U.S. patent application number 17/028210 was filed with the patent office on 2021-08-26 for fcgammariib-specific fc region variant.
This patent application is currently assigned to Chugai Seiyaku Kabushiki Kaisha. The applicant listed for this patent is Chugai Seiyaku Kabushiki Kaisha. Invention is credited to Tomoyuki Igawa, Shojiro Kadono, Hitoshi Katada, Futa Mimoto.
Application Number | 20210261648 17/028210 |
Document ID | / |
Family ID | 1000005568440 |
Filed Date | 2021-08-26 |
United States Patent
Application |
20210261648 |
Kind Code |
A1 |
Katada; Hitoshi ; et
al. |
August 26, 2021 |
FCgammaRIIB-SPECIFIC FC REGION VARIANT
Abstract
An objective of the present invention is to provide an Fc region
variant with enhanced Fc.gamma.RIIb-binding activity, and/or
enhanced binding selectivity to Fc.gamma.RIIb compared to
Fc.gamma.RIIa (type R), as compared to those of a polypeptide
containing an Fc region to which an amino acid alteration(s) has
not been introduced; a polypeptide which includes the Fc region
variant; a pharmaceutical composition containing the polypeptide;
preventing therapeutic or preventive agent for immunological
inflammatory diseases that includes the pharmaceutical composition;
a production method thereof; and a method of enhancing
Fc.gamma.RIIb-binding activity and also enhancing binding
selectivity to Fc.gamma.RIIb compared to Fc.gamma.RIIa (type R). It
was found that a polypeptide containing an antibody Fc region
variant that contains an amino acid sequence in which an amino-acid
alteration at position 238 (EU numbering) is combined with other
specific amino-acid alterations enhances Fc.gamma.RIIb-binding
activity, and/or enhances binding selectivity to Fc.gamma.RIIb
compared to Fc.gamma.RIIa (type R).
Inventors: |
Katada; Hitoshi; (Shizuoka,
JP) ; Kadono; Shojiro; (Kanagawa, JP) ;
Mimoto; Futa; (Shizuoka, JP) ; Igawa; Tomoyuki;
(Shizuoka, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chugai Seiyaku Kabushiki Kaisha |
Tokyo |
|
JP |
|
|
Assignee: |
Chugai Seiyaku Kabushiki
Kaisha
Tokyo
JP
|
Family ID: |
1000005568440 |
Appl. No.: |
17/028210 |
Filed: |
September 22, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14423269 |
Feb 23, 2015 |
10919953 |
|
|
PCT/JP2013/072507 |
Aug 23, 2013 |
|
|
|
17028210 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/28 20130101;
C07K 2317/53 20130101; C07K 2317/94 20130101; C07K 2317/52
20130101; C07K 2317/524 20130101; C07K 2317/72 20130101; C07K
16/303 20130101; Y02A 50/30 20180101; C07K 2319/30 20130101; C07K
2317/92 20130101; C07K 16/00 20130101; C07K 2317/71 20130101; A61K
2039/505 20130101; A61K 9/0019 20130101; C07K 16/2866 20130101 |
International
Class: |
C07K 16/00 20060101
C07K016/00; C07K 16/30 20060101 C07K016/30; C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 24, 2012 |
JP |
2012-185868 |
Claims
1. An Fc region variant in which amino acid at position 238
according to EU numbering and at least one amino acid selected from
those at positions 233, 234, 235, 237, 264, 265, 266, 267, 268,
269, 271, 272, 274, 296, 326, 327, 330, 331, 332, 333, 334, 355,
356, 358, 396, 409, and 419 according to EU numbering are altered
to other amino acids, wherein binding activity of the variant to
Fc.gamma. receptors [KD value for Fc.gamma.RIIb of a polypeptide
comprising an Fc region to which an amino acid alteration(s) has
not been introduced]/[KD value for Fc.gamma.RIIb of a polypeptide
comprising the Fc region variant] has a value which is 15.0 or
greater.
2. The Fc region variant of claim 1, wherein the amino acids at
positions 238, 268, and 271 according to EU numbering are altered
to other amino acids, and in addition, at least one amino acid
selected from amino acids at positions 233, 237, 264, 267, 272,
296, 327, 330, 332, and 396 according to EU numbering are altered
to other amino acids.
3. An Fc region variant whose amino acid at position 238 according
to EU numbering is Asp, and which comprises at least one amino acid
selected from the amino acid group consisting of Asp at amino acid
position 233, Tyr at amino acid position 234, Phe at amino acid
position 235, Asp at amino acid position 237, Ile at amino acid
position 264, Glu at amino acid position 265, Phe, Leu, or Met at
amino acid position 266, Ala, Glu, Gly, or Gln at amino acid
position 267, Asp, Gln, or Glu at amino acid position 268, Asp at
amino acid position 269, Gly at amino acid position 271, Asp, Phe,
Ile, Met, Asn, Pro, or Gln at amino acid position 272, Gln at amino
acid position 274, Asp or Phe at amino acid position 296, Ala or
Asp at amino acid position 326, Gly at amino acid position 327,
Lys, Arg, or Ser at amino acid position 330, Ser at amino acid
position 331, Lys, Arg, Ser, or Thr at amino acid position 332,
Lys, Arg, Ser, or Thr at amino acid position 333, Arg, Ser, or Thr
at amino acid position 334, Ala or Gln at amino acid position 355,
Glu at amino acid position 356, Met at amino acid position 358,
Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr at amino acid position 396, Arg at amino
acid position 409, and Glu at amino acid position 419, wherein
binding activity of the variant to Fc.gamma. receptors [KD value
for Fc.gamma.RIIb of a polypeptide comprising an Fc region to which
an amino acid alteration(s) has not been introduced]/[KD value for
Fc.gamma.RIIb of a polypeptide comprising the Fc region variant]
has a value which is 15.0 or greater.
4. The Fc region variant of claim 3, wherein the amino acid at
position 238 is Asp, the amino acid at position 268 is Asp or Glu,
and the amino acid at position 271 is Gly, according to EU
numbering, and wherein the Fc region variant further comprises at
least one amino acid selected from the amino acid group consisting
of Asp at amino acid position 233, Asp at amino acid position 237,
Ile at amino acid position 264, Ala or Gly at amino acid position
267, Asp or Pro at amino acid position 272, Asp at amino acid
position 296, Gly at amino acid position 327, Arg at amino acid
position 330, Thr at amino acid position 332, and Leu or Met at
amino acid position 396.
5. The Fc region variant of claim 1, wherein the value for [KD
value for Fc.gamma.RIIb of a polypeptide comprising an Fc region to
which an amino acid alteration(s) has not been introduced]/[KD
value for Fc.gamma.RIIb of a polypeptide comprising an Fc region
variant] is 50.0 or more.
6. The Fc region variant of claim 1, wherein the value for [KD
value for Fc.gamma.RIIb of a polypeptide comprising an Fc region to
which an amino acid alteration(s) has not been introduced]/[KD
value for Fc.gamma.RIIb of a polypeptide comprising an Fc region
variant] is 100.0 or more.
7. The Fc region variant of claim 1, wherein the value for [KD
value for Fc.gamma.RIIa (type R) of a polypeptide comprising an Fc
region variant]/[KD value for Fc.gamma.RIIb of a polypeptide
comprising an Fc region variant] is 10.0 or greater.
8. The Fc region variant of claim 1, wherein the value for [KD
value for Fc.gamma.RIIa (type R) of a polypeptide comprising an Fc
region variant]/[KD value for Fc.gamma.RIIb of a polypeptide
comprising an Fc region variant] is 20.0 or greater.
9. The Fc region variant of claim 1, wherein the Fc region variant
comprises any one of the following set of amino acid alterations of
(a) to (x): (a) amino acid alterations at positions 238, 233, 237,
268, 271, 296, and 330 (EU numbering) of an Fc region; (b) amino
acid alterations at positions 238, 237, 268, 271, 296, and 330 (EU
numbering) of an Fc region; (c) amino acid alterations at positions
238, 233, 237, 268, 271, 296, 330, and 332 (EU numbering) of an Fc
region; (d) amino acid alterations at positions 238, 233, 237, 264,
267, 268, 271, and 330 (EU numbering) of an Fc region; (e) amino
acid alterations at positions 238, 233, 237, 267, 268, 271, 296,
330, and 332 (EU numbering) of an Fc region; (f) amino acid
alterations at positions 238, 237, 267, 268, 271, 296, 330, and 332
(EU numbering) of an Fc region; (g) amino acid alterations at
positions 238, 233, 237, 268, 271, 296, 327, and 330 (EU numbering)
of an Fc region; (h) amino acid alterations at positions 238, 233,
237, 264, 267, 268, and 271 (EU numbering) of an Fc region; (i)
amino acid alterations at positions 238, 233, 237, 264, 267, 268,
271, 296, and 330 (EU numbering) of an Fc region; (j) amino acid
alterations at positions 238, 233, 237, 264, 267, 268, 271, 296,
330, and 396 (EU numbering) of an Fc region; (k) amino acid
alterations at positions 238, 237, 264, 267, 268, 271, and 330 (EU
numbering) of an Fc region; (l) amino acid alterations at positions
238, 237, 264, 267, 268, 271, 296, and 330 (EU numbering) of an Fc
region; (m) amino acid alterations at positions 238, 264, 267, 268,
and 271 (EU numbering) of an Fc region; (n) amino acid alterations
at positions 238, 264, 267, 268, 271, and 296 (EU numbering) of an
Fc region; (o) amino acid alterations at positions 238, 237, 267,
268, 271, 296, and 330 (EU numbering) of an Fc region; (p) amino
acid alterations at positions 238, 233, 237, 264, 267, 268, 271,
330, and 396 (EU numbering) of the Fc region; (q) amino acid
alterations at positions 238, 233, 237, 264, 267, 268, 271, 296,
327, 330, and 396 (EU numbering) of an Fc region; (r) amino acid
alterations at positions 238, 233, 237, 264, 267, 268, 271, 272,
and 296 (EU numbering) of an Fc region; (s) amino acid alterations
at positions 238, 237, 264, 267, 268, 271, 272, and 330 (EU
numbering) of an Fc region; (t) amino acid alterations at positions
238, 237, 264, 267, 268, 271, 272, 296, and 330 (EU numbering) of
an Fc region; (u) amino acid alterations at positions 238, 233,
264, 267, 268, and 271 (EU numbering) of an Fc region; (v) amino
acid alterations at positions 238, 237, 267, 268, 271, 296, and 330
(EU numbering) of an Fc region; (w) amino acid alterations at
positions 238, 264, 267, 268, 271, 272, and 296 (EU numbering) of
an Fc region; and (x) amino acid alterations at positions 238, 233,
264, 267, 268, 271, and 296 (EU numbering) of an Fc region.
