, and GMO. "A" shows a polypeptide complex in which the
CH1/CL interface association is regulated, and Knobs-into-Holes
(KiH) modifications are introduced. "B" shows a polypeptide complex
that has no regulation of the CH1/CL interface association or
introduction of KiH modifications. The domain with cross-hatched
lines represents the H-chain variable region of the anti-cancer
antigen (GPC3 or EpCAM) antibody; the domain with diagonal lines
represents the L-chain variable region of the anti-cancer antigen
(GPC3 or EpCAM) antibody; the domain with dotted lines represents
the H-chain variable region of the anti-CD3 antibody; the closed
domain represents the L-chain variable region of the anti-CD3
antibody; the open domain represents the antibody constant region;
the cross represents a silent Fc mutation; the star represents a
mutation promoting heteromeric Fc association; and the
doughnut-shaped symbol represents a mutation regulating the CH1/CL
interface interaction.
[0054] FIG. 25 is a graph showing comparison of the cytotoxic
activities of GM1, GM2, and GMO. Closed triangle
(.tangle-solidup.), open square (.quadrature.), and open circle
(.smallcircle.) represent the cytotoxic activities of GM1, GM2, and
GM3, respectively.
[0055] FIG. 26 is a graph showing the cytotoxic activity of EGFR
ERY17-2. Closed triangle (.tangle-solidup.) represents the
cytotoxic activity of EGFR ERY17-2.
MODE FOR CARRYING OUT THE INVENTION
[0056] The definitions below are provided to aid understanding of
the present invention.
Antibody
[0057] Herein, "antibody" refers to a natural immunoglobulin or an
immunoglobulin produced by partial or complete synthesis.
Antibodies can be isolated from natural sources such as
naturally-occurring plasma and serum, or culture supernatants of
antibody-producing hybridomas. Alternatively, antibodies can be
partially or completely synthesized using techniques such as
genetic recombination. Preferred antibodies include, for example,
antibodies of an immunoglobulin isotype or subclass belonging
thereto. Known human immunoglobulins include antibodies of the
following nine classes (isotypes): IgG1, IgG2, IgG3, IgG4, IgA1,
IgA2, IgD, IgE, and IgM. Of these isotypes, antibodies of the
present invention include IgG1, IgG2, IgG3, and IgG4.
[0058] Methods for producing an antibody with desired binding
activity are known to those skilled in the art. Below is an example
that describes a method for producing an antibody (anti-GPC3
antibody) that binds to GPC3, which belongs to the GPI-anchored
receptor family (Int J Cancer. (2003) 103(4), 455-65). Antibodies
that bind to an antigen other than GPC3 can also be produced
according to the example described below.
[0059] Anti-GPC3 antibodies can be obtained as polyclonal or
monoclonal antibodies using known methods. The anti-GPC3 antibodies
preferably produced are monoclonal antibodies derived from mammals.
Such mammal-derived monoclonal antibodies include antibodies
produced by hybridomas or host cells transformed with an expression
vector carrying an antibody gene by genetic engineering
techniques.
[0060] Monoclonal antibody-producing hybridomas can be produced
using known techniques, for example, as described below.
Specifically, mammals are immunized by conventional immunization
methods using a GPC3 protein as a sensitizing antigen. Resulting
immune cells are fused with known parental cells by conventional
cell fusion methods. Then, hybridomas producing an anti-GPC3
antibody can be selected by screening for monoclonal
antibody-producing cells using conventional screening methods.
[0061] Specifically, monoclonal antibodies are prepared as
mentioned below. First, the GPC3 gene whose nucleotide sequence is
disclosed in RefSeq accession number NM_001164617.1 (SEQ ID NO: 1)
can be expressed to produce a GPC3 protein shown in RefSeq
accession number NP_001158089.1 (SEQ ID NO: 2), which will be used
as a sensitizing antigen for antibody preparation. That is, a gene
sequence encoding GPC3 is inserted into a known expression vector,
and appropriate host cells are transformed with this vector. The
desired human GPC3 protein is purified from the host cells or their
culture supernatants by known methods. For example, to prepare
soluble GPC3 from culture supernatants, amino acids at positions
564 to 580 that form the hydrophobic region corresponding to the
GPI-anchor sequence used to anchor GPC3 on the cell membrane are
deleted from the GPC3 polypeptide sequence of SEQ ID NO: 2, and
then the resulting protein is expressed instead of the GPC3 protein
of SEQ ID NO: 2. Alternatively, it is possible to use a purified
natural GPC3 protein as a sensitizing antigen.
[0062] The purified GPC3 protein can be used as a sensitizing
antigen for immunization of mammals. A partial GPC3 peptide may
also be used as a sensitizing antigen. In this case, a partial
peptide can be prepared by chemical synthesis based on the amino
acid sequence of human GPC3, or by inserting a partial GPC3 gene
into an expression vector for expression. Alternatively, a partial
peptide can be produced by degrading a GPC3 protein with a
protease. The length and region of the partial GPC3 peptide are not
limited to particular embodiments. A preferred region can be
arbitrarily selected from the amino acid sequence at amino acid
positions 564 to 580 in the amino acid sequence of SEQ ID NO: 2.
The number of amino acids forming a peptide to be used as a
sensitizing antigen is preferably at least five or more, six or
more, or seven or more. More specifically, a peptide of 8 to 50
residues, more preferably 10 to 30 residues can be used as a
sensitizing antigen.
[0063] For sensitizing antigen, alternatively it is possible to use
a fusion protein prepared by fusing a desired partial polypeptide
or peptide of the GPC3 protein with a different polypeptide. For
example, antibody Fc fragments and peptide tags are preferably used
to produce fusion proteins to be used as sensitizing antigens.
Vectors for expression of such fusion proteins can be constructed
by fusing in frame genes encoding two or more desired polypeptide
fragments and inserting the fusion gene into an expression vector
as described above. Methods for producing fusion proteins are
described in Molecular Cloning 2nd ed. (Sambrook, J et al.,
Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab.
Press). Methods for preparing GPC3 to be used as a sensitizing
antigen, and immunization methods using GPC3 are specifically
described in WO 2003/000883, WO 2004/022754, and WO
2006/006693.
[0064] There is no particular limitation on the mammals to be
immunized with the sensitizing antigen. However, it is preferable
to select the mammals by considering their compatibility with the
parent cells to be used for cell fusion. In general, rodents such
as mice, rats, and hamsters, rabbits, and monkeys are preferably
used.
[0065] The above animals are immunized with a sensitizing antigen
by known methods. Generally performed immunization methods include,
for example, intraperitoneal or subcutaneous injection of a
sensitizing antigen into mammals. Specifically, a sensitizing
antigen is appropriately diluted with PBS (Phosphate-Buffered
Saline), physiological saline, or the like. If desired, a
conventional adjuvant such as Freund's complete adjuvant is mixed
with the antigen, and the mixture is emulsified. Then, the
sensitizing antigen is administered to a mammal several times at 4-
to 21-day intervals. Appropriate carriers may be used in
immunization with the sensitizing antigen. In particular, when a
low-molecular-weight partial peptide is used as the sensitizing
antigen, it is sometimes desirable to couple the sensitizing
antigen peptide to a carrier protein such as albumin or keyhole
limpet hemocyanin for immunization.
[0066] Alternatively, hybridomas producing a desired antibody can
be prepared using DNA immunization as mentioned below. DNA
immunization is an immunization method that confers
immunostimulation by expressing a sensitizing antigen in an animal
immunized as a result of administering a vector DNA constructed to
allow expression of an antigen protein-encoding gene in the animal.
As compared to conventional immunization methods in which a protein
antigen is administered to animals to be immunized, DNA
immunization is expected to be superior in that:
[0067] immunostimulation can be provided while retaining the
structure of a membrane protein such as GPC3; and
[0068] there is no need to purify the antigen for immunization.
[0069] In order to prepare a monoclonal antibody of the present
invention using DNA immunization, first, a DNA expressing a GPC3
protein is administered to an animal to be immunized. The
GPC3-encoding DNA can be synthesized by known methods such as PCR.
The obtained DNA is inserted into an appropriate expression vector,
and then this is administered to an animal to be immunized.
Preferably used expression vectors include, for example,
commercially-available expression vectors such as pcDNA3.1. Vectors
can be administered to an organism using conventional methods. For
example, DNA immunization is performed by using a gene gun to
introduce expression vector-coated gold particles into cells in the
body of an animal to be immunized. Antibodies that recognized GPC3
can also be produced by the methods described in WO
2003/104453.
[0070] After immunizing a mammal as described above, an increase in
the titer of a GPC3-binding antibody is confirmed in the serum.
Then, immune cells are collected from the mammal, and then
subjected to cell fusion. In particular, splenocytes are preferably
used as immune cells.
[0071] A mammalian myeloma cell is used as a cell to be fused with
the above-mentioned immunocyte. The myeloma cells preferably
comprise a suitable selection marker for screening. A selection
marker confers characteristics to cells for their survival (or
death) under a specific culture condition. Hypoxanthine-guanine
phosphoribosyltransferase deficiency (hereinafter abbreviated as
HGPRT deficiency) and thymidine kinase deficiency (hereinafter
abbreviated as TK deficiency) are known as selection markers. Cells
with HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine
sensitivity (hereinafter abbreviated as HAT sensitivity).
HAT-sensitive cells cannot synthesize DNA in a HAT selection
medium, and are thus killed. However, when the cells are fused with
normal cells, they can continue DNA synthesis using the salvage
pathway of the normal cells, and therefore they can grow even in
the HAT selection medium.
[0072] HGPRT-deficient and TK-deficient cells can be selected in a
medium containing 6-thioguanine, 8-azaguanine (hereinafter
abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal
cells are killed because they incorporate these pyrimidine analogs
into their DNA. Meanwhile, cells that are deficient in these
enzymes can survive in the selection medium, since they cannot
incorporate these pyrimidine analogs. In addition, a selection
marker referred to as G418 resistance provided by the
neomycin-resistant gene confers resistance to 2-deoxystreptamine
antibiotics (gentamycin analogs). Various types of myeloma cells
that are suitable for cell fusion are known.
[0073] For example, myeloma cells including the following cells can
be preferably used:
P3(P3.times.63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550);
P3.times.63Ag8U.1 (Current Topics in Microbiology and Immunology
(1978)81, 1-7);
[0074] NS-1 (C. Eur. J. Immunol. (1976)6 (7), 511-519);
MPC-11 (Cell (1976) 8 (3), 405-415);
SP2/0 (Nature (1978) 276 (5685), 269-270);
[0075] FO (J. Immunol. Methods (1980) 35 (1-2), 1-21);
S194/5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323);
R210 (Nature (1979) 277 (5692), 131-133), etc.
[0076] Cell fusions between the immunocytes and myeloma cells are
essentially carried out using known methods, for example, a method
by Kohler and Milstein et al (Methods Enzymol. (1981) 73:
3-46).
[0077] More specifically, cell fusion can be carried out, for
example, in a conventional culture medium in the presence of a cell
fusion-promoting agent. The fusion-promoting agents include, for
example, polyethylene glycol (PEG) and Sendai virus (HVJ). If
required, an auxiliary substance such as dimethyl sulfoxide is also
added to improve fusion efficiency.
[0078] The ratio of immunocytes to myeloma cells may be determined
at one's own discretion, preferably, for example, one myeloma cell
for every one to ten immunocytes. Culture media to be used for cell
fusions include, for example, media that are suitable for the
growth of myeloma cell lines, such as RPMI1640 medium and MEM
medium, and other conventional culture medium used for this type of
cell culture. In addition, serum supplements such as fetal calf
serum (FCS) may be preferably added to the culture medium.
[0079] For cell fusion, predetermined amounts of the above immune
cells and myeloma cells are mixed well in the above culture medium.
Then, a PEG solution (for example, the average molecular weight is
about 1,000 to 6,000) prewarmed to about 37.degree. C. is added
thereto at a concentration of generally 30% to 60% (w/v). This is
gently mixed to produce desired fusion cells (hybridomas). Then, an
appropriate culture medium mentioned above is gradually added to
the cells, and this is repeatedly centrifuged to remove the
supernatant. Thus, cell fusion agents and such which are
unfavorable to hybridoma growth can be removed.
[0080] The hybridomas thus obtained can be selected by culture
using a conventional selective medium, for example, HAT medium (a
culture medium containing hypoxanthine, aminopterin, and
thymidine). Cells other than the desired hybridomas (non-fused
cells) can be killed by continuing culture in the above HAT medium
for a sufficient period of time. Typically, the period is several
days to several weeks. Then, hybridomas producing the desired
antibody are screened and singly cloned by conventional limiting
dilution methods.
[0081] The hybridomas thus obtained can be selected using a
selection medium based on the selection marker possessed by the
myeloma used for cell fusion. For example, HGPRT- or TK-deficient
cells can be selected by culture using the HAT medium (a culture
medium containing hypoxanthine, aminopterin, and thymidine).
Specifically, when HAT-sensitive myeloma cells are used for cell
fusion, cells successfully fused with normal cells can selectively
proliferate in the HAT medium. Cells other than the desired
hybridomas (non-fused cells) can be killed by continuing culture in
the above HAT medium for a sufficient period of time. Specifically,
desired hybridomas can be selected by culture for generally several
days to several weeks. Then, hybridomas producing the desired
antibody are screened and singly cloned by conventional limiting
dilution methods.
[0082] Desired antibodies can be preferably selected and singly
cloned by screening methods based on known antigen/antibody
reaction. For example, a GPC3-binding monoclonal antibody can bind
to GPC3 expressed on the cell surface. Such a monoclonal antibody
can be screened by fluorescence activated cell sorting (FACS). FACS
is a system that assesses the binding of an antibody to cell
surface by analyzing cells contacted with a fluorescent antibody
using laser beam, and measuring the fluorescence emitted from
individual cells.
[0083] To screen for hybridomas that produce a monoclonal antibody
of the present invention by FACS, GPC3-expressing cells are first
prepared. Cells preferably used for screening are mammalian cells
in which GPC3 is forcedly expressed. As control, the activity of an
antibody to bind to cell-surface GPC3 can be selectively detected
using non-transformed mammalian cells as host cells. Specifically,
hybridomas producing an anti-GPC3 monoclonal antibody can be
isolated by selecting hybridomas that produce an antibody which
binds to cells forced to express GPC3, but not to host cells.
[0084] Alternatively, the activity of an antibody to bind to
immobilized GPC3-expressing cells can be assessed based on the
principle of ELISA. For example, GPC3-expressing cells are
immobilized to the wells of an ELISA plate. Culture supernatants of
hybridomas are contacted with the immobilized cells in the wells,
and antibodies that bind to the immobilized cells are detected.
When the monoclonal antibodies are derived from mouse, antibodies
bound to the cells can be detected using an anti-mouse
immunoglobulin antibody. Hybridomas producing a desired antibody
having the antigen-binding ability are selected by the above
screening, and they can be cloned by a limiting dilution method or
the like.
[0085] Monoclonal antibody-producing hybridomas thus prepared can
be passaged in a conventional culture medium, and stored in liquid
nitrogen for a long period.
[0086] The above hybridomas are cultured by a conventional method,
and desired monoclonal antibodies can be prepared from the culture
supernatants. Alternatively, the hybridomas are administered to and
grown in compatible mammals, and monoclonal antibodies are prepared
from the ascites. The former method is suitable for preparing
antibodies with high purity.
[0087] Antibodies encoded by antibody genes that are cloned from
antibody-producing cells such as the above hybridomas can also be
preferably used. A cloned antibody gene is inserted into an
appropriate vector, and this is introduced into a host to express
the antibody encoded by the gene. Methods for isolating antibody
genes, inserting the genes into vectors, and transforming host
cells have already been established, for example, by Vandamme et
al. (Eur. J. Biochem. (1990) 192(3), 767-775). Methods for
producing recombinant antibodies are also known as described
below.
[0088] For example, a cDNA encoding the variable region (V region)
of an anti-GPC3 antibody is prepared from hybridoma cells
expressing the anti-GPC3 antibody. For this purpose, total RNA is
first extracted from hybridomas. Methods used for extracting mRNAs
from cells include, for example:
[0089] the guanidine ultracentrifugation method (Biochemistry
(1979) 18(24), 5294-5299), and
[0090] the AGPC method (Anal. Biochem. (1987) 162(1), 156-159)
[0091] Extracted mRNAs can be purified using the mRNA Purification
Kit (GE Healthcare Bioscience) or such. Alternatively, kits for
extracting total mRNA directly from cells, such as the QuickPrep
mRNA Purification Kit (GE Healthcare Bioscience), are also
commercially available. mRNAs can be prepared from hybridomas using
such kits. cDNAs encoding the antibody V region can be synthesized
from the prepared mRNAs using a reverse transcriptase. cDNAs can be
synthesized using the AMV Reverse Transcriptase First-strand cDNA
Synthesis Kit (Seikagaku Co.) or such. Furthermore, the SMART RACE
cDNA amplification kit (Clontech) and the PCR-based 5'-RACE method
(Proc. Natl. Acad. Sci. USA (1988) 85(23), 8998-9002; Nucleic Acids
Res. (1989) 17(8), 2919-2932) can be appropriately used to
synthesize and amplify cDNAs. In such a cDNA synthesis process,
appropriate restriction enzyme sites described below may be
introduced into both ends of a cDNA.
[0092] The cDNA fragment of interest is purified from the resulting
PCR product, and then this is ligated to a vector DNA. A
recombinant vector is thus constructed, and introduced into E. coli
or such. After colony selection, the desired recombinant vector can
be prepared from the colony-forming E. coli. Then, whether the
recombinant vector has the cDNA nucleotide sequence of interest is
tested by a known method such as the dideoxy nucleotide chain
termination method.
[0093] The 5'-RACE method which uses primers to amplify the
variable region gene is conveniently used for isolating the gene
encoding the variable region. First, a 5'-RACE cDNA library is
constructed by cDNA synthesis using RNAs extracted from hybridoma
cells as a template. A commercially available kit such as the SMART
RACE cDNA amplification kit is appropriately used to synthesize the
5'-RACE cDNA library.
[0094] The antibody gene is amplified by PCR using the prepared
5'-RACE cDNA library as a template. Primers for amplifying the
mouse antibody gene can be designed based on known antibody gene
sequences. The nucleotide sequences of the primers vary depending
on the immunoglobulin subclass. Therefore, it is preferable that
the subclass is determined in advance using a commercially
available kit such as the Iso Strip mouse monoclonal antibody
isotyping kit (Roche Diagnostics).
[0095] Specifically, for example, primers that allow amplification
of genes encoding .gamma.1, .gamma.2a, .gamma.2b, and .gamma.3
heavy chains and .kappa. and .lamda. light chains are used to
isolate mouse IgG-encoding genes. In general, a primer that anneals
to a constant region site close to the variable region is used as a
3'-side primer to amplify an IgG variable region gene. Meanwhile, a
primer attached to a 5' RACE cDNA library construction kit is used
as a 5'-side primer.
[0096] PCR products thus amplified are used to reshape
immunoglobulins composed of a combination of heavy and light
chains. A desired antibody can be selected using the GPC3-binding
activity of a reshaped immunoglobulin as an indicator. For example,
when the objective is to isolate an antibody against GPC3, it is
more preferred that the binding of the antibody to GPC3 is
specific. A GPC3-binding antibody can be screened, for example, by
the following steps:
[0097] (1) contacting a GPC3-expressing cell with an antibody
comprising the V region encoded by a cDNA isolated from a
hybridoma;
[0098] (2) detecting the binding of the antibody to the
GPC3-expressing cell; and
[0099] (3) selecting an antibody that binds to the GPC3-expressing
cell.
[0100] Methods for detecting the binding of an antibody to
GPC3-expressing cells are known. Specifically, the binding of an
antibody to GPC3-expressing cells can be detected by the
above-described techniques such as FACS. Immobilized samples of
GPC3-expressing cells are appropriately used to assess the binding
activity of an antibody.
[0101] Preferred antibody screening methods that use the binding
activity as an indicator also include panning methods using phage
vectors. Screening methods using phage vectors are advantageous
when the antibody genes are isolated from heavy-chain and
light-chain subclass libraries from a polyclonal
antibody-expressing cell population. Genes encoding the heavy-chain
and light-chain variable regions can be linked by an appropriate
linker sequence to form a single-chain Fv (scFv). Phages presenting
scFv on their surface can be produced by inserting a gene encoding
scFv into a phage vector. The phages are contacted with an antigen
of interest. Then, a DNA encoding scFv having the binding activity
of interest can be isolated by collecting phages bound to the
antigen. This process can be repeated as necessary to enrich scFv
having the binding activity of interest.
[0102] After isolation of the cDNA encoding the V region of the
anti-GPC3 antibody of interest, the cDNA is digested with
restriction enzymes that recognize the restriction sites introduced
into both ends of the cDNA. Preferred restriction enzymes recognize
and cleave a nucleotide sequence that occurs in the nucleotide
sequence of the antibody gene at a low frequency. Furthermore, a
restriction site for an enzyme that produces a sticky end is
preferably introduced into a vector to insert a single-copy
digested fragment in the correct orientation. The cDNA encoding the
V region of the anti-GPC3 antibody is digested as described above,
and this is inserted into an appropriate expression vector to
construct an antibody expression vector. In this case, if a gene
encoding the antibody constant region (C region) and a gene
encoding the above V region are fused in-frame, a chimeric antibody
is obtained. Herein, "chimeric antibody" means that the origin of
the constant region is different from that of the variable region.
Thus, in addition to mouse/human heterochimeric antibodies,
human/human allochimeric antibodies are included in the chimeric
antibodies of the present invention. A chimeric antibody expression
vector can be constructed by inserting the above V region gene into
an expression vector that already has the constant region.
Specifically, for example, a recognition sequence for a restriction
enzyme that excises the above V region gene can be appropriately
placed on the 5' side of an expression vector carrying a DNA
encoding a desired antibody constant region (C region). A chimeric
antibody expression vector is constructed by fusing in frame the
two genes digested with the same combination of restriction
enzymes.
[0103] To produce an anti-GPC3 monoclonal antibody, antibody genes
are inserted into an expression vector so that the genes are
expressed under the control of an expression regulatory region. The
expression regulatory region for antibody expression includes, for
example, enhancers and promoters. Furthermore, an appropriate
signal sequence may be attached to the amino terminus so that the
expressed antibody is secreted to the outside of cells. In the
Examples described below, a peptide having the amino acid sequence
MGWSCIILFLVATATGVHS (SEQ ID NO: 72) is used as a signal sequence.
Meanwhile, other appropriate signal sequences may be attached. The
expressed polypeptide is cleaved at the carboxyl terminus of the
above sequence, and the resulting polypeptide is secreted to the
outside of cells as a mature polypeptide. Then, appropriate host
cells are transformed with the expression vector, and recombinant
cells expressing the anti-GPC3 antibody-encoding DNA are
obtained.
[0104] DNAs encoding the antibody heavy chain (H chain) and light
chain (L chain) are separately inserted into different expression
vectors to express the antibody gene. An antibody molecule having
the H and L chains can be expressed by co-transfecting the same
host cell with vectors into which the H-chain and L-chain genes are
respectively inserted. Alternatively, host cells can be transformed
with a single expression vector into which DNAs encoding the H and
L chains are inserted (see WO 94/11523).
[0105] There are various known host cell/expression vector
combinations for antibody preparation by introducing isolated
antibody genes into appropriate hosts. All of these expression
systems are applicable to isolation of the antigen-binding domains
and CD3-binding domains of the present invention. Appropriate
eukaryotic cells used as host cells include animal cells, plant
cells, and fungal cells. Specifically, the animal cells include,
for example, the following cells.
(1) mammalian cells: CHO, COS, myeloma, baby hamster kidney (BHK),
HeLa, Vero, or such; (2) amphibian cells: Xenopus oocytes, or such;
and (3) insect cells: sf9, sf21, Tn5, or such.
[0106] In addition, as a plant cell, an antibody gene expression
system using cells derived from the Nicotiana genus such as
Nicotiana tabacum is known. Callus cultured cells can be
appropriately used to transform plant cells.
[0107] Furthermore, the following cells can be used as fungal
cells:
yeasts: the Saccharomyces genus such as Saccharomyces cerevisiae,
and the Pichia genus such as Pichia pastoris; and filamentous
fungi: the Aspergillus genus such as Aspergillus niger.
[0108] Furthermore, antibody gene expression systems that utilize
prokaryotic cells are also known. For example, when using bacterial
cells, E. coli cells, Bacillus subtilis cells, and such can
suitably be utilized in the present invention. Expression vectors
carrying the antibody genes of interest are introduced into these
cells by transfection. The transfected cells are cultured in vitro,
and the desired antibody can be prepared from the culture of
transformed cells.
[0109] In addition to the above-described host cells, transgenic
animals can also be used to produce a recombinant antibody. That
is, the antibody can be obtained from an animal into which the gene
encoding the antibody of interest is introduced. For example, the
antibody gene can be constructed as a fusion gene by inserting in
frame into a gene that encodes a protein produced specifically in
milk. Goat .beta.-casein or such can be used, for example, as the
protein secreted in milk. DNA fragments containing the fused gene
inserted with the antibody gene is injected into a goat embryo, and
then this embryo is introduced into a female goat. Desired
antibodies can be obtained as a protein fused with the milk protein
from milk produced by the transgenic goat born from the
embryo-recipient goat (or progeny thereof). In addition, to
increase the volume of milk containing the desired antibody
produced by the transgenic goat, hormones can be administered to
the transgenic goat as necessary (Ebert, K. M. et al,
Bio/Technology (1994) 12 (7), 699-702).
[0110] When a polypeptide complex described herein is administered
to human, an antigen-binding domain derived from a genetically
recombinant antibody that has been artificially modified to reduce
the heterologous antigenicity against human and such, can be
appropriately used as the antigen-binding domain of the complex.
Such genetically recombinant antibodies include, for example,
humanized antibodies. These modified antibodies are appropriately
produced by known methods.
[0111] An antibody variable region used to produce the
antigen-binding domain of a polypeptide complex described herein is
generally formed by three complementarity-determining regions
(CDRs) that are separated by four framework regions (FRs). CDR is a
region that substantially determines the binding specificity of an
antibody. The amino acid sequences of CDRs are highly diverse. On
the other hand, the FR-forming amino acid sequences often have high
identity even among antibodies with different binding
specificities. Therefore, generally, the binding specificity of a
certain antibody can be introduced to another antibody by CDR
grafting.
[0112] A humanized antibody is also called a reshaped human
antibody. Specifically, humanized antibodies prepared by grafting
the CDR of a non-human animal antibody such as a mouse antibody to
a human antibody and such are known. Common genetic engineering
techniques for obtaining humanized antibodies are also known.
Specifically, for example, overlap extension PCR is known as a
method for grafting a mouse antibody CDR to a human FR. In overlap
extension PCR, a nucleotide sequence encoding a mouse antibody CDR
to be grafted is added to primers for synthesizing a human antibody
FR. Primers are prepared for each of the four FRs. It is generally
considered that when grafting a mouse CDR to a human FR, selecting
a human FR that has high identity to a mouse FR is advantageous for
maintaining the CDR function. That is, it is generally preferable
to use a human FR comprising an amino acid sequence which has high
identity to the amino acid sequence of the FR adjacent to the mouse
CDR to be grafted.
[0113] Nucleotide sequences to be ligated are designed so that they
will be connected to each other in frame. Human FRs are
individually synthesized using the respective primers. As a result,
products in which the mouse CDR-encoding DNA is attached to the
individual FR-encoding DNAs are obtained. Nucleotide sequences
encoding the mouse CDR of each product are designed so that they
overlap with each other. Then, complementary strand synthesis
reaction is conducted to anneal the overlapping CDR regions of the
products synthesized using a human antibody gene as template. Human
FRs are ligated via the mouse CDR sequences by this reaction.
[0114] The full length V region gene, in which three CDRs and four
FRs are ultimately ligated, is amplified using primers that anneal
to its 5'- or 3'-end, which are added with suitable restriction
enzyme recognition sequences. An expression vector for humanized
antibody can be produced by inserting the DNA obtained as described
above and a DNA that encodes a human antibody C region into an
expression vector so that they will ligate in frame. After the
recombinant vector is transfected into a host to establish
recombinant cells, the recombinant cells are cultured, and the DNA
encoding the humanized antibody is expressed to produce the
humanized antibody in the cell culture (see, European Patent
Publication No. EP 239400 and International Patent Publication No.
WO 1996/002576).
[0115] By qualitatively or quantitatively measuring and evaluating
the antigen-binding activity of the humanized antibody produced as
described above, one can suitably select human antibody FRs that
allow CDRs to form a favorable antigen-binding site when ligated
through the CDRs. Amino acid residues in FRs may be substituted as
necessary, so that the CDRs of a reshaped human antibody form an
appropriate antigen-binding site. For example, amino acid sequence
mutations can be introduced into FRs by applying the PCR method
used for grafting a mouse CDR into a human FR. More specifically,
partial nucleotide sequence mutations can be introduced into
primers that anneal to the FR. Nucleotide sequence mutations are
introduced into the FRs synthesized by using such primers. Mutant
FR sequences having the desired characteristics can be selected by
measuring and evaluating the activity of the amino acid-substituted
mutant antibody to bind to the antigen by the above-mentioned
method (Sato, K. et al, Cancer Res. (1993) 53: 851-856).
[0116] Alternatively, desired human antibodies can be obtained by
immunizing transgenic animals having the entire repertoire of human
antibody genes (see WO 1993/012227; WO 1992/003918; WO 1994/002602;
WO 1994/025585; WO 1996/034096; WO 1996/033735) by DNA
immunization.
[0117] Furthermore, techniques for preparing human antibodies by
panning using human antibody libraries are also known. For example,
the V region of a human antibody is expressed as a single-chain
antibody (scFv) on phage surface by the phage display method.
Phages expressing an scFv that binds to the antigen can be
selected. The DNA sequence encoding the human antibody V region
that binds to the antigen can be determined by analyzing the genes
of selected phages. The DNA sequence of the scFv that binds to the
antigen is determined. An expression vector is prepared by fusing
the V region sequence in frame with the C region sequence of a
desired human antibody, and inserting this into an appropriate
expression vector. The expression vector is introduced into cells
appropriate for expression such as those described above. The human
antibody can be produced by expressing the human antibody-encoding
gene in the cells. These methods are already known (see WO
1992/001047; WO 1992/020791; WO 1993/006213; WO 1993/011236; WO
1993/019172; WO 1995/001438; WO 1995/015388).
Antigen-Binding Domain
[0118] Herein, "antigen-binding domain" refers to an antibody
portion which comprises a region that specifically binds and is
complementary to the whole or a portion of an antigen. When the
molecular weight of an antigen is large, an antibody can only bind
to a particular portion of the antigen. The particular portion is
called "epitope". An antigen-binding domain can be provided from
one or more antibody variable domains. Preferably, the
antigen-binding domains contain both the antibody light chain
variable region (VL) and antibody heavy chain variable region (VH).
Such preferable antigen-binding domains include, for example,
"single-chain Fv (scFv)", "single-chain antibody", "Fv",
"single-chain Fv2 (scFv2)", "Fab", and "F (ab')2".
[0119] The antigen-binding domains of polypeptide complexes of the
present invention may bind to the same epitope. The epitope can be
present in a protein comprising the amino acid sequence of SEQ ID
NO: 2 or 4. Alternatively, the antigen-binding domains of
polypeptide complexes of the present invention may individually
bind to different epitopes. The epitope can be present in a protein
comprising the amino acid sequence of SEQ ID NO: 2 or 4.
Specificity
[0120] "Specific" means that a molecule that binds specifically to
one or more binding partners does not show any significant binding
to molecules other than the partners. Furthermore, "specific" is
also used when an antigen-binding domain is specific to a
particular epitope of multiple epitopes contained in an antigen.
When an epitope bound by an antigen-binding domain is contained in
multiple different antigens, a polypeptide complex containing the
antigen-binding domain can bind to various antigens that have the
epitope.
Antigen
[0121] Herein, there is no particular limitation on the antigen,
and it is possible to use any antigen except for CD3. Such antigens
include, for example, receptors, cancer antigens, MHC antigens, and
differentiation antigens. The receptors include, for example, those
belonging to the hematopoietic growth factor receptor family,
cytokine receptor family, tyrosine kinase receptor family,
serine/threonine kinase receptor family, TNF receptor family, G
protein-coupled receptor family, GPI-anchored receptor family,
tyrosine phosphatase receptor family, adhesion factor family, and
hormone receptor family. Receptors belonging to these receptor
families and their characteristics are described in various
documents, for example, reviews such as Cooke B A., King R J B.,
van der Molen H J. ed. New Comprehensive Biochemistry Vol. 18B
"Hormones and their Actions Part II" pp. 1-46 (1988) Elsevier
Science Publishers BV.; and SAIBO KOGAKU (Cell Technology)
Supplementary Volume Handbook Series "Secchaku Inshi Handbook
(Handbook for Adhesion factors)" M. Miyasaka Ed. (1994) Shujunsha,
Tokyo, Japan; and Patthy (Cell (1990) 61(1), 13-14); Ullrich et
al., (Cell (1990) 61(2), 203-212); Massague (Cell (1992) 69(6),
1067-1070); Miyajima et al., (Annu. Rev. Immunol. (1992) 10,
295-331); Taga et al., (FASEB J. (1992) 6, 3387-3396); Fantl et
al., (Annu. Rev. Biochem. (1993), 62, 453-481), Smith et al., (Cell
(1994) 76(6) 959-962), Flower DR. Biochim. Biophys. Acta, Flower
(Biochim. Biophys. Acta (1999) 1422(3) 207-234.
[0122] Specifically, receptors belonging to the above receptor
families preferably include, for example, human and mouse
erythropoietin (EPO) receptors (Blood (1990) 76(1), 31-35; Cell
(1989) 57(2), 277-285), human and mouse granulocyte colony
stimulating factor (G-CSF) receptors (Proc. Natl. Acad. Sci. USA.
(1990) 87(22), 8702-8706; mG-CSFR, Cell (1990) 61(2), 341-350),
human and mouse thrombopoietin (TPO) receptors (Proc. Natl. Acad.
Sci. USA (1992) 89(12), 5640-5644; EMBO J. (1993) 12(7), 2645-53),
human and mouse insulin receptors (Nature (1985) 313(6005),
756-761), human and mouse Flt-3 ligand receptors (Proc. Natl. Acad.
Sci. USA (1994) 91(2), 459-463), human and mouse platelet-derived
growth factor (PDGF) receptors (Proc. Natl. Acad. Sci. USA. (1988)
85(10), 3435-3439), human and mouse interferon (IFN)-.alpha. and
-.beta. receptors (Cell (1990) 60(2), 225-234; and Cell (1994)
77(3), 391-400), human and mouse leptin receptors, human and mouse
growth hormone (GH) receptors, human and mouse interleukin (IL)-10
receptors, human and mouse insulin-like growth factor (IGF)-I
receptors, human and mouse leukemia inhibitory factor (LIF)
receptors, and human and mouse ciliary neurotrophic factor (CNTF)
receptors.
[0123] Cancer antigens are antigens expressed as cells become
malignant, and are also called "tumor-specific antigens".