10. The Fc region variant of claim 1, wherein the Fc region variant
comprises any one of the following amino acid sequences of (a) to
(x): (a) an amino acid sequence in which the amino acid at position
238 is Asp, the amino acid at position 233 is Asp, the amino acid
at position 237 is Asp, the amino acid at position 268 is Asp, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, and the amino acid at position 330 is Arg, according to EU
numbering, in an Fc region; (b) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 237
is Asp, the amino acid at position 268 is Asp or Glu, the amino
acid at position 271 is Gly, the amino acid at position 296 is Asp,
and the amino acid at position 330 is Arg, according to EU
numbering, in an Fc region; (c) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 233
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 268 is Asp, the amino acid at position 271 is Gly, the
amino acid at position 296 is Asp, the amino acid at position 330
is Arg, and the amino acid at position 332 is Thr, according to EU
numbering, in an Fc region; (d) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 233
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 264 is Ile, the amino acid at position 267 is Gly or Ala,
the amino acid at position 268 is Glu, the amino acid at position
271 is Gly, and the amino acid at position 330 is Arg, according to
EU numbering, in an Fc region; (e) an amino acid sequence in which
the amino acid at position 238 is Asp, the amino acid at position
233 is Asp, the amino acid at position 237 is Asp, the amino acid
at position 267 is Ala, the amino acid at position 268 is Glu, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, the amino acid at position 330 is Arg, and the amino acid
at position 332 is Thr, according to EU numbering, in an Fc region;
(f) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 267 is Ala, the amino acid at position 268 is Glu, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, the amino acid at position 330 is Arg, and the amino acid
at position 332 is Thr, according to EU numbering, in an Fc region;
(g) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 268 is Asp, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, the amino acid at position 327 is Gly, and the amino acid
at position 330 is Arg, according to EU numbering, in an Fc region;
(h) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, and the amino acid at position 271 is Gly, according to EU
numbering, in an Fc region; (i) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 233
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 264 is Ile, the amino acid at position 267 is Ala, the
amino acid at position 268 is Glu, the amino acid at position 271
is Gly, the amino acid at position 296 is Asp, and the amino acid
at position 330 is Arg, according to EU numbering, in an Fc region;
(j) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, the amino acid at position 271 is Gly, the amino acid at
position 296 is Asp, the amino acid at position 330 is Arg, and the
amino acid at position 396 is Met or Leu, according to EU
numbering, in an Fc region; (k) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 237
is Asp, the amino acid at position 264 is Ile, the amino acid at
position 267 is Ala, the amino acid at position 268 is Glu, the
amino acid at position 271 is Gly, and the amino acid at position
330 is Arg, according to EU numbering, in an Fc region; (l) an
amino acid sequence in which the amino acid at position 238 is Asp,
the amino acid at position 237 is Asp, the amino acid at position
264 is Ile, the amino acid at position 267 is Ala, the amino acid
at position 268 is Glu, the amino acid at position 271 is Gly, the
amino acid at position 296 is Asp, and the amino acid at position
330 is Arg, according to EU numbering, in an Fc region; (m) an
amino acid sequence in which the amino acid at position 238 is Asp,
the amino acid at position 264 is Ile, the amino acid at position
267 is Ala, the amino acid at position 268 is Glu, and the amino
acid at position 271 is Gly, according to EU numbering, in an Fc
region; (n) an amino acid sequence in which the amino acid at
position 238 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, the amino acid at position 271 is Gly, and the amino acid
at position 296 is Asp, according to EU numbering, in an Fc region;
(o) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 267 is Ala or Gly, the amino acid at position 268 is Glu,
the amino acid at position 271 is Gly, the amino acid at position
296 is Asp, and the amino acid at position 330 is Arg, according to
EU numbering, in an Fc region; (p) an amino acid sequence in which
the amino acid at position 238 is Asp, the amino acid at position
233 is Asp, the amino acid at position 237 is Asp, the amino acid
at position 264 is Ile, the amino acid at position 267 is Ala, the
amino acid at position 268 is Glu, the amino acid at position 271
is Gly, the amino acid at position 330 is Arg, and the amino acid
at position 396 is Met or Leu, according to EU numbering, in an Fc
region; (q) an amino acid sequence in which the amino acid at
position 238 is Asp, the amino acid at position 233 is Asp, the
amino acid at position 237 is Asp, the amino acid at position 264
is Ile, the amino acid at position 267 is Ala, the amino acid at
position 268 is Glu, the amino acid at position 271 is Gly, the
amino acid at position 296 is Asp, the amino acid at position 327
is Gly, the amino acid at position 330 is Arg, and the amino acid
at position 396 is Met, according to EU numbering, in an Fc region;
(r) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, the amino acid at position 271 is Gly, the amino acid at
position 272 is Asp, and the amino acid at position 296 is Asp,
according to EU numbering, in an Fc region; (s) an amino acid
sequence in which the amino acid at position 238 is Asp, the amino
acid at position 237 is Asp, the amino acid at position 264 is Ile,
the amino acid at position 267 is Ala, the amino acid at position
268 is Glu, the amino acid at position 271 is Gly, the amino acid
at position 272 is Pro, and the amino acid at position 330 is Arg,
according to EU numbering, in an Fc region; (t) an amino acid
sequence in which the amino acid at position 238 is Asp, the amino
acid at position 237 is Asp, the amino acid at position 264 is Ile,
the amino acid at position 267 is Ala, the amino acid at position
268 is Glu, the amino acid at position 271 is Gly, the amino acid
at position 272 is Pro, the amino acid at position 296 is Asp, and
the amino acid at position 330 is Arg, according to EU numbering,
in an Fc region; (u) an amino acid sequence in which the amino acid
at position 238 is Asp, the amino acid at position 233 is Asp, the
amino acid at position 264 is Ile, the amino acid at position 267
is Ala, the amino acid at position 268 is Glu, and the amino acid
at position 271 is Gly, according to EU numbering, in an Fc region;
(v) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 267 is Gly, the amino acid at position 268 is Asp, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, and the amino acid at position 330 is Arg, according to EU
numbering, in an Fc region; (w) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 264
is Ile, the amino acid at position 267 is Ala, the amino acid at
position 268 is Glu, the amino acid at position 271 is Gly, the
amino acid at position 272 is Asp, and the amino acid at position
296 is Asp, according to EU numbering, in an Fc region; and (x) an
amino acid sequence in which the amino acid at position 238 is Asp,
the amino acid at position 233 is Asp, the amino acid at position
264 is Ile, the amino acid at position 267 is Ala, the amino acid
at position 268 is Glu, the amino acid at position 271 is Gly, and
the amino acid at position 296 is Asp, according to EU numbering,
in an Fc region.
11. An Fc region variant consisting of any one amino acid sequence
selected from among SEQ ID NOs: 43 to 68, SEQ ID NO: 70, SEQ ID NO:
71, and SEQ ID NOs: 75 to 77.
12. A polypeptide comprising at least two Fc region variants of
claim 1, wherein the two Fc region variants are associated.
13. The polypeptide of claim 12, wherein the amino acid sequences
of the two associated Fc region variants in the polypeptide are the
same.
14. The polypeptide of claim 12, wherein the amino acid sequences
of the two associated Fc region variants in the polypeptide are
different.
15. The polypeptide of claim 14, wherein the amino acid sequences
of the two associated Fc region variants have different amino
acid(s) at at least one amino acid position selected from positions
235, 236, 237, 238, and 239 according to EU numbering in the Fc
region variant.
16. The polypeptide of claim 15, wherein one of the amino acid
sequences of the two associated Fc region variants is an amino acid
sequence comprising at least one amino acid selected from Asp, Gln,
Glu, or Thr at amino acid position 235, Asn at amino acid position
236, Phe or Trp at amino acid position 237, Glu, Gly, or Asn at
amino acid position 238, and Asp or Glu at amino acid position 239
according to EU numbering.
17. The polypeptide of claim 12, wherein the polypeptide comprising
the Fc region variant is an IgG antibody.
18. The polypeptide of claim 12, wherein the polypeptide comprising
the Fc region variant is an Fc fusion protein molecule.
19. A pharmaceutical composition comprising the polypeptide of
claim 12.
20. A method of producing a pharmaceutical composition, the method
comprising: expressing nucleic acid that encodes the polypeptide of
claim 17; collecting the polypeptide, and preparing a
pharmaceutical composition comprising the polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 14/423,269, filed on Feb. 23, 2015 (now U.S.
Pat. No. 10,919,953), which is the National Stage of International
Application No. PCT/JP2013/072507, filed on Aug. 23, 2013, which
claims the benefit of Japanese Application No. 2012-185868, filed
on Aug. 24, 2012.
TECHNICAL FIELD
[0002] The present invention relates to Fc region variants
introduced with amino acid alteration(s) into an antibody Fc
region, which have enhanced Fc.gamma.RIIb-binding activity, and/or
enhanced binding selectivity to Fc.gamma.RIIb compared to
Fc.gamma.RIIa (type R), as compared to an Fc region to which an
amino acid alteration(s) has not been introduced; polypeptides
comprising the Fc region variants; and pharmaceutical compositions
comprising the polypeptides.
BACKGROUND ART
[0003] Antibodies are drawing attention as pharmaceuticals since
they are highly stable in blood and have few side effects
(Non-patent Documents 1 and 2). Almost all therapeutic antibodies
currently on the market are antibodies of the human IgG1 subclass.
One of the known functions of IgG class antibodies is
antibody-dependent cell-mediated cytotoxicity (hereinafter denoted
as ADCC activity) (Non-patent Document 3). For an antibody to
exhibit ADCC activity, the antibody Fc region must bind to an
Fc.gamma. receptor (hereinafter denoted as Fc.gamma.R) which is an
antibody-binding receptor present on the surface of effector cells
such as killer cells, natural killer cells, and activated
macrophages.
[0004] In humans, the Fc.gamma.RIa (CD64A), Fc.gamma.RIIa (CD32A),
Fc.gamma.RIIb (CD32B), Fc.gamma.RIIIa (CD16A), and Fc.gamma.RIIIb
(CD16B) isoforms have been reported as the Fc.gamma.R protein
family, and the respective allotypes have also been reported
(Non-patent Document 7). Fc.gamma.RIa, Fc.gamma.RIIa, and
Fc.gamma.RIIIa are called activating Fc.gamma.R since they have
immunologically active functions, and Fc.gamma.RIIb is called
inhibitory Fc.gamma.R since it has immunosuppressive functions
(Non-patent Document 8).
[0005] In the binding between the Fc region and Fc.gamma.R, several
amino acid residues in the antibody hinge region and CH2 domain,
and a sugar chain attached to Asn at position 297 (EU numbering)
bound to the CH2 domain have been shown to be important (Non-patent
Documents 4, 5, and 6). Various variants having Fc.gamma.R-binding
properties, mainly antibodies with mutations introduced into these
sites, have been studied so far; and Fc region variants having
higher binding activities towards activating Fc.gamma.R have been
obtained (Patent Documents 1, 2, 3, and 4).
[0006] When activating Fc.gamma.R is cross-linked with an immune
complex, it phosphorylates immunoreceptor tyrosine-based activating
motifs (ITAMs) contained in the intracellular domain or FcR common
.gamma.-chain (an interaction partner), activates a signal
transducer SYK, and triggers inflammatory immune response by
initiating an activation signal cascade (Non-patent Document
9).
[0007] Fc.gamma.RIIb is the only Fc.gamma.R expressed on B cells
(Non-patent Document 10). Interaction of the antibody Fc region
with Fc.gamma.RIIb has been reported to suppress the primary immune
response of B cells (Non-patent Document 11). Furthermore, it is
reported that when Fc.gamma.RIIb on B cells and a B cell receptor
(BCR) are cross-linked via an immune complex in blood, B cell
activation is suppressed, and antibody production by B cells is
suppressed (Non-patent Document 12). In this immunosuppressive
signal transduction mediated by BCR and Fc.gamma.RIIb, the
immunoreceptor tyrosine-based inhibitory motif (ITIM) contained in
the intracellular domain of Fc.gamma.RIIb is necessary (Non-patent
Documents 13 and 14). When ITIM is phosphorylated upon signaling,
SH2-containing inositol polyphosphate 5-phosphatase (SHIP) is
recruited, transduction of other activating Fc.gamma.R signal
cascades is inhibited, and inflammatory immune response is
suppressed (Non-patent Document 15). Furthermore, aggregation of
Fc.gamma.RIIb alone has been reported to transiently suppress
calcium influx due to BCR cross-linking and B cell proliferation in
a BCR-independent manner without inducing apoptosis of
IgM-producing B cells (Non-patent Document 16).
[0008] Furthermore, Fc.gamma.RIIb is also expressed on dendritic
cells, macrophages, activated neutrophils, mast cells, and
basophils. Fc.gamma.RIIb inhibits the functions of activating
Fc.gamma.R such as phagocytosis and release of inflammatory
cytokines in these cells, and suppresses inflammatory immune
responses (Non-patent Document 8).
[0009] The importance of immunosuppressive functions of
Fc.gamma.RIIb has been elucidated so far through studies using
Fc.gamma.RIIb knockout mice. There are reports that in
Fc.gamma.RIIb knockout mice, humoral immunity is not appropriately
regulated (Non-Patent Document 17), sensitivity towards
collagen-induced arthritis (CIA) is increased (Non-patent Document
18), lupus-like symptoms are presented, and Goodpasture's
syndrome-like symptoms are presented (Non-patent Document 19).
[0010] Furthermore, regulatory inadequacy of Fc.gamma.RIIb has been
reported to be related to human autoimmnue diseases. For example,
the relationship between genetic polymorphism in the transmembrane
region and promoter region of Fc.gamma.RIIb, and the frequency of
development of systemic lupus erythematosus (SLE) (Non-patent
Documents 20, 21, 22, 23, and 24), and decrease of Fc.gamma.RIIb
expression on the surface of B cells in SLE patients (Non-patent
Document 25 and 26) have been reported.
[0011] From mouse models and clinical findings as such,
Fc.gamma.RIIb is considered to play the role of controlling
autoimmune diseases and inflammatory diseases through involvement
in particular with B cells, and it is a promising target molecule
for controlling autoimmune diseases and inflammatory diseases.
[0012] IgG1, mainly used as a commercially available therapeutic
antibody, is known to bind not only to Fc.gamma.RIIb, but also
strongly to activating Fc.gamma.R (Non-patent Document 27). It may
be possible to develop therapeutic antibodies having greater
immunosuppressive properties compared with those of IgG1, by
utilizing an Fc region with enhanced Fc.gamma.RIIb binding, or
improved Fc.gamma.RIIb-binding selectivity compared with activating
Fc.gamma.R. For example, it has been suggested that the use of an
antibody having a variable region that binds to BCR and an Fc with
enhanced Fc.gamma.RIIb binding may inhibit B cell activation
(Non-patent Document 28). It has been reported that crosslinking
Fc.gamma.RIIb on B cells and IgE bound to a B-cell receptor
suppresses differentiation of B cells into plasma cells, which as a
result causes suppression of IgE production; and in human
PBMC-transplanted mice, human IgG and IgM concentrations are
maintained whereas the human IgE concentration is decreased
(Non-patent Document 29). Besides IgE, it has been reported that
when Fc.gamma.RIIB and CD79b which is a constituent molecule of a
B-cell receptor complex are cross-linked by an antibody, B cell
proliferation is suppressed in vitro, and arthritis symptoms are
alleviated in the collagen arthritis model (Non-patent Document
30).
[0013] Besides B cells, it has been reported that crosslinking of
Fc.epsilon.RI and Fc.gamma.RIIb on mast cells using molecules, in
which the Fc portion of an IgG with enhanced Fc.gamma.RIIb binding
is fused to the Fc portion of IgE that binds to an IgE receptor
Fc.epsilon.RI, causes Fc.gamma.RIIb phosphorylation of
Fc.gamma.RIIb, thereby suppressing Fc.epsilon.RI-dependent calcium
influx. This suggests that inhibition of degranulation via
Fc.gamma.RIIb stimulation is possible by enhancing Fc.gamma.RIIb
binding (Non-patent Document 31).
[0014] Accordingly, an antibody having an Fc with improved
Fc.gamma.RIIb-binding activity is suggested to be promising as a
therapeutic agent for inflammatory diseases such as autoimmune
diseases.
[0015] Furthermore, it has been reported that activation of
macrophages and dendritic cells via Toll-like receptor 4 due to LPS
stimulation is suppressed in the presence of an antibody-antigen
immune complex, and this effect is also suggested to be actions of
the immune complex via Fc.gamma.RIIb (Non-patent Documents 32 and
33). Therefore, use of antibodies with enhanced Fc.gamma.RIIb
binding is expected to enable enhancement of TLR-mediated
activation signal-suppressing actions; thus such antibodies have
been suggested as being promising as therapeutic agents for
inflammatory diseases such as autoimmune diseases.
[0016] Furthermore, mutants with enhanced Fc.gamma.RIIb binding
have been suggested to be promising therapeutic agents for cancer,
as well as therapeutic agents for inflammatory diseases such as
autoimmune diseases. So far, Fc.gamma.RIIb has been found to play
an important role in the agonistic activity of agonist antibodies
against the anti-TNF receptor superfamily. Specifically, it has
been suggested that interaction with Fc.gamma.RIIb is required for
the agonistic activity of antibodies against CD40, DR4, DR5, CD30,
and CD137, which are included in the TNF receptor family
(Non-patent Documents 34, 35, 36, 37, 38, 39 and 40). Non-patent
Document 34 shows that the use of antibodies with enhanced
Fc.gamma.RIIb binding enhances the anti-tumor effect of anti-CD40
antibodies. Accordingly, antibodies with enhanced Fc.gamma.RIIb are
expected to have an effect of enhancing agonistic activity of
agonist antibodies including antibodies against the anti-TNF
receptor superfamily.