Furthermore, abnormal sugar chains that are expressed on cell
surface or protein molecules when the cells become cancerous are
also cancer antigens, and are called "cancer-associated
carbohydrate antigen". Such cancer antigens include, for example,
GPC3 (Int J Cancer. (2003) 103(4), 455-65), Epithelial Cell
Adhesion Molecule (EpCAM) (Proc. Natl. Acad. Sci. USA. (1989)
86(1), 27-31), Epidermal Growth Factor Receptor (EGFR), CA19-9,
CA15-3, and sialyl SSEA-1 (SLX). GPC3 belongs to the
above-mentioned GPI-anchored receptor family and is expressed in
several types of cancers including liver cancer. EpCAM is expressed
in multiple types of cancers including lung cancer, and its
polynucleotide and polypeptide sequences are disclosed in RefSeq
accession numbers NM_002354.2 (SEQ ID NO: 3) and NP_002345.2 (SEQ
ID NO: 4), respectively).
[0124] Generally, WIC antigens are categorized into MHC class I and
class II antigens. The WIC class I antigens include HLA-A, -B, -C,
-E, -F, -G, and -H, while the MHC class II antigens include HLA-DR,
-DQ, and -DP.
[0125] Differentiation antigens include CD1, CD2, CD4, CD5, CD6,
CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18,
CD19, CD20, CD21, CD23, CD25, CD28, CD29, CD30, CD32, CD33, CD34,
CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, CD43, CD44, CD45,
CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54,
CD55, CD56, CD57, CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69,
CD71, CD73, CD95, CD102, CD106, CD122, CD126, and CDw130.
Epitope
[0126] "Epitope" means an antigenic determinant in an antigen, and
refers to an antigen site to which the antigen-binding domain of a
polypeptide complex disclosed herein binds. Thus, for example, the
epitope can be defined according to its structure. Alternatively,
the epitope may be defined according to the antigen-binding
activity of a polypeptide complex that recognizes the epitope. When
the antigen is a peptide or polypeptide, the epitope can be
specified by the amino acid residues forming the epitope.
Alternatively, when the epitope is a sugar chain, the epitope can
be specified by its specific sugar chain structure.
[0127] A linear epitope is an epitope that contains an epitope
whose primary amino acid sequence is recognized. Such a linear
epitope typically contains at least three and most commonly at
least five, for example, about 8 to 10 or 6 to 20 amino acids in
its specific sequence.
[0128] In contrast to the linear epitope, "conformational epitope"
is an epitope in which the primary amino acid sequence containing
the epitope is not the only determinant of the recognized epitope
(for example, the primary amino acid sequence of a conformational
epitope is not necessarily recognized by an epitope-defining
antibody). Conformational epitopes may contain a greater number of
amino acids compared to linear epitopes. A conformational
epitope-recognizing antibody recognizes the three-dimensional
structure of a peptide or protein. For example, when a protein
molecule folds and forms a three-dimensional structure, amino acids
and/or polypeptide main chains that form a conformational epitope
become aligned, and the epitope is made recognizable by the
antibody. Methods for determining epitope conformations include,
for example, X ray crystallography, two-dimensional nuclear
magnetic resonance, site-specific spin labeling, and electron
paramagnetic resonance, but are not limited thereto. See, for
example, Epitope Mapping Protocols in Methods in Molecular Biology
(1996), Vol. 66, Morris (ed.).
[0129] Examples of a method for assessing the epitope binding by a
test polypeptide complex containing a GPC3 antigen-binding domain
are described below. According to the examples below, methods for
assessing the epitope binding by a test polypeptide complex
containing an antigen-binding domain for an antigen other than
GPC3, can also be appropriately conducted.
[0130] For example, whether a test polypeptide complex containing a
GPC3 antigen-binding domain recognizes a linear epitope in the GPC3
molecule can be confirmed for example as mentioned below. A linear
peptide comprising an amino acid sequence forming the extracellular
domain of GPC3 is synthesized for the above purpose. The peptide
can be synthesized chemically, or obtained by genetic engineering
techniques using a region encoding the amino acid sequence
corresponding to the extracellular domain in a GPC3 cDNA. Then, a
test polypeptide complex containing a GPC3 antigen-binding domain
is assessed for its binding activity towards a linear peptide
comprising the amino acid sequence forming the extracellular
domain. For example, an immobilized linear peptide can be used as
an antigen by ELISA to evaluate the binding activity of the
polypeptide complex towards the peptide. Alternatively, the binding
activity towards a linear peptide can be assessed based on the
level that the linear peptide inhibits the binding of the
polypeptide complex to GPC3-expressing cells. These tests can
demonstrate the binding activity of the polypeptide complex towards
the linear peptide.
[0131] Whether a test polypeptide complex containing a GPC3
antigen-binding domain recognizes a conformational epitope can be
assessed as follows. GPC3-expressing cells are prepared for the
above purpose. A test polypeptide complex containing a GPC3
antigen-binding domain can be determined to recognize a
conformational epitope when it strongly binds to GPC3-expressing
cells upon contact, but does not substantially bind to an
immobilized linear peptide comprising an amino acid sequence
forming the extracellular domain of GPC3. Herein, "not
substantially bind" means that the binding activity is 80% or less,
generally 50% or less, preferably 30% or less, and particularly
preferably 15% or less compared to the binding activity towards
cells expressing human GPC3.
[0132] Methods for assaying the binding activity of a test
polypeptide complex containing a GPC3 antigen-binding domain
towards GPC3-expressing cells include, for example, the methods
described in Antibodies: A Laboratory Manual (Ed Harlow, David
Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically,
the assessment can be performed based on the principle of ELISA or
fluorescence activated cell sorting (FACS) using GPC3-expressing
cells as antigen.
[0133] In the ELISA format, the binding activity of a test
polypeptide complex containing a GPC3 antigen-binding domain
towards GPC3-expressing cells can be assessed quantitatively by
comparing the levels of signal generated by enzymatic reaction.
Specifically, a test polypeptide complex is added to an ELISA plate
onto which GPC3-expressing cells are immobilized. Then, the test
polypeptide complex bound to the cells is detected using an
enzyme-labeled antibody that recognizes the test polypeptide
complex. Alternatively, when FACS is used, a dilution series of a
test polypeptide complex is prepared, and the antibody binding
titer for GPC3-expressing cells can be determined to compare the
binding activity of the test polypeptide complex towards
GPC3-expressing cells.
[0134] The binding of a test polypeptide complex towards an antigen
expressed on the surface of cells suspended in buffer or the like
can be detected using a flow cytometer. Known flow cytometers
include, for example, the following devices:
FACSCanto.TM. II
FACSAria.TM.
FACSArray.TM.
FACSVantage.TM. SE
[0135] FACSCalibur.TM. (all are trade names of BD Biosciences)
EPICS ALTRA HyPerSort
Cytomics FC 500
EPICS XL-MCL ADC EPICS XL ADC
[0136] Cell Lab Quanta/Cell Lab Quanta SC (all are trade names of
Beckman Coulter)
[0137] Preferable methods for assaying the binding activity of a
test polypeptide complex containing a GPC3 antigen-binding domain
towards an antigen include, for example, the following method.
First, GPC3-expressing cells are reacted with a test polypeptide
complex, and then this is stained with an FITC-labeled secondary
antibody that recognizes the polypeptide complex. The test
polypeptide complex is appropriately diluted with a suitable buffer
to prepare the complex at a desired concentration. For example, the
complex can be used at a concentration within the range of 10
.mu.g/ml to 10 ng/ml. Then, the fluorescence intensity and cell
count are determined using FACSCalibur (BD). The fluorescence
intensity obtained by analysis using the CELL QUEST Software (BD),
i.e., the Geometric Mean value, reflects the quantity of antibody
bound to cells. That is, the binding activity of a test polypeptide
complex, which is represented by the quantity of the test
polypeptide complex bound, can be determined by measuring the
Geometric Mean value.
[0138] Whether a test polypeptide complex containing a GPC3
antigen-binding domain shares a common epitope with another
polypeptide complex can be assessed based on the competition
between the two complexes for the same epitope. The competition
between polypeptide complexes can be detected by cross-blocking
assay or the like. For example, the competitive ELISA assay is a
preferred cross-blocking assay.
[0139] Specifically, in cross-blocking assay, the GPC3 protein
immobilized to the wells of a microtiter plate is pre-incubated in
the presence or absence of a candidate competitor polypeptide
complex, and then a test polypeptide complex is added thereto. The
quantity of test polypeptide complex bound to the GPC3 protein in
the wells is indirectly correlated with the binding ability of a
candidate competitor polypeptide complex that competes for the
binding to the same epitope. That is, the greater the affinity of
the competitor polypeptide complex for the same epitope, the lower
the binding activity of the test polypeptide complex towards the
GPC3 protein-coated wells.
[0140] The quantity of the test polypeptide complex bound to the
wells via the GPC3 protein can be readily determined by labeling
the polypeptide complex in advance. For example, a biotin-labeled
polypeptide complex is measured using an avidin/peroxidase
conjugate and appropriate substrate. In particular, cross-blocking
assay that uses enzyme labels such as peroxidase is called
"competitive ELISA assay". The polypeptide complex can also be
labeled with other labeling substances that enable detection or
measurement. Specifically, radiolabels, fluorescent labels, and
such are known.
[0141] When the candidate competitor polypeptide complex can block
the binding by a test polypeptide complex containing a GPC3
antigen-binding domain by at least 20%, preferably at least 20 to
50%, and more preferably at least 50% compared to the binding
activity in a control experiment conducted in the absence of the
competitor polypeptide complex, the test polypeptide complex is
determined to substantially bind to the same epitope bound by the
competitor polypeptide complex, or compete for the binding to the
same epitope.
[0142] When the structure of an epitope bound by a test polypeptide
complex containing a GPC3 antigen-binding domain has already been
identified, whether the test and control polypeptide complexes
share a common epitope can be assessed by comparing the binding
activities of the two polypeptide complexes towards a peptide
prepared by introducing amino acid mutations into the peptide
forming the epitope.
[0143] To measure the above binding activities, for example, the
binding activities of test and control polypeptide complexes
towards a linear peptide into which a mutation is introduced are
compared in the above ELISA format. Besides the ELISA methods, the
binding activity towards the mutant peptide bound to a column can
be determined by flowing test and control polypeptide complexes in
the column, and then quantifying the polypeptide complex eluted in
the elution solution. Methods for adsorbing a mutant peptide to a
column, for example, in the form of a GST fusion peptide, are
known.
[0144] Alternatively, when the identified epitope is a
conformational epitope, whether test and control polypeptide
complexes share a common epitope can be assessed by the following
method. First, GPC3-expressing cells and cells expressing GPC3 with
a mutation introduced into the epitope are prepared. The test and
control polypeptide complexes are added to a cell suspension
prepared by suspending these cells in an appropriate buffer such as
PBS. Then, the cell suspensions are appropriately washed with a
buffer, and an FITC-labeled antibody that recognizes the test and
control polypeptide complexes is added thereto. The fluorescence
intensity and number of cells stained with the labeled antibody are
determined using FACSCalibur (BD). The test and control polypeptide
complexes are appropriately diluted using a suitable buffer, and
used at desired concentrations. For example, they may be used at a
concentration within the range of 10 .mu.g/ml to 10 ng/ml. The
fluorescence intensity determined by analysis using the CELL QUEST
Software (BD), i.e., the Geometric Mean value, reflects the
quantity of labeled antibody bound to cells. That is, the binding
activities of the test and control polypeptide complexes, which are
represented by the quantity of labeled antibody bound, can be
determined by measuring the Geometric Mean value.
[0145] In the above method, whether a polypeptide complex does "not
substantially bind to cells expressing mutant GPC3" can be
assessed, for example, by the following method. First, the test and
control polypeptide complexes bound to cells expressing mutant GPC3
are stained with a labeled antibody. Then, the fluorescence
intensity of the cells is determined. When FACSCalibur is used for
fluorescence detection by flow cytometry, the determined
fluorescence intensity can be analyzed using the CELL QUEST
Software. From the Geometric Mean values in the presence and
absence of the polypeptide complex, the comparison value
(.DELTA.Geo-Mean) can be calculated according to the following
formula to determine the ratio of increase in fluorescence
intensity as a result of the binding by the polypeptide
complex.
.DELTA.Geo-Mean=Geo-Mean (in the presence of the polypeptide
complex)/Geo-Mean (in the absence of the polypeptide complex)
[0146] The Geometric Mean comparison value (.DELTA.Geo-Mean value
for the mutant GPC3 molecule) determined by the above analysis,
which reflects the quantity of a test polypeptide complex bound to
cells expressing mutant GPC3, is compared to the .DELTA.Geo-Mean
comparison value that reflects the quantity of the test polypeptide
complex bound to GPC3-expressing cells. In this case, the
concentrations of the test polypeptide complex used to determine
the .DELTA.Geo-Mean comparison values for GPC3-expressing cells and
cells expressing mutant GPC3 are particularly preferably adjusted
to be equal or substantially equal. A polypeptide complex that has
been confirmed to recognize an epitope in GPC3 is used as a control
polypeptide complex.
[0147] If the .DELTA.Geo-Mean comparison value of a test
polypeptide complex for cells expressing mutant GPC3 is smaller
than the .DELTA.Geo-Mean comparison value of the test polypeptide
complex for GPC3-expressing cells by at least 80%, preferably 50%,
more preferably 30%, and particularly preferably 15%, then the test
polypeptide complex "does not substantially bind to cells
expressing mutant GPC3". The formula for determining the Geo-Mean
(Geometric Mean) value is described in the CELL QUEST Software
User's Guide (BD biosciences). When the comparison shows that the
comparison values are substantially equivalent, the epitope for the
test and control polypeptide complexes can be determined to be the
same.
Variable Fragment (Fv)
[0148] Herein, the term "variable fragment (Fv)" refers to the
minimum unit of an antibody-derived antigen-binding domain that is
composed of a pair of the antibody light chain variable region (VL)
and antibody heavy chain variable region (VH). In 1988, Skerra and
Pluckthun found that homogeneous and active antibodies can be
prepared from the E. coli periplasm fraction by inserting an
antibody gene downstream of a bacterial signal sequence and
inducing expression of the gene in E. coli (Science (1988)
240(4855), 1038-1041). In the Fv prepared from the periplasm
fraction, VH associates with VL in a manner so as to bind to an
antigen.
[0149] Herein, Fv preferably includes, for example, a pair of Fv
which is a polypeptide complex or such comprising:
(1) a bivalent antigen-binding domain which is a bivalent scFv,
wherein one monovalent scFv of the bivalent scFv is linked to one
polypeptide forming an Fc domain by a heavy-chain Fv fragment
forming a CD3-binding domain, and the other monovalent scFv is
linked to the other polypeptide forming an Fc domain by a
light-chain Fv fragment forming a CD3-binding domain; (2) a domain
comprising an Fc domain that has no Fc.gamma. receptor-binding
activity, and which is derived from amino acids forming the Fc
domain of IgG1, IgG2a, IgG3, or IgG4; and (3) at least a monovalent
CD3-binding domain, wherein the light-chain and heavy-chain Fv
fragments associate to form a CD3-binding domain such that it can
bind to the CD3 antigen. scFv, Single-Chain Antibody, and
Sc(Fv)2
[0150] Herein, the terms "scFv", "single-chain antibody", and
"sc(Fv)2" all refer to an antibody fragment of a single polypeptide
chain that contains variable regions derived from the heavy and
light chains, but not the constant region. In general, a
single-chain antibody also contains a polypeptide linker between
the VH and VL domains, which enables formation of a desired
structure that is thought to allow antigen binding. The
single-chain antibody is discussed in detail by Pluckthun in "The
Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and
Moore, eds., Springer-Verlag, New York, 269-315 (1994)". See also
International Patent Publication WO 1988/001649; U.S. Pat. Nos.
4,946,778 and 5,260,203. In a particular embodiment, the
single-chain antibody can be bispecific and/or humanized.
[0151] scFv is an antigen-binding domain in which VH and VL forming
Fv are linked together by a peptide linker (Proc. Natl. Acad. Sci.
U.S.A. (1988) 85(16), 5879-5883). VH and VL can be retained in
close proximity by the peptide linker.
[0152] sc(Fv)2 is a single-chain antibody in which four variable
regions of two VL and two VH are linked by linkers such as peptide
linkers to form a single chain (J Immunol. Methods (1999) 231(1-2),
177-189). The two VH and two VL may be derived from different
monoclonal antibodies. Such sc(Fv)2 preferably includes, for
example, a bispecific sc(Fv)2 that recognizes two epitopes present
in a single antigen as disclosed in the Journal of Immunology
(1994) 152(11), 5368-5374. sc(Fv)2 can be produced by methods known
to those skilled in the art. For example, sc(Fv)2 can be produced
by linking scFv by a linker such as a peptide linker.
[0153] Herein, the form of an antigen-binding domain forming an
sc(Fv)2 include an antibody in which the two VH units and two VL
units are arranged in the order of VH, VL, VH, and VL
([VH]-linker-[VL]-linker-[VH]-linker-[VL]) beginning from the N
terminus of a single-chain polypeptide. The order of the two VH
units and two VL units is not limited to the above form, and they
may be arranged in any order. Example order of the form is listed
below.
[VL]-linker-[VH]-linker-[VH]-linker-[VL]
[VH]-linker-[VL]-linker-[VL]-linker-[VH]
[VH]-linker-[VH]-linker-[VL]-linker-[VL]
[VL]-linker-[VL]-linker-[VH]-linker-[VH]
[VL]-linker-[VH]-linker-[VL]-linker-[VH]
[0154] The molecular form of sc(Fv)2 is also described in detail in
WO 2006/132352. According to these descriptions, those skilled in
the art can appropriately prepare desired sc(Fv)2 to produce the
polypeptide complexes disclosed herein.
[0155] Furthermore, the polypeptide complexes of the present
invention may be conjugated with a carrier polymer such as PEG or
an organic compound such as an anticancer agent. Alternatively, a
sugar chain addition sequence is preferably inserted into the
polypeptide complexes such that the sugar chain produces a desired
effect.
[0156] The linkers to be used for linking the variable regions of
an antibody comprise arbitrary peptide linkers that can be
introduced by genetic engineering, synthetic linkers, and linkers
disclosed in, for example, Protein Engineering, 9(3), 299-305,
1996. However, peptide linkers are preferred in the present
invention. The length of the peptide linkers is not particularly
limited, and can be suitably selected by those skilled in the art
according to the purpose. The length is preferably five amino acids
or more (without particular limitation, the upper limit is
generally 30 amino acids or less, preferably 20 amino acids or
less), and particularly preferably 15 amino acids. When sc(Fv)2
contains three peptide linkers, their length may be all the same or
different.
[0157] For example, such peptide linkers include:
TABLE-US-00001 Ser Gly Ser Gly.cndot.Gly.cndot.Ser
Ser.cndot.Gly.cndot.Gly Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID
NO: 5) Ser.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID NO: 6)
Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID NO: 7)
Ser.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID NO: 8)
Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID NO:
9) Ser.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID
NO: 10)
Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser
(SEQ ID NO: 11)
Ser.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly
(SEQ ID NO: 12) (Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ
ID NO: 7))n (Ser.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID
NO: 8))n
where n is an integer of 1 or larger. The length or sequences of
peptide linkers can be selected accordingly by those skilled in the
art depending on the purpose.
[0158] Synthetic linkers (chemical crosslinking agents) is
routinely used to crosslink peptides, and for example:
N-hydroxy succinimide (NHS), disuccinimidyl suberate (DSS),
bis(sulfosuccinimidyl) suberate (BS.sup.3), dithiobis(succinimidyl
propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP),
ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol
bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl
tartrate (DST), di sulfosuccinimidyl tartrate (sulfo-DST),
bis[2-(succinimidoxycarbonyloxy)ethyl] sulfone (BSOCOES), and
bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl] sulfone
(sulfo-BSOCOES). These crosslinking agents are commercially
available.
[0159] In general, three linkers are required to link four antibody
variable regions together. The linkers to be used may be of the
same type or different types.
Fab, F(ab')2, and Fab'
[0160] "Fab" consists of a single light chain, and a CH1 domain and
variable region from a single heavy chain. The heavy chain of Fab
molecule cannot form disulfide bonds with another heavy chain
molecule.
[0161] "F(ab')2" or "Fab" is produced by treating an immunoglobulin
(monoclonal antibody) with a protease such as pepsin and papain,
and refers to an antibody fragment generated by digesting an
immunoglobulin (monoclonal antibody) at near the disulfide bonds
present between the hinge regions in each of the two H chains. For
example, papain cleaves IgG upstream of the disulfide bonds present
between the hinge regions in each of the two H chains to generate
two homologous antibody fragments, in which an L chain comprising
VL (L-chain variable region) and CL (L-chain constant region) is
linked to an H-chain fragment comprising VH (H-chain variable
region) and CH.gamma.1 (.gamma.1 region in an H-chain constant
region) via a disulfide bond at their C-terminal regions. Each of
these two homologous antibody fragments is called Fab'.
[0162] "F(ab')2" consists of two light chains and two heavy chains
comprising the constant region of a CH1 domain and a portion of CH2
domains so that disulfide bonds are formed between the two heavy
chains. The F(ab')2 forming a polypeptide complex disclosed herein
can be preferably produced as follows. A whole monoclonal antibody
or such comprising a desired antigen-binding domain is partially
digested with a protease such as pepsin; and Fc fragments are
removed by adsorption onto a Protein A column. The protease is not
particularly limited, as long as it can cleave the whole antibody
in a selective manner to produce F(ab')2 under an appropriate setup
enzyme reaction condition such as pH. Such proteases include, for
example, pepsin and ficin.
Fc Domain
[0163] An Fc domain that forms a polypeptide complex disclosed
herein can be preferably produced in the following manner. An
antibody such as a monoclonal antibody is partially digested with a
protease such as pepsin. Then, the resulting fragment is adsorbed
onto a Protein A or Protein G column, and eluted with an
appropriate elution buffer. The protease is not particularly
limited, as long as it can cleave antibodies such as monoclonal
antibodies under an appropriate setup enzyme reaction condition
such as pH. Such proteases include, for example, pepsin and
ficin.
[0164] The polypeptide complexes described herein comprise an Fc
domain with reduced Fc.gamma. receptor-binding activity, which
includes amino acids forming the Fc domain of IgG1, IgG2, IgG3, or
IgG4.
[0165] Antibody isotype is determined according to the structure of
the constant region. Constant regions of the isotypes IgG1, IgG2,
IgG3, and IgG4 are called C.gamma.1, C.gamma.2, C.gamma.3, and
C.gamma.4, respectively. The amino acid sequences of Fc domain
polypeptides forming human C.gamma.1, C.gamma.2, C.gamma.3, and
C.gamma.4 are exemplified in SEQ ID NO: 23, 24, 25, and 26,
respectively. The relationship between amino acid residues forming
each amino acid sequence and Kabat's EU numbering (herein also
referred to as EU INDEX) are shown in FIG. 18.
[0166] The Fc domain refers to the region besides F(ab')2 which
comprises two light chains and two heavy chains comprising a
portion of the constant region that comprises a CH1 domain and a
region between the CH1 and CH2 domains so that disulfide bonds are
formed between the two heavy chains. The Fc domain forming a
polypeptide complex disclosed herein can be preferably produced as
follows. A monoclonal IgG1, IgG2, IgG3, or IgG4 antibody or the
like is partially digested with a protease such as pepsin, followed
by elution of the fraction adsorbed onto a Protein A column. The
protease is not particularly limited, as long as it can cleave the
whole antibody in a selective manner to produce F(ab')2 in an
appropriate setup enzyme reaction condition such as pH. Such
proteases include, for example, pepsin and ficin.
Fc.gamma. Receptor
[0167] Fc.gamma. receptor refers to a receptor capable of binding
to the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4
antibodies, and includes all members belonging to the family of
proteins substantially encoded by an Fc.gamma. receptor gene. In
human, the family includes Fc.gamma.RI (CD64) including isoforms
Fc.gamma.RIa, Fc.gamma.RIb and Fc.gamma.RIc; Fc.gamma.RII (CD32)
including isoforms Fc.gamma.RIIa (including allotype H131 and
R131), Fc.gamma.RIIb (including Fc.gamma.RIIb-1 and
Fc.gamma.RIIb-2), and Fc.gamma.RIIc; and Fc.gamma.RIII (CD16)
including isoform Fc.gamma.RIIIa (including allotype V158 and F158)
and Fc.gamma.RIIIb (including allotype Fc.gamma.RIIIb-NA1 and
Fc.gamma.RIIIb-NA2); as well as all unidentified human Fc.gamma.Rs,
Fc.gamma.R isoforms, and allotypes thereof. However, Fc.gamma.
receptor is not limited to these examples. Without being limited
thereto, Fc.gamma.R includes those derived from humans, mice, rats,
rabbits, and monkeys. Fc.gamma.R may be derived from any organisms.
Mouse Fc.gamma.R includes, without being limited to, Fc.gamma.RI
(CD64), Fc.gamma.RII (CD32), Fc.gamma.RIII (CD16), and
Fc.gamma.RIII-2 (CD16-2), as well as all unidentified mouse
Fc.gamma.Rs, Fc.gamma.R isoforms, and allotypes thereof. Such
preferred Fc.gamma. receptors include, for example, human
Fc.gamma.I (CD64), Fc.gamma.IIA (CD32), Fc.gamma.IIB (CD32),
Fc.gamma.IIIA (CD16), and/or Fc.gamma.IIIB (CD16). The
polynucleotide sequence and amino acid sequence of Fc.gamma.l are
shown in SEQ ID NOs: 13 (NM_000566.3) and 14 (NP_000557.1),
respectively; the polynucleotide sequence and amino acid sequence
of Fc.gamma.IIA are shown in SEQ ID NOs: 15 (BC020823.1) and 16
(AAH20823.1), respectively; the polynucleotide sequence and amino
acid sequence of Fc.gamma.IIB are shown in SEQ ID NOs: 17
(BC146678.1) and 18 (AAI46679.1), respectively; the polynucleotide
sequence and amino acid sequence of Fc.gamma.IIIA are shown in SEQ
ID NOs: 19 (BC033678.1) and 20 (AAH33678.1), respectively; and the
polynucleotide sequence and amino acid sequence of Fc.gamma.IIIB
are shown in SEQ ID NOs: 21 (BC128562.1) and 22 (AAI28563.1),
respectively (RefSeq accession number is shown in each
parentheses). Whether an Fc.gamma. receptor has binding activity to
the Fc domain of a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody
can be assessed by ALPHA screen (Amplified Luminescent Proximity
Homogeneous Assay), surface plasmon resonance (SPR)-based BIACORE
method, and others (Proc. Natl. Acad. Sci. USA (2006) 103(11),
4005-4010), in addition to the above-described FACS and ELISA
formats.
[0168] Meanwhile, "Fc ligand" or "effector ligand" refers to a
molecule and preferably a polypeptide that binds to an antibody Fc
domain, forming an Fc/Fc ligand complex. The molecule may be
derived from any organisms. The binding of an Fc ligand to Fc
preferably induces one or more effector functions. Such Fc ligands
include, but are not limited to, Fc receptors, Fc.gamma.R,
Fc.alpha.R, Fc.epsilon.t, FcRn, C1q, and C3, mannan-binding lectin,
mannose receptor, Staphylococcus Protein A, Staphylococcus Protein
G, and viral Fc.gamma.Rs. The Fc ligands also include Fc receptor
homologs (FcRH) (Davis et al., (2002) Immunological Reviews 190,
123-136), which are a family of Fc receptors homologous to
Fc.gamma.R. The Fc ligands also include unidentified molecules that
bind to Fc.
Fc.gamma. Receptor-Binding Activity
[0169] The impaired binding activity of Fc domain to any of the
Fc.gamma. receptors Fc.gamma.l, Fc.gamma.IIA, Fc.gamma.IIB,
Fc.gamma.IIA, and/or Fc.gamma.IIIB can be assessed by using the
above-described FACS and ELISA formats as well as ALPHA screen
(Amplified Luminescent Proximity Homogeneous Assay) and surface
plasmon resonance (SPR)-based BIACORE method (Proc. Natl. Acad.
Sci. USA (2006) 103(11), 4005-4010).
[0170] ALPHA screen is performed by the ALPHA technology based on
the principle described below using two types of beads: donor and
acceptor beads. A luminescent signal is detected only when
molecules linked to the donor beads interact biologically with
molecules linked to the acceptor beads and when the two beads are
located in close proximity. Excited by laser beam, the
photosensitizer in a donor bead converts oxygen around the bead
into excited singlet oxygen. When the singlet oxygen diffuses
around the donor beads and reaches the acceptor beads located in
close proximity, a chemiluminescent reaction within the acceptor
beads is induced. This reaction ultimately results in light
emission. If molecules linked to the donor beads do not interact
with molecules linked to the acceptor beads, the singlet oxygen
produced by donor beads do not reach the acceptor beads and
chemiluminescent reaction does not occur.
[0171] For example, a biotin-labeled polypeptide complex is
immobilized to the donor beads and glutathione S-transferase
(GST)-tagged Fc.gamma. receptor is immobilized to the acceptor
beads. In the absence of a polypeptide complex comprising a
competitive mutant Fc domain, Fc.gamma. receptor interacts with a
polypeptide complex comprising a wild-type Fc domain, inducing a
signal of 520 to 620 nm as a result. The polypeptide complex having
a non-tagged mutant Fc domain competes with the polypeptide complex
comprising a wild-type Fc domain for the interaction with Fc.gamma.
receptor. The relative binding affinity can be determined by
quantifying the reduction of fluorescence as a result of
competition. Methods for biotinylating the polypeptide complexes
such as antibodies using Sulfo-NHS-biotin or the like are known.
Appropriate methods for adding the GST tag to an Fc.gamma. receptor
include methods that involve fusing polypeptides encoding Fc.gamma.
and GST in-frame, expressing the fused gene using cells introduced
with a vector carrying the gene, and then purifying using a
glutathione column. The induced signal can be preferably analyzed,
for example, by fitting to a one-site competition model based on
nonlinear regression analysis using software such as GRAPHPAD PRISM
(GraphPad; San Diego).
[0172] One of the substances for observing their interaction is
immobilized as a ligand onto the gold thin layer of a sensor chip.
When light is shed on the rear surface of the sensor chip so that
total reflection occurs at the interface between the gold thin
layer and glass, the intensity of reflected light is partially
reduced at a certain site (SPR signal). The other substance for
observing their interaction is injected as an analyte onto the
surface of the sensor chip. The mass of immobilized ligand molecule
increases when the analyte binds to the ligand. This alters the
refraction index of solvent on the surface of the sensor chip. The
change in refraction index causes a positional shift of SPR signal
(conversely, the dissociation shifts the signal back to the
original position). In the Biacore system, the amount of shift
described above (i.e., the change of mass on the sensor chip
surface) is plotted on the vertical axis, and thus the change of
mass over time is shown as measured data (sensorgram). Kinetic
parameters (association rate constant (ka) and dissociation rate
constant (kd)) are determined from the curve of sensorgram, and
affinity (KD) is determined from the ratio between these two
constants. Inhibition assay is preferably used in the BIACORE
methods. Examples of such inhibition assay are described in Proc.
Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.
[0173] Herein, "Fc.gamma. receptor-binding activity is reduced"
means, for example, that based on the above-described analysis
method the competitive activity of a test polypeptide complex is
50% or less, preferably 45% or less, 40% or less, 35% or less, 30%
or less, 20% or less, or 15% or less, and particularly preferably
10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or
less, 4% or less, 3% or less, 2% or less, or 1% or less than the
competitive activity of a control polypeptide complex.
[0174] Polypeptide complexes comprising the Fc domain of a
monoclonal IgG1, IgG2, IgG3, or IgG4 antibody can be appropriately
used as control polypeptide complexes. The Fc domain structures are
shown in SEQ ID NOs: 23 (A is added to the N terminus of RefSeq
accession number AAC82527.1), 24 (A is added to the N terminus of
RefSeq accession number AAB59393.1), 25 (A is added to the N
terminus of RefSeq accession number CAA27268.1), and 26 (A is added
to the N terminus of RefSeq accession number AAB59394.1).
Furthermore, when a polypeptide complex comprising an Fc domain
mutant of an antibody of a particular isotype is used as a test
substance, the effect of the mutation of the mutant on the
Fc.gamma. receptor-binding activity is assessed using as a control
a polypeptide complex comprising an Fc domain of the same isotype.
As described above, polypeptide complexes comprising an Fc domain
mutant whose Fc.gamma. receptor-binding activity has been judged to
be reduced are appropriately prepared.
[0175] Such known mutants include, for example, mutants having a
deletion of amino acids 231A-238S (EU numbering) (WO 2009/011941),
as well as mutants C226S, C229S, P238S, (C220S) (J. Rheumatol
(2007) 34, 11); C226S and C229S (Hum. Antibod. Hybridomas (1990)
1(1), 47-54); C226S, C229S, E233P, L234V, and L235A (Blood (2007)
109, 1185-1192).
[0176] Specifically, the preferred polypeptide complexes include
those comprising an Fc domain with a substitution of the amino acid
at position 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238,
239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299,
300, 325, 327, 328, 329, 330, 331, or 332 (EU numbering) in the
amino acids forming the Fc domain of an antibody of a particular
isotype. The isotype of antibody from which the Fc domain
originates is not particularly limited, and it is possible to use
an appropriate Fc domain derived from a monoclonal IgG1, IgG2,
IgG3, or IgG4 antibody. It is preferable to use Fc domains derived
from IgG1 antibodies.
[0177] The preferred polypeptide complexes include, for example,
those comprising an Fc domain which has any one of the
substitutions shown below, whose positions are specified according
to EU numbering (each number represents the position of an amino
acid residue in the EU numbering; and the one-letter amino acid
symbol before the number represents the amino acid residue before
substitution, while the one-letter amino acid symbol after the
number represents the amino acid residue before the substitution)
in the amino acids forming the Fc domain of IgG1 antibody:
[0178] (a) L234F, L235E, P331S;
[0179] (b) C226S, C229S, P238S;
[0180] (c) C226S, C229S; or
[0181] (d) C226S, C229S, E233P, L234V, L235A;
as well as those having an Fc domain which has a deletion of the
amino acid sequence at positions 231 to 238.
[0182] Furthermore, the preferred polypeptide complexes also
include those comprising an Fc domain that has any one of the
substitutions shown below, whose positions are specified according
to EU numbering in the amino acids forming the Fc domain of an IgG2
antibody:
[0183] (e) H268Q, V309L, A330S, and P331S;
[0184] (f) V234A;
[0185] (g) G237A;
[0186] (h) V234A and G237A;
[0187] (i) A235E and G237A; or
[0188] (j) V234A, A235E, and G237A. Each number represents the
position of an amino acid residue in EU numbering; and the
one-letter amino acid symbol before the number represents the amino
acid residue before substitution, while the one-letter amino acid
symbol after the number represents the amino acid residue before
the substitution.
[0189] Furthermore, the preferred polypeptide complexes also
include those comprising an Fc domain that has any one of the
substitutions shown below, whose positions are specified according
to EU numbering in the amino acids forming the Fc domain of an IgG3
antibody:
[0190] (k) F241A;
[0191] (l) D265A; or
[0192] (m) V264A. Each number represents the position of an amino
acid residue in EU numbering; and the one-letter amino acid symbol
before the number represents the amino acid residue before
substitution, while the one-letter amino acid symbol after the
number represents the amino acid residue before the
substitution.
[0193] Furthermore, the preferred polypeptide complexes also
include those comprising an Fc domain that has any one of the
substitutions shown below, whose positions are specified according
to EU numbering in the amino acids forming the Fc domain of an IgG4
antibody:
[0194] (n) L235A, G237A, and E318A;
[0195] (o) L235E; or
[0196] (p) F234A and L235A. Each number represents the position of
an amino acid residue in EU numbering; and the one-letter amino
acid symbol before the number represents the amino acid residue
before substitution, while the one-letter amino acid symbol after
the number represents the amino acid residue before the
substitution.
[0197] The other preferred polypeptide complexes include, for
example, those comprising an Fc domain in which any amino acid at
position 233, 234, 235, 236, 237, 327, 330, or 331 (EU numbering)
in the amino acids forming the Fc domain of an IgG1 antibody is
substituted with an amino acid of the corresponding position in EU
numbering in the corresponding IgG2 or IgG4.
[0198] The preferred polypeptide complexes also include, for
example, those comprising an Fc domain in which any one or more of
the amino acids at positions 234, 235, and 297 (EU numbering) in
the amino acids forming the Fc domain of an IgG1 antibody is
substituted with other amino acids. The type of amino acid after
substitution is not particularly limited; however, the polypeptide
complexes comprising an Fc domain in which any one or more of the
amino acids at positions 234, 235, and 297 are substituted with
alanine are particularly preferred.
[0199] The preferred polypeptide complexes also include, for
example, those comprising an Fc domain in which an amino acid at
position 265 (EU numbering) in the amino acids forming the Fc
domain of an IgG1 antibody is substituted with another amino acid.
The type of amino acid after substitution is not particularly
limited; however, polypeptide complexes comprising an Fc domain in
which an amino acid at position 265 is substituted with alanine are
particularly preferred.
Fc Domain Derived from Bispecific Antibody
[0200] Herein, appropriate Fc domains derived from bispecific
antibody are also used as an Fc domain with reduced Fc.gamma.
receptor-binding activity. A bispecific antibody is an antibody
having two different specificities. The IgG-type bispecific
antibody can be secreted from hybrid hybridoma (quadroma) produced
by fusing two types of IgG antibody-producing hybridomas (Milstein
C et al., Nature (1983) 305, 537-540).
[0201] Alternatively, the IgG-type bispecific antibody can also be
secreted by introducing a total of four genes, genes of the L
chains and H chains forming two types of IgGs of interest, into
cells and co-expressing them. However, in theory, there are ten
combinations of IgG H chains and L chains produced by such methods.
It is difficult to purify IgG consisting of a desired combination
of H chain and L chain from ten types of IgGs. In addition, in
theory the amount of secreted IgG with a desired combination is
also significantly reduced, which requires large scale culture.
This further increases the production cost.
[0202] In this case, an appropriate amino acid substitution can be
introduced into the CH3 domain forming an H-chain Fc domain in
order to preferentially secrete IgG with a heterologous combination
of H chains. Specifically, this method is conducted by substituting
an amino acid having a larger side chain (knob (which means
"bulge")) for an amino acid in the CH3 domain of one of the H
chains, and substituting an amino acid having a smaller side chain
(hole (which means "void")) for an amino acid in the CH3 domain of
the other H chain so that the knob is placed in the hole. This
promotes heteromeric H chain formation and simultaneously inhibits
homomeric H chain formation (WO 1996/027011; Ridgway J B et al.,
Protein Engineering (1996) 9, 617-621; Merchant A M et al., Nature
Biotechnology (1998) 16, 677-681).
[0203] On the other hand, with respect to the L chain, the L-chain
variable region is less polymorphic than the H-chain variable
region, and thus obtaining a common L chain that can confer binding
ability to both H chains is expected. Efficient expression of a
bispecific IgG can be achieved by introducing the genes of such a
common L chain and two H chains into cells to express the IgG
(Nature Biotechnology (1998) 16, 677-681). However, it is difficult
to realize this idea because the probability that two types of
antibodies containing the same L chain are randomly selected is
low. Thus, a method for selecting a common L chain which shows
strong binding ability to any different H chains is proposed (WO
2004/065611).
[0204] Furthermore, there are also known techniques for producing a
bispecific antibody by applying methods for controlling polypeptide
association, or association of polypeptide-formed heteromeric
multimers to the association between the two polypeptides that form
an Fc domain. Specifically, methods for controlling polypeptide
association may be employed to produce a bispecific antibody (WO
2006/106905), in which amino acid residues forming the interface
between two polypeptides that form the Fc domain are altered to
inhibit the association between Fc domains having the same sequence
and to allow the formation of polypeptide complexes formed by two
Fc domains of different sequences.
[0205] The above-described two polypeptides forming an Fc domain
derived from a bispecific antibody can be appropriately used as a
domain encompassing an Fc domain of the present invention. More
specifically, such preferred two polypeptides forming an Fc domain
include those in which amino acids at positions 349 and 366 (EU
numbering) in the amino acid sequence of one polypeptide are
cysteine and tryptophan, respectively, and amino acids at positions
356, 366, 368, and 407 (EU numbering) in the amino acid sequence of
the other polypeptide are cysteine, serine, alanine, and valine,
respectively.
[0206] In another embodiment, preferred domains encompassing an Fc
domain of the present invention include two polypeptides forming an
Fc domain, in which amino acid at position 409 (EU numbering) in
the amino acid sequence of one polypeptide is aspartic acid, and
amino acid at position 399 (EU numbering) in the amino acid
sequence of the other polypeptide is lysine. In the above
embodiment, amino acid at position 409 may be glutamic acid instead
of aspartic acid, while the amino acid at position 399 may be
arginine instead of lysine. Furthermore, aspartic acid at position
360 or 392 can also be preferably combined with lysine at position
399.
[0207] In another embodiment, preferred domains encompassing an Fc
domain of the present invention include two polypeptides forming an
Fc domain, in which the amino acid at position 370 (EU numbering)
in the amino acid sequence of one polypeptide is glutamic acid, and
the amino acid at position 357 (EU numbering) in the amino acid
sequence of the other polypeptide is lysine.
[0208] In still another embodiment, preferred domains encompassing
an Fc domain of the present invention include two polypeptides
forming an Fc domain, in which amino acid at position 439 (EU
numbering) in the amino acid sequence of one polypeptide is
glutamic acid, and amino acid at position 356 (EU numbering) in the
amino acid sequence of the other polypeptide is lysine.
[0209] In addition, preferred domains encompassing an Fc domain of
the present invention include embodiments with a combination
thereof:
two polypeptides forming an Fc domain, in which amino acids at
positions 409 and 370 (EU numbering) in the amino acid sequence of
one polypeptide are aspartic acid and glutamic acid, respectively,
and amino acids at positions 399 and 357 (EU numbering) in the
amino acid sequence of the other polypeptide are both lysine (in
this embodiment, glutamic acid at position 370 may be replaced with
aspartic acid, and glutamic acid at position 370 may be replaced
with aspartic acid at position 392); two polypeptides forming an Fc
domain, in which amino acids at positions 409 and 439 (EU
numbering) in the amino acid sequence of one polypeptide are
aspartic acid and glutamic acid, respectively, and amino acids at
positions 399 and 356 (EU numbering) in the amino acid sequence of
the other polypeptide are both lysine (in this embodiment, glutamic
acid position at 439 may be replaced with aspartic acid at position
360, 392, or 439); two polypeptides forming an Fc domain, in which
amino acids at positions 370 and 439 (EU numbering) in the amino
acid sequence of one polypeptide are both glutamic acid, and amino
acids at positions 357 and 356 (EU numbering) in the amino acid
sequence of the other polypeptide are both lysine; and two
polypeptides forming an Fc domain, in which amino acids at
positions 409, 370, and 439 (EU numbering) in the amino acid
sequence of one polypeptide are aspartic acid, glutamic acid, and
glutamic acid, respectively, and amino acids at positions 399, 357,
and 356 (EU numbering) in the amino acid sequence of the other
polypeptide are all lysine (in this embodiment, the amino acid at
position 370 may not be substituted with glutamic acid;
alternatively, instead of substituting amino acid at position 370
with glutamic acid, glutamic acid at position 439 may be replaced
with aspartic acid, or glutamic acid at position 439 may be
replaced with aspartic acid at position 392).
[0210] In yet another embodiment, preferred domains encompassing an
Fc domain of the present invention include two polypeptides forming
an Fc domain, in which amino acid at position 356 (EU numbering) in
the amino acid sequence of one polypeptide is lysine, and amino
acids at positions 435 and 439 (EU numbering) in the amino acid
sequence of the other polypeptide are arginine and glutamic acid,
respectively.
[0211] When the above-described two polypeptides forming an Fc
domain derived from a bispecific antibody are used as a domain
encompassing an Fc domain of the present invention, antigen-binding
domains and/or CD3-binding domains of the present invention can be
arranged in a desired combination.
Fc Domain with Reduced C-Terminal Heterogeneity
[0212] Herein, Fc domains with improved Fc domain C-terminal
heterogeneity in addition to the above-described characteristic are
appropriately used as Fc domains with reduced Fc.gamma.
receptor-binding activity. More specifically, the present invention
provides Fc domains in which glycine and lysine at positions 446
and 447 (EU numbering), respectively, in the amino acid sequences
of two polypeptides forming an Fc domain derived from IgG1, IgG2,
IgG3, or IgG4 are deleted. T cell receptor complex-binding
domain
[0213] Herein, "T cell receptor complex-binding domain" refers to a
portion of a T cell receptor complex antibody, which comprises a
region that specifically binds and is complementary to the whole or
a portion of a T cell receptor complex. Such T cell receptor
complex may be T cell receptor itself, or an adaptor molecule that
together with the T cell receptor forms the T cell receptor
complex. A preferred adaptor is CD3.
T Cell Receptor-Binding Domain
[0214] Herein, "T cell receptor-binding domain" refers to a portion
of a T cell receptor antibody, which comprises a region that
specifically binds and is complementary to the whole or a portion
of a T cell receptor.
[0215] It is possible to use the variable region or constant region
of a T cell receptor. However, preferred epitopes to which a
CD3-binding domain binds are those located in the constant region.
Sequences of the constant region include, for example, those of the
T cell receptor a chain (RefSeq accession number CAA26636.1; SEQ ID
NO: 67), T cell receptor .beta. chain (RefSeq accession number
C25777; SEQ ID NO: 68), T cell receptor yl chain (RefSeq accession
number A26659; SEQ ID NO: 69), T cell receptor .gamma.2 chain
(RefSeq accession number AAB63312.1; SEQ ID NO: 70), and T cell
receptor .delta. chain (RefSeq accession number AAA61033.1; SEQ ID
NO: 71).
CD3-Binding Domain
[0216] Herein, "CD3-binding domain" refers to a portion of a CD3
antibody, which comprises a region that specifically binds and is
complementary to the whole or a portion of CD3. The CD3-binding
domain can be derived from one or more antibody variable domains.
Preferably, the CD3-binding domain includes both CD3 antibody light
chain variable region (VL) and CD3 antibody heavy chain variable
region (VH). Such preferred CD3-binding domains include, for
example, "single-chain Fv (scFv)", "single chain antibody", "Fv",
"single-chain Fv2 (scFv2)", "Fab", and "F(ab')2".
[0217] The CD3-binding domain of the present invention may bind to
any epitope, as long as the epitope is located within the .gamma.
chain, .delta. chain, or .epsilon. chain sequence forming human
CD3. In the present invention, preferred CD3-binding domains
include those comprising a CD3 antibody light chain variable region
(VL) and a CD3 antibody heavy chain variable region (VH), which
bind to an epitope in the extracellular domain of the 6 chain of a
human CD3 complex. Such preferred CD3-binding domains include those
comprising a CD3 antibody light chain variable region (VL) and a
CD3 antibody heavy chain variable region (VH) of antibody OKT3
(Proc. Natl. Acad. Sci. USA (1980) 77, 4914-4917) or various known
CD3 antibodies. Furthermore, such appropriate CD3-binding domains
include those derived from a CD3 antibody with desired
characteristics, which are obtained by immunizing a desired animal
with the .gamma. chain, .delta. chain, or .epsilon. chain forming
human CD3 by the above-described methods. Appropriate anti-CD3
antibodies from which a CD3-binding domain is derived include human
antibodies and antibodies appropriately humanized as described
above. The structures of .gamma. chain, .delta. chain, and
.epsilon. chain forming CD3 are shown as polynucleotide sequences
in SEQ ID NOs: 27 (NM_000073.2), 29 (NM_000732.4), and 31
(NM_000733.3), respectively, and as polypeptide sequences in SEQ ID
NO: 28 (NP_000064.1), 30 (NP_000723.1), and 32 (NP_000724.1),
respectively (RefSeq accession number is shown in parentheses).
Polypeptide Complex
[0218] The structure of a polypeptide complex of the present
invention is not limited, as long as it contains
[0219] (1) an antigen-binding domain;
[0220] (2) a domain comprising an Fc domain with reduced Fc.gamma.
receptor-binding activity; and
[0221] (3) a T cell receptor complex-binding domain, as described
above.
In the present invention, the preferred T cell receptor
complex-binding domain is a T cell receptor-binding domain or
CD3-binding domain. Each of the domains described above may be
linked directly via peptide linkage. For example, (1) F(ab')2 is
used as the antigen-binding domain, and (2) an Fc domain with
reduced Fc.gamma. receptor-binding activity is used as the domain
comprising an Fc domain with reduced Fc.gamma. receptor-binding
activity. In this case, when the antigen-binding domain of (1) is
linked via a peptide bond to the domain comprising an Fc domain of
(2), the linked polypeptide forms an antibody structure. Such an
antibody may be prepared by purifying the culture media of the
above-described hybridoma or purifying culture media of desired
host cells stably carrying the polynucleotide that encodes the
antibody-forming polypeptide.
[0222] When the CD3-binding domain of (3) is linked to the antibody
structure, the CD3-binding domain may be linked via peptide bond to
the C terminus of the constant region of the antibody structure. In
another embodiment, the CD3-binding domain is linked via peptide
bond to the N terminus of the heavy chain variable region or light
chain variable region of the antibody structure. In the other
embodiment, the CD3-binding domain may be linked via peptide bond
to the C terminus of the light chain constant region of the
antibody structure. The CD3-binding domain to be linked may have
any desired structure; however, such an appropriate CD3-binding
domain includes preferably Fv, and more preferably scFv. The
valency of the CD3-binding domain that binds to the antibody
structure is not limited. To link a divalent CD3-binding domain to
the antibody structure, a monovalent CD3-binding domain may be
linked via peptide bond to the respective C termini of two Fc
domains forming the constant region of the antibody structure.
Alternatively, to link a divalent CD3-binding domain to the
antibody structure, a divalent scFv (i.e., sc(Fv)2) may be linked
via peptide bond to the C terminus of one of the two Fc domains. In
this case, the polypeptide complex in which a divalent scFv (i.e.,
sc(Fv)2) is linked to the C terminus of only one of the two Fc
domains forming the constant region of the antibody structure is
efficiently produced by using an above-described Fc domain derived
from a bispecific antibody. Alternatively, to link a monovalent
CD3-binding domain to the antibody structure, a monovalent scFv may
be linked via peptide bond to the C terminus of one of the two Fc
domains. In this case, a polypeptide complex of the present
invention in which a monovalent scFv is linked to the C terminus of
only one of the two Fc domains forming the constant region of the
antibody structure is efficiently produced by using an
above-described Fc domain derived from a bispecific antibody.
[0223] Furthermore, when the CD3-binding domain of (3) is linked
via peptide bond to the C terminus of the constant region of the
antibody structure, the appropriate polypeptide complexes include
those in which the heavy chain Fv fragment forming the CD3-binding
domain is linked to the C terminus of one constant region (CH3
domain) forming the Fc domain, and the light chain Fv fragment
forming the CD3-binding domain is linked to the C terminus of the
other constant region (CH3 domain) forming the Fc domain. In this
case, an appropriate linker such as Gly-Gly-Gly-Gly-Ser (SEQ ID NO:
7) is inserted to link the heavy chain or light chain Fv fragment
to the C terminus of the constant region (CH3 domain). The number
of repeats in the linker is not limited; however, it is selected
from 1 to 10, preferably 2 to 8, or more preferably 2 to 6.
Specifically, it is possible to insert an appropriate linker in
which the number of [Gly-Gly-Gly-Gly-Ser] (SEQ ID NO: 7) repeats is
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0224] Alternatively, when a polypeptide complex is produced in
which the heavy chain Fv fragment forming the CD3-binding domain is
linked to the C terminus of one constant region (CH3 domain)
forming the Fc domain and the light chain Fv fragment of the
CD3-binding domain is linked to the C terminus of the other
constant region (CH3 domain) forming the Fc domain, appropriate
alterations of amino acid residues that allow formation of
disulfide bonds between the heavy chain Fv fragment and light chain
Fv fragment can be used to enhance the association between the
heavy chain Fv fragment and light chain Fv fragment.
[0225] In another embodiment, when a polypeptide complex of the
present invention in which the heavy chain Fv fragment forming the
CD3-binding domain is linked to the C terminus of one constant
region (CH3 domain) forming the Fc domain and the light chain Fv
fragment forming the CD3-binding domain is linked to the C terminus
of the other constant region (CH3 domain) forming the Fc domain is
produced, antibody CH1 and CL domains can be linked to each of the
heavy chain Fv fragment and light chain Fv fragment to enhance the
association between the heavy chain Fv fragment and light chain Fv
fragment.
[0226] In still another embodiment, in order to link a divalent
CD3-binding domain to the antibody structure, a monovalent
CD3-binding domain may be linked via peptide bond to the respective
C termini of two light chain constant regions or the respective N
termini of the light chain variable regions of the antibody
structure. Alternatively, in order to link a divalent CD3-binding
domain to the antibody structure, a divalent scFv (i.e., sc(Fv)2)
may be linked via peptide bond to the respective C termini of two
light chain constant regions or the respective N termini of light
chain variable regions. In this case, polypeptide complexes in
which a divalent scFv (i.e., sc(Fv)2) is linked to the C or N
terminus of one of the two light chain variable regions of the
antibody structure can be efficiently produced by using an
above-described Fc domain derived from a bispecific antibody.
Alternatively, in order to link a monovalent CD3-binding domain to
the antibody structure, a monovalent scFv may be linked via peptide
bond to the C or N terminus of one of the two light chain variable
regions. In this case, polypeptide complexes of the present
invention in which a monovalent scFv is linked to the N or C
terminus of one light chain variable region of the two light chain
variable regions of the antibody structure can be efficiently
produced by using an above-described Fc domain derived from
bispecific antibody.
[0227] In another embodiment, in order to link a divalent
CD3-binding domain to the antibody structure, a monovalent
CD3-binding domain may be linked via peptide bond to the respective
N termini of two heavy chain variable regions of the antibody
structure. Alternatively, in order to link a divalent CD3-binding
domain to the antibody structure, a divalent scFv (i.e., sc(Fv)2)
may be linked via peptide bond to the N terminus of one of the two
heavy chain variable regions. In this case, polypeptide complexes
in which a divalent scFv (i.e., sc(Fv)2) is linked to the N
terminus of only one of two heavy chain variable regions of the
antibody structure can be efficiently produced by using an
above-described Fc domain derived from bispecific antibody.
Alternatively, in order to link a monovalent CD3-binding domain to
the antibody structure, a monovalent scFv may be linked via peptide
bond to the N terminus of one of the two heavy chain variable
regions. In this case, polypeptide complexes of the present
invention in which a monovalent scFv is linked to the N terminus of
one of the two heavy chain variable regions of the antibody
structure can be efficiently produced by using an above-described
Fc domain derived from a bispecific antibody.
[0228] Furthermore, an above-described polypeptide complex can be
produced by linking each domain directly via peptide bond or by
peptide binding via a peptide linker. In this case, the linker to
be used includes the linker described above as an example and
appropriate linkers with a peptide tag, for example, His-tag,
HA-tag, myc-tag, or FLAG-tag. In addition, it is preferred to use
the property of mutual binding based on hydrogen bonding, disulfide
linkage, covalent bonding, or ionic interaction, or a combination
thereof. For example, it is possible to employ the affinity between
antibody CH1 and CL, or the above-described Fc domains derived from
a bispecific antibody may be used for the hetermomeric association
of Fc domains. Moreover, interdomain disulfide bonds can be
preferably used as described in the Examples.
[0229] In another structure of the polypeptide complex of the
present invention, for example, a monovalent Fv and a monovalent
Fab are preferably used as (1) the antigen binding domain. In this
case, the following structure is used. The heavy chain Fv fragment
(VH) or light chain Fv fragment (VL) of monovalent Fv is linked via
peptide bond to the heavy chain CH1 domain. The heavy chain CH1
domain is linked via peptide bond to one of (2) the two Fc domains
with reduced Fc.gamma. receptor-binding activity which form the
polypeptide complex of the present invention. The other VL or VH
fragment of the monovalent Fv is linked via peptide bond to the
light chain CH domain which is linked via disulfide bond to the
heavy chain CH1 domain. Thus, VH and VL respectively linked to the
termini of heavy chain CH1 domain and light chain CL domain form an
antibody-binding domain. sc(Fv)2 which forms both the (1)
antibody-binding domain and (3) CD3-binding domain may be linked
via peptide bond to the N terminus of the other Fc domain of the
two described above. In this case, a polypeptide complex having a
structure in which the heavy chain CH1 domain is linked via peptide
bond to one of the two Fc domains forming the polypeptide complex,
and sc(Fv)2 is linked via peptide bond to the other Fc domain can
be produced by using an above-described Fc domain derived from a
bispecific antibody. The above-described polypeptide complex can be
produced by linking each domain directly via peptide bond or by
peptide binding via a peptide linker. In this case, the linker to
be used includes the linkers described above as an example and
appropriate linkers with a peptide tag, for example, His-tag,
HA-tag, myc-tag, or FLAG-tag. In another preferred structure of the
polypeptide complex of the present invention, for example, a
divalent scFv is also used as (1) the antigen-binding domain. In an
embodiment of the structure, it is also possible to produce a
polypeptide complex in which one of the divalent scFvs is linked
via peptide bond through VH forming (3) the CD3-binding domain to
one of the two (2) Fc domains with reduced Fc.gamma.
receptor-binding activity, and the other divalent scFv is linked
via peptide bond through VL forming (3) the CD3-binding domain to
one of the two (2) Fc domains with reduced Fc.gamma.
receptor-binding activity. In this case, it is possible to use an
above-described Fc domain derived from a bispecific antibody. The
above-described polypeptide complex can be produced by linking each
domain directly via peptide bond or by peptide binding via a
peptide linker. In this case, the linker to be used includes the
linkers described above as an example and appropriate linkers with
a peptide tag, for example, His-tag, HA-tag, myc-tag, or
FLAG-tag.
[0230] In another embodiment of the structure where a divalent scFv
is used as (1) the antigen-binding domain, it is possible to
produce a polypeptide complex in which one of the divalent scFv is
linked via peptide bond scFv forming (3) the CD3-binding domain to
one of the two (2) Fc domains with reduced Fc.gamma.
receptor-binding activity, and the other divalent scFv is linked
via peptide bond to the other (2) Fc domain with reduced Fc.gamma.
receptor. In this case, a polypeptide complex in which scFv forming
the antigen-binding domain is linked via peptide bond through scFv
forming the CD3-binding domain to one of the two Fc domains forming
the polypeptide complex, and scFv forming the antigen-binding
domain is linked via peptide bond to the other Fc domain can be
produced by using an above-described Fc domain derived from a
bispecific antibody. The above-described polypeptide complex can be
produced by linking each domain directly via peptide bond or by
peptide binding via a peptide linker. In this case, the linker to
be used includes the linkers described above as an example and
appropriate linkers with a peptide tag, for example, His-tag,
HA-tag, myc-tag, or FLAG-tag.
[0231] In another preferred structure of the polypeptide complex of
the present invention, for example, both antigen-binding domain and
T cell receptor complex-binding domain are each a structure of
monovalent Fab. In an embodiment of the structure, it is possible
to produce a polypeptide complex in which a heavy chain Fv fragment
of a monovalent Fab forming the antigen-binding domain is linked
through a CH1 domain to one polypeptide forming an Fc domain and a
light chain Fv fragment of the Fab is linked to a CL domain, while
a heavy chain Fv fragment of Fab forming the T cell
receptor-binding domain is linked through a CH1 domain to the other
polypeptide forming the Fc domain and a light chain Fv fragment of
the Fab is linked to a CL domain.
[0232] In another embodiment of the structure, it is also possible
to produce a polypeptide complex in which a heavy chain Fv fragment
of a monovalent Fab forming the antigen-binding domain is linked
through a CH1 domain to one polypeptide forming an Fc domain and a
light chain Fv fragment of the Fab is linked to a CL domain, while
a light chain Fv fragment of an Fab forming the T cell
receptor-binding domain is linked through a CH1 domain to the other
polypeptide forming the Fc domain and a heavy chain Fv fragment of
the Fab is linked to a CL domain. Alternatively, it is also
possible to produce a polypeptide complex in which a heavy chain Fv
fragment of monovalent Fab forming the T cell receptor-binding
domain is linked through a CH1 domain to one polypeptide forming an
Fc domain and a light chain Fv fragment of the Fab is linked to a
CL domain, while a light chain Fv fragment of an Fab forming the
antigen-binding domain is linked through a CH1 domain to the other
polypeptide forming an Fc domain and a heavy chain Fv fragment of
the Fab is linked to a CL domain.
[0233] In another embodiment of the structure, it is also possible
to produce a polypeptide complex in which a heavy chain Fv fragment
of a monovalent Fab forming the antigen-binding domain is linked
through a CH1 domain to one polypeptide forming an Fc domain and a
light chain Fv fragment of the Fab is linked to a CL domain, while
a heavy chain Fv fragment of Fab forming the T cell
receptor-binding domain is linked through a CL domain to the other
polypeptide forming an Fc domain and a light chain Fv fragment of
the Fab is linked to a CH1 domain. Alternatively, it is also
possible to produce a polypeptide complex in which a heavy chain Fv
fragment of a monovalent Fab forming the T cell receptor-binding
domain is linked through a CH1 domain to one polypeptide forming an
Fc domain and a light chain Fv fragment of the Fab is linked to a
CL domain, while a heavy chain Fv fragment of Fab forming the
antigen-binding domain is linked through a CL domain to the other
polypeptide forming an Fc domain and a light chain Fv fragment of
the Fab is linked to a CH1 domain.
[0234] In an embodiment of another structure of the polypeptide
complex of the present invention in which both antigen-binding
domain and T cell receptor complex-binding domain are each a
structure of monovalent Fab, the preferred polypeptides of the
present invention include those that have:
(1) an antigen-binding domain in which a heavy chain Fv fragment of
a monovalent Fab structure that binds to an antigen is linked
through a CH1 domain to one of the above-described polypeptides
forming an Fc domain and a light chain Fv fragment of the Fab
structure is linked to a CL domain; and (2) a T cell receptor
complex-binding domain in which a heavy chain Fv fragment of a
monovalent Fab structure that binds to an T cell receptor complex
is linked through a CH1 domain to the other polypeptide forming an
Fc domain and a light chain Fv fragment of the Fab structure is
linked to a CL domain; and in which the electric charges of the CH1
and CL domains are controlled so that the heavy chain Fv fragment
of the antigen-binding domain is associated with the light chain Fv
fragment of the antigen-binding domain, or the heavy chain Fv
fragment of the T cell receptor-binding domain is associated with
the light chain Fv fragment of the T cell receptor-binding domain.
In this embodiment, the structure (structure with controlled
association) of the polypeptide complex is not limited to a
particular structure, as long as the electric charges of the CH1
and CL domains are controlled so that the heavy chain Fv fragment
of the antigen-binding domain is associated with the light chain Fv
fragment of the antigen-binding domain, or the heavy chain Fv
fragment of the T cell receptor-binding domain is associated with
the light chain Fv fragment of the T cell receptor-binding
domain.
[0235] In an embodiment of the structure with controlled
association, it is possible to produce a polypeptide complex in
which amino acid residues of the CH1 domain linked to the heavy
chain Fv fragment of the T cell receptor complex-binding domain
have the same electric charges as amino acid residues of the CL
domain linked to the light chain Fv fragment of the antigen-binding
domain.
[0236] In an embodiment of the structure with controlled
association, it is possible to produce a polypeptide complex in
which amino acid residues of the CH1 domain linked to the heavy
chain Fv fragment of the antigen-binding domain have the same
electric charges as amino acid residues of the CL domain linked to
the light chain Fv fragment of the T cell receptor complex-binding
domain.
[0237] In an embodiment of the structure with controlled
association, it is possible to produce a polypeptide complex in
which amino acid residues of the CH1 domain linked to the heavy
chain Fv fragment of the T cell receptor complex-binding domain
have the same electric charges as amino acid residues of the CL
domain linked to the light chain Fv fragment of the antigen-binding
domain, and amino acid residues of the CH1 domain linked to the
heavy chain Fv fragment of antigen-binding domain have the same
electric charges as amino acid residues of the CL domain linked to
the light chain Fv fragment of the T cell receptor complex-binding
domain.
[0238] In another embodiment of the structure with controlled
association, it is possible to produce a polypeptide complex in
which amino acid residues of the CH1 domain linked to the heavy
chain Fv fragment of the T cell receptor complex-binding domain
have the same electric charges as amino acid residues of the CL
domain linked to the light chain Fv fragment of the antigen-binding
domain, and amino acid residues of the CH1 domain linked to the
heavy chain Fv fragment of the T cell receptor complex-binding
domain have electric charges opposite to those of amino acid
residues of the CL domain linked to the light chain Fv fragment of
the T cell receptor-binding domain.
[0239] In still another embodiment of the structure with controlled
association, it is possible to produce a polypeptide complex in
which amino acid residues of the CH1 domain linked to the heavy
chain Fv fragment of the T cell receptor complex-binding domain
have the same electric charges as amino acid residues of the CL
domain linked to the light chain Fv fragment of the antigen-binding
domain; amino acid residues of the CH1 domain linked to the heavy
chain Fv fragment of the antigen-binding domain have the same
electric charges as amino acid residues of the CL domain linked to
the light chain Fv fragment of the T cell receptor complex-binding
domain; and amino acid residues of the CH1 domain linked to the
heavy chain Fv fragment of the T cell receptor complex-binding
domain have electric charges opposite to those of amino acid
residues of the CL domain linked to the light chain Fv fragment of
the T cell receptor-binding domain.
[0240] In yet another embodiment of the structure with controlled
association, it is possible to produce a polypeptide complex in
which amino acid residues of the CH1 domain linked to the heavy
chain Fv fragment of the antigen-binding domain have the same
electric charges as amino acid residues of the CL domain linked to
the light chain Fv fragment of the T cell receptor complex-binding
domain, and amino acid residues of the CH1 domain linked to the
heavy chain Fv fragment of the antigen-binding domain have electric
charges opposite to those of amino acid residues of the CL domain
linked to the light chain Fv fragment of the antigen-binding
domain.
[0241] In an alternative embodiment of the structure with
controlled association, it is possible to produce a polypeptide
complex in which amino acid residues of the CH1 domain linked to
the heavy chain Fv fragment of the T cell receptor complex-binding
domain have the same electric charges as amino acid residues of the
CL domain linked to the light chain Fv fragment of the
antigen-binding domain; amino acid residues of the CH1 domain
linked to the heavy chain Fv fragment of the antigen-binding domain
have the same electric charges as amino acid residues of the CL
domain linked to the light chain Fv fragment of the T cell receptor
complex-binding domain; and amino acid residues of the CH1 domain
linked to the heavy chain Fv fragment of the antigen-binding domain
have electric charges opposite to those of amino acid residues of
the CL domain linked to the light chain Fv fragment of the
antigen-binding domain.
[0242] In still yet another embodiment of the structure with
controlled association, it is possible to produce a polypeptide
complex in which amino acid residues of the CH1 domain linked to
the heavy chain Fv fragment of the T cell receptor complex-binding
domain have the same electric charges as amino acid residues of the
CL domain linked to the light chain Fv fragment of the
antigen-binding domain; amino acid residues of the CH1 domain
linked to heavy chain Fv fragment of the antigen-binding domain
have the same electric charges as amino acid residues of the CL
domain linked to the light chain Fv fragment of the T cell receptor
complex-binding domain; amino acid residues of the CH1 domain
linked to the heavy chain Fv fragment of the T cell receptor
complex-binding domain have electric charges opposite to those of
amino acid residues of the CL domain linked to the light chain Fv
fragment of the T cell receptor-binding domain; and amino acid
residues of the CH1 domain linked to the heavy chain Fv fragment of
the antigen-binding domain have electric charges opposite to those
of amino acid residues of the CL domain linked to the light chain
Fv fragment of the antigen-binding domain.
Control of Electric Charges of CH1 and CL Domains
[0243] To obtain a bispecific polypeptide complex that recognizes
an epitope of the T cell receptor-binding domain by the heavy and
light chains of the T cell receptor-binding domain, and an epitope
of an antigen by the heavy and light chains of the antigen-binding
domain, theoretically ten types of polypeptide complex molecules
are produced if each of the four chains is expressed when producing
the polypeptide complex.
[0244] Nevertheless, the desired polypeptide complex molecule can
be preferentially obtained, for example, by controlling the domains
so as to inhibit the association between the heavy chain of the T
cell receptor-binding domain and the light chain of the
antigen-binding domain, and/or the association between the heavy
chain of the antigen-binding domain and the light chain of the T
cell receptor-binding domain.
[0245] Examples include alterations of amino acid residues that
form an interface between the heavy chain CH1 of the T cell
receptor-binding domain and the light chain CL of the
antigen-binding domain to positively charged amino acid residues,
and alterations of amino acid residues that form an interface
between the heavy chain CH1 of the antigen-binding domain and the
light chain CL of the T cell receptor-binding domain to negatively
charged amino acid residues. As a result of such alterations,
undesired association between the heavy chain CH1 of the T cell
receptor-binding domain and the light chain CL of the
antigen-binding domain is inhibited because the electric charges of
the amino acid residues that form the interface are both positive,
and undesired association between the heavy chain CH1 of the
antigen-binding domain and the light chain CL of the T cell
receptor-binding domain is also inhibited because the electric
charges of the amino acid residues that form the interface are both
negative. A desired polypeptide complex of the present invention
can be efficiently produced as a result of the desired association
between the heavy chain CH1 of T cell receptor-binding domain and
the light chain CL of the T cell receptor-binding domain as well as
the desired association between the heavy chain CH1 of the
antigen-binding domain and the light chain CL of the
antigen-binding domain. Furthermore, the desired association
between the heavy light chains of the T cell receptor-binding
domains is preferably promoted because the amino acid residues that
form the interface have opposite electric charges from each other.
The desired association between the heavy and light chains of the
antigen-binding domains is also preferably promoted because the
amino acid residues that form the interface have opposite electric
charges from each other. This allows efficient production of a
polypeptide complex of the present invention with the desired
association.
[0246] Furthermore, control of the association in the present
invention can also be used to inhibit the association between CH1s
(the heavy chains of the T cell receptor-binding domain and
antigen-binding domain) or between CLs (the light chains of the T
cell receptor-binding domain and antigen-binding domain).
[0247] Those skilled in the art can appropriately find in a desired
polypeptide complex whose association is controlled according to
the present invention the types of amino acid residues locating in
close proximity at the CH1/CL interface upon association.
[0248] Furthermore, by using public databases or such, those
skilled in the art can appropriately find usable antibody CH1 and
CL sequences in an organism such as human, monkey, mouse, or
rabbit. More specifically, the amino acid sequence information of
CH1 and CL can be obtained by methods described below in the
Examples.