[0017] In addition, it has been shown that cell proliferation is
suppressed when using an antibody that recognizes Kit, a type of
receptor tyrosine kinase (RTK), to crosslink Fc.gamma.RIIb and Kit
on Kit-expressing cells. Similar effects have been reported even in
cases where this Kit is constitutively activated and has mutations
that cause oncogenesis (Non-patent Document 41). Therefore, it is
expected that use of antibodies with enhanced Fc.gamma.RIIb binding
may enhance inhibitory effects on cells expressing RTK having
constitutively activated mutations.
[0018] Antibodies having an Fc with improved Fc.gamma.RIIb-binding
activity have been reported (Non-patent Document 28). In this
Document, Fc.gamma.RIIb-binding activity was improved by adding
alterations such as S267E/L328F, G236D/S267E, and S239D/S267E to an
antibody Fc region. Among them, the antibody introduced with the
S267E/L328F mutation most strongly binds to Fc.gamma.RIIb, and
maintains the same level of binding to Fc.gamma.RIa and
Fc.gamma.RIIa type H in which a residue at position 131 of
Fc.gamma.RIIa is His as that of a naturally-occurring IgG1.
However, another report shows that this alteration enhances the
binding to Fc.gamma.RIIa type R in which a residue at position 131
of Fc.gamma.RIIa is Arg several hundred times to the same level of
Fc.gamma.RIIb binding, which means the Fc.gamma.RIIb-binding
selectivity is not improved in comparison with type-R Fc.gamma.RIIa
(Patent Document 5).
[0019] Only the effect of enhancing Fc.gamma.RIIa binding and not
the enhancement of Fc.gamma.RIIb binding is considered to have
influence on cells such as platelets which express Fc.gamma.RIIa
but do not express Fc.gamma.RIIb (Non-patent Document 8). For
example, the group of patients who were administered bevacizumab,
an antibody against VEGF, is known to have an increased risk for
thromboembolism (Non-patent Document 42). Furthermore,
thromboembolism has been observed in a similar manner in clinical
development tests of antibodies against the CD40 ligand, and the
clinical study was discontinued (Non-patent Document 43). In both
cases of these antibodies, later studies using animal models and
such have suggested that the administered antibodies aggregate
platelets via Fc.gamma.RIIa binding on the platelets, and form
blood clots (Non-patent Documents 44 and 45). In systemic lupus
erythematosus which is an autoimmune disease, platelets are
activated via an Fc.gamma.RIIa-dependent mechanism, and platelet
activation has been reported to correlate with the severity of
symptoms (Non-patent Document 46). Administering an antibody with
enhanced Fc.gamma.RIIa binding to such patients who already have a
high risk for developing thromboembolism will increase the risk for
developing thromboembolism, thus is extremely dangerous.
[0020] Furthermore, antibodies with enhanced Fc.gamma.RIIa binding
have been reported to enhance macrophage-mediated antibody
dependent cellular phagocytosis (ADCP) (Non-patent Document 47).
When antigens to be bound by the antibodies are phagocytized by
macrophages, antibodies themselves are considered to be also
phagocytized at the same time. When antibodies are administered as
pharmaceuticals, it is supposed that peptide fragments derived from
the administered antibodies are likely to be also presented as an
antigen, thereby increasing the risk of production of antibodies
against therapeutic antibodies (anti-therapeutic antibodies). More
specifically, enhancing Fc.gamma.RIIa binding will increase the
risk of production of antibodies against the therapeutic
antibodies, and this will remarkably decrease their value as
pharmaceuticals. Furthermore, Fc.gamma.RIIb on dendritic cells have
been suggested to contribute to peripheral tolerance by inhibiting
dendritic cell activation caused by immune complexes formed between
antigens and antibodies, or by suppressing antigen presentation to
T cells via activating Fc.gamma. receptors (Non-patent Document
48). Since Fc.gamma.RIIa is also expressed on dendritic cells, when
antibodies having an Fc with enhanced selective binding to
Fc.gamma.RIIb are used as pharmaceuticals, antigens are not readily
presented by dendritic cells and such due to enhanced selective
binding to Fc.gamma.RIIb, and risk of anti-drug antibody production
can be relatively decreased. Such antibodies may be useful in that
regard as well.
[0021] More specifically, the value as pharmaceuticals will be
considerably reduced when Fc.gamma.RIIa binding is enhanced, which
leads to increased risk of thrombus formation via platelet
aggregation and increased risk of anti-therapeutic antibody
production due to an increased immunogenicity.
[0022] From such a viewpoint, the aforementioned Fc variant with
enhanced Fc.gamma.RIIb binding shows remarkably enhanced type-R
Fc.gamma.RIIa binding compared with that of a naturally-occurring
IgG1. Therefore, its value as a pharmaceutical for patients
carrying type-R Fc.gamma.RIIa is considerably reduced. Types H and
R of Fc.gamma.RIIa are observed in Caucasians and African-Americans
with approximately the same frequency (Non-patent Documents 49 and
50). Therefore, when this Fc variant was used for treatment of
autoimmune diseases, the number of patients who can safely use it
while enjoying its effects as a pharmaceutical will be limited.
[0023] Furthermore, in dendritic cells deficient in Fc.gamma.RIIb
or dendritic cells in which the interaction between Fc.gamma.RIIb
and the antibody Fc portion is inhibited by an anti-Fc.gamma.RIIb
antibody, dendritic cells have been reported to mature (Non-patent
Documents 51 and 52). This report suggests that Fc.gamma.RIIb is
actively suppressing maturation of dendritic cells in a steady
state where inflammation and such are not taking place and
activation does not take place. Fc.gamma.RIIa is expressed on the
dendritic cell surface in addition to Fc.gamma.RIIb; therefore,
even if binding to inhibitory Fc.gamma.RIIb is enhanced and if
binding to activating Fc.gamma.R such as Fc.gamma.RIIa is also
enhanced, maturation of dendritic cells may be promoted as a
result. More specifically, improving not only the
Fc.gamma.RIIb-binding activity but also the ratio of
Fc.gamma.RIIb-binding activity relative to Fc.gamma.RIIa-binding
activity is considered to be important in providing antibodies with
an immunosuppressive action.
[0024] Therefore, when considering generation of pharmaceuticals
that utilize the Fc.gamma.RIIb binding-mediated immunosuppressive
action, there is a need for an Fc variant that not only has
enhanced Fc.gamma.RIIb-binding activity, but also has binding to
both Fc.gamma.RIIa, types H and R allotypes, which is maintained at
a similar level or is weakened to a lower level than that of a
naturally-occurring IgG1.
[0025] Meanwhile, cases where amino acid alterations were
introduced into the Fc region to increase the Fc.gamma.RIIb-binding
selectivity have been reported so far (Non-patent Document 53).
However, all variants said to have improved Fc.gamma.RIIb
selectivity as reported in this document showed decreased
Fc.gamma.RIIb binding compared with that of a naturally-occurring
IgG1. Therefore, it is considered to be difficult for these
variants to actually induce an Fc.gamma.RIIb-mediated
immunosuppressive reaction more strongly than IgG1.
[0026] Furthermore, since Fc.gamma.RIIb plays an important role in
the agonist antibodies mentioned above, enhancing their binding
activity is expected to enhance the agonistic activity. However,
when Fc.gamma.RIIa binding is similarly enhanced, unintended
activities such as ADCC activity and ADCP activity will be
exhibited, and this may cause side effects. Also from such
viewpoint, it is preferable to be able to selectively enhance
Fc.gamma.RIIb-binding activity.
[0027] From these results, in producing therapeutic antibodies to
be used for treating autoimmune diseases and cancer utilizing
Fc.gamma.RIIb, it is important that compared with those of a
naturally-occurring IgG, the activities of binding to both
Fc.gamma.RIIa allotypes are maintained or decreased, and
Fc.gamma.RIIb binding is enhanced. However, Fc.gamma.RIIb shares
93% sequence identity in the extracellular region with that of
Fc.gamma.RIIa which is one of the activating Fc.gamma.Rs, and they
are very similar structurally. There are allotypes of
Fc.gamma.RIIa, H type and R type, in which the amino acid at
position 131 is His (type H) or Arg (type R), and yet each of them
reacts differently with the antibodies (Non-patent Document 54).
Therefore, the difficult problem may be producing an Fc region
variant with enhanced selective Fc.gamma.RIIb binding as compared
to each allotype of Fc.gamma.RIIa, which involves distinguishing
highly homologous sequences between Fc.gamma.RIIa and
Fc.gamma.RIIb. In fact, variants having sufficient binding activity
and selectivity to Fc.gamma.RIIb have not been obtained so far.
Patent Document 5 reports variants with enhanced
Fc.gamma.RIIb-binding activity; however, the degree of enhancement
is low, and there is a demand for development of variants having
properties similar to those described above.
PRIOR ART DOCUMENTS
Patent Documents
[0028] [Patent Document 1] WO 2000/42072 [0029] [Patent Document 2]
WO 2006/019447 [0030] [Patent Document 3] WO 2004/99249 [0031]
[Patent Document 4] WO 2004/29207 [0032] [Patent Document 5]
US2009/0136485
Non-Patent Documents
[0032] [0033] [Non-patent Document 1] Nat Biotechnol, 23,
1073-1078, 2005 [0034] [Non-patent Document 2] Eur J Pharm
Biopharm, 59(3), 389-96. 2005 [0035] [Non-patent Document 3] Chem
Immunol, 65, 88-110, 1997 [0036] [Non-patent Document 4] J Biol
Chem, 276, 16478-16483, 2001 [0037] [Non-patent Document 5] Eur J
Immunol 23, 1098-1104, 1993 [0038] [Non-patent Document 6]
Immunology, 86, 319-324, 1995 [0039] [Non-patent Document 7]
Immunol Lett, 82, 57-65, 2002 [0040] [Non-patent Document 8] Nat
Rev Immunol, 10, 328-343, 2010 [0041] [Non-patent Document 9] Nat
Rev Immunol, 8, 34-47, 2008 [0042] [Non-patent Document 10] Eur J
Immunol 19, 1379-1385, 1989 [0043] [Non-patent Document 11] J Exp
Med 129, 1183-1201, 1969 [0044] [Non-patent Document 12] Immunol
Lett 88, 157-161, 2003 [0045] [Non-patent Document 13] Science,
256, 1808-1812, 1992 [0046] [Non-patent Document 14] Nature, 368,
70-73, 1994 [0047] [Non-patent Document 15] Science, 290, 84-89,
2000 [0048] [Non-patent Document 16] J Imunol, 181, 5350-5359, 2008
[0049] [Non-patent Document 17] J Immunol, 163, 618-622, 1999
[0050] [Non-patent Document 18] J Exp Med, 189, 187-194, 1999
[0051] [Non-patent Document 19] J Exp Med, 191, 899-906, 2000
[0052] [Non-patent Document 20] Hum, Genet, 117, 220-227, 2005
[0053] [Non-patent Document 21] J Biol Chem, 282, 1738-1746, 2007
[0054] [Non-patent Document 22] Arthritis Rheum, 54, 3908-3917,
2006 [0055] [Non-patent Document 23] Nat Med, 11, 1056-1058, 2005
[0056] [Non-patent Document 24] J Immunol, 176, 5321-5328, 2006
[0057] [Non-patent Document 25] J Exp Med, 203, 2157-2164, 2006
[0058] [Non-patent Document 26] J Immunol. 178, 3272-3280, 2007
[0059] [Non-patent Document 27] Blood, 113, 3716-3725, 2009 [0060]
[Non-patent Document 28] Mol Immunol, 45, 3926-3933, 2008 [0061]
[Non-patent Document 29] J Allergy Clin Immunol, 2012, 129:
1102-1115 [0062] [Non-patent Document 30] Arthritis Rheum, 62,
1933-1943, 2010 [0063] [Non-patent Document 31] Immunol let, doi:
10.1016/j.imlet.2012.01.008) [0064] [Non-patent Document 32] J.
Immunol, 2009, 183: 4509-4520 [0065] [Non-patent Document 33] J.
Immunol, 2009, 182: 554-562 [0066] [Non-patent Document 34]
Science, 333, 1030-1034, 2011 [0067] [Non-patent Document 35]
Cancer Cell 19, 101-113, 2011 [0068] [Non-patent Document 36] J
Clin Invest, 122 (3), 1066-1075, 2012 [0069] [Non-patent Document
37] J Immunol 171, 562-, 2003 [0070] [Non-patent Document 38]
Blood, 108, 705-, 2006 [0071] [Non-patent Document 39] J Immunol
166, 4891, 2001 [0072] [Non-patent Document 40] doi:
10.1073/pnas.1208698109 [0073] [Non-patent Document 41] Immunol
let, 2002, 143: 28-33 [0074] [Non-patent Document 42] J Natl Cancer
Inst, 99, 1232-1239, 2007 [0075] [Non-patent Document 43] Arthritis
Rheum, 48, 719-727, 2003 [0076] [Non-patent Document 44] J Thromb
Haemost, 7, 171-181, 2008 [0077] [Non-patent Document 45] J
Immunol, 185, 1577-1583, 2010 [0078] [Non-patent Document 46] Sci
Transl Med, vol 2, issue 47, 47-63, 2010 [0079] [Non-patent
Document 47] Mol Cancer Ther 7, 2517-2527, 2008 [0080] [Non-patent
Document 48] J. Immunol, 2007, 178: 6217-6226 [0081] [Non-patent
Document 49] J Clin Invest, 97, 1348-1354, 1996 [0082] [Non-patent
Document 50] Arthritis Rheum, 41, 1181-1189, 1998 [0083]
[Non-patent Document 51] J Clin Invest 115, 2914-2923, 2005 [0084]
[Non-patent Document 52] Proc Natl Acad Sci USA, 102, 2910-2915,
2005 [0085] [Non-patent Document 53] Mol Immunol, 40, 585-593, 2003
[0086] [Non-patent Document 54] J Exp Med, 172, 19-25, 1990
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0087] The present invention was achieved in view of the above
circumstances. An objective of the present invention is to provide
an Fc region variant by introducing an amino acid alteration(s)
into an antibody Fc region, which variant has enhanced
Fc.gamma.RIIb-binding activity, and/or enhanced binding selectivity
to Fc.gamma.RIIb compared to Fc.gamma.RIIa (type R), as compared to
when no amino acid alteration has been introduced into the Fc
region; a polypeptide comprising the Fc region variant; and a
pharmaceutical composition containing the polypeptide.
Means for Solving the Problems
[0088] The present inventors carried out dedicated research on: an
Fc region variant with enhanced Fc.gamma.RIIb-binding and enhanced
binding selectivity to Fc.gamma.RIIb compared to Fc.gamma.RIIa
(type R), as compared to when the Fc region is unaltered, by
introducing amino acid alteration(s) into the Fc region; and a
polypeptide comprising the Fc region variant. As a result, the
present inventors found that Fc.gamma.RIIb-binding activity is
enhanced and/or binding selectivity to Fc.gamma.RIIb compared to
Fc.gamma.RIIa (type R) is enhanced by combining an Fc region
variant in which the amino acid at position 238 (EU numbering) in
the Fc Region has been altered with other amino acid
alteration(s).
[0089] More specifically, the present invention relates to the
following:
[1] an Fc region variant in which amino acid at position 238
according to EU numbering and at least one amino acid selected from
those at positions 233, 234, 235, 237, 264, 265, 266, 267, 268,
269, 271, 272, 274, 296, 326, 327, 330, 331, 332, 333, 334, 355,
356, 358, 396, 409, and 419 according to EU numbering are altered
to other amino acids, wherein binding activity of the variant to
Fc.gamma. receptors [KD value for Fc.gamma.RIIb of a polypeptide
comprising an Fc region to which an amino acid alteration(s) has
not been introduced]/[KD value for Fc.gamma.RIIb of a polypeptide
comprising the Fc region variant] has a value which is 15.0 or
greater; [2] the Fc region variant of [1], wherein the amino acids
at positions 238, 268, and 271 according to EU numbering are
altered to other amino acids, and in addition, at least one amino
acid selected from amino acids at positions 233, 237, 264, 267,
272, 296, 327, 330, 332, and 396 according to EU numbering are
altered to other amino acids; [3] an Fc region variant whose amino
acid at position 238 according to EU numbering is Asp, and which
comprises at least one amino acid selected from the amino acid
group consisting of Asp at amino acid position 233, Tyr at amino
acid position 234, Phe at amino acid position 235, Asp at amino
acid position 237, Ile at amino acid position 264, Glu at amino
acid position 265, Phe, Leu, or Met at amino acid position 266,
Ala, Glu, Gly, or Gln at amino acid position 267, Asp, Gln, or Glu
at amino acid position 268, Asp at amino acid position 269, Gly at
amino acid position 271, Asp, Phe, Ile, Met, Asn, Pro, or Gln at
amino acid position 272, Gln at amino acid position 274, Asp or Phe
at amino acid position 296, Ala or Asp at amino acid position 326,
Gly at amino acid position 327, Lys, Arg, or Ser at amino acid
position 330, Ser at amino acid position 331, Lys, Arg, Ser, or Thr
at amino acid position 332, Lys, Arg, Ser, or Thr at amino acid
position 333, Arg, Ser, or Thr at amino acid position 334, Ala or
Gln at amino acid position 355, Glu at amino acid position 356, Met
at amino acid position 358, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys,
Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr at amino acid
position 396, Arg at amino acid position 409, and Glu at amino acid
position 419, wherein binding activity of the variant to Fc.gamma.