[0249] Specifically, as shown in the Examples described below,
specific examples of combinations of amino acid residues locating
in close proximity (facing or contacting) at the CH1/CL interface
upon association between CH1 and CL which are respectively linked
to the VH and VL forming the T cell receptor-binding domain or
antigen-binding domain include:
[0250] lysine (K) at position 147 (EU numbering) in CH1 (for
example, position 147 in the amino acid sequence of SEQ ID NO: 1)
and threonine (T) at position 180 (EU numbering) in the facing
(contacting) CL;
[0251] lysine (K) at position 147 (EU numbering) in CH1 and serine
(S) at position 131 (EU numbering) in the facing (contacting)
CL;
[0252] lysine (K) at position 147 (EU numbering) in CH1 and
threonine (T) at position 164 (EU numbering) in the facing
(contacting) CL;
[0253] lysine (K) at position 147 (EU numbering) in CH1 and
asparagine (N) at position 138 (EU numbering) in CL, which face
(contact with) each other;
[0254] lysine (K) at position 147 (EU numbering) in CH1 and
glutamic acid (E) at position 123 (EU numbering) in the facing
(contacting) CL;
[0255] glutamine (Q) at position 175 (EU numbering) in CH1 and
glutamine (Q) at position 160 (EU numbering) in the facing
(contacting) CL; or
[0256] lysine (K) at position 213 (EU numbering) in CH1 and
glutamic acid (E) at position 123 (EU numbering) in the facing
(contacting) CL.
[0257] These positions are numbered according to the document by
Kabat et al. (Kabat E A et al., 1991. Sequence of Proteins of
Immunological Interest. NIH).
[0258] Herein, the numbers indicated by EU numbering are assigned
according to EU numbering (Sequences of proteins of immunological
interest, NIH Publication No. 91-3242). In the present invention,
"amino acid residue at position X (EU numbering)" and "amino acid
at position X (EU numbering)" (where X is an arbitrary number) are
interchangeable with "amino acid residue corresponding to position
X (EU numbering)", "amino acid corresponding to position X (EU
numbering)".
[0259] As described in the Examples below, a desired polypeptide
complex can be preferentially obtained by altering the amino acid
residues and conducting the methods of the present invention.
[0260] The amino acid residues described above are known to be
highly conserved in human and mouse (J. Mol. Recognit. (2003) 16,
113-120). Thus, the association between CH1 and CL in the constant
region of a polypeptide complex of the present invention other than
the polypeptide complexes described in the Examples can also be
controlled by altering amino acid residues corresponding to the
above-described amino acid residues.
[0261] Specifically, the present invention provides polypeptide
complexes with controlled association between the heavy chain and
light chain, in which one, two or more pairs selected from the
group consisting of the pairs of amino acid residues described in
(a) to (f) below have the same electric charges:
[0262] (a) the amino acid residue at position 147 (EU numbering) in
CH1 and the amino acid residue at position 180 (EU numbering) in
CL;
[0263] (b) the amino acid residue at position 147 (EU numbering) in
CH1 and the amino acid residue at position 131 (EU numbering) in
CL;
[0264] (c) the amino acid residue at position 147 (EU numbering) in
CH1 and the amino acid residue at position 164 (EU numbering) in
CL;
[0265] (d) the amino acid residue at position 147 (EU numbering) in
CH1 and the amino acid residue at position 138 (EU numbering) in
CL;
[0266] (e) the amino acid residue at position 147 (EU numbering) in
CH1 and the amino acid residue at position 123 (EU numbering) in
CL; and
[0267] (f) the amino acid residue at position 175 (EU numbering) in
CH1 and the amino acid residue at position 160 (EU numbering) in
CL.
[0268] Furthermore, in another embodiment, the present invention
provides antibodies in which the amino acid residues in the pair of
amino acid residues described in (g) below have the same electric
charges:
[0269] (g) the amino acid residue at position 213 (EU numbering) in
CH1 and the amino acid residue at position 123 (EU numbering) in
CL.
[0270] The amino acid residues in each of the pairs described above
are located in close proximity with each other upon association, as
described in the EXAMPLES below. By homology modeling or other
methods using commercially available software, those skilled in the
art can appropriately find in a desired CH1 or CL amino acid
positions corresponding to the amino acid residues described in (a)
to (g) above, and can appropriately alter amino acid residues at
those positions.
[0271] Such an "electrically charged amino acid residue" in an
above-described antibody is preferably selected, for example, from
the amino acid residues belonging to the group (X) or (Y) described
below:
[0272] (X) glutamic acid (E) and aspartic acid (D); and
[0273] (Y) lysine (K), arginine (R) and histidine (H).
[0274] In the above-described polypeptide complexes, "has the same
electric charge" means that, for example, all of two or more amino
acid residues belongs to one of groups (X) and (Y) described above.
On the other hand, "has an opposite electric charge" means that,
for example, at least one of two or more amino acid residues has an
amino acid residue that belongs to one of groups (X) and (Y)
described above, while the other amino acid residues have an amino
acid residues that belongs to the other group.
[0275] Methods of producing the above-described polypeptide
complexes and methods of the present invention for controlling the
association by altering the amino acid residues in groups (a) to
(g) above to amino acid residues having the same electric charge
are also preferred embodiments of the present invention.
[0276] In the present invention, amino acid residues "to be
altered" are not limited to the above-described amino acid residues
of the constant region. By homology modeling or other methods using
commercially available software, those skilled in the art can
appropriately identify amino acid residues that form an interface
in a mutant polypeptide or heteromeric multimer and appropriately
alter amino acid residues at those positions to control
association.
[0277] In the techniques for inhibiting undesired association
between the heavy chain and light chain by introducing charge
repulsion at the interface between the heavy chain and light chain
variable regions, amino acid residues in contact with each other at
the interface between the heavy chain variable region (VH) and
light chain variable region (VL) include, for example, glutamine
(Q) at position 39 (for example, position 39 in the amino acid
sequence of SEQ ID NO: 6 in WO 2006/106905) in the heavy chain
variable region FR2 and glutamine (Q) at position 38 (for example,
position 44 in the amino acid sequence of SEQ ID NO: 8 in WO
2006/106905) in the facing (contacting) light chain variable region
FR2. Such preferred amino acid residues also include, for example,
leucine (L) at position 45 (for example, position 45 in the amino
acid sequence of SEQ ID NO: 6 in WO 2006/106905) in the heavy chain
variable region FR2 and proline (P) at position 44 (for example,
position 44 in the amino acid sequence of SEQ ID NO: 8 in WO
2006/106905) in the facing light chain variable region FR2. These
positions are numbered according to the document by Kabat et al.
(Kabat E A et al. 1991. Sequence of Proteins of Immunological
Interest. NIH).
[0278] The amino acid residues described above are known to be
highly conserved in human and mouse (J. Mol. Recognit. (2003) 16,
113-120). Thus, the association between VH and VL in antibody
variable regions other than the polypeptide complexes described in
the Examples can also be controlled by altering amino acid residues
corresponding to the above-described amino acid residues.
[0279] More specifically, such antibodies having heavy chain and
light chain variable regions include those in which the amino acid
residues of (1) and (2), or (3) and (4) described below have the
same electric charges:
[0280] (1) the amino acid residue corresponding to position 39 (EU
numbering) in the heavy chain variable region;
[0281] (2) the amino acid residue corresponding to position 38 (EU
numbering) in the light chain variable region;
[0282] (3) the amino acid residue corresponding to position 45 (EU
numbering) in the heavy chain variable region;
[0283] (4) the amino acid residue corresponding to position 44 (EU
numbering) in the light chain variable region.
[0284] The amino acid residues of (1) and (2), or (3) and (4)
described above are located in close proximity to each other upon
association. By homology modeling or other methods using
commercially available software, those skilled in the art can
appropriately identify in a desired heavy chain or light chain
variable region amino acid positions corresponding to the amino
acid residues described in (1) to (4) above, and can appropriately
alter amino acid residues at those positions.
[0285] In an above-described antibody, the "electrically charged
amino acid residue" is preferably selected, for example, from the
amino acid residues belonging to the group (X) or (Y) below:
[0286] (X) glutamic acid (E) and aspartic acid (D); and
[0287] (Y) lysine (K), arginine (R), and histidine (H).
[0288] In human and mouse, generally, the amino acid residues of
(1) to (4) described above are:
[0289] (1) glutamine (Q);
[0290] (2) glutamine (Q);
[0291] (3) leucine (L); and
[0292] (4) proline (P), respectively.
In a preferred embodiment of the present invention, the
above-described amino acid residues are altered (for example,
substitution with a charged amino acid). The types of amino acid
residues (1) to (4) described above are not limited to those
described above. These amino acids may be any other amino acids
corresponding to those described above. For example, in the case of
human, an amino acid corresponding to the amino acid at position 38
(EU numbering) in light chain variable region may be histidine (H).
By referring to published documents (for example, J. Mol. Recognit.
(2003) 16, 113-120) or the like, those skilled in the art can find
the type of amino acid residue corresponding to an arbitrary
position in the light chain, and thus can appropriately alter the
amino acid residue (for example, substitution with a charged amino
acid).
[0293] In techniques for inhibiting undesired association between
the heavy chain and light chain by substituting electrically
charged polar amino acids for amino acid residues that form the
hydrophobic core at the interface between the heavy chain and light
chain variable regions, preferred amino acid residues capable of
forming the hydrophobic core at the interface between the heavy
chain variable region (VH) and light chain variable region (VL)
include, for example, leucine (L) at position 45 in the heavy chain
variable region and proline (P) at position 44 in the facing light
chain variable region.
[0294] In general, a "hydrophobic core" refers to a portion where
side chains of hydrophobic amino acids assemble inside the
associated polypeptide. Hydrophobic amino acids include, for
example, alanine, isoleucine, leucine, methionine, phenylalanine,
proline, tryptophan, and valine. Meanwhile, amino acid residues
other than the hydrophobic amino acids (for example, tyrosin) can
also be involved in the formation of hydrophobic core. Together
with the hydrophilic surface from outward exposure of the side
chains of hydrophilic amino acids, the hydrophobic core serves as a
driving force to promote the association of water-soluble
polypeptides. When hydrophobic amino acids of two different domains
are present on the molecular surface and exposed to water
molecules, the entropy is increased, which results in an increase
in the free energy. Therefore, the two domains associate with each
other to decrease free energy for stabilization. The hydrophobic
amino acids at the interface are buried inside of the molecule to
form a hydrophobic core.
[0295] It is considered that when the hydrophobic amino acids that
form a hydrophobic core in the polypeptide association are altered
to polar amino acids with electric charge, the formation of
hydrophobic core is inhibited. This results in inhibition of the
polypeptide association.
[0296] Other known technologies are also applicable to the
polypeptide complexes of the present invention. For example, to
promote the association of the first VH (VH1) and first VL (VL1),
and/or second VH (VH2) and second VL (VL2), in addition to the
"alterations" of the present invention, amino acids in one of the H
chain variable regions are substituted with those having a larger
side chain (knob; bulge) and amino acids in the other H chain
variable region are substituted with those having a smaller side
chain (hole; void) so that the knob is placed in the hole. This
promotes the association of VH1 and VL1, and/or VH2 and VL2,
resulting in further inhibition of the association between VH1 and
VL2 and/or between VH2 and VL1 polypeptides (WO 1996/027011;
Ridgway J B et al., Protein Engineering (1996) 9, 617-621; Merchant
A M et al., Nature Biotechnology (1998) 16, 677-681).
[0297] In the production of an above-described polypeptide complex,
each domain may be linked directly via peptide bond or by peptide
binding via a peptide linker. In this case, the linker to be used
includes the linker described above as an example and appropriate
linkers with a peptide tag, for example, His-tag, HA-tag, myc-tag,
or FLAG-tag. In addition, it is preferable to use the property of
mutual binding based on hydrogen bonding, disulfide bond, covalent
bonding, or ionic interaction, or a combination thereof. For
example, it is possible to employ the affinity between antibody CH1
and CL, or the above-described Fc domains derived from a bispecific
antibody may be used for heteromeric association of Fc domains.
Moreover, interdomain disulfide bonds can be preferably used as
described in the EXAMPLES.
[0298] The polypeptide complexes of the present invention include,
for example, the embodiments shown in FIGS. 17, 19, and 24.
[0299] The polypeptide complexes of the present invention can be
produced by the same methods as the above-described methods for
producing recombinant antibodies.
[0300] Furthermore, the present invention relates to
polynucleotides encoding the polypeptide complex of the present
invention. A polypeptide complex of the present invention can be
inserted into any expression vectors. An appropriate host is
transformed with the expression vector to obtain cells expressing
the polypeptide complex. The polypeptide complex encoded by the
polynucleotide can be obtained by culturing cells expressing the
polypeptide complex and collecting the expression product from the
culture supernatant. Specifically, the present invention relates to
vectors carrying a polynucleotide encoding the polypeptide complex
of the present invention, cells containing the vectors, and methods
of producing the polypeptide complex of the present invention, in
which the cells are cultured and the polypeptide complex is
collected from the culture supernatant. Those described above can
be obtained by the same technologies described above for the
recombinant antibodies.
Pharmaceutical Composition
[0301] In another aspect, the present invention provides
pharmaceutical compositions which comprise as an active ingredient
a polypeptide complex comprising:
[0302] (1) an antigen-binding domain;
[0303] (2) a domain comprising an Fc domain with reduced Fc.gamma.
receptor-binding activity; and
[0304] (3) a CD3-binding domain.
The present invention also relates to therapeutic agents to induce
cellular cytotoxicity, which comprise the above-described complex
as an active ingredient (therapeutic agents for inducing cellular
cytotoxicity), cell growth-suppressing agents, and anticancer
agents. The pharmaceutical compositions of the present invention
can be used as a therapeutic or preventive agent for cancer. The
therapeutic agents for inducing cellular cytotoxicity, cell
growth-suppressing agents, and anticancer agents of the present
invention are preferably administered to subjects with cancer or a
likelihood of cancer recurrence.
[0305] In the present invention, the therapeutic agents for
inducing cellular cytotoxicity, cell growth-suppressing agents, and
anticancer agents, which comprise as an active ingredient a
polypeptide complex comprising:
[0306] (1) an antigen-binding domain;
[0307] (2) a domain encompassing an Fc domain with reduced
Fc.gamma. receptor-binding activity; and
[0308] (3) a CD3-binding domain
may also be described as a method for preventing or treating
cancer, which comprises the step of administering the polypeptide
complex to a subject, or use of the polypeptide complex in
producing therapeutic agents for inducing cellular cytotoxicity,
cell growth-suppressing agents, or anticancer agents.
[0309] Herein, "comprising as an active ingredient a polypeptide
complex comprising:
[0310] (1) an antigen-binding domain;
[0311] (2) a domain encompassing an Fc domain with reduced
Fc.gamma. receptor-binding activity; and
[0312] (3) a CD3-binding domain"
means comprising the polypeptide complex as a major active
ingredient; however, the content ratio of the polypeptide complex
is not limited.
[0313] A pharmaceutical composition of the present invention, a
therapeutic agent for inducing cellular cytotoxicity, a cell
growth-suppressing agent, or an anticancer agent of the present
invention may be formulated with different types of polypeptide
complexes, if needed. For example, the cytotoxic action against
cells expressing an antigen can be enhanced by a cocktail of
multiple polypeptide complexes of the present invention that bind
to the same antigen. Alternatively, the therapeutic effect can be
increased by formulating a polypeptide complex of the present
invention comprising an antigen-binding domain that binds to a
cancer antigen in combination with other polypeptide complexes of
the present invention comprising an antigen-binding domain against
a different antigen.
[0314] If necessary, the polypeptide complexes of the present
invention may be encapsulated in microcapsules (microcapsules made
from hydroxymethylcellulose, gelatin, poly[methylmethacrylate], and
the like), and made into components of colloidal drug delivery
systems (liposomes, albumin microspheres, microemulsions,
nano-particles, and nano-capsules) (for example, see "Remington's
Pharmaceutical Science 16th edition", Oslo Ed. (1980)). Moreover,
methods for preparing agents as sustained-release agents are known,
and these can be applied to the polypeptide complexes of the
present invention (J. Biomed. Mater. Res. (1981) 15, 267-277;
Chemtech. (1982) 12, 98-105; U.S. Pat. No. 3,773,719; European
Patent Application (EP) Nos. EP58481 and EP133988; Biopolymers
(1983) 22, 547-556).
[0315] The pharmaceutical compositions, cell growth-suppressing
agents, or anticancer agents of the present invention may be
administered either orally or parenterally to patients. Parental
administration is preferred. Specifically, such administration
methods include injection, nasal administration, transpulmonary
administration, and percutaneous administration. Injections
include, for example, intravenous injections, intramuscular
injections, intraperitoneal injections, and subcutaneous
injections. For example, pharmaceutical compositions, therapeutic
agents for inducing cellular cytotoxicity, cell growth-suppressing
agents, or anticancer agents of the present invention can be
administered locally or systemically by injection. Furthermore,
appropriate administration methods can be selected according to the
patient's age and symptoms. The administered dose can be selected,
for example, from the range of 0.0001 mg to 1,000 mg per kg of body
weight for each administration. Alternatively, the dose can be
selected, for example, from the range of 0.001 mg/body to 100,000
mg/body per patient. However, the dose of a pharmaceutical
composition of the present invention is not limited to these
doses.
[0316] The pharmaceutical compositions of the present invention can
be formulated according to conventional methods (for example,
Remington's Pharmaceutical Science, latest edition, Mark Publishing
Company, Easton, U.S.A.), and may also contain pharmaceutically
acceptable carriers and additives. Examples include, but are not
limited to, surfactants, excipients, coloring agents, flavoring
agents, preservatives, stabilizers, buffers, suspension agents,
isotonic agents, binders, disintegrants, lubricants, fluidity
promoting agents, and corrigents, and other commonly used carriers
can be suitably used. Specific examples of the carriers include
light anhydrous silicic acid, lactose, crystalline cellulose,
mannitol, starch, carmellose calcium, carmellose sodium,
hydroxypropyl cellulose, hydroxypropyl methylcellulose,
polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin,
medium-chain triglyceride, polyoxyethylene hardened castor oil 60,
saccharose, carboxymethyl cellulose, corn starch, inorganic salt,
and such.
[0317] The present invention also provides methods for damaging
cells expressing a cancer antigen or for suppressing the cell
growth by contacting the cells expressing the cancer antigen with a
polypeptide complex of the present invention that binds to the
cancer antigen. Monoclonal antibodies that bind to the cancer
antigen are described above as a cancer antigen-binding polypeptide
complex of the present invention, which is included in the
therapeutic agents for inducing cellular cytotoxicity, cell
growth-suppressing agents, and anticancer agents of the present
invention. Cells to which a cancer antigen-binding polypeptide
complex of the present invention binds are not particularly
limited, as long as they express the cancer antigen. Specifically,
in the present invention, the preferred cancer antigen-expressing
cells include ovary cancer cells, prostate cancer cells, breast
cancer cells, uterine cancer cells, liver cancer cells, lung cancer
cells, pancreatic cancer cells, stomach cancer cells, urinary
bladder cancer cells, and colon cancer cells. When the cancer
antigen is GPC3, cells are not limited as long as they are cancer
cells expressing GPC3. However, the preferred cancer cells include
hepatocarcinoma cells, lung cancer cells, and ovary cancer
cells.
[0318] In the present invention, "contact" can be carried out, for
example, by adding a cancer antigen-binding polypeptide complex of
the present invention to culture media of cells expressing the
cancer antigen cultured in vitro. In this case, a polypeptide
complex to be added can be used in an appropriate form, such as a
solution or solid prepared by lyophilization or the like. When the
polypeptide complex of the present invention is added as an aqueous
solution, the solution may be a pure aqueous solution containing
the polypeptide complex alone or a solution containing, for
example, an above-described surfactant, excipient, coloring agent,
flavoring agent, preservative, stabilizer, buffering agent,
suspending agent, isotonizing agent, binder, disintegrator,
lubricant, fluidity accelerator, and corrigent. The added
concentration is not particularly limited; however, the final
concentration in a culture medium is preferably in a range of 1
pg/ml to 1 g/ml, more preferably 1 ng/ml to 1 mg/ml, and still more
preferably 1 .mu.g/ml to 1 mg/ml.
[0319] In another embodiment of the present invention, "contact"
can also be carried out by administration to nonhuman animals
transplanted with cancer antigen-expressing cells in vivo or to
animals having cancer cells expressing the cancer antigen
endogenously. The administration method may be oral or parenteral.
Parenteral administration is particularly preferred. Specifically,
the parenteral administration method includes injection, nasal
administration, pulmonary administration, and percutaneous
administration. Injections include, for example, intravenous
injections, intramuscular injections, intraperitoneal injections,
and subcutaneous injections. For example, pharmaceutical
compositions, therapeutic agents for inducing cellular
cytotoxicity, cell growth-suppressing agents, or anticancer agents
of the present invention can be administered locally or
systemically by injection. Furthermore, an appropriate
administration method can be selected according to the age and
symptoms of an animal subject. When the polypeptide complex is
administered as an aqueous solution, the solution may be a pure
aqueous solution containing the polypeptide complex alone or a
solution containing, for example, an above-described surfactant,
excipient, coloring agent, flavoring agent, preservative,
stabilizer, buffering agent, suspending agent, isotonizing agent,
binder, disintegrator, lubricant, fluidity accelerator, and
corrigent. The administered dose can be selected, for example, from
the range of 0.0001 to 1,000 mg per kg of body weight for each
administration. Alternatively, the dose can be selected, for
example, from the range of 0.001 to 100,000 mg/body for each
patient. However, the dose of a polypeptide complex of the present
invention is not limited to these examples.
[0320] The methods described below are preferably used as a method
for assessing or determining cellular cytotoxicity caused by
contacting a polypeptide complex of the present invention with
antigen-expressing cells to which the antigen-binding domain
forming the polypeptide complex of the present invention binds. The
methods for assessing or determining the cytotoxic activity in
vitro include methods for determining the activity of cytotoxic T
cells or the like. Whether a polypeptide complex of the present
invention has the activity of inducing T-cell mediated cellular
cytotoxicity can be determined by known methods (see, for example,
Current protocols in Immunology, Chapter 7. Immunologic studies in
humans, Editor, John E, Coligan et al., John Wiley & Sons,
Inc., (1993)). In the cytotoxicity assay, a polypeptide complex
whose antigen-binding domain binds to an antigen different from
that recognized by the antigen-binding domain of the polypeptide
complex of the present invention and which is not expressed in the
cells is used as a control polypeptide complex. The control
polypeptide complex is assayed in the same manner. Then, the
activity is assessed by testing whether a polypeptide complex of
the present invention exhibits a stronger cytotoxic activity than
that of a control polypeptide complex.
[0321] Meanwhile, the in vivo cytotoxic activity is assessed or
determined, for example, by the following procedure. Cells
expressing the antigen to which the antigen-binding domain forming
a polypeptide complex of the present invention binds are
transplanted intracutaneously or subcutaneously to a nonhuman
animal subject. Then, from the day of transplantation or
thereafter, a test polypeptide complex is administered into vein or
peritoneal cavity every day or at intervals of several days. The
tumor size is measured over time. Difference in the change of tumor
size can be defined as the cytotoxic activity. As in an in vitro
assay, a control polypeptide complex is administered. The
polypeptide complex of the present invention can be judged to have
cytotoxic activity when the tumor size is smaller in the group
administered with the polypeptide complex of the present invention
than in the group administered with the control polypeptide
complex.
[0322] An MTT method and measurement of isotope-labeled thymidine
uptake into cells are preferably used to assess or determine the
effect of contact with a polypeptide complex of the present
invention to suppress the growth of cells expressing an antigen to
which the antigen-binding domain forming the polypeptide complex
binds. Meanwhile, the same methods described above for assessing or
determining the in vivo cytotoxic activity can be used preferably
to assess or determine the activity of suppressing cell growth in
vivo.
[0323] The present invention also provides kits for use in a method
of the present invention, which contain a polypeptide complex of
the present invention or a polypeptide complex produced by a method
of the present invention. The kits may be packaged with an
additional pharmaceutically acceptable carrier or medium, or
instruction manual describing how to use the kits, etc.
[0324] In addition, the present invention relates to polypeptide
complexes of the present invention or polypeptide complexes
produced by a method of the present invention for use in a method
of the present invention.
[0325] All prior-art documents cited herein are incorporated herein
by reference.
EXAMPLES
[0326] Hereinbelow, the present invention is specifically described
with reference to the Examples, but it is not to be construed as
being limited thereto.
[Example 1] Construction and Assessment of GPC3 ERY2
(1) Outline
[0327] There is a well-known method for prolonging the blood
half-life of a protein administered in vivo, which is based on the
FcRn-mediated recycling using a protein of interest conjugated to
an antibody Fc domain. However, the conjugation of a natural type
of Fc to BiTE could lead to induction of various cytokines, since a
single molecule would bind to a T cell via the anti-CD3 scFv of its
BiTE moiety and simultaneously to the FcgR (Fc.gamma. receptor) on
the cellular membrane of, for example, a NK cell or macrophage via
its Fc domain, and the resulting cross-linking would activate these
cells in a cancer antigen-independent manner. Thus, a molecule
termed ERY2, in which a BiTE is linked via a polypeptide linker to
an Fc domain having reduced Fc.gamma. receptor-binding activity
(silent Fc), was prepared, and the activity of ERY2 was assessed by
comparing it to that of the BiTE. The scFv of an anti-CD3 epsilon
antibody was linked via a short peptide linker to the scFv of an
antibody against Glypican 3 (GPC3), which is a GPI-anchored protein
known to be expressed at a high level in liver cancer cells, to
produce BiTE against GPC3 (GPC3 BiTE) (FIG. 17A). This was then
linked to a silent Fc to produce an ERY2 against GPC3 (GPC3 ERY2)
(FIG. 17C). Furthermore, a normal IgG-type anti-GPC3 antibody was
constructed for comparison. The IgG-type anti-GPC3 antibody was
prepared as an antibody with reduced fucose content in its sugar
chain moiety, i.e., a low-fucose antibody, which is known to have
an enhanced ADCC activity.
(2) Construction of GPC3 BiTE
[0328] By PCR amplification using an expression vector for an
anti-GPC3 antibody as a template, cDNAs each encoding an H chain
variable region (anti-GPC3 VH) or an L chain variable region
(anti-GPC3 VL) were obtained. PCR was performed using primers
containing appropriate additional sequences and the above cDNAs as
templates to construct a cDNA fragment encoding an anti-GPC3 scFv
having an amino acid sequence in which the anti-GPC3 VH and
anti-GPC3 VL were linked together via a linker with three repeats
of Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7).
[0329] Furthermore, a series of oligonucleotides were prepared each
of which had a nucleotide sequence encoding a partial sequence of
the H chain variable region (M12 VH) or L chain variable region
(M12 VL) of an anti-CD3 antibody (M12), and had complementary
sequences at the ends. The oligonucleotides were designed such that
they would be linked together via the complementary sequence
portions by polymerase reaction to synthesize a polynucleotide
corresponding to the H chain variable region (M12 VH) and L chain
variable region (M12 VL). The oligonucleotides were mixed and then
assembled together by PCR to give two cDNAs encoding the amino acid
sequences of respective variable regions. PCR was performed using
primers containing appropriate additional sequences and the above
cDNAs as templates to produce a cDNA fragment encoding M12 scFv
having an amino sequence in which M12 VL and M12 VH were linked
together via a linker having three repeats of Gly-Gly-Gly-Gly-Ser
(SEQ ID NO: 7).
[0330] Next, by PCR using primers containing appropriate additional
sequences and the cDNA fragments each encoding anti-GPC3 scFv or
M12 scFv as templates, a cDNA fragment was constructed which
encoded an amino acid sequence in which anti-GPC3 scFv and M12 scFv
were linked together via a linker composed of Gly-Gly-Gly-Gly-Ser
(SEQ ID NO: 7) and its C terminus had a His tag (eight histidines)
(the sequence of SEQ ID NO: 33 without its amino terminal 19 amino
acids).
[0331] Using primers containing appropriate additional sequences
and as a template the cDNA fragment encoding the amino acid
sequence of SEQ ID NO: 33 lacking its amino terminal 19 amino
acids, PCR was performed to produce a cDNA fragment in which an
EcoRI cleavage sequence, kozac sequence, and a nucleotide sequence
encoding a secretion signal sequence were attached to the 5' end of
the above cDNA fragment and a NotI cleavage sequence to the 3' end.
The resulting cDNA fragment was cleaved with EcoRI and NotI, and
inserted into an mammalian cell expression vector to obtain an
expression vector for GPC3 BiTE (SEQ ID NO: 33; the mature sequence
does not contain the amino terminal 19 amino acids, which serve as
a signal sequence).
[0332] The vector was introduced into CHO DG44 cells by
electroporation. After limiting dilution, the cells were cultured
in the presence of 1 mg/ml Geneticin to isolate drug-resistant cell
lines. The culture supernatant of the obtained cell lines was
analyzed by Western blotting using an anti-His tag antibody to
select a cell line expressing GPC3 BiTE.
[0333] The culture supernatant obtained by large scale cell culture
of the above-described cell line was loaded onto an SP Sepharose FF
column (GE Healthcare). After washing the column, a fraction
containing GPC3 BiTE was eluted with a NaCl concentration gradient.
The fraction was loaded onto a HisTrap HP column (GE Healthcare).
After washing the column, a fraction containing GPC3 BiTE was
eluted with an imidazole concentration gradient. The fraction was
concentrated by ultrafiltration, and then the concentrate was
loaded onto a Superdex 200 column (GE Healthcare). Only a monomeric
GPC3 BiTE fraction was collected to obtain purified GPC3 BiTE.
(3) Construction of GPC3 ERY2
[0334] PCR using primers containing the same appropriate additional
sequences as in the above-described method and a method well known
to those skilled in the art such as a method using the QuikChange
Site-Directed Mutagenesis Kit (Stratagene) were performed to
produce expression vectors to which a polynucleotide encoding GPC3
ERY2_Hk (SEQ ID NO: 34; the mature sequence does not contain the
amino terminal 19 amino acids, which serve as a signal sequence) or
GPC3 ERY2_Hh (SEQ ID NO: 35; the mature sequence does not contain
the amino terminal 19 amino acids, which serve as a signal
sequence) was inserted.
[0335] These expression vectors were co-introduced into
FreeStyle293-F cells (Invitrogen) to express GPC3 ERY2 transiently.
The resulting culture supernatant was loaded onto an Anti FLAG M2
column (Sigma). After washing, the column was eluted with 0.1 mg/ml
FLAG peptide (Sigma). A fraction containing GPC3 ERY2 was loaded
onto a HisTrap HP column (GE Healthcare). After washing, the column
was eluted with an imidazole concentration gradient. A fraction
containing GPC3 ERY2 was concentrated by ultrafiltration, and the
concentrate was loaded onto a Superdex 200 column (GE Healthcare).
Only a monomeric GPC3 ERY2 fraction was collected from the eluate
to obtain purified GPC3 ERY2.
(4) Construction of Low-Fucose Anti-GPC3 Antibody
[0336] An expression vector for an anti-GPC3 antibody (that is
referred to as humanized GC33 antibody in WO 2006/006693) was
introduced into GDP fucose-knockout CHO DXB11 cells (Cancer Sci.
(2010) 101(10), 2227-33) by electroporation. After limiting
dilution, the cells were cultured in the presence of 0.5 mg/ml
Geneticin to select drug resistant lines, and a cell line
expressing a low-fucose anti-GPC3 antibody was obtained. From the
culture supernatant obtained by culturing these cells, an antibody
fraction was prepared by conventional affinity purification using
Hitrap.RTM. Protein A (Pharmacia). Then, the antibody fraction was
subjected to gel filtration purification using Superdex 20026/60
(Pharmacia). A monomer fraction was collected from the eluate to
obtain a low-fucose anti-GPC3 antibody.
(5) Cytotoxicity Assay Using Human Peripheral Blood Mononuclear
Cells
(5-1) Preparation of Human Peripheral Blood Mononuclear Cell (PBMC)
Suspension
[0337] From healthy volunteers (adult), 50 ml of peripheral blood
was collected using syringes to which 100 .mu.l of 1,000 units/ml
heparin solution (Novo-Heparin 5000 units for injection; Novo
Nordisk) had been added in advance. The peripheral blood was
diluted two fold with PBS(-), divided into four equal aliquots, and
added to Leucosep lymphocyte separation tubes (Cat. No. 227290;
Greiner bio-one) that had been injected with 15 ml of Ficoll-Paque
PLUS and centrifuged in advance. After centrifugation (2,150 rpm,
10 minutes, room temperature) of the separation tubes, a
mononuclear cell fraction layer was collected. The cells in the
mononuclear cell fraction were washed once with Dulbecco's Modified
Eagle's Medium (SIGMA) containing 10% FBS (hereinafter referred to
as 10% FBS/D-MEM), and then the cell density was adjusted to
4.times.10.sup.6 cells/ml using 10% FBS/D-MEM. The cell suspension
thus prepared was used as a human PBMC suspension in subsequent
experiments.
(5-2) Cytotoxic Activity Assay
[0338] The cytotoxic activity was assessed based on the cell growth
inhibition rate determined using xCELLigence real-time cell
analyzer (Roche Diagnostics). The target cell used was SK-pca13a
cell line established by forcedly expressing human GPC3 in SK-HEP-1
cell line. SK-pca13a cells were detached from dishes, and seeded
onto an E-Plate 96 (Roche Diagnostics) plate at 1.times.10.sup.4
cells/well (100 .mu.l/well). Then viable cell assay was started
using xCELLigence real-time cell analyzer. On the following day,
the plate was removed from the xCELLigence real-time cell analyzer,
and 50 .mu.l of each antibody prepared at various concentrations
(0.004, 0.04, 0.4, and 4 nM) was added to the plate. After 15
minutes of reaction at room temperature, 50 .mu.l of human PBMC
suspension (2.times.10.sup.5 cells/well) prepared in (5-1) was
added. The plate was placed in the xCELLigence real-time cell
analyzer again to start viable cell assay. The reaction was carried
out under 5% carbon dioxide gas at 37.degree. C. The cell growth
inhibition rate (%) was determined according to the formula shown
below using the Cell Index value at 72 hours after the addition of
human PBMCs. The Cell Index value used in the calculation was
normalized such that the Cell Index value immediately before the
addition of antibody was taken as 1.
Cell growth inhibition rate (%)=(A-B).times.100/(A-1)
A denotes the mean Cell Index value for the no-antibody well (the
target cell and human PBMC only), while B denotes the mean Cell
Index value for each well. The measurement was carried out in
triplicate.
[0339] The cytotoxic activity of GPC3 BiTE, GPC3 ERY2, and IgG-type
anti-GPC3 antibody was measured using PBMCs (peripheral blood
mononuclear cells) prepared from human blood as effector cells.
GPC3 BiTE showed a very strong activity (FIG. 1). This activity was
much stronger than that of the low-fucose anti-GPC3 antibody. Thus,
GPC3 BiTE may serve as an excellent cancer therapeutic agent that
exceeds the IgG-type antibody. On the other hand, the activity of
GPC3 ERY2 was not as strong as that of GPC3 BiTE, although it was
greater than that of the IgG-type anti-GPC3 antibody. This suggests
that the mere addition of Fc to BiTE does not enable creation of a
desired molecule.
[Example 2] Construction and Assessment of GPC3 ERY5, GPC3 ERY6,
and GPC3 ERY7
[0340] Next, in an attempt to improve the specific activity, the
cancer antigen (GPC3)-binding domain was made bivalent to enhance
the cancer cell-binding activity. Another anti-GPC3 scFv was added
to GPC3 ERY2 to construct GPC3 ERY5 (FIG. 17D). Furthermore,
instead of the scFv, a Fab-type GPC3-binding domain was added to
produce GPC3 ERY7 (FIG. 17F). In addition, GPC3 ERY6 (FIG. 17E) was
also constructed in which the anti-CD3 epsilon scFv of GPC3 ERY5
was split into two arms.