receptors [KD value for Fc.gamma.RIIb of a polypeptide comprising
an Fc region to which an amino acid alteration(s) has not been
introduced]/[KD value for Fc.gamma.RIIb of a polypeptide comprising
the Fc region variant] has a value which is 15.0 or greater; [4]
the Fc region variant of [3], wherein the amino acid at position
238 is Asp, the amino acid at position 268 is Asp or Glu, and the
amino acid at position 271 is Gly, according to EU numbering, and
wherein the Fc region variant further comprises at least one amino
acid selected from the amino acid group consisting of Asp at amino
acid position 233, Asp at amino acid position 237, Ile at amino
acid position 264, Ala or Gly at amino acid position 267, Asp or
Pro at amino acid position 272, Asp at amino acid position 296, Gly
at amino acid position 327, Arg at amino acid position 330, Thr at
amino acid position 332, and Leu or Met at amino acid position 396;
[5] the Fc region variant of any one of [1] to [4], wherein the
value for [KD value for Fc.gamma.RIIb of a polypeptide comprising
an Fc region to which an amino acid alteration(s) has not been
introduced]/[KD value for Fc.gamma.RIIb of a polypeptide comprising
an Fc region variant] is 50.0 or more; [6] the Fc region variant of
any one of [1] to [4], wherein the value for [KD value for
Fc.gamma.RIIb of a polypeptide comprising an Fc region to which an
amino acid alteration(s) has not been introduced]/[KD value for
Fc.gamma.RIIb of a polypeptide comprising an Fc region variant] is
100.0 or more; [7] the Fc region variant of any one of [1] to [6],
wherein the value for [KD value for Fc.gamma.RIIa (type R) of a
polypeptide comprising an Fc region variant]/[KD value for
Fc.gamma.RIIb of a polypeptide comprising an Fc region variant] is
10.0 or greater; [8] the Fc region variant of any one of [1] to
[6], wherein the value for [KD value for Fc.gamma.RIIa (type R) of
a polypeptide comprising an Fc region variant]/[KD value for
Fc.gamma.RIIb of a polypeptide comprising an Fc region variant] is
20.0 or greater; [9] The Fc region variant of any one of [1] to
[8], wherein the Fc region variant comprises any one of the
following set of amino acid alterations of (a) to (x): (a) amino
acid alterations at positions 238, 233, 237, 268, 271, 296, and 330
(EU numbering) of an Fc region; (b) amino acid alterations at
positions 238, 237, 268, 271, 296, and 330 (EU numbering) of an Fc
region; (c) amino acid alterations at positions 238, 233, 237, 268,
271, 296, 330, and 332 (EU numbering) of an Fc region; (d) amino
acid alterations at positions 238, 233, 237, 264, 267, 268, 271,
and 330 (EU numbering) of an Fc region; (e) amino acid alterations
at positions 238, 233, 237, 267, 268, 271, 296, 330, and 332 (EU
numbering) of an Fc region; (f) amino acid alterations at positions
238, 237, 267, 268, 271, 296, 330, and 332 (EU numbering) of an Fc
region; (g) amino acid alterations at positions 238, 233, 237, 268,
271, 296, 327, and 330 (EU numbering) of an Fc region; (h) amino
acid alterations at positions 238, 233, 237, 264, 267, 268, and 271
(EU numbering) of an Fc region; (i) amino acid alterations at
positions 238, 233, 237, 264, 267, 268, 271, 296, and 330 (EU
numbering) of an Fc region; (j) amino acid alterations at positions
238, 233, 237, 264, 267, 268, 271, 296, 330, and 396 (EU numbering)
of an Fc region; (k) amino acid alterations at positions 238, 237,
264, 267, 268, 271, and 330 (EU numbering) of an Fc region; (l)
amino acid alterations at positions 238, 237, 264, 267, 268, 271,
296, and 330 (EU numbering) of an Fc region; (m) amino acid
alterations at positions 238, 264, 267, 268, and 271 (EU numbering)
of an Fc region; (n) amino acid alterations at positions 238, 264,
267, 268, 271, and 296 (EU numbering) of an Fc region; (o) amino
acid alterations at positions 238, 237, 267, 268, 271, 296, and 330
(EU numbering) of an Fc region; (p) amino acid alterations at
positions 238, 233, 237, 264, 267, 268, 271, 330, and 396 (EU
numbering) of the Fc region; (q) amino acid alterations at
positions 238, 233, 237, 264, 267, 268, 271, 296, 327, 330, and 396
(EU numbering) of an Fc region; (r) amino acid alterations at
positions 238, 233, 237, 264, 267, 268, 271, 272, and 296 (EU
numbering) of an Fc region; (s) amino acid alterations at positions
238, 237, 264, 267, 268, 271, 272, and 330 (EU numbering) of an Fc
region; (t) amino acid alterations at positions 238, 237, 264, 267,
268, 271, 272, 296, and 330 (EU numbering) of an Fc region; (u)
amino acid alterations at positions 238, 233, 264, 267, 268, and
271 (EU numbering) of an Fc region; (v) amino acid alterations at
positions 238, 237, 267, 268, 271, 296, and 330 (EU numbering) of
an Fc region; (w) amino acid alterations at positions 238, 264,
267, 268, 271, 272, and 296 (EU numbering) of an Fc region; and (x)
amino acid alterations at positions 238, 233, 264, 267, 268, 271,
and 296 (EU numbering) of an Fc region; [10] the Fc region variant
of any one of [1] to [8], wherein the Fc region variant comprises
any one of the following amino acid sequences of (a) to (x): (a) an
amino acid sequence in which the amino acid at position 238 is Asp,
the amino acid at position 233 is Asp, the amino acid at position
237 is Asp, the amino acid at position 268 is Asp, the amino acid
at position 271 is Gly, the amino acid at position 296 is Asp, and
the amino acid at position 330 is Arg, according to EU numbering,
in an Fc region; (b) an amino acid sequence in which the amino acid
at position 238 is Asp, the amino acid at position 237 is Asp, the
amino acid at position 268 is Asp or Glu, the amino acid at
position 271 is Gly, the amino acid at position 296 is Asp, and the
amino acid at position 330 is Arg, according to EU numbering, in an
Fc region; (c) an amino acid sequence in which the amino acid at
position 238 is Asp, the amino acid at position 233 is Asp, the
amino acid at position 237 is Asp, the amino acid at position 268
is Asp, the amino acid at position 271 is Gly, the amino acid at
position 296 is Asp, the amino acid at position 330 is Arg, and the
amino acid at position 332 is Thr, according to EU numbering, in an
Fc region; (d) an amino acid sequence in which the amino acid at
position 238 is Asp, the amino acid at position 233 is Asp, the
amino acid at position 237 is Asp, the amino acid at position 264
is Ile, the amino acid at position 267 is Gly or Ala, the amino
acid at position 268 is Glu, the amino acid at position 271 is Gly,
and the amino acid at position 330 is Arg, according to EU
numbering, in an Fc region; (e) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 233
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 267 is Ala, the amino acid at position 268 is Glu, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, the amino acid at position 330 is Arg, and the amino acid
at position 332 is Thr, according to EU numbering, in an Fc region;
(f) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 267 is Ala, the amino acid at position 268 is Glu, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, the amino acid at position 330 is Arg, and the amino acid
at position 332 is Thr, according to EU numbering, in an Fc region;
(g) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 268 is Asp, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, the amino acid at position 327 is Gly, and the amino acid
at position 330 is Arg, according to EU numbering, in an Fc region;
(h) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, and the amino acid at position 271 is Gly, according to EU
numbering, in an Fc region; (i) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 233
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 264 is Ile, the amino acid at position 267 is Ala, the
amino acid at position 268 is Glu, the amino acid at position 271
is Gly, the amino acid at position 296 is Asp, and the amino acid
at position 330 is Arg, according to EU numbering, in an Fc region;
(j) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, the amino acid at position 271 is Gly, the amino acid at
position 296 is Asp, the amino acid at position 330 is Arg, and the
amino acid at position 396 is Met or Leu, according to EU
numbering, in an Fc region; (k) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 237
is Asp, the amino acid at position 264 is Ile, the amino acid at
position 267 is Ala, the amino acid at position 268 is Glu, the
amino acid at position 271 is Gly, and the amino acid at position
330 is Arg, according to EU numbering, in an Fc region; (l) an
amino acid sequence in which the amino acid at position 238 is Asp,
the amino acid at position 237 is Asp, the amino acid at position
264 is Ile, the amino acid at position 267 is Ala, the amino acid
at position 268 is Glu, the amino acid at position 271 is Gly, the
amino acid at position 296 is Asp, and the amino acid at position
330 is Arg, according to EU numbering, in an Fc region; (m) an
amino acid sequence in which the amino acid at position 238 is Asp,
the amino acid at position 264 is Ile, the amino acid at position
267 is Ala, the amino acid at position 268 is Glu, and the amino
acid at position 271 is Gly, according to EU numbering, in an Fc
region; (n) an amino acid sequence in which the amino acid at
position 238 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, the amino acid at position 271 is Gly, and the amino acid
at position 296 is Asp, according to EU numbering, in an Fc region;
(o) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 267 is Ala or Gly, the amino acid at position 268 is Glu,
the amino acid at position 271 is Gly, the amino acid at position
296 is Asp, and the amino acid at position 330 is Arg, according to
EU numbering, in an Fc region; (p) an amino acid sequence in which
the amino acid at position 238 is Asp, the amino acid at position
233 is Asp, the amino acid at position 237 is Asp, the amino acid
at position 264 is Ile, the amino acid at position 267 is Ala, the
amino acid at position 268 is Glu, the amino acid at position 271
is Gly, the amino acid at position 330 is Arg, and the amino acid
at position 396 is Met or Leu, according to EU numbering, in an Fc
region; (q) an amino acid sequence in which the amino acid at
position 238 is Asp, the amino acid at position 233 is Asp, the
amino acid at position 237 is Asp, the amino acid at position 264
is Ile, the amino acid at position 267 is Ala, the amino acid at
position 268 is Glu, the amino acid at position 271 is Gly, the
amino acid at position 296 is Asp, the amino acid at position 327
is Gly, the amino acid at position 330 is Arg, and the amino acid
at position 396 is Met, according to EU numbering, in an Fc region;
(r) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, the amino acid at position 271 is Gly, the amino acid at
position 272 is Asp, and the amino acid at position 296 is Asp,
according to EU numbering, in an Fc region; (s) an amino acid
sequence in which the amino acid at position 238 is Asp, the amino
acid at position 237 is Asp, the amino acid at position 264 is Ile,
the amino acid at position 267 is Ala, the amino acid at position
268 is Glu, the amino acid at position 271 is Gly, the amino acid
at position 272 is Pro, and the amino acid at position 330 is Arg,
according to EU numbering, in an Fc region; (t) an amino acid
sequence in which the amino acid at position 238 is Asp, the amino
acid at position 237 is Asp, the amino acid at position 264 is Ile,
the amino acid at position 267 is Ala, the amino acid at position
268 is Glu, the amino acid at position 271 is Gly, the amino acid
at position 272 is Pro, the amino acid at position 296 is Asp, and
the amino acid at position 330 is Arg, according to EU numbering,
in an Fc region; (u) an amino acid sequence in which the amino acid
at position 238 is Asp, the amino acid at position 233 is Asp, the
amino acid at position 264 is Ile, the amino acid at position 267
is Ala, the amino acid at position 268 is Glu, and the amino acid
at position 271 is Gly, according to EU numbering, in an Fc region;
(v) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 267 is Gly, the amino acid at position 268 is Asp, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, and the amino acid at position 330 is Arg, according to EU
numbering, in an Fc region; (w) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 264
is Ile, the amino acid at position 267 is Ala, the amino acid at
position 268 is Glu, the amino acid at position 271 is Gly, the
amino acid at position 272 is Asp, and the amino acid at position
296 is Asp, according to EU numbering, in an Fc region; and (x) an
amino acid sequence in which the amino acid at position 238 is Asp,
the amino acid at position 233 is Asp, the amino acid at position
264 is Ile, the amino acid at position 267 is Ala, the amino acid
at position 268 is Glu, the amino acid at position 271 is Gly, and
the amino acid at position 296 is Asp, according to EU numbering,
in an Fc region; [11] an Fc region variant consisting of any one
amino acid sequence selected from among SEQ ID NOs: 43 to 68, SEQ
ID NO: 70, SEQ ID NO: 71, and SEQ ID NOs: 75 to 77; [12] a
polypeptide comprising at least two Fc region variants of any one
of [1] to [11], wherein the two Fc region variants are associated;
[13] the polypeptide of [12], wherein the amino acid sequences of
the two associated Fc region variants in the polypeptide are the
same; [14] the polypeptide of [12], wherein the amino acid
sequences of the two associated Fc region variants in the
polypeptide are different; [15] the polypeptide of [14], wherein
the amino acid sequences of the two associated Fc region variants
have different amino acid(s) at at least one amino acid position
selected from positions 235, 236, 237, 238, and 239 according to EU
numbering in the Fc region variant; [16] the polypeptide of [15],
wherein one of the amino acid sequences of the two associated Fc
region variants is an amino acid sequence comprising at least one
amino acid selected from Asp, Gln, Glu, or Thr at amino acid
position 235, Asn at amino acid position 236, Phe or Trp at amino
acid position 237, Glu, Gly, or Asn at amino acid position
238, and Asp or Glu at amino acid position 239 according to EU
numbering; [17] the polypeptide of any one of [12] to [16], wherein
the polypeptide comprising the Fc region variant is an IgG
antibody; [18] the polypeptide of any one of [12] to [16], wherein
the polypeptide comprising the Fc region variant is an Fc fusion
protein molecule; and [19] a pharmaceutical composition comprising
the polypeptide of any one of [12] to [18].
[0090] Furthermore, the present invention relates to a method of
enhancing Fc.gamma.RIIb-binding activity of an Fc region and
enhancing binding selectivity to Fc.gamma.RIIb compared to
Fc.gamma.RIIa (type R), by introducing an amino acid alteration(s)
into the Fc region of the present invention. The present invention
also relates to a method of suppressing production of antibodies
against a polypeptide containing an Fc region, by introducing an
amino acid alteration(s) of the present invention into the Fc
region.
[0091] The present invention also relates to a therapeutic or
preventive agent for immune inflammatory diseases that comprises a
polypeptide of the present invention. Furthermore, the present
invention relates to a method for treating or preventing immune
inflammatory diseases, which comprises the step of administering a
polypeptide of the present invention to a subject. In addition, the
present invention relates to a kit for use in the method of the
present invention for treating or preventing immune inflammatory
diseases, which comprises a polypeptide of the present invention.
The present invention also relates to use of a polypeptide of the
present invention in the production of a therapeutic or preventive
agent for immune inflammatory diseases. Furthermore, the present
invention relates to a polypeptide of the present invention for use
in the method for treating or preventing immune inflammatory
diseases of the present invention.
[0092] The present invention relates to an activation inhibitor for
B cells, mast cells, dendritic cells, and/or basophils, which
comprises a polypeptide of the present invention. Furthermore, the
present invention relates to a method of inhibiting activation of B
cells, mast cells, dendritic cells, and/or basophils, which
comprises administering a polypeptide of the present invention to a
subject. The present invention also relates to a kit for use in the
method of inhibiting activation of B cells, mast cells, dendritic
cells, and/or basophils, which comprises a polypeptide of the
present invention. The present invention relates to a use of a
polypeptide of the present invention in producing activation
inhibitors for B cells, mast cells, dendritic cells, and/or
basophils. The present invention also relates to a polypeptide of
the present invention for use in the method of the present
invention of inhibiting activation of B cells, mast cells,
dendritic cells, and/or basophils.
[0093] Furthermore, the present invention relates to a therapeutic
agent for diseases in which a protein necessary for an organism is
deficient, wherein the agent comprises a polypeptide of the present
invention. The present invention also relates to a method for
treating diseases in which a protein necessary for an organism is
deficient, which comprises administering a polypeptide of the
present invention to a subject. Furthermore, the present invention
relates to a kit for use in the method of the present invention for
treating diseases in which a protein necessary for an organism is
deficient, wherein the kit comprises a polypeptide of the present
invention. The present invention relates to use of a polypeptide of
the present invention in producing a therapeutic agent for diseases
in which a protein necessary for an organism is deficient. The
present invention also relates to a polypeptide of the present
invention for use in the method of the present invention for
treating diseases in which a protein necessary for an organism is
deficient.
[0094] In addition, the present invention relates to an agent for
suppressing virus proliferation, which comprises a polypeptide of
the present invention. The present invention also relates to a
method of suppressing virus proliferation, which comprises
administering a polypeptide of the present invention to a subject.
Furthermore, the present invention relates to a kit of the present
invention for use in the method of suppressing virus proliferation,
wherein the kit comprises a polypeptide of the present invention.
The present invention relates to use of a polypeptide of the
present invention in producing an agent for suppressing virus
proliferation. The present invention also relates to a polypeptide
of the present invention for use in the method of the present
invention of suppressing virus proliferation.
Effects of the Invention
[0095] Fc region variants with enhanced Fc.gamma.RIIb-binding
activity and/or enhanced binding selectivity to Fc.gamma.RIIb
compared to Fc.gamma.RIIa (type R), as compared to when the Fc
region is unaltered, are provided by the present invention. By
using the polypeptides containing the Fc region variants, it is
possible to enhance inhibitory signals of inflammatory immune
responses mediated by phosphorylation of ITIM of Fc.gamma.RIIb.
Also, by conferring an Fc region with the property of selective
Fc.gamma.RIIb binding, it may be possible to suppress anti-drug
antibody production. Also, by using an Fc region variant of the
present invention as a polypeptide having human FcRn-binding
activity under an acidic pH range condition and comprising an
antigen-binding domain in which an antigen-binding activity of an
antigen-binding molecule changes depending on the ion concentration
conditions, it is possible to promote elimination of antigens that
bind to the polypeptide, which are present in plasma.
BRIEF DESCRIPTION OF THE DRAWINGS
[0096] FIG. 1 is a graph showing the results of evaluating platelet
aggregation due to the omalizumab-G1d-3/IgE immune complex in a
platelet aggregation assay using platelets derived from a donor
having Fc.gamma.RIIa (R/H) polymorphism.
[0097] FIG. 2 is a graph showing the results of evaluating platelet
aggregation due to the omalizumab-G1d-3/IgE immune complex in a
platelet aggregation assay using platelets derived from a donor
having Fc.gamma.RIIa (H/H) polymorphism.
[0098] FIG. 3 is a graph showing the results of evaluating CD62p
expression on a washed platelet membrane surface. The black-filled
curve indicates the result obtained when reaction with PBS was
followed by stimulation by ADP addition, and the unfilled curve
indicates the result obtained when reaction with an immune complex
was followed by stimulation with ADP.
[0099] FIG. 4 is a graph showing the results of evaluating active
integrin expression on a washed platelet membrane surface. The
black-filled curve indicates the result obtained when reaction with
PBS was followed by stimulation by ADP addition, and the unfilled
curve indicates the result obtained when reaction with an immune
complex was followed by stimulation with ADP.
[0100] FIG. 5 shows the Fc(P208)/Fc.gamma.RIIb extracellular region
complex as determined by X-ray crystal structure analysis. For each
of the Fc-region CH2 domain and CH3 domain, the portion shown on
the left was defined as domain A and the portion shown on the right
was defined as domain B.
[0101] FIG. 6 shows a comparison made by superimposing the X-ray
crystal structure of the Fc(P208)/Fc.gamma.RIIb extracellular
region complex and the structure of the Fc(WT)/Fc.gamma.RIIa
extracellular region complex (PDB code: 3RY6), with respect to Fc
CH2 domain A by least square fitting based on Ca atom pair
distances. In the figure, the Fc(P208)/Fc.gamma.RIIb extracellular
region complex structure is depicted using thick lines and the
Fc(WT)/Fc.gamma.RIIa extracellular region complex structure is
depicted using thin lines. Regarding the structure of the
Fc(WT)/Fc.gamma.RIIa extracellular region complex, only the Fc
portion CH2 domain A is depicted.