[0341] Specifically, a method known to those skilled in the art,
such as PCR using primers containing the same appropriate
additional sequences as in the above-described method, was
performed to produce a series of expression vectors into which a
polynucleotide encoding GPC3 ERY5_Hh, GPC3 ERY6_Hk, GPC3 ERY6_Hh,
GPC3 ERY7_Hh, or GPC3 ERY7_L was inserted.
[0342] The following combinations of expression vectors were
introduced into FreeStyle293-F cells to express each designed
molecule transiently.
A. Designed Molecule: GPC3 ERY5
[0343] Polypeptides encoded by polynucleotides inserted in
expression vectors: GPC3 ERY5_Hh (SEQ ID NO: 36; the mature
sequence does not contain the amino terminal 19 amino acids, which
serves as a signal sequence) and GPC3 ERY2_Hk
B. Designed Molecule: GPC3 ERY6
[0344] Polypeptides encoded by polynucleotides inserted in
expression vectors: GPC3 ERY6_Hk (SEQ ID NO: 37; the mature
sequence does not contain the amino terminal 19 amino acids, which
serves as a signal sequence) and GPC3 ERY6_Hh (SEQ ID NO: 38; the
mature sequence does not contain the amino terminal 19 amino acids,
which serves as a signal sequence)
C. Designed Molecule: GPC3 ERY7
[0345] Polypeptides encoded by polynucleotides inserted in
expression vectors: GPC3 ERY7_Hh (SEQ ID NO: 39; the mature
sequence does not contain the amino terminal 19 amino acids, which
serves as a signal sequence), GPC3 ERY7_L (SEQ ID NO: 40; the
mature sequence does not contain the amino terminal 19 amino acids,
which serves as a signal sequence), and GPC3 ERY2_Hk
[0346] The resulting culture supernatant was loaded onto an Anti
FLAG M2 column (Sigma). After washing, the column was eluted with
0.1 mg/ml FLAG peptide (Sigma). A fraction containing the designed
molecule was loaded onto a HisTrap HP column (GE Healthcare). After
washing, the column was eluted with an imidazole concentration
gradient. A fraction containing the designed molecule was
concentrated by ultrafiltration. Then, the fraction was loaded onto
a Superdex 200 column (GE Healthcare). Only a monomer fraction was
collected from the eluate to obtain each purified designed
molecule.
[0347] These polypeptide complexes were compared to GPC3 BiTE in
terms of the cytotoxic activity. The result showed that the
cytotoxic activity of these polypeptide complexes was not as much
as that of GPC3 BiTE (FIGS. 2 to 4). This finding suggests that the
addition of Fc to the BiTE structure or its mimetic structure and
the configuration that allows bivalent binding to a cancer antigen
do not enable creation of a desired molecule.
[Example 3] Construction and Assessment of GPC3 ERY8-2, GPC3
ERY9-1, and GPC3 ERY10-1
(1) Construction of GPC3 ERY8-2, GPC3 ERY9-1, and GPC3 ERY10-1
[0348] Next, molecules having no BiTE structure but possessing the
desired activity were designed. An anti-cancer antigen (GPC3) IgG
was used as a backbone, and a molecule in which an anti-CD3 epsilon
scFv was added to this backbone was constructed. The IgG Fc used as
a backbone was a silent Fc having reduced FcgR (Fc.gamma.
receptor)-binding activity, as in the above-described cases. GPC3
ERY8-2 (FIG. 17G), GPC3 ERY10-1 (FIG. 17I), and GPC3 ERY9-1 (FIG.
17H) were constructed in which the anti-CD3 epsilon scFv was
attached to the N terminus of the H chain, C terminus of the H
chain, and C terminus of the L chain of the anti-GPC3 antibody IgG,
respectively.
[0349] Specifically, by a method known to those skilled in the art,
such as PCR using primers containing the same appropriate
additional sequences as in the above-described method, a series of
expression vectors were constructed into which a polynucleotide
encoding GPC3 ERY8-2_Hk (SEQ ID NO: 41; the mature sequence does
not contain the amino terminal 19 amino acids, which serve as a
signal sequence), GPC3 ERY8-2_Hh (SEQ ID NO: 42; the mature
sequence does not contain the amino terminal 19 amino acids, which
serve as a signal sequence), GPC3 ERY9-1_H (SEQ ID NO: 43; the
mature sequence does not contain the amino terminal 19 amino acids,
which serve as a signal sequence), GPC3 ERY9-1_L-His (SEQ ID NO:
44; the mature sequence does not contain the amino terminal 19
amino acids, which serve as a signal sequence), GPC3 ERY9-1_L-FLAG
(SEQ ID NO: 45; the mature sequence does not contain the amino
terminal 19 amino acids, which serve as a signal sequence), or GPC3
ERY10-1_Hh (SEQ ID NO: 46; the mature sequence does not contain the
amino terminal 19 amino acids, which serve as a signal sequence)
was inserted.
[0350] The following combinations of expression vectors were
introduced into FreeStyle293-F cells to express each designed
molecule transiently.
D. Designed Molecule: GPC3 ERY8-2
[0351] Polypeptides encoded by polynucleotides inserted in
expression vectors: GPC3 ERY8-2_Hk (SEQ ID NO: 41; the mature
sequence does not contain the amino terminal 19 amino acids, which
serve as a signal sequence), GPC3 ERY8-2_Hh (SEQ ID NO: 42; the
mature sequence does not contain the amino terminal 19 amino acids,
which serve as a signal sequence), and GPC3 ERY7_L
E. Designed Molecule: GPC3 ERY9-1
[0352] Polypeptides encoded by polynucleotides inserted in
expression vectors: GPC3 ERY9-1_H (SEQ ID NO: 43; the mature
sequence does not contain the amino terminal 19 amino acids, which
serve as a signal sequence), GPC3 ERY9-1_L-His (SEQ ID NO: 44; the
mature sequence does not contain the amino terminal 19 amino acids,
which serves as a signal sequence), and GPC3 ERY9-1_L-FLAG (SEQ ID
NO: 45; the mature sequence does not contain the amino terminal 19
amino acids, which serve as a signal sequence)
F. Designed Molecule: GPC3 ERY10-1
[0353] Polypeptides encoded by polynucleotides inserted in
expression vectors: GPC3 ERY10-1_Hh (SEQ ID NO: 46; the mature
sequence does not contain the amino terminal 19 amino acids, which
serve as a signal sequence) and GPC3 ERY8-2_Hk, GPC3 ERY7_L
[0354] The resulting culture supernatant was loaded onto an Anti
FLAG M2 column (Sigma). After washing, the column was eluted with
0.1 mg/ml FLAG peptide (Sigma). A fraction containing the designed
molecule was loaded onto a HisTrap HP column (GE Healthcare). After
washing, the column was eluted with an imidazole concentration
gradient. A fraction containing the designed molecule was
concentrated by ultrafiltration. Then, the fraction was loaded onto
a Superdex 200 column (GE Healthcare). Only a monomer fraction was
collected from the eluate to obtain each purified designed
molecule.
[0355] These molecules were assessed for the in vitro cytotoxic
activity. The result revealed that all molecules exhibited a
cytotoxic activity comparable to or greater than that of GPC3 BiTE
(FIG. 5). In particular, GPC3 ERY9-1 and GPC3 ERY10-1 were found to
clearly have a greater cytotoxic activity than GPC3 BiTE. The
present invention for the first time demonstrates that molecules
made by adding an anti-CD3 epsilon scFv to an anti-cancer antigen
IgG also have a cytotoxic activity comparable to or greater than
that of BiTE. Particularly, it is surprising that molecules such as
GPC3 ERY9-1 and GPC3 ERY10-1 clearly exhibited a greater cytotoxic
activity than BiTE although there was a large distance between
their cancer antigen-binding domain and CD3 epsilon-binding
domain.
(2) Assessment of the In Vivo Efficacy of GPC3 ERY8-2 and GPC3
ERY10-1:
[0356] GPC3 ERY8-2 and GPC3 ERY10-1, which were demonstrated to
have a cytotoxic activity comparable to or greater than that of
GPC3 BiTE in the in vitro assay described in (1), were assessed for
the in vivo efficacy. Cells of GPC3-expressing human lung cancer
cell line PC-10 were mixed with human PBMCs, and then transplanted
to NOD scid mice. The mice were treated by administering GPC3
ERY8-2 or GPC3 ERY10-1 (referred to as pre-mix model).
[0357] Specifically, the efficacy test for GPC3 ERY8-2 using the
PC-10 pre-mix model was conducted as follows. PBMCs were isolated
from blood collected from healthy volunteers. NK cells were removed
from the PBMCs using CD56 MicroBeads, human (MCAS Miltenyi biotec).
Human lung squamous carcinoma cell line PC-10 (Immuno-Biological
Laboratories Co., Ltd.) (5.times.10.sup.6 cells), human PBMCs
without NK cells (4.5.times.10.sup.6 cells), and Matrigel Basement
Membrane Matrix (BD) were mixed, and then transplanted
subcutaneously to the inguinal region of NOD scid mice (CLEA Japan
Inc.; female, 7W). The day of transplantation was designated day 0.
On the day before transplantation, an anti-asialo GM1 antibody
(Wako Pure Chemical Industries) was intraperitoneally administered
to the mice at 0.2 mg/head. After two hours of transplantation, GPC
ERY8-2 was intraperitoneally administered at 30 .mu.g/head. GPC
ERY8-2 was administered five times in total during the period of
days 0 to 4.
[0358] Furthermore, the efficacy test for GPC3 ERY10-1 using the
PC-10 pre-mix model was conducted as follows. PBMCs were isolated
from blood collected from healthy volunteers. NK cells were removed
from the PBMCs using CD56 MicroBeads, human (MCAS Miltenyi biotec).
Human lung squamous carcinoma cell line PC-10 (Immuno-Biological
Laboratories Co., Ltd.) (5.times.10.sup.6 cells), human PBMCs
without NK cells (4.5.times.10.sup.6 cells), and Matrigel Basement
Membrane Matrix (BD) were mixed, and then transplanted
subcutaneously to the inguinal region of NOD scid mice (CLEA Japan
Inc.; female, 7W). The day of transplantation was designated day 0.
On the day before transplantation, an anti-asialo-GM1 antibody
(Wako Pure Chemical Industries) was intraperitoneally administered
to the mice at 0.2 mg/head. After two hours of transplantation, GPC
ERY10-1 was intraperitoneally administered at 30 .mu.g/head. GPC
ERY10-1 was administered 13 times in total during the periods of
days 0 to 4, days 7 to 11, and days 14 to 16.
[0359] The result showed that in the GPC3 ERY8-2 and GPC3 ERY10-1
administration groups the tumor growth was clearly suppressed as
compared to the solvent (PBS) administration group (FIGS. 6 and
7).
[0360] Furthermore, GPC3 ERY10-1 was also assessed for the in vivo
efficacy using an alternative model. Specifically, T cells were
grown by culturing human PBMCs in vitro and then introduced into
NOD scid mice which had developed tumors originating from
transplanted PC-10. The mice were treated by administering GPC3
ERY10-1 (referred to as T cell transfer model).
[0361] Specifically, the efficacy test for GPC3 ERY10-1 using the
PC-10 T cell transfer model was conducted as follows. T cell
expansion culture was carried out using T cell activation/expansion
kit/human (MACS Miltenyi biotec) and PBMCs isolated from blood
collected from healthy volunteers. Human lung squamous carcinoma
cell line PC-10 cells (Immuno-Biological Laboratories Co., Ltd.)
(1.times.10.sup.7 cells) were mixed with Matrigel Basement Membrane
Matrix (BD), and then transplanted subcutaneously to the inguinal
region of NOD scid mice (CLEA Japan Inc.; female, 7W). The day of
transplantation was designated day 0. On the day before
transplantation and days 6, 8, 12, 16, and 20, an anti-asialo-GM1
antibody (Wako Pure Chemical Industries) was intraperitoneally
administered to the mice at 0.2 mg/head. On day 6 of
transplantation, the mice were grouped by the tumor size and body
weight, and then T cells prepared by expansion culture as described
above were transplanted at 1.times.10.sup.7 cells/head into the
peritoneal cavity. After two hours of transplantation, GPC ERY10-1
was intraperitoneally administered at 30 .mu.g/head. GPC ERY10-1
was administered five times in total on days 7, 8, 12, 16, and
17.
[0362] The result showed that the GPC3 ERY10-1 administration group
of this model also exhibited a clear anti-tumor effect as compared
to the solvent administration group (FIG. 8).
[0363] The finding described above demonstrates that a series of
molecules in which one scFv of an anti-CD3 epsilon antibody is
added to an IgG backbone having a silent Fc exhibit a clear in vivo
anti-tumor effect.
(3) Assessment of Plasma Retention
[0364] In order to assess whether molecules such as GPC3 ERY8-2,
GPC3 ERY9-1, and GPC3 ERY10-1 have a considerably longer half-life
in plasma than GPC3 BiTE, GPC3 ERY9-1 and GPC3 ERY10-1 were
administered at 30 .mu.g/head to NOD scid mice to which no cancer
cells had been transplanted, and their plasma concentrations were
measured over time.
[0365] Specifically, PK analysis was carried out in the following
manner. GPC3 ERY9-1 and GPC3 ERY10-1 were intraperitoneally
administered to NOD scid mice (CLEA Japan Inc.; female, 8W) at 30
.mu.g/head. Blood was collected from the buccal vein of the mice
using hematocrit capillaries (Terumo) at 15 minutes, two hours, 1
day, 2 days, and 7 days after administration. Plasma was prepared
from the blood.
[0366] GPC3 ERY9-1 and GPC3 ERY10-1 were appropriately diluted and
added to GPC3-expressing Ba/F3 cells (GPC3/BaF) or human CD3
epsilon-expressing Ba/F3 cells (CD3/BaF) to allow GPC3 ERY9-1 or
GPC3 ERY10-1 to react with GPC3/BaF and CD3/BaF. After washing
these cells, an FITC-labeled secondary antibody was added for
further reaction. After washing the cells, the fluorescent
intensity of the label on the cells was measured using Epics XL
flow cytometer (Beckman coulter) to prepare a calibration curve for
each antibody.
[0367] Blood was collected over time from the mice that had been
given GPC3 ERY9-1 or GPC3 ERY10-1. Plasma was prepared from the
blood and diluted appropriately. In the same manner as for the
preparation of calibration curves described above, the plasma
samples were reacted with GPC3/BaF or CD3/BaF to determine the
amount of plasma GPC3 ERY9-1 and GPC3 ERY10-1 bound to each cell.
The plasma concentration of each antibody was calculated using
determined values and the calibration curves described above.
[0368] The result showed that the blood concentration of both GPC3
ERY9-1 and GPC3 ERY10-1 remained higher than 10 nM after two days
of the administration (FIGS. 9 and 10). This finding demonstrates
that molecules such as GPC3 ERY9-1 and GPC3 ERY10-1 have a
significantly improved plasma half-life as compared to BiTE.
(4) Effect of Silent Fc on Cancer Antigen-Independent Cytokine
Induction
(4-1) Construction of GPC3 ERY15-1 Having FcgR-Binding Fc
[0369] GPC3 ERY15-1 having an FcgR-binding Fc (FIG. 17J) was
constructed to test whether molecules such as GPC3 ERY8-2, GPC3
ERY9-1, and GPC3 ERY10-1 would induce cytokines in a cancer
antigen-independent manner.
[0370] Specifically, as in the above-described method, PCR using
primers containing appropriate additional sequences and a method
known to those skilled in the art such as a method using QuikChange
Site-Directed Mutagenesis Kit (Stratagene) were performed to
construct expression vectors into which a polynucleotide encoding
GPC3 ERY15-1_Hh (SEQ ID NO: 47; the mature sequence does not
contain the amino terminal 19 amino acids, which serve as a signal
sequence) or GPC3 ERY15-1_Hk (SEQ ID NO: 48; the mature sequence
does not contain the amino terminal 19 amino acids, which serve as
a signal sequence) was inserted.
[0371] The expression vectors for GPC3 ERY15-1_Hh (SEQ ID NO: 47;
the mature sequence does not contain the amino terminal 19 amino
acids, which serve as a signal sequence), GPC3 ERY15-1_Hk (SEQ ID
NO: 48; the mature sequence does not contain the amino terminal 19
amino acids, which serve as a signal sequence), and GPC3 ERY7_L
were co-introduced into FreeStyle293-F cells to express GPC3
ERY15-1 transiently. The resulting culture supernatant was loaded
onto an Anti FLAG M2 column (Sigma). After washing, the column was
eluted with 0.1 mg/ml FLAG peptide (Sigma). A fraction containing
GPC3 ERY15-1 was loaded onto a HisTrap HP column (GE Healthcare).
After washing, the column was eluted with an imidazole
concentration gradient. A fraction containing GPC3 ERY15-1 was
concentrated by ultrafiltration. Then, the fraction was loaded onto
a Superdex 200 column (GE Healthcare). Only a monomeric GPC3
ERY15-1 fraction was collected from the eluate to obtain purified
GPC3 ERY15-1.
(4-2) Assay for Cancer Antigen-Independent Cytokine-Inducing
Ability
[0372] The cancer antigen-independent cytokine-inducing ability of
GPC3 ERY15-1 was compared to those of GPC3 BiTE, GPC3 ERY9-1, GPC3
ERY10-1, and catumaxomab. By using the method described above,
PBMCs were prepared from blood collected from healthy volunteers.
Fifty .mu.1 of each antibody adjusted to 40 nM was added to 50
.mu.l of human PBMC suspension (2.times.10.sup.5 cells/well), and
then 100 .mu.l of 10% FBS/D-MEM was added thereto. The reaction
mixture was incubated under 5% carbon dioxide gas at 37.degree. C.
After 72 hours of incubation, the culture supernatant was
collected, and cytokines secreted in the culture supernatant were
quantified by Cytometric Beads Array (CBA) assay using Human
Th1/Th2/Th17 Kit (BD). The assay was carried out in triplicate by
the method according to the appended protocol.
[0373] As a result, GPC3 ERY15-1 and catumaxomab, which have a
FcgR-binding Fc, showed clear cytokine induction. In contrast, no
cytokine induction was observed for GPC3 BiTE, which has no Fc, and
GPC3 ERY9-1 and GPC3 ERY10-1, which possess a silent Fc (FIG. 11).
This result suggests that molecules having a silent Fc such as GPC3
ERY8-2, GPC3 ERY9-1, and GPC3 ERY10-1 are highly safe molecules
which do not induce cytokines in a cancer antigen-independent
manner.
[Example 4] Construction and Assessment of GPC3 ERY18 L1, L2, L3,
L4, and S1
[0374] Molecules having a CD3-binding domain different from the
scFv structure were assessed. GPC3 ERY18 (FIG. 17K) was constructed
in which the VH and VL domains of an anti-CD3 antibody were linked
to the C termini of the two H chains of an anti-cancer antigen
(GPC3) IgG. In this construction, a series of molecules (GPC3 ERY18
L1, L2, L3, and L4) having one to four linker units
(Gly-Gly-Gly-Gly-Ser) at the junction were produced. At the same
time, another molecule (GPC3 ERY18 S1) was constructed in which
amino acids at appropriate positions were substituted with Cys to
allow introduction of a disulfide bond.
[0375] Specifically, a method known to those skilled in the art,
such as PCR using primers containing appropriate additional
sequences as in the method described above, was performed to
construct a series of expression vectors into which a
polynucleotide encoding GPC3 ERY18 L1_Hh (SEQ ID NO: 49; the mature
sequence does not contain the amino terminal 19 amino acids, which
serve as a signal sequence), GPC3 ERY18 L1_Hk (SEQ ID NO: 50; the
mature sequence does not contain the amino terminal 19 amino acids,
which serve as a signal sequence), GPC3 ERY18 L2_Hh (SEQ ID NO: 51;
the mature sequence does not contain the amino terminal 19 amino
acids, which serve as a signal sequence), GPC3 ERY18 L2_Hk (SEQ ID
NO:52; the mature sequence does not contain the amino terminal 19
amino acids, which serve as a signal sequence), GPC3 ERY18 L3_Hh
(SEQ ID NO: 53; the mature sequence does not contain the amino
terminal 19 amino acids, which serve as a signal sequence), GPC3
ERY18 L3_Hk (SEQ ID NO: 54; the mature sequence does not contain
the amino terminal 19 amino acids, which serve as a signal
sequence), GPC3 ERY18 L4_Hh (SEQ ID NO: 55; the mature sequence
does not contain the amino terminal 19 amino acids, which serve as
a signal sequence), GPC3 ERY18 L4_Hk (SEQ ID NO: 56; the mature
sequence does not contain the amino terminal 19 amino acids, which
serve as a signal sequence), GPC3 ERY18 S1_Hh (SEQ ID NO: 57; the
mature sequence does not contain the amino terminal 19 amino acids,
which serve as a signal sequence), or GPC3 ERY18 S1_Hk (SEQ ID NO:
58; the mature sequence does not contain the amino terminal 19
amino acids, which serve as a signal sequence) was inserted.
[0376] The following combinations of expression vectors were
introduced into FreeStyle293-F cells to express each designed
molecule transiently.
G. Designed Molecule: GPC3 ERY18 L1
[0377] Expression vectors: GPC3 ERY18 L1_Hh (SEQ ID NO: 49; the
mature sequence does not contain the amino terminal 19 amino acids,
which serve as a signal sequence), GPC3 ERY18 L1_Hk (SEQ ID NO: 50;
the mature sequence does not contain the amino terminal 19 amino
acids, which serve as a signal sequence), and GPC3 ERY7 L
H. Designed Molecule: GPC3 ERY18 L2
[0378] Expression vectors: GPC3 ERY18 L2_Hh (SEQ ID NO: 51; the
mature sequence does not contain the amino terminal 19 amino acids,
which serve as a signal sequence), GPC3 ERY18 L2_Hk (SEQ ID NO: 52;
the mature sequence does not contain the amino terminal 19 amino
acids, which serve as a signal sequence), and GPC3 ERY7 L
I. Designed Molecule: GPC3 ERY18 L3
[0379] Expression vectors: GPC3 ERY18 L3_Hh (SEQ ID NO: 53; the
mature sequence does not contain the amino terminal 19 amino acids,
which serve as a signal sequence), GPC3 ERY18 L3_Hk (SEQ ID NO: 54;
the mature sequence does not contain the amino terminal 19 amino
acids, which serve as a signal sequence), and GPC3 ERY7 L
J. Designed Molecule: GPC3 ERY18 L4
[0380] Expression vectors: GPC3 ERY18 L4_Hh (SEQ ID NO: 55; the
mature sequence does not contain the amino terminal 19 amino acids,
which serve as a signal sequence), GPC3 ERY18 L4_Hk (SEQ ID NO: 56;
the mature sequence does not contain the amino terminal 19 amino
acids, which serve as a signal sequence), and GPC3 ERY7_L
K. Designed Molecule: GPC3 ERY18 S1
[0381] Expression vectors: GPC3 ERY18 S1_Hh (SEQ ID NO: 57; the
mature sequence does not contain the amino terminal 19 amino acids,
which serve as a signal sequence), GPC3 ERY18 S1_Hk (SEQ ID NO: 58;
the mature sequence does not contain the amino terminal 19 amino
acids, which serve as a signal sequence), and GPC3 ERY7 L
[0382] The resulting culture supernatant was loaded onto an Anti
FLAG M2 column (Sigma). After washing, the column was eluted with
0.1 mg/ml FLAG peptide (Sigma). A fraction containing the designed
molecule was loaded onto a HisTrap HP column (GE Healthcare). After
washing, the column was eluted with an imidazole concentration
gradient. A fraction containing the designed molecule was
concentrated by ultrafiltration. Then, the fraction was loaded onto
a Superdex 200 column (GE Healthcare). Only a monomer fraction was
collected from the eluate to obtain each purified designed
molecule.
[0383] GPC3 ERY18 L1, GPC3 ERY18L2, GPC3 ERY18L3, GPC3 ERY18L4, and
GPC3 ERY18S1 molecules were each assessed for the in vitro
cytotoxic activity (FIGS. 12 and 13). The result showed that all
molecules except GPC3 ERY18 L1 had an activity comparable to that
of GPC3 ERY10-1. This result demonstrates that molecules having a
non-scFv structure have a comparable cytotoxic activity. The
structure where the VH and VL domains of CD3 antibody are each
linked to the C termini of two H chains of an anti-cancer antigen
(GPC3) IgG is expected to contribute to stabilization of the
polypeptide complexes of the present invention.
[Example 5] Construction and Assessment of GPC3 ERY19-3
[0384] Next, molecules having a Fab-like CD3-binding domain were
assessed. GPC3 ERY19-3 (FIG. 17L) was constructed in which the VH
and CH1 domains, and VL and CL domains of CD3 antibody were each
linked to the C termini of the two H chains of an anti-cancer
antigen (GPC3) IgG antibody. Specifically, by a method known to
those skilled in the art, such as PCR using primers containing
appropriate additional sequences in the same manner as in the
method described above, expression vectors were constructed into
which a polynucleotide encoding GPC3 ERY19-3_Hh (SEQ ID NO: 59; the
mature sequence does not contain the amino terminal 19 amino acids,
which serve as a signal sequence) or GPC3 ERY19-3_Hk (SEQ ID NO:
60; the mature sequence does not contain the amino terminal 19
amino acids, which serve as a signal sequence) was inserted.
[0385] The expression vectors for GPC3 ERY19-3_Hh (SEQ ID NO: 59;
the mature sequence does not contain the amino terminal 19 amino
acids, which serve as a signal sequence), GPC3 ERY19-3_Hk (SEQ ID
NO: 60; the mature sequence does not contain the amino terminal 19
amino acids, which serve as a signal sequence), and GPC3 ERY7_L
were co-introduced into FreeStyle293-F cells to express GPC3
ERY19-3 transiently. The resulting culture supernatant was loaded
onto a HiTrap rProtein A FF column (GE Healthcare). After washing,
the column was eluted with an acid. A fraction containing GPC3
ERY19-3 was concentrated by ultrafiltration, and then loaded onto a
Superdex 200 column (GE Healthcare). Only a monomeric GPC3 ERY19-3
fraction was collected from the eluate to obtain purified GPC3
ERY19-3.
[0386] The GPC3 ERY19-3 molecule was assessed for the in vitro
cytotoxic activity. The result showed that the molecule had an
activity comparable to GPC3 BiTE (FIG. 14). The CD3-binding domain
with a Fab-like structure is expected to contribute to
stabilization of the polypeptide complex molecules of the present
invention.
[Example 6] Preparation of Polypeptide Complexes Using a Protein a
Purification Step Alone by Introduction of Mutation into the CH3
Domain of GPC3 ERY 10-1
(1) Outline
[0387] In GPC3 ERY10-1 prepared in Example 3, the CH3 domain has
the knobs-into-holes structure. The desired GPC3 ERY10-1 molecule,
in which the two H chains were heteromerically associated together,
was purified by two types of affinity purification using the His
tag and FLAG tag attached to the C terminus of each H chain. If the
GPC3 ERY10-1 molecule is produced as a pharmaceutical, Protein A
chromatography is first performed on the culture supernatant of
GPC3 ERY10-1-expressing cells to purify a polypeptide complex
having a Fc domain. This step has to be followed by an additional
chromatographic purification step using His tag affinity
chromatography and FLAG tag affinity chromatography. This results
in increased costs for the purification process. Thus, this Example
examined molecular modifications that enable purification of the
desired GPC3 ERY10-1 molecule having the two heteromerically
associated H chains by Protein A chromatography only without using
a His tag and FLAG tag.
[0388] Specifically, modifications to eliminate the Protein A
binding in one of the two H chains were examined. As a result of
such modifications, when non-Protein A-binding H chains are
homomerically associated, the molecule cannot bind to Protein A and
therefore passes through Protein A chromatography. On the other
hand, a molecule in which a non-Protein A-binding H chain is
heteromerically associated with a Protein A-binding H chain, and a
molecule in which Protein A-binding H chains are homomerically
associated, may be separated using Protein A chromatography based
on difference in the affinity for Protein A. However, in the
antibody Fc domain, the binding site for Protein A overlaps with
the binding site for FcRn, which is crucial for the plasma
retention of antibody. Thus, it is necessary to selectively reduce
the Protein A-binding activity only, while maintaining the
FcRn-binding activity. As such a modification, substitution of His
at position 435 (EU numbering) with Arg was discovered. The
combination of this mutation with the mutations described in WO
2006/106905 (substituting Asp at position 356 (EU numbering) in one
of the H chains with Lys, and Lys at position 439 (EU numbering) in
the other H chain with Glu), which promote heteromeric association
of the two H chains, was tested as to whether it could enable
purification of polypeptide complexes such as GPC3 ERY10-1 using
Protein A chromatography only.
(2) Construction of Antibody Gene Expression Vectors and Expression
of Respective Antibodies
[0389] For the antibody H chain variable region, a gene encoding
GC33(2)H (anti-human Glypican-3 antibody H chain variable region,
SEQ ID NO: 61; the mature sequence does not contain the amino
terminal 19 amino acids, which serve as a signal sequence) was
constructed by a method known to those skilled in the art.
Similarly, for the antibody L chain, a gene encoding GC33-k0
(anti-human Glypican-3 antibody L chain, SEQ ID NO: 62; the mature
sequence does not contain the amino terminal 19 amino acids, which
serve as a signal sequence) was constructed by a method known to
those skilled in the art. Next, for the antibody H chain constant
region, the genes described below were constructed by a method
known to those skilled in the art.
L. Designed Molecule: LALA-G1d
[0390] LALA-G1d (SEQ ID NO: 63; the mature sequence does not
contain the amino terminal 19 amino acids, which serve as a signal
sequence), in which Leu at positions 234 and 235 (EU numbering)
have been substituted with Ala, Asn at position 297 has been
substituted with Ala, and Gly and Lys at the C-terminal have been
removed in the sequence of IgG1
M. Designed Molecule: LALA-G1d-CD3
[0391] LALA-G1d-CD3 (SEQ ID NO: 64; the mature sequence does not
contain the amino terminal 19 amino acids, which serve as a signal
sequence), in which a CD3 scFv (anti-human CD3 antibody H chain
variable region and anti-human CD3 antibody L chain variable region
are linked together via a polypeptide linker) has been linked to
the C terminus of LALA-G1d (SEQ ID NO: 63; the mature sequence does
not contain the amino terminal 19 amino acids, which serve as a
signal sequence)
N. Designed Molecule: LALA-G3S3E-G1d
[0392] LALA-G3S3E-G1d (SEQ ID NO: 65; the mature sequence does not
contain the amino terminal 19 amino acids, which serve as a signal
sequence), in which His at position 435 (EU numbering) has been
substituted with Arg, and Lys at position 439 (EU numbering) has
been substituted with Glu in the sequence of LALA-G1d (SEQ ID NO:
63; the mature sequence does not contain the amino terminal 19
amino acids, which serve as a signal sequence)
O. Designed Molecule: LALA-S3K-G1d-CD3
[0393] LALA-S3K-G1d-CD3 (SEQ ID NO: 66; the mature sequence does
not contain the amino terminal 19 amino acids, which serve as a
signal sequence), in which Asp at position 356 (EU numbering) has
been substituted with Lys in the sequence of LALA-G1d-CD3 (SEQ ID
NO: 64; the mature sequence does not contain the amino terminal 19
amino acids, which serve as a signal sequence)
[0394] Anti-human GPC3 antibody H chain genes NTA1L and NTA1R were
constructed by linking LALA-G1d-CD3 or LALA-G1d downstream of
GC33(2)H, respectively. Meanwhile, anti-human GPC3 antibody H chain
genes NTA2L and NTA2R were constructed by linking LALA-S3K-G1d-CD3
or LALA-G3S3E-G1d downstream of GC33(2)H, respectively.
[0395] Expression vectors for NTA1L, NTA1R, NTA2L, NTA2R (H
chains), and GC33-k0 (L chain) were constructed by inserting each
gene into an animal cell expression vector. These vectors were
combined as shown below and introduced into FreeStyle293 cells
(Invitrogen) by a method known to those skilled in the art to
transiently express the polypeptide complexes described below. As
shown below, the polypeptide complexes are referred to by the names
of introduced genes combined in the order of [first H chain/second
H chain/L chain].
NTA1L/NTA1R/GC33-k0
NTA2L/NTA2R/GC33-k0
(3) Purification of Expressed Samples and Assessment for
Heteromeric Complex Formation
[0396] Culture supernatant of FreeStyle293 cells (hereinafter
referred to as CM) containing a polypeptide complex shown below was
used as a sample.
NTA1L/NTA1R/GC33-k0
NTA2L/NTA2R/GC33-k0
[0397] CM was filtered through a .PHI.0.22-.mu.m filter and loaded
onto a rProtein A Sepharose Fast Flow column (GE Healthcare)
equilibrated with D-PBS. Washing steps 1 and 2, and elution step 1
were carried out using the buffers shown in Table 1. The loading
amount of CM was adjusted so that the loading amount of antibody
was 20 mg/ml resin. Eluted fractions were collected and analyzed by
size exclusion chromatography to identify the components in the
fractions.
TABLE-US-00002 TABLE 1 EQUILIBRATION D-PBS WASH 1 1 mM sodium
acetate, 150 mM NaCl, pH 6.5 WASH 2 0.3 mM HCl, 150 mM NaCl, pH 3.7
ELUTION 1 2 mM HCl, pH 2.7
[0398] The result of size exclusion chromatography analysis of each
eluted fraction is shown in FIG. 15 and Table 2. The values
indicate percent area of elution peak. When NTA1L/NTA1R/GC33-k0 or
NTA2L/NTA2R/GC33-k0 was expressed, the anti-GPC3 homomeric antibody
(NTA1L/GC33-k0 or NTA2L/GC33-k0) was almost undetectable in CM.
Meanwhile, the anti-GPC3 homomeric antibody (NTA2R/GC33-k0) was
only about 2% in CM where NTA2L/NTA2R/GC33-k0 was expressed,
whereas it was about 76% in CM where NTA1L/NTA1R/GC33-k0 was
expressed. This result demonstrates that, when His at position 435
(EU numbering) is substituted with Arg and, in order to allow
efficient formation of the heteromeric molecule of the respective H
chains, Asp at position 356 (EU numbering) in the polypeptide
sequence of one of the H chains is substituted with Lys and Lys at
position 439 (EU numbering) in the polypeptide sequence of the
other H chain is substituted with Glu, heteromeric polypeptide
complexes having the same molecular form as GPC3 ERY10-1 can be
efficiently purified with a purity of more than 98% by only the
purification step using Protein A.
TABLE-US-00003 TABLE 2 CD3 GPC3 homomeric Heteromeric homomeric
antibody antibody antibody NTA1L/NTA1R/GC33-k0 0.7 23.5 75.8
NTA2L/NTA2R/GC33-k0 -- 98.2 1.8
[Example 7] Construction and Assessment of GPC3 ERY 17-2 and GPC3
ERY 17-3
(1) Construction of GPC3 ERY 17-2 and GPC3 ERY 17-3
[0399] Next, a molecule was constructed by using an anti-cancer
antigen (GPC3) IgG as a backbone and substituting one of the Fabs
with a CD3 epsilon-binding domain. As in the above-described cases,
the Fc of the backbone IgG was silent Fc having a reduced FcgR
(Fc.gamma. receptor)-binding activity. For the CD3 epsilon-binding
domains, the VH and VL domains of anti-CD3 epsilon Fab were
exchanged to produce GPC3 ERY17-2 (FIG. 19, diagram A), and the CH1
and CL domains were exchanged to produce GPC3 ERY17-3 (FIG. 19,
diagram B).
[0400] Specifically, by a method known to those skilled in the art,
such as PCR using primers containing the same appropriate
additional sequences as in the method described above, a series of
expression vectors were constructed into which a polynucleotide
encoding ERY17-2_Hh (SEQ ID NO: 73; the mature sequence does not
contain the amino terminal 19 amino acids, which serve as a signal
sequence), ERY17-2_L (SEQ ID NO: 74; the mature sequence does not
contain the amino terminal 19 amino acids, which serve as a signal
sequence), ERY17-3_Hh (SEQ ID NO: 75; the mature sequence does not
contain the amino terminal 19 amino acids, which serve as a signal
sequence), or ERY17-3_L (SEQ ID NO: 76; the mature sequence does
not contain the amino terminal 19 amino acids, which serve as a
signal sequence) was inserted.
[0401] The following combinations of expression vectors were
introduced into FreeStyle293-F cells to express each designed
molecule transiently.
P. Designed Molecule: GPC3 ERY17-2
[0402] Polypeptides encoded by polynucleotides inserted in
expression vectors: GPC3 ERY8-2_Hk, GPC3 ERY7_L, ERY17-2_Hh (SEQ ID
NO: 73; the mature sequence does not contain the amino terminal 19
amino acids, which serve as a signal sequence), and ERY17-2_L (SEQ
ID NO: 74; the mature sequence does not contain the amino terminal
19 amino acids, which serve as a signal sequence)
Q. Designed Molecule: GPC3 ERY17-3
[0403] Polypeptides encoded by polynucleotides inserted in
expression vectors: GPC3 ERY8-2_Hk, GPC3 ERY7_L, ERY17-3_Hh (SEQ ID
NO: 75; the mature sequence does not contain the amino terminal 19
amino acids, which serve as a signal sequence), and ERY17-3_L (SEQ
ID NO: 76; the mature sequence does not contain the amino terminal
19 amino acids, which serve as a signal sequence)
(2) Purification of GPC3 ERY 17-2 and GPC3 ERY 17-3
[0404] The resulting culture supernatant was loaded onto an Anti
FLAG M2 column (Sigma). After washing, the column was eluted with
0.1 mg/ml FLAG peptide (Sigma). A fraction containing the designed
molecule was loaded onto a HisTrap HP column (GE Healthcare). After
washing, the column was eluted with an imidazole concentration
gradient. A fraction containing the designed molecule was
concentrated by ultrafiltration. Then, the fraction was loaded onto
a Superdex 200 column (GE Healthcare). Only a monomer fraction was
collected from the eluate to obtain each purified designed
molecule.
(3) Cytotoxic Activity of GPC3 ERY 17-2 and GPC3 ERY 17-3
[0405] GPC3 ERY 17-2 and GPC3 ERY 17-3 were assessed for the in
vitro cytotoxic activity (FIG. 20). The result showed that both
molecules clearly exhibited a greater cytotoxic activity than GPC3
BiTE. Thus, the present invention for the first time demonstrates
that molecules in which an anti-cancer antigen IgG is used as a
backbone and one of the Fabs is substituted with a CD3
epsilon-binding domain exhibit a cytotoxic activity comparable to
or greater than that of BiTE.
(4) Efficacy Test for GPC3 ERY17-2 Using PC-10 T Cell Transfer
Model
[0406] GPC3 ERY17-2, which was demonstrated to have a cytotoxic
activity comparable to or greater than that of GPC3 BiTE in the in
vitro assay, was assessed for the in vivo efficacy using the PC-10
T cell transfer model. Specifically, the efficacy test of GPC3
ERY17-2 using the PC-10 T cell transfer model was carried out as
follows. T cell expansion culture was carried out using T cell
activation/expansion kit/human (MACS Miltenyi biotec) and PBMCs
isolated from blood collected from healthy volunteers. Human lung
squamous carcinoma cell line PC-10 cells (Immuno-Biological
Laboratories Co., Ltd.) (1.times.10.sup.7 cells) were mixed with
Matrigel Basement Membrane Matrix (BD), and then transplanted
subcutaneously to the inguinal region of NOD scid mice (CLEA Japan
Inc.; female, 7W). The day of transplantation was designated day 0.
On the day before transplantation and days 13, 17, 21, and 25, an
anti-asialo-GM1 antibody (Wako Pure Chemical Industries) was
intraperitoneally administered to the mice at 0.2 mg/head. On day
13 after transplantation, the mice were grouped by the tumor size
and body weight. On day 14 after transplantation, T cells prepared
by expansion culture as described above were transplanted at
3.times.10.sup.7 cells/head to the peritoneal cavity. After two
hours of transplantation, GPC ERY17-2 was intravenously
administered at 30 .mu.g/head. GPC ERY17-2 was administered five
times in total on days 14, 15, 16, 17, and 18.
[0407] The result showed that a clear anti-tumor effect was also
observed in the GPC3 ERY17-2 administration group of this model, as
compared to the solvent administration group (FIG. 21).
[0408] The finding described above demonstrates that molecules in
which an anti-cancer antigen IgG is used as a backbone and one of
the Fabs is substituted with a CD3 epsilon-binding domain produce a
clear anti-tumor effect in vivo.
[Example 8] Construction and Assessment of GPC3 ERY17-2-M20
(1) Construction of GPC3 ERY17-2-M20
[0409] Next, a molecule retaining the desired activity even after
alterations to the CD3 epsilon-binding domain was constructed. GPC3
ERY17-2-M20 (FIG. 19, diagram A) was constructed in which the
sequence of the CD3 epsilon-binding domain was altered.
Specifically, using as a template an expression vector for an
anti-CD3 antibody (M20), a method known to a person skilled in the
art such as PCR using primers containing the same appropriate
sequences as in the above-described methods was performed to
produce a series of expression vectors into which a polynucleotide
encoding ERY17-2-M20_Hh (SEQ ID NO: 77; the mature sequence does
not contain the amino terminal 19 amino acids, which serve as a
signal sequence) or ERY17-2-M20_L (SEQ ID NO: 78; the mature
sequence does not contain the amino terminal 19 amino acids, which
serve as a signal sequence) was inserted.
(2) Purification of GPC3 ERY17-2-M20
[0410] The expression vectors for GPC3 ERY8-2_Hk, GPC3 ERY7_L,
ERY17-2-M20_Hh (SEQ ID NO: 77), and ERY17-2-M20_L (SEQ ID NO: 78)
were co-introduced into FreeStyle293-F cells to express GPC3
ERY17-2-M20 transiently. The resulting culture supernatant was
filtered through a .PHI.0.22-.mu.m filter, and then loaded onto an
equilibrated rProtein A Sepharose Fast Flow column (GE Healthcare).
Purified GPC3 ERY17-2-M20 was obtained by washing steps 1 and 2,
and elution step 1 using the buffers shown in Table 3.
TABLE-US-00004 TABLE 3 FreeStyle 293 Expression Medium
(Invitrogen), EQUILIBRATION 1% Pen Strep (Invitrogen) WASH 1 1 mM
sodium acetate, 150 mM NaCl, pH 6.5 WASH 2 0.3 mM HCl, 150 mM NaCl,
pH 3.7 ELUTION 1 2 mM HCl, pH 2.7
(3) Cytotoxic Activity of GPC3 ERY17-2-M20
[0411] GPC3 ERY17-2-M20 was tested for the in vitro cytotoxic
activity, and showed a cytotoxic activity comparable to that of
GPC3 ERY17-2 (FIG. 22). This finding demonstrates that even
molecules having an altered sequence in the CD3 epsilon-binding
domain have a comparable cytotoxic activity.
[Example 9] Construction and Assessment of EpCAM ERY17-2 and EpCAM
ERY17-3
(1) Construction of EpCAM ERY17-2 and EpCAM ERY17-3
[0412] Next, molecules targeting a different cancer antigen but
retaining the desired activity were constructed. EpCAM ERY17-2
(FIG. 19, diagram A), in which the anti-GPC3 Fab in GPC3 ERY17-2
was replaced with an anti-EpCAM Fab, and EpCAM ERY17-3 (FIG. 19,
diagram B), in which the anti-GPC3 Fab in GPC3 ERY17-3 was replaced
with an anti-EpCAM Fab, were produced. Specifically, using as a
template an expression vector for an anti-EpCAM antibody, a method
known to those skilled in the art such as PCR using primers
containing the same appropriate sequences as in the above-described
method was performed to produce a series of expression vectors into
which a polynucleotide encoding EpCAM ERY17_Hk (SEQ ID NO: 79; the
mature sequence does not contain the amino terminal 19 amino acids,
which serve as a signal sequence) or EpCAM ERY17_L (SEQ ID NO: 80;
the mature sequence does not contain the amino terminal 19 amino
acids, which serve as a signal sequence) was inserted.
[0413] The following combinations of expression vectors were
introduced into FreeStyle293-F cells to express each designed
molecule transiently.
R. Designed Molecule: EpCAM ERY17-2
[0414] Polypeptides encoded by polynucleotides inserted in
expression vectors: EpCAM ERY17_Hk (SEQ ID NO: 79; the mature
sequence does not contain the amino terminal 19 amino acids, which
serve as a signal sequence), EpCAM ERY17_L (SEQ ID NO: 80; the
mature sequence does not contain the amino terminal 19 amino acids,
which serve as a signal sequence), ERY17-2_Hh, and ERY17-2_L
S. Designed Molecule: EpCAM ERY17-3
[0415] Polypeptides encoded by polynucleotides inserted in
expression vectors: EpCAM ERY17_Hk, EpCAM ERY17_L, ERY17-3_Hh, and
ERY17-3_L
(2) Purification of EpCAM ERY17-2 and EpCAM ERY17-3
[0416] The resulting culture supernatant was loaded onto an Anti
FLAG M2 column (Sigma). After washing, the column was eluted with
0.1 mg/ml FLAG peptide (Sigma). A fraction containing the designed
molecule was loaded onto a HisTrap HP column (GE Healthcare). After
washing, the column was eluted with an imidazole concentration
gradient. A fraction containing the designed molecule was
concentrated by ultrafiltration. Then, the fraction was loaded onto
a Superdex 200 column (GE Healthcare). Only a monomer fraction was
collected from the eluate to obtain each purified designed
molecule.
(3) Cytotoxic Activity of EpCAM ERY17-2 and EpCAM ERY17-3
[0417] EpCAM ERY17-2 and EpCAM ERY17-3 were tested for the in vitro
cytotoxic activity, and both showed a strong cytotoxic activity
(FIG. 23). Thus, the present invention demonstrates that molecules
in which an anti-cancer antigen IgG is used as a backbone and one
of the Fabs is substituted with a CD3 epsilon-binding domain have a
cytotoxic activity even when the type of cancer antigen has been
changed.
[Example 10] Construction and Assessment of Bispecific Antibodies
with Modulated CH1/CL Interfacial Association
(1) Design of Bispecific Antibody
[0418] By introducing mutations into each of the CH1 and CL domains
of an bispecific antibody and thereby modulating the CH1/CL
interfacial association with the use of electric charge repulsion
at the CH1/CL interface, specific association may be allowed to
occur between the anti-GPC3 H chain and L chain and between the
anti-CD3 H chain and L chain. In order to modulate the CH1/CL
interfacial association using electric charge repulsion, amino acid
residues in CH1 of the H chains or CL of the L chains were
substituted with Lys, which is positively charged, or with Glu,
which is negatively charged.
(2) Construction of Expression Vectors for Antibody Genes and
Expression of Respective Antibodies
[0419] A bispecific antibody (FIG. 24A) was created by modulating
the CH1/CL interfacial association of anti-CD3 antibody M12 (H
chain, SEQ ID NO: 81; L chain, SEQ ID NO: 82) and anti-GPC3
antibody GC33(2) (H chain, SEQ ID NO: 83; L chain, SEQ ID NO: 84)
and further introducing Knobs-into-Holes (KiH) modifications (WO
1996/027011; Ridgway J B et al. (Protein Engineering (1996) 9,
617-621), Merchant A M et al. (Nat. Biotechnol. (1998) 16,
677-681)) into them to avoid the H chains from associating with
each other. A control bispecific antibody (FIG. 24B), in which
neither the CH1/CL interfacial association modulation nor the
Knobs-into-Holes (KiH) modifications were introduced, was also
constructed. Specifically, expression vectors having as an insert a
polynucleotide encoding M12_TH2h (SEQ ID NO: 85), in which several
amino acids in CH1 of the H chain of M12 (SEQ ID NO: 81) were
substituted with Lys, or M12_TL17 (SEQ ID NO: 86), in which several
amino acids in CL of the L chain (SEQ ID NO: 82) were substituted
with Glu, were constructed by a method known to those skilled in
the art. Likewise, expression vectors having as an insert a
polynucleotide encoding GC33(2)_TH13k (SEQ ID NO: 87) or
GC33(2)_TH15k (SEQ ID NO: 88), in which several amino acids in CH1
of the H chain of GC33(2) (SEQ ID NO: 83) were substituted with
Glu, or GC33(2)_TL16 (SEQ ID NO: 89) or GC33(2)_TL19 (SEQ ID NO:
90), in which several amino acids in CL of the L chain (SEQ ID NO:
84) were substituted with Lys, were constructed by a method known
to those skilled in the art.
[0420] The following combinations of expression vectors were
introduced into FreeStyle293-F cells to express each designed
molecule transiently.
T. Designed Molecule: GM1
[0421] Expression vectors: M12_TH2h (SEQ ID NO: 85), M12_TL17 (SEQ
ID NO: 86), GC33(2)_TH13k (SEQ ID NO: 87), and GC33(2)_TL16 (SEQ ID
NO: 89)
U. Designed Molecule: GM2
[0422] Expression vectors: M12_TH2h (SEQ ID NO: 85), M12_TL17 (SEQ
ID NO: 86), GC33(2)_TH15k (SEQ ID NO: 88), and GC33(2)_TL19 (SEQ ID
NO: 90)
V. Designed Molecule: GMO
[0423] Expression vectors: H chain of M12 (SEQ ID NO: 81), L chain
of M12 (SEQ ID NO: 82), H chain of GC33(2) (SEQ ID NO: 83), and L
chain of GC33(2) (SEQ ID NO: 84)
[0424] From the resulting culture supernatant, antibodies were
purified by a method known to those skilled in the art using
rProtein A Sepharose.TM. Fast Flow (GE Healthcare).
(3) Cytotoxic Activity of GM1, GM2, and GMO
[0425] Polypeptide complexes GM1, GM2, and GMO were assessed for
the in vitro cytotoxic activity. The result showed that GM1 and GM2
exhibited a comparable cytotoxic activity, and this activity was
clearly greater than that of GMO (FIG. 25). Thus, the present
invention demonstrates that the combination of the modulation of
CH1/CL interfacial association and the KiH modifications allows
efficient production of bispecific antibodies.
[Example 11] Construction and Assessment of EGFR ERY17-2
(1) Construction of EGFR ERY17-2
[0426] Furthermore, a molecule having the desired activity that
targets another cancer antigen was prepared. EGFR ERY17-2 (FIG. 19,
diagram A) was constructed by replacing the anti-GPC3 Fab of GPC3
ERY17-2 with the anti-EGFR Fab. Specifically, using as a template
an expression vector for an anti-EGFR antibody, a method known to
those skilled in the art such as PCR using primers containing the
same appropriate sequences as in the above-described method was
performed to produce a series of expression vectors into which the
polynucleotide encoding EGFR ERY17_Hk (SEQ ID NO: 91: the mature
sequence does not contain the amino terminal 19 amino acids, which
serve as a signal sequence) or EGFR ERY17_L (SEQ ID NO: 92: the
mature sequence does not contain the amino terminal 19 amino acids,
which serve as a signal sequence) was inserted.
[0427] The following combinations of expression vectors were
introduced into FreeStyle293-F cells to express each designed
molecule transiently.
W. Designed Molecule: EGFR ERY17-2
[0428] Polypeptides encoded by polynucleotides inserted in
expression vectors: EGFR ERY17_Hk (SEQ ID NO: 91: the mature
sequence does not contain the amino terminal 19 amino acids, which
serve as a signal sequence), EGFR ERY17_L (SEQ ID NO: 92: the
mature sequence does not contain the amino terminal 19 amino acids,
which serve as a signal sequence), ERY17-2_Hh, and ERY17-2_L.
(2) Purification of EGFR ERY17-2
[0429] The resulting culture supernatants were loaded onto an Anti
FLAG M2 column (Sigma). After washing, the column was eluted with
0.1 mg/mL FLAG peptide (Sigma). A fraction containing the designed
molecule was loaded onto a HisTrap HP column (GE Healthcare). After
washing, the column was eluted with an imidazole concentration
gradient. A fraction containing the designed molecule was
concentrated by ultrafiltration. Then, the fraction was loaded onto
a Superdex 200 column (GE Healthcare). Only a monomer fraction was
collected from the eluate to obtain each purified designed
molecule.
(3) Cytotoxic Activity of EGFR ERY17-2
[0430] EGFR ERY17-2 was tested for the in vitro cytotoxic activity,
and it showed a strong cytotoxic activity (FIG. 26). Thus, the
present invention demonstrates that molecules in which an
anti-cancer antigen IgG is used as a backbone and one of the Fabs
is substituted with a CD3 epsilon-binding domain have a cytotoxic
activity even when the cancer antigen is GPC3 or EpCAM or the type
of cancer antigen has been further changed.
INDUSTRIAL APPLICABILITY
[0431] The present invention provides novel polypeptide complexes
that retain the strong anti-tumor activity of BiTE and have a long
half-life in blood, as well as excellent safety properties that
result in no induction of cancer antigen-independent cytokine storm
or such. When the antigen-binding domain of a polypeptide complex
of the present invention is substituted, therapeutic agents that
comprise the polypeptide complex as an active ingredient for
inducing cellular cytotoxicity, can target and damage various cells
including cancer cells. Thus, various cancers can be treated or
prevented. This allows desirable treatments that are highly safe
and convenient, and reduce the physical burden for patients.
Sequence CWU 1
1
9211812DNAHomo sapiens 1atggccggga ccgtgcgcac cgcgtgcttg gtggtggcga
tgctgctcag cttggacttc 60ccgggacagg cgcagccccc gccgccgccg ccggacgcca
cctgtcacca agtccgctcc 120ttcttccaga gactgcagcc cggactcaag
tgggtgccag aaactcccgt gccaggatca 180gatttgcaag tatgtctccc
taagggccca acatgctgct caagaaagat ggaagaaaaa 240taccaactaa
cagcacgatt gaacatggaa cagctgcttc agtctgcaag tatggagctc
300aagttcttaa ttattcagaa tgctgcggtt ttccaagagg cctttgaaat
tgttgttcgc 360catgccaaga actacaccaa tgccatgttc aagaacaact
acccaagcct gactccacaa 420gcttttgagt ttgtgggtga atttttcaca
gatgtgtctc tctacatctt gggttctgac 480atcaatgtag atgacatggt
caatgaattg tttgacagcc tgtttccagt catctatacc 540cagctaatga
acccaggcct gcctgattca gccttggaca tcaatgagtg cctccgagga
600gcaagacgtg acctgaaagt atttgggaat ttccccaagc ttattatgac
ccaggtttcc 660aagtcactgc aagtcactag gatcttcctt caggctctga
atcttggaat tgaagtgatc 720aacacaactg atcacctgaa gttcagtaag
gactgtggcc gaatgctcac cagaatgtgg 780tactgctctt actgccaggg
actgatgatg gttaaaccct gtggcggtta ctgcaatgtg 840gtcatgcaag
gctgtatggc aggtgtggtg gagattgaca agtactggag agaatacatt
900ctgtcccttg aagaacttgt gaatggcatg tacagaatct atgacatgga
gaacgtactg 960cttggtctct tttcaacaat ccatgattct atccagtatg
tccagaagaa tgcaggaaag 1020ctgaccacca ctgaaactga gaagaaaata
tggcacttca aatatcctat cttcttcctg 1080tgtatagggc tagacttaca
gattggcaag ttatgtgccc attctcaaca acgccaatat 1140agatctgctt
attatcctga agatctcttt attgacaaga aagtattaaa agttgctcat
1200gtagaacatg aagaaacctt atccagccga agaagggaac taattcagaa
gttgaagtct 1260ttcatcagct tctatagtgc tttgcctggc tacatctgca
gccatagccc tgtggcggaa 1320aacgacaccc tttgctggaa tggacaagaa
ctcgtggaga gatacagcca aaaggcagca 1380aggaatggaa tgaaaaacca
gttcaatctc catgagctga aaatgaaggg ccctgagcca 1440gtggtcagtc
aaattattga caaactgaag cacattaacc agctcctgag aaccatgtct
1500atgcccaaag gtagagttct ggataaaaac ctggatgagg aagggtttga
aagtggagac 1560tgcggtgatg atgaagatga gtgcattgga ggctctggtg
atggaatgat aaaagtgaag 1620aatcagctcc gcttccttgc agaactggcc
tatgatctgg atgtggatga tgcgcctgga 1680aacagtcagc aggcaactcc
gaaggacaac gagataagca cctttcacaa cctcgggaac 1740gttcattccc
cgctgaagct tctcaccagc atggccatct cggtggtgtg cttcttcttc
1800ctggtgcact ga 18122603PRTHomo sapiens 2Met Ala Gly Thr Val Arg
Thr Ala Cys Leu Val Val Ala Met Leu Leu1 5 10 15Ser Leu Asp Phe Pro
Gly Gln Ala Gln Pro Pro Pro Pro Pro Pro Asp 20 25 30Ala Thr Cys His
Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly 35 40 45Leu Lys Trp
Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu Gln Val 50 55 60Cys Leu
Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys65 70 75
80Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala
85 90 95Ser Met Glu Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe
Gln 100 105 110Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr
Thr Asn Ala 115 120 125Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro
Gln Ala Phe Glu Phe 130 135 140Val Gly Glu Phe Phe Thr Asp Val Ser
Leu Tyr Ile Leu Gly Ser Asp145 150 155 160Ile Asn Val Asp Asp Met
Val Asn Glu Leu Phe Asp Ser Leu Phe Pro 165 170 175Val Ile Tyr Thr
Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu 180 185 190Asp Ile
Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe 195 200
205Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser Leu Gln
210 215 220Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu
Val Ile225 230 235 240Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp
Cys Gly Arg Met Leu 245 250 255Thr Arg Met Trp Tyr Cys Ser Tyr Cys
Gln Gly Leu Met Met Val Lys 260 265 270Pro Cys Gly Gly Tyr Cys Asn
Val Val Met Gln Gly Cys Met Ala Gly 275 280 285Val Val Glu Ile Asp
Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu 290 295 300Glu Leu Val
Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu305 310 315
320Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln Tyr Val Gln Lys
325 330 335Asn Ala Gly Lys Leu Thr Thr Thr Glu Thr Glu Lys Lys Ile
Trp His 340 345 350Phe Lys Tyr Pro Ile Phe Phe Leu Cys Ile Gly Leu
Asp Leu Gln Ile 355 360 365Gly Lys Leu Cys Ala His Ser Gln Gln Arg
Gln Tyr Arg Ser Ala Tyr 370 375 380Tyr Pro Glu Asp Leu Phe Ile Asp
Lys Lys Val Leu Lys Val Ala His385 390 395 400Val Glu His Glu Glu
Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile Gln 405 410 415Lys Leu Lys
Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr Ile 420 425 430Cys
Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn Gly 435 440
445Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly Met
450 455 460Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro
Glu Pro465 470 475 480Val Val Ser Gln Ile Ile Asp Lys Leu Lys His
Ile Asn Gln Leu Leu 485 490 495Arg Thr Met Ser Met Pro Lys Gly Arg
Val Leu Asp Lys Asn Leu Asp 500 505 510Glu Glu Gly Phe Glu Ser Gly
Asp Cys Gly Asp Asp Glu Asp Glu Cys 515 520 525Ile Gly Gly Ser Gly
Asp Gly Met Ile Lys Val Lys Asn Gln Leu Arg 530 535 540Phe Leu Ala
Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro Gly545 550 555
560Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe His
565 570 575Asn Leu Gly Asn Val His Ser Pro Leu Lys Leu Leu Thr Ser
Met Ala 580 585 590Ile Ser Val Val Cys Phe Phe Phe Leu Val His 595
6003945DNAHomo sapiens 3atggcgcccc cgcaggtcct cgcgttcggg cttctgcttg
ccgcggcgac ggcgactttt 60gccgcagctc aggaagaatg tgtctgtgaa aactacaagc
tggccgtaaa ctgctttgtg 120aataataatc gtcaatgcca gtgtacttca
gttggtgcac aaaatactgt catttgctca 180aagctggctg ccaaatgttt
ggtgatgaag gcagaaatga atggctcaaa acttgggaga 240agagcaaaac
ctgaaggggc cctccagaac aatgatgggc tttatgatcc tgactgcgat
300gagagcgggc tctttaaggc caagcagtgc aacggcacct ccatgtgctg
gtgtgtgaac 360actgctgggg tcagaagaac agacaaggac actgaaataa
cctgctctga gcgagtgaga 420acctactgga tcatcattga actaaaacac
aaagcaagag aaaaacctta tgatagtaaa 480agtttgcgga ctgcacttca
gaaggagatc acaacgcgtt atcaactgga tccaaaattt 540atcacgagta
ttttgtatga gaataatgtt atcactattg atctggttca aaattcttct
600caaaaaactc agaatgatgt ggacatagct gatgtggctt attattttga
aaaagatgtt 660aaaggtgaat ccttgtttca ttctaagaaa atggacctga
cagtaaatgg ggaacaactg 720gatctggatc ctggtcaaac tttaatttat
tatgttgatg aaaaagcacc tgaattctca 780atgcagggtc taaaagctgg
tgttattgct gttattgtgg ttgtggtgat agcagttgtt 840gctggaattg
ttgtgctggt tatttccaga aagaagagaa tggcaaagta tgagaaggct
900gagataaagg agatgggtga gatgcatagg gaactcaatg cataa 9454314PRTHomo
sapiens 4Met Ala Pro Pro Gln Val Leu Ala Phe Gly Leu Leu Leu Ala
Ala Ala1 5 10 15Thr Ala Thr Phe Ala Ala Ala Gln Glu Glu Cys Val Cys
Glu Asn Tyr 20 25 30Lys Leu Ala Val Asn Cys Phe Val Asn Asn Asn Arg
Gln Cys Gln Cys 35 40 45Thr Ser Val Gly Ala Gln Asn Thr Val Ile Cys
Ser Lys Leu Ala Ala 50 55 60Lys Cys Leu Val Met Lys Ala Glu Met Asn
Gly Ser Lys Leu Gly Arg65 70 75 80Arg Ala Lys Pro Glu Gly Ala Leu
Gln Asn Asn Asp Gly Leu Tyr Asp 85 90 95Pro Asp Cys Asp Glu Ser Gly
Leu Phe Lys Ala Lys Gln Cys Asn Gly 100 105 110Thr Ser Met Cys Trp
Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp 115 120 125Lys Asp Thr
Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile 130 135 140Ile
Ile Glu Leu Lys His Lys Ala Arg Glu Lys Pro Tyr Asp Ser Lys145 150
155 160Ser Leu Arg Thr Ala Leu Gln Lys Glu Ile Thr Thr Arg Tyr Gln
Leu 165 170 175Asp Pro Lys Phe Ile Thr Ser Ile Leu Tyr Glu Asn Asn
Val Ile Thr 180 185 190Ile Asp Leu Val Gln Asn Ser Ser Gln Lys Thr
Gln Asn Asp Val Asp 195 200 205Ile Ala Asp Val Ala Tyr Tyr Phe Glu
Lys Asp Val Lys Gly Glu Ser 210 215 220Leu Phe His Ser Lys Lys Met
Asp Leu Thr Val Asn Gly Glu Gln Leu225 230 235 240Asp Leu Asp Pro
Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala 245 250 255Pro Glu
Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile Ala Val Ile 260 265
270Val Val Val Val Ile Ala Val Val Ala Gly Ile Val Val Leu Val Ile
275 280 285Ser Arg Lys Lys Arg Met Ala Lys Tyr Glu Lys Ala Glu Ile
Lys Glu 290 295 300Met Gly Glu Met His Arg Glu Leu Asn Ala305
31054PRTArtificialan artificially synthesized sequence 5Gly Gly Gly
Ser164PRTArtificialan artificially synthesized sequence 6Ser Gly
Gly Gly175PRTArtificialan artificially synthesized sequence 7Gly
Gly Gly Gly Ser1 585PRTArtificialan artificially synthesized
sequence 8Ser Gly Gly Gly Gly1 596PRTArtificialan artificially
synthesized sequence 9Gly Gly Gly Gly Gly Ser1 5106PRTArtificialan
artificially synthesized sequence 10Ser Gly Gly Gly Gly Gly1
5117PRTArtificialan artificially synthesized sequence 11Gly Gly Gly
Gly Gly Gly Ser1 5127PRTArtificialan artificially synthesized
sequence 12Ser Gly Gly Gly Gly Gly Gly1 5131125DNAHomo sapiens
13atgtggttct tgacaactct gctcctttgg gttccagttg atgggcaagt ggacaccaca
60aaggcagtga tcactttgca gcctccatgg gtcagcgtgt tccaagagga aaccgtaacc
120ttgcactgtg aggtgctcca tctgcctggg agcagctcta cacagtggtt
tctcaatggc 180acagccactc agacctcgac ccccagctac agaatcacct
ctgccagtgt caatgacagt 240ggtgaataca ggtgccagag aggtctctca
gggcgaagtg accccataca gctggaaatc 300cacagaggct ggctactact
gcaggtctcc agcagagtct tcacggaagg agaacctctg 360gccttgaggt
gtcatgcgtg gaaggataag ctggtgtaca atgtgcttta ctatcgaaat
420ggcaaagcct ttaagttttt ccactggaat tctaacctca ccattctgaa
aaccaacata 480agtcacaatg gcacctacca ttgctcaggc atgggaaagc
atcgctacac atcagcagga 540atatctgtca ctgtgaaaga gctatttcca
gctccagtgc tgaatgcatc tgtgacatcc 600ccactcctgg aggggaatct
ggtcaccctg agctgtgaaa caaagttgct cttgcagagg 660cctggtttgc
agctttactt ctccttctac atgggcagca agaccctgcg aggcaggaac
720acatcctctg aataccaaat actaactgct agaagagaag actctgggtt
atactggtgc 780gaggctgcca cagaggatgg aaatgtcctt aagcgcagcc
ctgagttgga gcttcaagtg 840cttggcctcc agttaccaac tcctgtctgg
tttcatgtcc ttttctatct ggcagtggga 900ataatgtttt tagtgaacac
tgttctctgg gtgacaatac gtaaagaact gaaaagaaag 960aaaaagtggg
atttagaaat ctctttggat tctggtcatg agaagaaggt aatttccagc
1020cttcaagaag acagacattt agaagaagag ctgaaatgtc aggaacaaaa
agaagaacag 1080ctgcaggaag gggtgcaccg gaaggagccc cagggggcca cgtag
112514374PRTHomo sapiens 14Met Trp Phe Leu Thr Thr Leu Leu Leu Trp
Val Pro Val Asp Gly Gln1 5 10 15Val Asp Thr Thr Lys Ala Val Ile Thr
Leu Gln Pro Pro Trp Val Ser 20 25 30Val Phe Gln Glu Glu Thr Val Thr
Leu His Cys Glu Val Leu His Leu 35 40 45Pro Gly Ser Ser Ser Thr Gln
Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60Thr Ser Thr Pro Ser Tyr
Arg Ile Thr Ser Ala Ser Val Asn Asp Ser65 70 75 80Gly Glu Tyr Arg
Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95Gln Leu Glu
Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105 110Val
Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys 115 120
125Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe
130 135 140Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr
Asn Ile145 150 155 160Ser His Asn Gly Thr Tyr His Cys Ser Gly Met
Gly Lys His Arg Tyr 165 170 175Thr Ser Ala Gly Ile Ser Val Thr Val
Lys Glu Leu Phe Pro Ala Pro 180 185 190Val Leu Asn Ala Ser Val Thr
Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205Thr Leu Ser Cys Glu
Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220Leu Tyr Phe
Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn225 230 235
240Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly
245 250 255Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu
Lys Arg 260 265 270Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln
Leu Pro Thr Pro 275 280 285Val Trp Phe His Val Leu Phe Tyr Leu Ala
Val Gly Ile Met Phe Leu 290 295 300Val Asn Thr Val Leu Trp Val Thr
Ile Arg Lys Glu Leu Lys Arg Lys305 310 315 320Lys Lys Trp Asp Leu
Glu Ile Ser Leu Asp Ser Gly His Glu Lys Lys 325 330 335Val Ile Ser
Ser Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys 340 345 350Cys
Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu Gly Val His Arg Lys 355 360
365Glu Pro Gln Gly Ala Thr 37015951DNAHomo sapiens 15atgactatgg
agacccaaat gtctcagaat gtatgtccca gaaacctgtg gctgcttcaa 60ccattgacag
ttttgctgct gctggcttct gcagacagtc aagctgctcc cccaaaggct
120gtgctgaaac ttgagccccc gtggatcaac gtgctccagg aggactctgt
gactctgaca 180tgccaggggg ctcgcagccc tgagagcgac tccattcagt
ggttccacaa tgggaatctc 240attcccaccc acacgcagcc cagctacagg
ttcaaggcca acaacaatga cagcggggag 300tacacgtgcc agactggcca
gaccagcctc agcgaccctg tgcatctgac tgtgctttcc 360gaatggctgg
tgctccagac ccctcacctg gagttccagg agggagaaac catcatgctg
420aggtgccaca gctggaagga caagcctctg gtcaaggtca cattcttcca
gaatggaaaa 480tcccagaaat tctcccattt ggatcccacc ttctccatcc
cacaagcaaa ccacagtcac 540agtggtgatt accactgcac aggaaacata
ggctacacgc tgttctcatc caagcctgtg 600accatcactg tccaagtgcc
cagcatgggc agctcttcac caatgggggt cattgtggct 660gtggtcattg
cgactgctgt agcagccatt gttgctgctg tagtggcctt gatctactgc
720aggaaaaagc ggatttcagc caattccact gatcctgtga aggctgccca
atttgagcca 780cctggacgtc aaatgattgc catcagaaag agacaacttg
aagaaaccaa caatgactat 840gaaacagctg acggcggcta catgactctg
aaccccaggg cacctactga cgatgataaa 900aacatctacc tgactcttcc
tcccaacgac catgtcaaca gtaataacta a 95116316PRTHomo sapiens 16Met
Thr Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu1 5 10
15Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp
20 25 30Ser Gln Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro
Trp 35 40 45Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln
Gly Ala 50 55 60Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn
Gly Asn Leu65 70 75 80Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe
Lys Ala Asn Asn Asn 85 90 95Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly
Gln Thr Ser Leu Ser Asp 100 105 110Pro Val His Leu Thr Val Leu Ser
Glu Trp Leu Val Leu Gln Thr Pro 115 120 125His Leu Glu Phe Gln Glu
Gly Glu Thr Ile Met Leu Arg Cys His Ser 130 135 140Trp Lys Asp Lys
Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys145 150 155 160Ser
Gln Lys Phe Ser His Leu Asp Pro Thr Phe Ser Ile Pro Gln Ala 165 170
175Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr
180 185 190Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val
Pro Ser 195 200 205Met Gly Ser Ser Ser Pro Met Gly Val Ile Val Ala
Val Val Ile Ala 210 215 220Thr Ala Val Ala Ala Ile Val Ala Ala Val
Val Ala Leu Ile Tyr Cys225 230 235 240Arg Lys Lys Arg Ile Ser Ala
Asn Ser Thr Asp Pro Val Lys Ala Ala 245 250 255Gln Phe Glu
Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln 260 265 270Leu
Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met 275 280
285Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu
290 295 300Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn305 310
31517876DNAHomo sapiens 17atgggaatcc tgtcattctt acctgtcctt
gccactgaga gtgactgggc tgactgcaag 60tccccccagc cttggggtca tatgcttctg
tggacagctg tgctattcct ggctcctgtt 120gctgggacac ctgcagctcc
cccaaaggct gtgctgaaac tcgagcccca gtggatcaac 180gtgctccagg
aggactctgt gactctgaca tgccggggga ctcacagccc tgagagcgac
240tccattcagt ggttccacaa tgggaatctc attcccaccc acacgcagcc
cagctacagg 300ttcaaggcca acaacaatga cagcggggag tacacgtgcc
agactggcca gaccagcctc 360agcgaccctg tgcatctgac tgtgctttct
gagtggctgg tgctccagac ccctcacctg 420gagttccagg agggagaaac
catcgtgctg aggtgccaca gctggaagga caagcctctg 480gtcaaggtca
cattcttcca gaatggaaaa tccaagaaat tttcccgttc ggatcccaac
540ttctccatcc cacaagcaaa ccacagtcac agtggtgatt accactgcac
aggaaacata 600ggctacacgc tgtactcatc caagcctgtg accatcactg
tccaagctcc cagctcttca 660ccgatgggga tcattgtggc tgtggtcact
gggattgctg tagcggccat tgttgctgct 720gtagtggcct tgatctactg
caggaaaaag cggatttcag ccaatcccac taatcctgat 780gaggctgaca
aagttggggc tgagaacaca atcacctatt cacttctcat gcacccggat
840gctctggaag agcctgatga ccagaaccgt atttag 87618291PRTHomo sapiens
18Met Gly Ile Leu Ser Phe Leu Pro Val Leu Ala Thr Glu Ser Asp Trp1
5 10 15Ala Asp Cys Lys Ser Pro Gln Pro Trp Gly His Met Leu Leu Trp
Thr 20 25 30Ala Val Leu Phe Leu Ala Pro Val Ala Gly Thr Pro Ala Ala
Pro Pro 35 40 45Lys Ala Val Leu Lys Leu Glu Pro Gln Trp Ile Asn Val
Leu Gln Glu 50 55 60Asp Ser Val Thr Leu Thr Cys Arg Gly Thr His Ser
Pro Glu Ser Asp65 70 75 80Ser Ile Gln Trp Phe His Asn Gly Asn Leu
Ile Pro Thr His Thr Gln 85 90 95Pro Ser Tyr Arg Phe Lys Ala Asn Asn
Asn Asp Ser Gly Glu Tyr Thr 100 105 110Cys Gln Thr Gly Gln Thr Ser
Leu Ser Asp Pro Val His Leu Thr Val 115 120 125Leu Ser Glu Trp Leu
Val Leu Gln Thr Pro His Leu Glu Phe Gln Glu 130 135 140Gly Glu Thr
Ile Val Leu Arg Cys His Ser Trp Lys Asp Lys Pro Leu145 150 155
160Val Lys Val Thr Phe Phe Gln Asn Gly Lys Ser Lys Lys Phe Ser Arg
165 170 175Ser Asp Pro Asn Phe Ser Ile Pro Gln Ala Asn His Ser His
Ser Gly 180 185 190Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Leu
Tyr Ser Ser Lys 195 200 205Pro Val Thr Ile Thr Val Gln Ala Pro Ser
Ser Ser Pro Met Gly Ile 210 215 220Ile Val Ala Val Val Thr Gly Ile
Ala Val Ala Ala Ile Val Ala Ala225 230 235 240Val Val Ala Leu Ile
Tyr Cys Arg Lys Lys Arg Ile Ser Ala Asn Pro 245 250 255Thr Asn Pro
Asp Glu Ala Asp Lys Val Gly Ala Glu Asn Thr Ile Thr 260 265 270Tyr
Ser Leu Leu Met His Pro Asp Ala Leu Glu Glu Pro Asp Asp Gln 275 280
285Asn Arg Ile 29019765DNAHomo sapiens 19atgtggcagc tgctcctccc
aactgctctg ctacttctag tttcagctgg catgcggact 60gaagatctcc caaaggctgt
ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120gacagtgtga
ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg
180tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga
cgctgccaca 240gttgacgaca gtggagagta caggtgccag acaaacctct
ccaccctcag tgacccggtg 300cagctagaag tccatatcgg ctggctgttg
ctccaggccc ctcggtgggt gttcaaggag 360gaagacccta ttcacctgag
gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420tatttacaga
atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca
480aaagccacac tcaaagacag cggctcctac ttctgcaggg ggcttgttgg
gagtaaaaat 540gtgtcttcag agactgtgaa catcaccatc actcaaggtt
tgtcagtgtc aaccatctca 600tcattctttc cacctgggta ccaagtctct
ttctgcttgg tgatggtact cctttttgca 660gtggacacag gactatattt
ctctgtgaag acaaacattc gaagctcaac aagagactgg 720aaggaccata
aatttaaatg gagaaaggac cctcaagaca aatga 76520254PRTHomo sapiens
20Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1
5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu
Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys
Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe
His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile
Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln
Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His
Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys
Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn
Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly
Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155
160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val
165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile
Thr Gln 180 185 190Gly Leu Ser Val Ser Thr Ile Ser Ser Phe Phe Pro
Pro Gly Tyr Gln 195 200 205Val Ser Phe Cys Leu Val Met Val Leu Leu
Phe Ala Val Asp Thr Gly 210 215 220Leu Tyr Phe Ser Val Lys Thr Asn
Ile Arg Ser Ser Thr Arg Asp Trp225 230 235 240Lys Asp His Lys Phe
Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 25021702DNAHomo sapiens
21atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact
60gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagcgt gcttgagaag
120gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc
cacacagtgg 180tttcacaatg agagcctcat ctcaagccag gcctcgagct
acttcattga cgctgccaca 240gtcaacgaca gtggagagta caggtgccag
acaaacctct ccaccctcag tgacccggtg 300cagctagaag tccatatcgg
ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360gaagacccta
ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca
420tatttacaga atggcaaaga caggaagtat tttcatcata attctgactt
ccacattcca 480aaagccacac tcaaagatag cggctcctac ttctgcaggg
ggcttgttgg gagtaaaaat 540gtgtcttcag agactgtgaa catcaccatc
actcaaggtt tggcagtgtc aaccatctca 600tcattctctc cacctgggta
ccaagtctct ttctgcttgg tgatggtact cctttttgca 660gtggacacag
gactatattt ctctgtgaag acaaacattt ga 70222233PRTHomo sapiens 22Met
Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10
15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro
20 25 30Gln Trp Tyr Ser Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys
Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His
Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp
Ala Ala Thr65 70 75 80Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Thr
Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile
Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu
Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr
Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Asp Arg
Lys Tyr Phe His His Asn Ser Asp Phe His Ile Pro145 150 155 160Lys
Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170
175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Ser Pro Pro Gly
Tyr Gln 195 200 205Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala
Val Asp Thr Gly 210 215 220Leu Tyr Phe Ser Val Lys Thr Asn Ile225
23023330PRTArtificialan artificially synthesized sequence 23Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33024326PRTArtificialan artificially synthesized sequence 24Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr
Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro
Pro Cys Pro Ala Pro 100 105 110Pro Val Ala Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 115 120 125Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 130 135 140Val Ser His Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly145 150 155 160Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170
175Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
180 185 190Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro 195 200 205Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
Gln Pro Arg Glu 210 215 220Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn225 230 235 240Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270Thr Pro Pro
Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295
300Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu305 310 315 320Ser Leu Ser Pro Gly Lys 32525377PRTArtificialan
artificially synthesized sequence 25Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Thr
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg
Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105
110Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg
115 120 125Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro
Arg Cys 130 135 140Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys
Pro Arg Cys Pro145 150 155 160Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys 165 170 175Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190Val Val Asp Val Ser
His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220Gln
Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His225 230
235 240Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys 245 250 255Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys Gly Gln 260 265 270Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met 275 280 285Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 290 295 300Ser Asp Ile Ala Val Glu Trp
Glu Ser Ser Gly Gln Pro Glu Asn Asn305 310 315 320Tyr Asn Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345
350Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln
355 360 365Lys Ser Leu Ser Leu Ser Pro Gly Lys 370
37526327PRTArtificialan artificially synthesized sequence 26Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro
Ser Cys Pro Ala Pro 100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp Val Ser Gln Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280
285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser305 310 315 320Leu Ser Leu Ser Leu Gly Lys 32527549DNAHomo
sapiens 27atggaacagg ggaagggcct ggctgtcctc atcctggcta tcattcttct
tcaaggtact 60ttggcccagt caatcaaagg aaaccacttg gttaaggtgt atgactatca
agaagatggt 120tcggtacttc tgacttgtga tgcagaagcc aaaaatatca
catggtttaa agatgggaag 180atgatcggct tcctaactga agataaaaaa
aaatggaatc tgggaagtaa tgccaaggac 240cctcgaggga tgtatcagtg
taaaggatca cagaacaagt caaaaccact ccaagtgtat 300tacagaatgt
gtcagaactg cattgaacta aatgcagcca ccatatctgg ctttctcttt
360gctgaaatcg tcagcatttt cgtccttgct gttggggtct acttcattgc
tggacaggat 420ggagttcgcc agtcgagagc ttcagacaag cagactctgt
tgcccaatga ccagctctac 480cagcccctca aggatcgaga agatgaccag
tacagccacc ttcaaggaaa ccagttgagg 540aggaattga 54928182PRTHomo
sapiens 28Met Glu Gln Gly Lys Gly Leu Ala Val Leu Ile Leu Ala Ile
Ile Leu1 5 10 15Leu Gln Gly Thr Leu Ala Gln Ser Ile Lys Gly Asn His
Leu Val Lys 20 25 30Val Tyr Asp Tyr Gln Glu Asp Gly Ser Val Leu Leu
Thr Cys Asp Ala 35 40 45Glu Ala Lys Asn Ile Thr Trp Phe Lys Asp Gly
Lys Met Ile Gly Phe 50 55 60Leu Thr Glu Asp Lys Lys Lys Trp Asn Leu
Gly Ser Asn Ala Lys Asp65 70 75 80Pro Arg Gly Met Tyr Gln Cys Lys
Gly Ser Gln Asn Lys Ser Lys Pro 85 90 95Leu Gln Val Tyr Tyr Arg Met
Cys Gln Asn Cys Ile Glu Leu Asn Ala 100 105 110Ala Thr Ile Ser Gly
Phe Leu Phe Ala Glu Ile Val Ser Ile Phe Val 115 120 125Leu Ala Val
Gly Val Tyr Phe Ile Ala Gly Gln Asp Gly Val Arg Gln 130 135 140Ser
Arg Ala Ser Asp Lys Gln Thr Leu Leu Pro Asn Asp Gln Leu Tyr145 150
155 160Gln Pro Leu Lys Asp Arg Glu Asp Asp Gln Tyr Ser His Leu Gln
Gly 165 170 175Asn Gln Leu Arg Arg Asn 18029516DNAHomo sapiens
29atggaacata gcacgtttct ctctggcctg gtactggcta cccttctctc gcaagtgagc
60cccttcaaga tacctataga ggaacttgag gacagagtgt ttgtgaattg caataccagc
120atcacatggg tagagggaac ggtgggaaca ctgctctcag acattacaag
actggacctg 180ggaaaacgca tcctggaccc acgaggaata tataggtgta
atgggacaga tatatacaag 240gacaaagaat ctaccgtgca agttcattat
cgaatgtgcc agagctgtgt ggagctggat 300ccagccaccg tggctggcat
cattgtcact gatgtcattg ccactctgct ccttgctttg 360ggagtcttct
gctttgctgg acatgagact ggaaggctgt ctggggctgc cgacacacaa
420gctctgttga ggaatgacca ggtctatcag cccctccgag atcgagatga
tgctcagtac 480agccaccttg gaggaaactg ggctcggaac aagtga
51630171PRTHomo sapiens 30Met Glu His Ser Thr Phe Leu Ser Gly Leu
Val Leu Ala Thr Leu Leu1 5 10 15Ser Gln Val Ser Pro Phe Lys Ile Pro
Ile Glu Glu Leu Glu Asp Arg 20 25 30Val Phe Val Asn Cys Asn Thr Ser
Ile Thr Trp Val Glu Gly Thr Val 35 40 45Gly Thr Leu Leu Ser Asp Ile
Thr Arg Leu Asp Leu Gly Lys Arg Ile 50 55 60Leu Asp Pro Arg Gly Ile
Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys65 70 75 80Asp Lys Glu Ser
Thr Val Gln Val His Tyr Arg Met Cys Gln Ser Cys 85 90 95Val Glu Leu
Asp Pro Ala Thr Val Ala Gly Ile Ile Val Thr Asp Val 100 105 110Ile
Ala Thr Leu Leu Leu Ala Leu Gly Val Phe Cys Phe Ala Gly His 115 120
125Glu Thr Gly Arg Leu Ser Gly Ala Ala Asp Thr Gln Ala Leu Leu Arg
130 135 140Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala
Gln Tyr145 150 155 160Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys
165 17031624DNAHomo sapiens 31atgcagtcgg gcactcactg gagagttctg
ggcctctgcc tcttatcagt tggcgtttgg 60gggcaagatg gtaatgaaga aatgggtggt
attacacaga caccatataa agtctccatc 120tctggaacca cagtaatatt
gacatgccct cagtatcctg gatctgaaat actatggcaa 180cacaatgata
aaaacatagg cggtgatgag gatgataaaa acataggcag tgatgaggat
240cacctgtcac tgaaggaatt ttcagaattg gagcaaagtg gttattatgt
ctgctacccc 300agaggaagca aaccagaaga tgcgaacttt tatctctacc
tgagggcaag agtgtgtgag 360aactgcatgg agatggatgt gatgtcggtg
gccacaattg tcatagtgga catctgcatc 420actgggggct tgctgctgct
ggtttactac tggagcaaga atagaaaggc caaggccaag 480cctgtgacac
gaggagcggg tgctggcggc aggcaaaggg gacaaaacaa ggagaggcca
540ccacctgttc ccaacccaga ctatgagccc atccggaaag gccagcggga
cctgtattct 600ggcctgaatc agagacgcat ctga 62432207PRTHomo sapiens
32Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser1
5 10 15Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile
Thr 20 25 30Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile
Leu Thr 35 40 45Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His
Asn Asp Lys 50 55 60Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly
Ser Asp Glu Asp65 70 75 80His Leu Ser Leu Lys Glu Phe Ser Glu Leu
Glu Gln Ser Gly Tyr Tyr 85 90 95Val Cys Tyr Pro Arg Gly Ser Lys Pro
Glu Asp Ala Asn Phe Tyr Leu 100 105 110Tyr Leu Arg Ala Arg Val Cys
Glu Asn Cys Met Glu Met Asp Val Met 115 120 125Ser Val Ala Thr Ile
Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu 130 135 140Leu Leu Leu
Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys145 150 155
160Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn
165 170 175Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro
Ile Arg 180 185 190Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln
Arg Arg Ile 195 200 20533523PRTArtificialan artificially
synthesized sequence 33Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Glu Met His Trp Ile
Arg Gln Pro Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp
Pro Lys Thr Gly Asp Thr Ala Tyr Ser65 70 75 80Gln Lys Phe Lys Gly
Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met
Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr
Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr 115 120
125Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu
Ser Leu145 150 155 160Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln 165 170 175Ser Leu Val His Ser Asn Arg Asn Thr
Tyr Leu His Trp Tyr Gln Gln 180 185 190Lys Pro Gly Gln Ala Pro Arg
Leu Leu Ile Tyr Lys Val Ser Asn Arg 195 200 205Phe Ser Gly Val Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220Phe Thr Leu
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr225 230 235
240Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr
245 250 255Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gln Ala Val Val
Thr Gln 260 265 270Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val
Thr Leu Thr Cys 275 280 285Arg Ser Ser Thr Gly Ala Val Thr Thr Ser
Asn Tyr Ala Asn Trp Val 290 295 300Gln Glu Lys Pro Asp His Leu Phe
Thr Gly Leu Ile Gly Gly Thr Asn305 310 315 320Lys Arg Ala Pro Gly
Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly 325 330 335Asp Lys Ala
Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala 340 345 350Ile
Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly 355 360
365Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly
370 375 380Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Leu Glu Ser Gly
Gly Gly385 390 395 400Leu Val Gln Pro Lys Gly Ser Leu Lys Leu Ser
Cys Ala Ala Ser Gly 405 410 415Phe Thr Phe Asn Thr Tyr Ala Met Asn
Trp Val Arg Gln Ala Pro Gly 420 425 430Lys Gly Leu Glu Trp Val Ala
Arg Ile Arg Ser Lys Tyr Asn Asn Tyr 435 440 445Ala Thr Tyr Tyr Ala
Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg 450 455 460Asp Asp Ser
Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr465 470 475
480Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn
485 490 495Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr 500 505 510Val Ser Ala His His His His His His His His 515
52034753PRTArtificialan artificially synthesized sequence 34Met Gly
Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val
His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25
30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly
Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala
Tyr Ser65 70 75 80Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp
Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser
Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr
Thr Tyr Trp Gly Gln Gly Thr 115 120 125Leu Val Thr Val Ser Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly Ser
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu145 150 155 160Pro Val
Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln 165 170
175Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln
180 185 190Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser
Asn Arg 195 200 205Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp 210 215 220Phe Thr Leu Lys Ile Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr225 230 235 240Tyr Cys Ser Gln Asn Thr His
Val Pro Pro Thr Phe Gly Gln Gly Thr 245 250 255Lys Leu Glu Ile Lys
Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln 260 265 270Glu Ser Ala
Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys 275 280 285Arg
Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val 290 295
300Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr
Asn305 310 315 320Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly
Ser Leu Ile Gly 325 330 335Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala
Gln Thr Glu Asp Glu Ala 340 345 350Ile Tyr Phe Cys Ala Leu Trp Tyr
Ser Asn Leu Trp Val Phe Gly Gly 355 360 365Gly Thr Lys Leu Thr Val
Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly 370 375 380Ser Gly Gly Gly
Gly Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly385 390 395 400Leu
Val Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly 405 410
415Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly
420 425 430Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn
Asn Tyr 435 440 445Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe
Thr Ile Ser Arg 450 455 460Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln
Met Asn Asn Leu Lys Thr465 470 475 480Glu Asp Thr Ala Met Tyr Tyr
Cys Val Arg His Gly Asn Phe Gly Asn 485 490 495Ser Tyr Val Ser Trp
Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 500 505 510Val Ser Ala
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro 515 520 525Cys
Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro 530 535
540Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr545 550 555 560Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn 565 570 575Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg 580 585 590Glu Glu Gln Tyr Ala Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val 595 600 605Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 610 615 620Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys625 630 635 640Gly
Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp 645 650
655Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe
660 665 670Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu 675 680 685Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe 690 695 700Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly705 710 715 720Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr 725 730 735Thr Gln Lys Ser Leu
Ser Leu Ser Pro His His His His His His His 740 745
750His35257PRTArtificialan artificially synthesized sequence 35Met
Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10
15Val His Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
20 25 30Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
Pro 35 40 45Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr 50 55 60Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn65 70 75 80Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg 85 90 95Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val 100 105 110Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser 115 120 125Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 130 135 140Gly Gln Pro Arg
Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys145 150 155 160Glu Leu
Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe 165 170
175Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
180 185 190Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe 195 200 205Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 210 215 220Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr225 230 235 240Thr Gln Lys Ser Leu Ser Leu
Ser Pro Asp Tyr Lys Asp Asp Asp Asp 245 250
255Lys36514PRTArtificialan artificially synthesized sequence 36Met
Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10
15Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe 35 40 45Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln
Gly Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr
Ala Tyr Ser65 70 75 80Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala
Asp Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr
Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Thr Arg Phe Tyr Ser
Tyr Thr Tyr Trp Gly Gln Gly Thr 115 120 125Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly
Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu145 150 155 160Pro
Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln 165 170
175Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln
180 185 190Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser
Asn Arg 195 200 205Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp 210 215 220Phe Thr Leu Lys Ile Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr225 230 235 240Tyr Cys Ser Gln Asn Thr His
Val Pro Pro Thr Phe Gly Gln Gly Thr 245 250 255Lys Leu Glu Ile Lys
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 260 265 270Gly Gly Gly
Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 275 280 285Pro
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 290 295
300Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val305 310 315 320Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe 325 330 335Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro 340 345 350Arg Glu Glu Gln Tyr Ala Ser Thr
Tyr Arg Val Val Ser Val Leu Thr 355 360 365Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 370 375 380Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala385 390 395 400Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 405 410
415Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly
420 425 430Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro 435 440 445Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser 450 455 460Phe Phe Leu Val Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln465 470 475 480Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His 485 490 495Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp 500 505 510Asp
Lys37629PRTArtificialan artificially synthesized sequence 37Met Gly
Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val
His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25
30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly
Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala
Tyr Ser65 70 75 80Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp
Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser
Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr
Thr Tyr Trp Gly Gln Gly Thr 115 120 125Leu Val Thr Val Ser Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly Ser
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu145 150 155 160Pro Val
Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln 165 170
175Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln
180 185 190Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser
Asn Arg 195 200 205Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp 210 215 220Phe Thr Leu Lys Ile Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr225 230 235 240Tyr Cys Ser Gln Asn Thr His
Val Pro Pro Thr Phe Gly Gln Gly Thr 245 250 255Lys Leu Glu Ile Lys
Gly Gly Gly Gly Ser Glu Val Lys Leu Leu Glu 260 265 270Ser Gly Gly
Gly Leu Val Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys 275 280 285Ala
Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg 290 295
300Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser
Lys305 310 315 320Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val
Lys Asp Arg Phe 325 330 335Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile
Leu Tyr Leu Gln Met Asn 340 345 350Asn Leu Lys Thr Glu Asp Thr Ala
Met Tyr Tyr Cys Val Arg His Gly 355 360 365Asn Phe Gly Asn Ser Tyr
Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly 370 375 380Thr Leu Val Thr
Val Ser Ala Glu Pro Lys Ser Ser Asp Lys Thr His385 390 395 400Thr
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 405 410
415Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
420 425 430Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu 435 440 445Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys 450 455 460Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser
Thr Tyr Arg Val Val Ser465 470 475 480Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys 485 490 495Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 500 505 510Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro 515 520 525Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 530 535
540Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn545 550 555 560Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser 565 570 575Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg 580 585 590Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu 595 600 605His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro His His His 610 615 620His His His His
His62538613PRTArtificialan artificially synthesized sequence 38Met
Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10
15Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe 35 40 45Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln
Gly Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr
Ala Tyr Ser65 70 75 80Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala
Asp Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr
Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Thr Arg Phe Tyr Ser
Tyr Thr Tyr Trp Gly Gln Gly Thr 115 120 125Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly
Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu145 150 155 160Pro
Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln 165 170
175Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln
180 185 190Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser
Asn Arg 195 200 205Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp 210 215 220Phe Thr Leu Lys Ile Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr225 230 235 240Tyr Cys Ser Gln Asn Thr His
Val Pro Pro Thr Phe Gly Gln Gly Thr 245 250 255Lys Leu Glu Ile Lys
Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln 260 265 270Glu Ser Ala
Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys 275 280 285Arg
Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val 290 295
300Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr
Asn305 310 315 320Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly
Ser Leu Ile Gly 325 330 335Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala
Gln Thr Glu Asp Glu Ala 340 345 350Ile Tyr Phe Cys Ala Leu Trp Tyr
Ser Asn Leu Trp Val Phe Gly Gly 355 360 365Gly Thr Lys Leu Thr Val
Leu Glu Pro Lys Ser Ser Asp Lys Thr His 370 375 380Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val385 390 395 400Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 405 410
415Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
420 425 430Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 435 440 445Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr
Arg Val Val Ser 450 455 460Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys465 470 475 480Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 485 490 495Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 500 505 510Pro Ser Arg
Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 515 520 525Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 530 535
540Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser545 550 555 560Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
Asp Lys Ser Arg 565 570 575Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 580 585 590His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Asp Tyr Lys 595 600 605Asp Asp Asp Asp Lys
61039470PRTArtificialan artificially synthesized sequence 39Met Gly
Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val
His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25
30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly
Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala
Tyr Ser65 70 75 80Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp
Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser
Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr
Thr Tyr Trp Gly Gln Gly Thr 115 120 125Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro 130 135 140Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly145 150 155 160Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 165 170
175Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
180 185 190Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser 195 200 205Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser 210 215 220Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr225 230 235 240His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly Gly Pro Ser 245 250 255Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265 270Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 275 280 285Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 290 295
300Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val
Val305 310 315 320Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr 325 330 335Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr 340 345 350Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu 355 360 365Pro Pro Ser Arg Cys Glu
Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 370 375 380Ala Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser385 390 395 400Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 405 410
415Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
420 425 430Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 435 440 445Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Asp Tyr 450 455 460Lys Asp Asp Asp Asp Lys465
47040238PRTArtificialan artificially synthesized sequence 40Met Gly
Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val
His Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val 20 25
30Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
35 40 45Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys
Pro 50 55 60Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg
Phe Ser65 70 75 80Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr 85 90 95Leu Lys Ile Ser Arg Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr Cys 100 105 110Ser Gln Asn Thr His Val Pro Pro
Thr Phe Gly Gln Gly Thr Lys Leu 115 120 125Glu Ile Lys Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140Ser Asp Glu Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu145 150 155 160Asn
Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170
175Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
180 185 190Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala 195 200 205Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly 210 215 220Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys225 230 23541470PRTArtificialan artificially
synthesized sequence 41Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Glu Met His Trp Ile
Arg Gln Pro Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp
Pro Lys Thr Gly Asp Thr Ala Tyr Ser65 70 75 80Gln Lys Phe Lys Gly
Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met
Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr
Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr 115 120
125Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
130 135 140Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly145 150 155 160Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn 165 170 175Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 180 185 190Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr225 230 235
240His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
245 250 255Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg 260 265 270Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro 275 280 285Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 290 295 300Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Ala Ser Thr Tyr Arg Val Val305 310 315 320Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345 350Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 355 360
365Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys
370 375 380Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser385 390 395 400Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp 405 410 415Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 420 425 430Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 435 440 445Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro His His 450 455 460His His His
His His His465 47042734PRTArtificialan artificially synthesized
sequence 42Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala
Thr Gly1 5 10 15Val His Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu
Thr Thr Ser 20 25 30Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser
Thr Gly Ala Val 35 40 45Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu
Lys Pro Asp His Leu 50 55 60Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys
Arg Ala Pro Gly Val Pro65 70 75 80Ala Arg Phe Ser Gly Ser Leu Ile
Gly Asp Lys Ala Ala Leu Thr Ile 85 90 95Thr Gly Ala Gln Thr Glu Asp
Glu Ala Ile Tyr Phe Cys Ala Leu Trp 100 105 110Tyr Ser Asn Leu Trp
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 115 120 125Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 130 135 140Val
Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly Ser145 150
155 160Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
Ala 165 170 175Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Ala 180 185 190Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr
Tyr Tyr Ala Asp Ser 195 200 205Val Lys Asp Arg Phe Thr Ile Ser Arg
Asp Asp Ser Gln Ser Ile Leu 210 215 220Tyr Leu Gln Met Asn Asn Leu
Lys Thr Glu Asp Thr Ala Met Tyr Tyr225 230 235 240Cys Val Arg His
Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala 245 250 255Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly 260 265
270Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val
275 280 285Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
Val Ser 290 295 300Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu
Met His Trp Ile305 310 315 320Arg Gln Pro Pro Gly Gln Gly Leu Glu
Trp Ile Gly Ala Ile Asp Pro 325 330 335Lys Thr Gly Asp Thr Ala Tyr
Ser Gln Lys Phe Lys Gly Arg Val Thr 340 345 350Leu Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser 355 360 365Leu Thr Ser
Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser 370 375 380Tyr
Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser385 390
395 400Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr 405 410 415Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro 420 425 430Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val 435 440 445His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser 450 455 460Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile465 470 475 480Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 485 490 495Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 500 505
510Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
515 520 525Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val 530 535 540Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val545 550 555 560Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln 565 570 575Tyr Ala Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln 580 585 590Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 595 600 605Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 610 615 620Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr625 630
635 640Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro
Ser 645 650 655Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr 660 665 670Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Val 675 680 685Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe 690 695 700Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys705 710 715 720Ser Leu Ser Leu
Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys 725 73043462PRTArtificialan
artificially synthesized sequence 43Met Gly Trp Ser Cys Ile Ile Leu
Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Glu Met
His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly
Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser65 70 75 80Gln Lys
Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser 85 90 95Thr
Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr
115 120 125Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro 130 135 140Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly145 150 155 160Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn 165 170 175Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln 180 185 190Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr225 230
235 240His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
Ser 245 250 255Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg 260 265 270Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro 275 280 285Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 290 295 300Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Ala Ser Thr Tyr Arg Val Val305 310 315 320Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345
350Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
355 360 365Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys 370 375 380Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser385 390 395 400Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp 405 410 415Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser 420 425 430Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 435 440 445Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455
46044246PRTArtificialan artificially synthesized sequence 44Met Gly
Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val
His Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val 20 25
30Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
35 40 45Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys
Pro 50 55 60Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg
Phe Ser65 70 75 80Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr 85 90 95Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys 100 105 110Ser Gln Asn Thr His Val Pro Pro Thr
Phe Gly Gln Gly Thr Lys Leu 115 120 125Glu Ile Lys Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu145 150 155 160Asn Asn
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170
175Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
180 185 190Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala 195 200 205Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly 210 215 220Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys His His225 230 235 240His His His His His His
24545505PRTArtificialan artificially synthesized sequence 45Met Gly
Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val
His Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val 20 25
30Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
35 40 45Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys
Pro 50 55 60Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg
Phe Ser65 70 75 80Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr 85 90 95Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys 100 105 110Ser Gln Asn Thr His Val Pro Pro Thr
Phe Gly Gln Gly Thr Lys Leu 115 120 125Glu Ile Lys Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu145 150 155 160Asn Asn
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170
175Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
180 185 190Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala 195 200 205Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly 210 215 220Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys Gly Gly225 230 235 240Gly Gly Ser Gly Gly Gly Gly
Ser Gln Ala Val Val Thr Gln Glu Ser 245 250 255Ala Leu Thr Thr Ser
Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser 260 265 270Ser Thr Gly
Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu 275 280 285Lys
Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg 290 295
300Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp
Lys305 310 315 320Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp
Glu Ala Ile Tyr 325 330 335Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp
Val Phe Gly Gly Gly Thr 340 345 350Lys Leu Thr Val Leu Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 355 360 365Gly Gly Gly Ser Glu Val
Lys Leu Leu Glu Ser Gly Gly Gly Leu Val 370 375 380Gln Pro Lys Gly
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr385 390
395 400Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys
Gly 405 410 415Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn
Tyr Ala Thr 420 425 430Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr
Ile Ser Arg Asp Asp 435 440 445Ser Gln Ser Ile Leu Tyr Leu Gln Met
Asn Asn Leu Lys Thr Glu Asp 450 455 460Thr Ala Met Tyr Tyr Cys Val
Arg His Gly Asn Phe Gly Asn Ser Tyr465 470 475 480Val Ser Trp Phe
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 485 490 495Ala Asp
Tyr Lys Asp Asp Asp Asp Lys 500 50546729PRTArtificialan
artificially synthesized sequence 46Met Gly Trp Ser Cys Ile Ile Leu
Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Glu Met
His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly
Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser65 70 75 80Gln Lys
Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser 85 90 95Thr
Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr
115 120 125Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro 130 135 140Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly145 150 155 160Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn 165 170 175Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln 180 185 190Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr225 230
235 240His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
Ser 245 250 255Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg 260 265 270Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro 275 280 285Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 290 295 300Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Ala Ser Thr Tyr Arg Val Val305 310 315 320Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345
350Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
355 360 365Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu
Ser Cys 370 375 380Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser385 390 395 400Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp 405 410 415Ser Asp Gly Ser Phe Phe Leu
Val Ser Lys Leu Thr Val Asp Lys Ser 420 425 430Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 435 440 445Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 450 455 460Gly
Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Ser465 470
475 480Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Arg
Ser 485 490 495Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp
Val Gln Glu 500 505 510Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly
Gly Thr Asn Lys Arg 515 520 525Ala Pro Gly Val Pro Ala Arg Phe Ser
Gly Ser Leu Ile Gly Asp Lys 530 535 540Ala Ala Leu Thr Ile Thr Gly
Ala Gln Thr Glu Asp Glu Ala Ile Tyr545 550 555 560Phe Cys Ala Leu
Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr 565 570 575Lys Leu
Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 580 585
590Gly Gly Gly Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val
595 600 605Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly
Phe Thr 610 615 620Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly625 630 635 640Leu Glu Trp Val Ala Arg Ile Arg Ser
Lys Tyr Asn Asn Tyr Ala Thr 645 650 655Tyr Tyr Ala Asp Ser Val Lys
Asp Arg Phe Thr Ile Ser Arg Asp Asp 660 665 670Ser Gln Ser Ile Leu
Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp 675 680 685Thr Ala Met
Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr 690 695 700Val
Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser705 710
715 720Ala Asp Tyr Lys Asp Asp Asp Asp Lys 72547729PRTArtificialan
artificially synthesized sequence 47Met Gly Trp Ser Cys Ile Ile Leu
Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Glu Met
His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly
Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser65 70 75 80Gln Lys
Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser 85 90 95Thr
Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr
115 120 125Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro 130 135 140Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly145 150 155 160Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn 165 170 175Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln 180 185 190Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr225 230
235 240His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser 245 250 255Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg 260 265 270Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro 275 280 285Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 290 295 300Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val305 310 315 320Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345
350Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
355 360 365Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu
Ser Cys 370 375 380Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser385 390 395 400Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp 405 410 415Ser Asp Gly Ser Phe Phe Leu
Val Ser Lys Leu Thr Val Asp Lys Ser 420 425 430Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 435 440 445Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 450 455 460Gly
Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Ser465 470
475 480Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Arg
Ser 485 490 495Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp
Val Gln Glu 500 505 510Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly
Gly Thr Asn Lys Arg 515 520 525Ala Pro Gly Val Pro Ala Arg Phe Ser
Gly Ser Leu Ile Gly Asp Lys 530 535 540Ala Ala Leu Thr Ile Thr Gly
Ala Gln Thr Glu Asp Glu Ala Ile Tyr545 550 555 560Phe Cys Ala Leu
Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr 565 570 575Lys Leu
Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 580 585
590Gly Gly Gly Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val
595 600 605Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly
Phe Thr 610 615 620Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly625 630 635 640Leu Glu Trp Val Ala Arg Ile Arg Ser
Lys Tyr Asn Asn Tyr Ala Thr 645 650 655Tyr Tyr Ala Asp Ser Val Lys
Asp Arg Phe Thr Ile Ser Arg Asp Asp 660 665 670Ser Gln Ser Ile Leu
Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp 675 680 685Thr Ala Met
Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr 690 695 700Val
Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser705 710
715 720Ala Asp Tyr Lys Asp Asp Asp Asp Lys 72548470PRTArtificialan
artificially synthesized sequence 48Met Gly Trp Ser Cys Ile Ile Leu
Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Glu Met
His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly
Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser65 70 75 80Gln Lys
Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser 85 90 95Thr
Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr
115 120 125Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro 130 135 140Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly145 150 155 160Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn 165 170 175Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln 180 185 190Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr225 230
235 240His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser 245 250 255Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg 260 265 270Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro 275 280 285Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 290 295 300Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val305 310 315 320Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345
350Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu
355 360 365Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Trp Cys 370 375 380Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser385 390 395 400Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp 405 410 415Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser 420 425 430Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 435 440 445Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro His His 450 455 460His
His His His His His465 47049600PRTArtificialan artificially
synthesized sequence 49Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Glu Met His Trp Ile
Arg Gln Pro Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp
Pro Lys Thr Gly Asp Thr Ala Tyr Ser65 70 75 80Gln Lys Phe Lys Gly
Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met
Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr
Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr 115 120
125Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
130 135 140Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly145 150 155 160Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn 165 170 175Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 180 185 190Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr225 230 235
240His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
245 250 255Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg 260 265 270Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro 275 280 285Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 290 295 300Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Ala Ser Thr Tyr Arg Val Val305 310 315 320Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345 350Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 355 360
365Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys
370 375 380Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser385 390 395 400Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu
Asp 405 410 415Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
Asp Lys Ser 420 425 430Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala 435 440 445Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Gly 450 455 460Gly Gly Ser Glu Val Lys Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln465 470 475 480Pro Lys Gly Ser
Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 485 490 495Asn Thr
Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 500 505
510Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
515 520 525Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp
Asp Ser 530 535 540Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys
Thr Glu Asp Thr545 550 555 560Ala Met Tyr Tyr Cys Val Arg His Gly
Asn Phe Gly Asn Ser Tyr Val 565 570 575Ser Trp Phe Ala Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ala 580 585 590Asp Tyr Lys Asp Asp
Asp Asp Lys 595 60050584PRTArtificialan artificially synthesized
sequence 50Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala
Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro
Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr
Gly Asp Thr Ala Tyr Ser65 70 75 80Gln Lys Phe Lys Gly Arg Val Thr
Leu Thr Ala Asp Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser
Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Thr Arg
Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr 115 120 125Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 130 135 140Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly145 150
155 160Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn 165 170 175Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln 180 185 190Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser 195 200 205Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser 210 215 220Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr225 230 235 240His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 245 250 255Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265
270Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
275 280 285Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala 290 295 300Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr
Tyr Arg Val Val305 310 315 320Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr 325 330 335Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345 350Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 355 360 365Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 370 375 380Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser385 390
395 400Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp 405 410 415Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser 420 425 430Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala 435 440 445Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Gly 450 455 460Gly Gly Ser Gln Ala Val Val
Thr Gln Glu Ser Ala Leu Thr Thr Ser465 470 475 480Pro Gly Glu Thr
Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val 485 490 495Thr Thr
Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu 500 505
510Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro
515 520 525Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu
Thr Ile 530 535 540Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe
Cys Ala Leu Trp545 550 555 560Tyr Ser Asn Leu Trp Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 565 570 575His His His His His His His
His 58051605PRTArtificialan artificially synthesized sequence 51Met
Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10
15Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe 35 40 45Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln
Gly Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr
Ala Tyr Ser65 70 75 80Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala
Asp Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr
Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Thr Arg Phe Tyr Ser
Tyr Thr Tyr Trp Gly Gln Gly Thr 115 120 125Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 130 135 140Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly145 150 155 160Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 165 170
175Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
180 185 190Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser 195 200 205Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser 210 215 220Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr225 230 235 240His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly Gly Pro Ser 245 250 255Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265 270Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 275 280 285Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 290 295
300Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val
Val305 310 315 320Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr 325 330 335Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr 340 345 350Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu 355 360 365Pro Pro Ser Arg Cys Glu
Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 370 375 380Ala Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser385 390 395 400Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 405 410
415Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
420 425 430Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 435 440 445Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Gly 450 455 460Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
Lys Leu Leu Glu Ser Gly465 470 475 480Gly Gly Leu Val Gln Pro Lys
Gly Ser Leu Lys Leu Ser Cys Ala Ala 485 490 495Ser Gly Phe Thr Phe
Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala 500 505 510Pro Gly Lys
Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn 515 520 525Asn
Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile 530 535
540Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn
Leu545 550 555 560Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg
His Gly Asn Phe 565 570 575Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr
Trp Gly Gln Gly Thr Leu 580 585 590Val Thr Val Ser Ala Asp Tyr Lys
Asp Asp Asp Asp Lys 595 600 60552589PRTArtificialan artificially
synthesized sequence 52Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Glu Met His Trp Ile
Arg Gln Pro Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp
Pro Lys Thr Gly Asp Thr Ala Tyr Ser65 70 75 80Gln Lys Phe Lys Gly
Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met
Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr
Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr 115 120
125Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
130 135 140Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly145 150 155 160Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn 165 170 175Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 180 185 190Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr225 230 235
240His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
245 250 255Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg 260 265 270Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro 275 280 285Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 290 295 300Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Ala Ser Thr Tyr Arg Val Val305 310 315 320Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345 350Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 355 360
365Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys
370 375 380Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser385 390 395 400Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp 405 410 415Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 420 425 430Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 435 440 445Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 450 455 460Gly Gly Ser
Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Ser465 470 475
480Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser
485 490 495Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val
Gln Glu 500 505 510Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly
Thr Asn Lys Arg 515 520 525Ala Pro Gly Val Pro Ala Arg Phe Ser Gly
Ser Leu Ile Gly Asp Lys 530 535 540Ala Ala Leu Thr Ile Thr Gly Ala
Gln Thr Glu Asp Glu Ala Ile Tyr545 550 555 560Phe Cys Ala Leu Trp
Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr 565 570 575Lys Leu Thr
Val Leu His His His His His His His His 580 58553610PRTArtificialan
artificially synthesized sequence 53Met Gly Trp Ser Cys Ile Ile Leu
Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Glu Met
His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly
Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser65 70 75 80Gln Lys
Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser 85 90 95Thr
Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr
115 120 125Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro 130 135 140Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly145 150 155 160Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn 165 170 175Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln 180 185 190Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr225 230
235 240His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
Ser 245 250 255Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg 260 265 270Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro 275 280 285Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 290 295 300Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Ala Ser Thr Tyr Arg Val Val305 310 315 320Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345
350Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
355 360 365Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu
Ser Cys 370 375 380Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser385 390 395 400Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp 405 410 415Ser Asp Gly Ser Phe Phe Leu
Val Ser Lys Leu Thr Val Asp Lys Ser 420 425 430Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 435 440 445Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 450 455 460Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
Lys465 470 475 480Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys
Gly Ser Leu Lys 485 490 495Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Asn Thr Tyr Ala Met Asn 500 505 510Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val Ala Arg Ile 515 520 525Arg Ser Lys Tyr Asn Asn
Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys 530 535 540Asp Arg Phe Thr
Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu545 550 555 560Gln
Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val 565 570
575Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp
580 585 590Gly Gln Gly Thr Leu Val Thr Val Ser Ala Asp Tyr Lys Asp
Asp Asp 595 600 605Asp Lys 61054594PRTArtificialan artificially
synthesized sequence 54Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Glu Met His Trp Ile
Arg Gln Pro Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp
Pro Lys Thr Gly Asp Thr Ala Tyr Ser65 70 75 80Gln Lys Phe Lys Gly
Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met
Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr
Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr 115 120
125Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
130 135 140Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly145 150 155 160Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn 165 170 175Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 180 185 190Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr225 230 235
240His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
245 250 255Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg 260 265 270Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro 275 280 285Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 290 295 300Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Ala Ser Thr Tyr Arg Val Val305 310 315 320Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345 350Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 355 360
365Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys
370 375 380Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser385 390 395 400Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp 405 410 415Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 420 425 430Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 435 440 445Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 450 455 460Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val465 470 475
480Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr
485 490 495Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn
Tyr Ala 500 505 510Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr
Gly Leu Ile Gly 515 520 525Gly Thr Asn Lys Arg Ala Pro Gly Val Pro
Ala Arg Phe Ser Gly Ser 530 535 540Leu Ile Gly Asp Lys Ala Ala Leu
Thr Ile Thr Gly Ala Gln Thr Glu545 550 555 560Asp Glu Ala Ile Tyr
Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val 565 570 575Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu His His His His His His 580 585 590His
His55615PRTArtificialan artificially synthesized sequence 55Met Gly
Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val
His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25
30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly
Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala
Tyr Ser65 70 75 80Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp
Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser
Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr
Thr Tyr Trp Gly Gln Gly Thr 115 120 125Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro 130 135 140Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly145 150 155 160Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 165 170
175Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
180 185 190Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser 195 200 205Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser 210 215 220Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr225 230 235 240His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly Gly Pro Ser 245 250 255Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265 270Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 275 280 285Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 290 295
300Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val
Val305 310 315 320Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr 325 330 335Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr 340 345 350Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu 355 360 365Pro Pro Ser Arg Cys Glu
Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 370 375 380Ala Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser385 390 395 400Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 405 410
415Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
420 425 430Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 435 440 445Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Gly 450 455 460Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly465 470 475 480Gly Ser Glu Val Lys Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro 485 490 495Lys Gly Ser Leu Lys
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn 500 505 510Thr Tyr Ala
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 515 520 525Trp
Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr 530 535
540Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser
Gln545 550 555 560Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr
Glu Asp Thr Ala 565 570 575Met Tyr Tyr Cys Val Arg His Gly Asn Phe
Gly Asn Ser Tyr Val Ser 580 585 590Trp Phe Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ala Asp 595 600 605Tyr Lys Asp Asp Asp Asp
Lys 610 61556599PRTArtificialan artificially synthesized sequence
56Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1
5 10 15Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe 35 40 45Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly
Gln Gly Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp
Thr Ala Tyr Ser65 70 75 80Gln Lys Phe Lys Gly Arg Val Thr Leu Thr
Ala Asp Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu
Thr Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Thr Arg Phe Tyr
Ser Tyr Thr Tyr Trp Gly Gln Gly Thr 115 120 125Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 130 135 140Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly145 150 155
160Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
165 170 175Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln 180 185 190Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser 195 200 205Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser 210 215 220Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr225 230 235 240His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 245 250 255Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265 270Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 275 280
285Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
290 295 300Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg
Val Val305 310 315 320Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr 325 330 335Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr 340 345 350Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Cys Thr Leu 355 360 365Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 370 375 380Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser385 390 395
400Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
405 410 415Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser 420 425 430Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala 435 440 445Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Gly 450 455 460Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly465 470 475 480Gly Ser Gln Ala Val
Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro 485 490 495Gly Glu Thr
Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr 500 505 510Thr
Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe 515 520
525Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala
530 535 540Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr
Ile Thr545 550 555 560Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe
Cys Ala Leu Trp Tyr 565 570 575Ser Asn Leu Trp Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu His 580 585 590His His His His His His His
59557610PRTArtificialan artificially synthesized sequence 57Met Gly
Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val
His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25
30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly
Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala
Tyr Ser65 70 75 80Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp
Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser
Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr
Thr Tyr Trp Gly Gln Gly Thr 115 120 125Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro 130 135 140Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly145 150 155 160Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 165 170
175Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
180 185 190Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser 195 200 205Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser 210 215 220Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr225 230 235 240His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly Gly Pro Ser 245 250 255Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265 270Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 275 280 285Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 290 295
300Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val
Val305 310 315 320Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr 325 330 335Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr 340 345 350Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu 355 360 365Pro Pro Ser Arg Cys Glu
Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 370 375 380Ala Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser385 390 395 400Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 405 410
415Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
420 425 430Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 435 440 445Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Gly 450 455 460Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Lys465 470 475 480Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Lys Gly Ser Leu Lys 485 490 495Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Asn Thr
Tyr Ala Met Asn 500 505 510Trp Val Arg Gln Ala Pro Gly Lys Cys Leu
Glu Trp Val Ala Arg Ile 515 520 525Arg Ser Lys Tyr Asn Asn Tyr Ala
Thr Tyr Tyr Ala Asp Ser Val Lys 530 535 540Asp Arg Phe Thr Ile Ser
Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu545 550 555 560Gln Met Asn
Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val 565 570 575Arg
His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp 580 585
590Gly Gln Gly Thr Leu Val Thr Val Ser Ala Asp Tyr Lys Asp Asp Asp
595 600 605Asp Lys 61058594PRTArtificialan artificially synthesized
sequence 58Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala
Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro
Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr
Gly Asp Thr Ala Tyr Ser65 70 75 80Gln Lys Phe Lys Gly Arg Val Thr
Leu Thr Ala Asp Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser
Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Thr Arg
Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr 115 120 125Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 130 135 140Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly145 150
155 160Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn 165 170 175Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln 180 185 190Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser 195 200 205Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser 210 215 220Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr225 230 235 240His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 245 250 255Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265
270Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
275 280 285Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala 290 295 300Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr
Tyr Arg Val Val305 310 315 320Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr 325 330 335Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345 350Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 355 360 365Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 370 375 380Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser385 390
395 400Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp 405 410 415Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser 420 425 430Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala 435 440 445Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Gly 450 455 460Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gln Ala Val465 470 475 480Val Thr Gln Glu
Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr 485 490 495Leu Thr
Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala 500 505
510Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly
515 520 525Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser
Gly Ser 530 535 540Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly
Ala Gln Thr Glu545 550 555 560Asp Glu Ala Ile Tyr Phe Cys Ala Leu
Trp Tyr Ser Asn Leu Trp Val 565 570 575Phe Gly Cys Gly Thr Lys Leu
Thr Val Leu His His His His His His 580 585 590His
His59704PRTArtificialan artificially synthesized sequence 59Met Gly
Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val
His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25
30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly
Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala
Tyr Ser65 70 75 80Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp
Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser
Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr
Thr Tyr Trp Gly Gln Gly Thr 115 120 125Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro 130 135 140Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly145 150 155 160Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 165 170
175Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
180 185 190Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser 195 200 205Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser 210 215 220Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr225 230 235 240His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly Gly Pro Ser 245 250 255Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265 270Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 275 280 285Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 290 295
300Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val
Val305 310 315 320Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr 325 330 335Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr 340 345 350Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu 355 360 365Pro Pro Ser Arg Cys Glu
Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 370 375 380Ala Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser385 390 395 400Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 405 410
415Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
420 425 430Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 435 440 445Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Gly 450 455 460Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Lys465 470 475 480Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Lys Gly Ser Leu Lys 485 490 495Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn 500 505 510Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile 515 520 525Arg
Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys 530 535
540Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr
Leu545 550 555 560Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met
Tyr Tyr Cys Val 565 570 575Arg His Gly Asn Phe Gly Asn Ser Tyr Val
Ser Trp Phe Ala Tyr Trp 580 585 590Gly Gln Gly Thr Leu Val Thr Val
Ser Ala Ala Ser Thr Lys Gly Pro 595 600 605Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 610 615 620Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr625 630 635 640Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 645 650
655Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
660 665 670Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 675 680 685His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser 690 695 70060692PRTArtificialan artificially
synthesized sequence 60Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Glu Met His Trp Ile
Arg Gln Pro Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Ala Ile Asp
Pro Lys Thr Gly Asp Thr Ala Tyr Ser65 70 75 80Gln Lys Phe Lys Gly
Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met
Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr
Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr 115 120
125Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
130 135 140Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly145 150 155 160Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn 165 170 175Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 180 185 190Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr225 230 235
240His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
245 250 255Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg 260 265 270Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro 275 280 285Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 290 295 300Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Ala Ser Thr Tyr Arg Val Val305 310 315 320Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345 350Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 355 360
365Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys
370 375 380Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser385 390 395 400Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp 405 410 415Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 420 425 430Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 435 440 445Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 450 455 460Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val465 470 475
480Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr
485 490 495Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn
Tyr Ala 500 505 510Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr
Gly Leu Ile Gly 515 520 525Gly Thr Asn Lys Arg Ala Pro Gly Val Pro
Ala Arg Phe Ser Gly Ser 530 535 540Leu Ile Gly Asp Lys Ala Ala Leu
Thr Ile Thr Gly Ala Gln Thr Glu545 550 555 560Asp Glu Ala Ile Tyr
Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val 565 570 575Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu Arg Thr Val Ala Ala Pro 580 585 590Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 595 600
605Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
610 615 620Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln Glu625 630 635 640Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser Ser 645 650 655Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr Ala 660 665 670Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser Phe 675 680 685Asn Arg Gly Glu
69061115PRTArtificialan artificially synthesized sequence 61Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25
30Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys
Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln
Gly Thr Leu Val Thr 100 105 110Val Ser Ser 11562219PRTArtificialan
artificially synthesized sequence 62Asp Val Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys
Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Arg Asn Thr Tyr Leu
His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr
His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
110Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe 130 135 140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 210 21563328PRTArtificialan
artificially synthesized sequence 63Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10
15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Ala Ala Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro
32564587PRTArtificialan artificially synthesized sequence 64Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Ala Ala Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
Ser Gly Gly Gly 325 330 335Gly Ser Gln Ala Val Val Thr Gln Glu Ser
Ala Leu Thr Thr Ser Pro 340 345 350Gly Glu Thr Val Thr Leu Thr Cys
Arg Ser Ser Thr Gly Ala Val Thr 355 360 365Thr Ser Asn Tyr Ala Asn
Trp Val Gln Glu Lys Pro Asp His Leu Phe 370 375 380Thr Gly Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala385 390 395 400Arg
Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr 405 410
415Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr
420 425 430Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Gly 435 440 445Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val 450 455 460Lys Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Lys Gly Ser Leu465 470 475 480Lys Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asn Thr Tyr Ala Met 485 490 495Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg 500 505 510Ile Arg Ser
Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val 515 520 525Lys
Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr 530 535
540Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr
Cys545 550 555 560Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser
Trp Phe Ala Tyr 565 570 575Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ala 580 58565328PRTArtificialan artificially synthesized sequence
65Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Ala Ala Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155
160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg
Tyr Thr305 310 315 320Gln Glu Ser Leu Ser Leu Ser Pro
32566587PRTArtificialan artificially synthesized sequence 66Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Ala Ala Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Lys Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly
Ser Gly Gly Gly 325 330 335Gly Ser Gln Ala Val Val Thr Gln Glu Ser
Ala Leu Thr Thr Ser Pro 340 345 350Gly Glu Thr Val Thr Leu Thr Cys
Arg Ser Ser Thr Gly Ala Val Thr 355 360 365Thr Ser Asn Tyr Ala Asn
Trp Val Gln Glu Lys Pro Asp His Leu Phe 370 375 380Thr Gly Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala385 390 395 400Arg
Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr 405 410
415Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr
420 425 430Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Gly 435 440 445Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val 450 455 460Lys Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Lys Gly Ser Leu465 470 475 480Lys Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asn Thr Tyr Ala Met 485 490 495Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg 500 505 510Ile Arg Ser
Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val 515 520 525Lys
Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr 530 535
540Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr
Cys545 550 555 560Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser
Trp Phe Ala Tyr 565 570 575Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ala 580 58567141PRTHomo sapiens 67Asp Ile Gln Asn Pro Asp Pro Ala
Val Tyr Gln Leu Arg Asp Ser Lys1 5 10 15Ser Ser Asp Lys Ser Val Cys
Leu Phe Thr Asp Phe Asp Ser Gln Thr 20 25 30Asn Val Ser Gln Ser Lys
Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr 35 40 45Val Leu Asp Met Arg
Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala 50 55 60Trp Ser Asn Lys
Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser65 70 75 80Ile Ile
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp 85 90 95Val
Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe 100 105
110Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala
115 120 125Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 130
135 14068179PRTHomo sapiens 68Glu Asp Leu Lys Asn Val Phe Pro Pro
Glu Val Ala Val Phe Glu Pro1 5 10 15Ser Glu Ala Glu Ile Ser His Thr
Gln Lys Ala Thr Leu Val Cys Leu 20 25 30Ala Thr Gly Phe Tyr Pro Asp
His Val Glu Leu Ser Trp Trp Val Asn 35 40 45Gly Lys Glu Val His Ser
Gly Val Ser Thr Asp Pro Gln Pro Leu Lys 50 55 60Glu Gln Pro Ala Leu
Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu65 70 75 80Arg Val Ser
Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys 85 90 95Gln Val
Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp 100 105
110Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg
115 120 125Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val
Leu Ser 130 135 140Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala
Thr Leu Tyr Ala145 150 155 160Val Leu Val Ser Ala Leu Val Leu Met
Ala Met Val Lys Arg Lys Asp 165 170 175Ser Arg Gly69173PRTHomo
sapiens 69Asp Lys Gln Leu Asp Ala Asp Val Ser Pro Lys Pro Thr Ile
Phe Leu1 5 10 15Pro Ser Ile Ala Glu Thr Lys Leu Gln Lys Ala Gly Thr
Tyr Leu Cys 20 25 30Leu Leu Glu Lys Phe Phe Pro Asp Val Ile Lys Ile
His Trp Gln Glu 35 40 45Lys Lys Ser Asn Thr Ile Leu Gly Ser Gln Glu
Gly Asn Thr Met Lys 50 55 60Thr Asn Asp Thr Tyr Met Lys Phe Ser Trp
Leu Thr Val Pro Glu Lys65 70 75 80Ser Leu Asp Lys Glu His Arg Cys
Ile Val Arg His Glu Asn Asn Lys 85 90 95Asn Gly Val Asp Gln Glu Ile
Ile Phe Pro Pro Ile Lys Thr Asp Val 100 105 110Ile Thr Met Asp Pro
Lys Asp Asn Cys Ser Lys Asp Ala Asn Asp Thr 115 120 125Leu Leu Leu
Gln Leu Thr Asn Thr Ser Ala Tyr Tyr Met Tyr Leu Leu 130 135 140Leu
Leu Leu Lys Ser Val Val Tyr Phe Ala Ile Ile Thr Cys Cys Leu145 150
155 160Leu Arg Arg Thr Ala Phe Cys Cys Asn Gly Glu Lys Ser 165
17070204PRTHomo sapiens 70Lys Gln Leu Asp Ala Asp Val Ser Pro Lys
Pro Thr Ile Phe Leu Pro1 5 10 15Ser Ile Ala Glu Thr Lys Leu Gln Lys
Ala Gly Thr Tyr Leu Cys Leu 20 25 30Leu Glu Lys Phe Phe Pro Asp Ile
Ile Lys Ile His Trp Gln Glu Lys 35 40 45Lys Ser Asn Thr Ile Leu Gly
Ser Gln Glu Gly Asn Thr Met Lys Thr 50 55 60Asn Asp Thr Tyr Met Lys
Phe Ser Trp Leu Thr Val Pro Glu Glu Ser65 70 75 80Leu Asp Lys Glu
His Arg Cys Ile Val Arg His Glu Asn Asn Lys Asn 85 90 95Gly Ile Asp
Gln Glu Ile Ile Phe Pro Pro Ile Lys Thr Asp Val Thr 100 105 110Thr
Val Asp Pro Lys Asp Ser Tyr Ser Lys Asp Ala Asn Asp Val Thr 115
120
125Thr Val Asp Pro Lys Tyr Asn Tyr Ser Lys Asp Ala Asn Asp Val Ile
130 135 140Thr Met Asp Pro Lys Asp Asn Trp Ser Lys Asp Ala Asn Asp
Thr Leu145 150 155 160Leu Leu Gln Leu Thr Asn Thr Ser Ala Tyr Tyr
Met Tyr Leu Leu Leu 165 170 175Leu Leu Lys Ser Val Val Tyr Phe Ala
Ile Ile Thr Cys Cys Leu Leu 180 185 190Gly Arg Thr Ala Phe Cys Cys
Asn Gly Glu Lys Ser 195 20071177PRTHomo sapiens 71Pro Ser Tyr Thr
Gly Gly Tyr Ala Asp Lys Leu Ile Phe Gly Lys Gly1 5 10 15Thr Arg Val
Thr Val Glu Pro Arg Ser Gln Pro His Thr Lys Pro Ser 20 25 30Val Phe
Val Met Lys Asn Gly Thr Asn Val Ala Cys Leu Val Lys Glu 35 40 45Phe
Tyr Pro Lys Asp Ile Arg Ile Asn Leu Val Ser Ser Lys Lys Ile 50 55
60Thr Glu Phe Asp Pro Ala Ile Val Ile Ser Pro Ser Gly Lys Tyr Asn65
70 75 80Ala Val Lys Leu Gly Lys Tyr Glu Asp Ser Asn Ser Val Thr Cys
Ser 85 90 95Val Gln His Asp Asn Lys Thr Val His Ser Thr Asp Phe Glu
Val Lys 100 105 110Thr Asp Ser Thr Asp His Val Lys Pro Lys Glu Thr
Glu Asn Thr Lys 115 120 125Gln Pro Ser Lys Ser Cys His Lys Pro Lys
Ala Ile Val His Thr Glu 130 135 140Lys Val Asn Met Met Ser Leu Thr
Val Leu Gly Leu Arg Met Leu Phe145 150 155 160Ala Lys Thr Val Ala
Val Asn Phe Leu Leu Thr Ala Lys Leu Phe Phe 165 170
175Leu7219PRTArtificialsignal sequence 72Met Gly Trp Ser Cys Ile
Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His
Ser73466PRTArtificial Sequencean artificially synthesized sequence
73Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1
5 10 15Val His Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr
Ser 20 25 30Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly
Ala Val 35 40 45Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro
Asp His Leu 50 55 60Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala
Pro Gly Val Pro65 70 75 80Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp
Lys Ala Ala Leu Thr Ile 85 90 95Thr Gly Ala Gln Thr Glu Asp Glu Ala
Ile Tyr Phe Cys Ala Leu Trp 100 105 110Tyr Ser Asn Leu Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 115 120 125Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 130 135 140Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys145 150 155
160Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
165 170 175Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu 180 185 190Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr 195 200 205Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val 210 215 220Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro225 230 235 240Pro Cys Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 245 250 255Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 260 265 270Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 275 280
285Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
290 295 300Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val
Leu Thr305 310 315 320Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val 325 330 335Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala 340 345 350Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg 355 360 365Cys Glu Leu Thr Lys
Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly 370 375 380Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro385 390 395
400Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
405 410 415Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln 420 425 430Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His 435 440 445Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Asp Tyr Lys Asp Asp Asp 450 455 460Asp Lys46574251PRTArtificial
Sequencean artificially synthesized sequence 74Met Gly Trp Ser Cys
Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Glu
Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Lys Gly
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45Asn Thr
Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu
Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr65 70 75
80Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser
85 90 95Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp
Thr 100 105 110Ala Met Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn
Ser Tyr Val 115 120 125Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ala 130 135 140Ala Ser Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu145 150 155 160Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 165 170 175Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 180 185 190Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 195 200
205Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
210 215 220Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser225 230 235 240Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
245 25075484PRTArtificial Sequencean artificially synthesized
sequence 75Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala
Thr Gly1 5 10 15Val His Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln 20 25 30Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe 35 40 45Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr
Asn Asn Tyr Ala Thr Tyr65 70 75 80Tyr Ala Asp Ser Val Lys Asp Arg
Phe Thr Ile Ser Arg Asp Asp Ser 85 90 95Gln Ser Ile Leu Tyr Leu Gln
Met Asn Asn Leu Lys Thr Glu Asp Thr 100 105 110Ala Met Tyr Tyr Cys
Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val 115 120 125Ser Trp Phe
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 130 135 140Ala
Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu145 150
155 160Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 165 170 175Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln 180 185 190Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser 195 200 205Thr Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu 210 215 220Lys His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser225 230 235 240Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys Asp Lys Thr His Thr 245 250 255Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 260 265
270Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
275 280 285Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val 290 295 300Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr305 310 315 320Lys Pro Arg Glu Glu Gln Tyr Ala Ser
Thr Tyr Arg Val Val Ser Val 325 330 335Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys 340 345 350Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 355 360 365Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 370 375 380Ser
Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val385 390
395 400Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly 405 410 415Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp 420 425 430Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp 435 440 445Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His 450 455 460Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Asp Tyr Lys Asp465 470 475 480Asp Asp Asp
Lys76233PRTArtificial Sequencean artificially synthesized sequence
76Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1
5 10 15Val His Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr
Ser 20 25 30Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly
Ala Val 35 40 45Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro
Asp His Leu 50 55 60Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala
Pro Gly Val Pro65 70 75 80Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp
Lys Ala Ala Leu Thr Ile 85 90 95Thr Gly Ala Gln Thr Glu Asp Glu Ala
Ile Tyr Phe Cys Ala Leu Trp 100 105 110Tyr Ser Asn Leu Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 115 120 125Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 130 135 140Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys145 150 155
160Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
165 170 175Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu 180 185 190Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr 195 200 205Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val 210 215 220Asp Lys Lys Val Glu Pro Lys Ser
Cys225 23077466PRTArtificial Sequencean artificially synthesized
sequence 77Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala
Thr Gly1 5 10 15Val His Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu
Thr Val Ser 20 25 30Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser
Thr Gly Ala Val 35 40 45Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln Gln
Lys Pro Gly Gln Ala 50 55 60Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe
Leu Ala Pro Gly Thr Pro65 70 75 80Ala Arg Phe Ser Gly Ser Leu Leu
Gly Gly Lys Ala Ala Leu Thr Leu 85 90 95Ser Gly Val Gln Pro Glu Asp
Glu Ala Glu Tyr Tyr Cys Val Leu Trp 100 105 110Tyr Ser Asn Arg Trp
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 115 120 125Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 130 135 140Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys145 150
155 160Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu 165 170 175Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu 180 185 190Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr 195 200 205Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val 210 215 220Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro225 230 235 240Pro Cys Pro Ala
Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 245 250 255Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 260 265
270Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
275 280 285Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro 290 295 300Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
Ser Val Leu Thr305 310 315 320Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val 325 330 335Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala 340 345 350Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 355 360 365Cys Glu Leu
Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly 370 375 380Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro385 390
395 400Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser 405 410 415Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln 420 425 430Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His 435 440 445Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Asp Tyr Lys Asp Asp Asp 450 455 460Asp Lys46578251PRTArtificial
Sequencean artificially synthesized sequence 78Met Gly Trp Ser Cys
Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45Asn Lys
Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu
Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr65 70 75
80Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser
85 90 95Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp
Thr 100 105 110Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn
Ser Tyr Ile 115 120 125Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 130 135 140Ala Ser Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu145 150 155 160Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 165 170 175Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 180 185 190Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 195 200
205Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
210 215 220Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser225 230 235 240Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 245 25079475PRTArtificial Sequencean artificially synthesized
sequence 79Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala
Thr Gly1 5 10 15Val His Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala
Glu Leu Val 20 25 30Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Ala 35 40 45Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln
Arg Pro Gly His Gly 50 55 60Leu Glu Trp Ile Gly Asp Ile Phe Pro Gly
Ser Gly Asn Ile His Tyr65 70 75 80Asn Glu Lys Phe Lys Gly Lys Ala
Thr Leu Thr Ala Asp Lys Ser Ser 85 90 95Ser Thr Ala Tyr Met Gln Leu
Ser Ser Leu Thr Phe Glu Asp Ser Ala 100 105 110Val Tyr Phe Cys Ala
Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr 115 120 125Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150
155 160Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val 165 170 175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe 180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 245 250 255Ala Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265
270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val 290 295 300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Ala Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Cys
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 370 375 380Val
Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390
395 400Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr 405 410 415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu 420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu Ser Pro His His His His
His His His His465 470 47580239PRTArtificial Sequencean
artificially synthesized sequence 80Met Gly Trp Ser Cys Ile Ile Leu
Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Glu Leu Val Met
Thr Gln Ser Pro Ser Ser Leu Thr Val 20 25 30Thr Ala Gly Glu Lys Val
Thr Met Ser Cys Lys Ser Ser Gln Ser Leu 35 40 45Leu Asn Ser Gly Asn
Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys 50 55 60Pro Gly Gln Pro
Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu65 70 75 80Ser Gly
Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95Thr
Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr 100 105
110Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys
115 120 125Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro 130 135 140Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu145 150 155 160Leu Asn Asn Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp 165 170 175Asn Ala Leu Gln Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190Ser Lys Asp Ser Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205Ala Asp Tyr
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220Gly
Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230
23581453PRTArtificial Sequencean artificially synthesized sequence
81Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly1
5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr
Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp
Ser Gln Ser Ile65 70 75 80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr
Glu Asp Thr Ala Met Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly
Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ala Ala Ser Thr 115 120 125Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu145 150 155
160Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser 180 185 190Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys 195 200 205Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu 210 215 220Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro225 230 235 240Glu Ala Ala Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280
285Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp305 310 315 320Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu 325 330 335Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 340 345 350Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys385 390 395
400Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 420 425 430Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser 435 440 445Leu Ser Leu Ser Pro
45082216PRTArtificial Sequencean artificially synthesized sequence
82Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu1
5 10 15Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr
Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe
Thr Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro
Ala Arg Phe 50 55 60Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr
Ile Thr Gly Ala65 70 75 80Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys
Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Arg Thr Val 100 105 110Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn145 150 155
160Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
165 170 175Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys 180 185 190Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr 195 200 205Lys Ser Phe Asn Arg Gly Glu Cys 210
21583443PRTArtificial Sequencean artificially synthesized sequence
83Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser
Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp
Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala145 150 155
160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys 195 200 205Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280
285Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys305 310 315 320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser 340 345 350Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395
400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
44084219PRTArtificial Sequencean artificially synthesized sequence
84Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His
Ser 20 25 30Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly
Gln Ala 35 40 45Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155
160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 21585453PRTArtificial Sequencean artificially synthesized
sequence 85Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Lys Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Asn Thr Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala
Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg
Asp Asp Ser Gln Ser Ile65 70 75 80Leu Tyr Leu Gln Met Asn Asn Leu
Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95Tyr Cys Val Arg His Gly Asn
Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr 115 120 125Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu145 150
155 160Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His 165 170 175Thr Phe Pro Ala Val Leu Lys Ser Ser Gly Leu Tyr Ser
Leu Ser Ser 180 185 190Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys 195 200 205Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu 210 215 220Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro225 230 235 240Glu Ala Ala Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265
270Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr 290 295 300Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp305 310 315 320Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu 325 330 335Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys 355 360 365Asn
Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 370 375
380Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys385 390 395 400Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Val Ser 405 410 415Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser 420 425 430Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser 435 440 445Leu Ser Leu Ser Pro
45086216PRTArtificial Sequencean artificially synthesized sequence
86Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu1
5 10 15Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr
Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe
Thr Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro
Ala Arg Phe 50 55 60Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr
Ile Thr Gly Ala65 70 75 80Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys
Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Arg Thr Val 100 105 110Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125Ser Gly Thr Ala Glu
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn145 150 155
160Ser Glu Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
165 170 175Leu Ser Ser Thr Leu Glu Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys 180 185 190Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr 195 200 205Lys Ser Phe Asn Arg Gly Glu Cys 210
21587443PRTArtificial Sequencean artificially synthesized sequence
87Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser
Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp
Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140Glu Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala145 150 155
160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Glu Ser Ser Gly
165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys 195 200 205Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280
285Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys305 310 315 320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Cys Thr Leu Pro Pro Ser 340 345 350Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Trp Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395
400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
44088443PRTArtificial Sequencean artificially synthesized sequence
88Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser
Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp
Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140Glu Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala145 150 155
160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Glu Ser Ser Gly
165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys 195 200 205Val Asp Glu Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280
285Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys305 310 315 320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Cys Thr Leu Pro Pro Ser 340 345 350Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Trp Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395
400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
44089219PRTArtificial Sequencean artificially synthesized sequence
89Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His
Ser 20 25 30Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly
Gln Ala 35 40 45Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly
Thr Ala Lys Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155
160Ser Gly Asn Ser Lys Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Lys Leu Ser Lys Ala Asp
Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 21590219PRTArtificial Sequencean artificially synthesized
sequence 90Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
Val His Ser 20 25 30Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys
Pro Gly Gln Ala 35 40 45Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg
Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Lys 115 120 125Gln Leu Lys
Ser Gly Thr Ala Lys Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150
155 160Ser Gly Asn Ser Lys Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Lys Leu Ser Lys Ala
Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 210 21591474PRTArtificial Sequencean artificially
synthesized sequence 91Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Lys Gln Ser
Gly Pro Gly Leu Val Gln 20 25 30Pro Ser Gln Ser Leu Ser Ile Thr Cys
Thr Val Ser Gly Phe Ser Leu 35 40 45Thr Asn Tyr Gly Val His Trp Val
Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60Glu Trp Leu Gly Val Ile Trp
Ser Gly Gly Asn Thr Asp Tyr Asn Thr65 70 75 80Pro Phe Thr Ser Arg
Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln 85 90 95Val Phe Phe Lys
Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr 100 105 110Tyr Cys
Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp 115 120
125Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro
130 135 140Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr145 150 155 160Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr 165 170 175Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro 180 185 190Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220His Lys Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser225 230 235
240Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
245 250 255Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu 260 265 270Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser 275 280 285His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu 290 295 300Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Ala Ser Thr305 310 315 320Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360
365Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
370 375 380Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val385 390 395 400Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro 405 410 415Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr 420 425 430Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460Ser Pro His
His His His His His His His465 47092233PRTArtificial Sequencean
artificially synthesized sequence 92Met Gly Trp Ser Cys Ile Ile Leu
Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Asp Ile Leu Leu
Thr Gln Ser Pro Val Ile Leu Ser Val 20 25 30Ser Pro Gly Glu Arg Val
Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile 35 40 45Gly Thr Asn Ile His
Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg 50 55 60Leu Leu Ile Lys
Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg65 70 75 80Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser 85 90 95Val
Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn 100 105
110Trp Pro Thr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr
115 120 125Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu 130 135 140Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro145 150 155 160Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly 165 170 175Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220Thr
Lys Ser Phe Asn Arg Gly Glu Cys225 230
* * * * *