[0102] FIG. 7 shows the detailed structure around Asp at position
237 (EU numbering) in Fc portion CH2 domain A whose main chain
forms a hydrogen bond with Tyr at position 160 of Fc.gamma.RIIb in
the X-ray crystal structure of the Fc(P208)/Fc.gamma.RIIb
extracellular region complex.
[0103] FIG. 8 shows the structure of amino acid residues around the
side chain of Asp at position 237 (EU numbering) in Fc portion CH2
domain A whose main chain forms a hydrogen bond with Tyr at
position 160 of Fc.gamma.RIIb in the X-ray crystal structure of the
Fc(208)/Fc.gamma.RIIb extracellular region complex.
[0104] FIG. 9 shows an image of superimposing the X-ray crystal
structure of the Fc(P238D)/Fc.gamma.RIIb extracellular region
complex shown in Reference Example 7 and the X-ray crystal
structure of the Fc(P208)/Fc.gamma.RIIb extracellular region
complex, with respect to Fc portion CH2 domain B by least square
fitting based on Ca atom pair distances, to compare the region
around the loop of positions 266 to 271 according to EU numbering.
In the loop, Fc(P208) has an H268D alteration at position 268 (EU
numbering) and a P271G alteration at position 271 (EU numbering)
when compared to Fc(P238D).
[0105] FIG. 10 shows the structure around Ser239 of the Fc portion
CH2 domain B along with electron density obtained by X-ray crystal
structure analysis which uses 2Fo-Fc as the coefficient in the
X-ray crystal structure of the Fc(P208)/Fc.gamma.RIIb extracellular
region complex.
[0106] FIG. 11 shows a comparison made by superimposing the
three-dimensional structure of the Fc(P208)/Fc.gamma.RIIa type R
extracellular region complex and three-dimensional structure of the
Fc(P208)/Fc.gamma.RIIb extracellular region complex, which are
determined by X-ray crystal structure analysis, by least square
fitting based on Ca atom pair distances.
[0107] FIG. 12 shows a comparison of the X-ray crystal structure of
the Fc(P208)/Fc.gamma.RIIa type R extracellular region complex and
the X-ray crystal structure of the Fc(P208)/Fc.gamma.RIIb
extracellular region complex around Asp at position 237 (EU
numbering) in Fc portion CH2 domain A, along with electron density
obtained by X-ray crystal structure analysis which uses 2Fo-Fc as
the coefficient.
[0108] FIG. 13 shows a comparison of the X-ray crystal structure of
the Fc(P208)/Fc.gamma.RIIa type R extracellular region complex and
the X-ray crystal structure of the Fc(P208)/Fc.gamma.RIIb
extracellular region complex around Asp at position 237 (EU
numbering) in Fc portion CH2 domain B, along with electron density
obtained by X-ray crystal structure analysis which uses 2Fo-Fc as
the coefficient.
[0109] FIG. 14 shows a comparison of the constant region sequences
of G1d and G4d. In the figure, the boxed amino acids show portions
where the amino acid residues are different between G1d and
G4d.
[0110] FIG. 15 shows the values of the binding amount of each
variant to FcgRIIb on the horizontal axis, and the binding amount
of each variant to FcgRIIaR on the vertical axis. The alterations
indicated in the figure, G237W, G237F, G236N, P238G, P238N, P238E,
and P238D, refer to alterations introduced into GpH7-B3. A5/B3
refers to GpH7-A5/GpH7-B3/GpL16-k0 without any introduction of
alterations to both chains, and a variant containing P238D in only
one of the chains refers to GpH7-A5/GpH7-BF648/GpL16-k0.
[0111] FIG. 16 shows the KD values of each variant for FcgRIIb on
the horizontal axis, and the KD values of each variant for FcgRIIaR
on the vertical axis. IL6R-B3/IL6R-L and IL6R-G1d/IL6R-L in the
figure refer to antibodies having native human IgG sequences which
serve as a comparison control when evaluating each of the variants.
IL6R-BP264/IL6R-L is an original variant when producing each of the
variants. IL6R-BP404/IL6R-L is a variant introduced with L234Y into
both chains of IL6R-BP264/IL6R-L, which has improved FcgRIIb
binding compared to that of the IL6R-BP264/IL6R-L before
introducing alteration.
[0112] FIG. 17 shows comparison of Fc.gamma.RIa binding and
Fc.gamma.RIIb binding. Binding of the antibody with substitution of
Pro at position 238 (EU numbering) with Asp, and binding of the
antibody with substitution of Leu at position 328 (EU numbering)
with Glu have been labeled. "Mutation A" refers to an alteration
produced by substituting Pro at position 238 (EU numbering) with
Asp and "mutation B" refers to an alteration produced by
substituting Leu at position 328 (EU numbering) with Glu.
[0113] FIG. 18 shows comparison of Fc.gamma.RIIa type H binding and
Fc.gamma.RIIb binding. Binding of the antibody with substitution of
Pro at position 238 (EU numbering) with Asp, and binding of the
antibody with substitution of Leu at position 328 (EU numbering)
with Glu have been labeled. "Mutation A" refers to an alteration
produced by substituting Pro at position 238 (EU numbering) with
Asp, and "mutation B" refers to an alteration produced by
substituting Leu at position 328 (EU numbering) with Glu.
[0114] FIG. 19 shows comparison of Fc.gamma.RIIa type R binding and
Fc.gamma.RIIb binding. Binding of the antibody with substitution of
Pro at position 238 (EU numbering) with Asp, and binding of the
antibody with substitution of Leu at position 328 (EU numbering)
with Glu have been labeled. "Mutation A" refers to an alteration
produced by substituting Pro at position 238 (EU numbering) with
Asp, and "mutation B" refers to an alteration produced by
substituting Leu at position 328 (EU numbering) with Glu.
[0115] FIG. 20 shows comparison of Fc.gamma.RIIIa binding and
Fc.gamma.RIIb binding. Binding of the antibody with substitution of
Pro at position 238 (EU numbering) with Asp, and binding of the
antibody with substitution of Leu at position 328 (EU numbering)
with Glu have been labeled. "Mutation A" refers to an alteration
produced by substituting Pro at position 238 (EU numbering) with
Asp, and "mutation B" refers to an alteration produced by
substituting Leu at position 328 (EU numbering) with Glu.
[0116] FIG. 21 shows the relationship between the amino acid
residues constituting the constant regions of IgG1, IgG2, IgG3, and
IgG4, and EU numbering (herein, also referred to as EU INDEX).
[0117] FIG. 22 shows a graph in which the horizontal axis shows the
relative value of Fc.gamma.RIIb-binding activity of each PD
variant, and the vertical axis shows the relative value of
Fc.gamma.RIIa type R-binding activity of each PD variant. The value
for the amount of binding of each PD variant to each Fc.gamma.R was
divided by the value for the amount of binding of IL6R-F652/IL6R-L,
which is a control antibody prior to introduction of the alteration
(altered Fc with substitution of Pro at position 238 (EU numbering)
with Asp), to each Fc.gamma.R; and then the obtained value was
multiplied by 100, and used as the relative binding activity value
for each PD variant to each Fc.gamma.R. The F652 plot in the figure
shows the value for IL6R-F652/IL6R-L.
[0118] FIG. 23 shows a graph in which the vertical axis shows the
relative value of Fc.gamma.RIIb-binding activity of variants
produced by introducing each alteration into GpH7-B3 which does not
have the P238D alteration, and the horizontal axis shows the
relative value of Fc.gamma.RIIb-binding activity of variants
produced by introducing each alteration into IL6R-F652 which has
the P238D alteration. The value for the amount of Fc.gamma.RIIb
binding of each variant was divided by the value for the amount of
Fc.gamma.RIIb binding of the pre-altered antibody; and then the
obtained value was multiplied by 100, and used as the value of
relative binding activity. Here, region A contains alterations that
exhibit the effect of enhancing Fc.gamma.RIIb binding in both cases
where an alteration is introduced into GpH7-B3 which does not have
P238D and where an alteration is introduced into IL6R-F652 which
has P238D. Region B contains alterations that exhibit the effect of
enhancing Fc.gamma.RIIb binding when introduced into GpH7-B3 which
does not have P238D, but do not exhibit the effect of enhancing
Fc.gamma.RIIb binding when introduced into IL6R-F652 which has
P238D.
[0119] FIG. 24 shows a crystal structure of the
Fc(P238D)/Fc.gamma.RIIb extracellular region complex.
[0120] FIG. 25 shows an image of superimposing the crystal
structure of the Fc(P238D)/Fc.gamma.RIIb extracellular region
complex and the model structure of the Fc(WT)/Fc.gamma.RIIb
extracellular region complex, with respect to the Fc.gamma.RIIb
extracellular region and the Fc CH2 domain A by least square
fitting based on Ca atom pair distances.
[0121] FIG. 26 shows comparison of the detailed structure around
P238D after superimposing the crystal structure of the
Fc(P238D)/Fc.gamma.RIIb extracellular region complex and the model
structure of the Fc(WT)/Fc.gamma.RIIb extracellular region complex
with respect to the only Fc CH2 domain A or the only Fc CH2 domain
B by least square fitting based on Ca atom pair distances.
[0122] FIG. 27 shows that a hydrogen bond is found between the main
chain of Gly at position 237 (EU numbering) in Fc CH2 domain A, and
Tyr at position 160 in Fc.gamma.RIIb in the crystal structure of
the Fc(P238D)/Fc.gamma.RIIb extracellular region complex.
[0123] FIG. 28 shows that an electrostatic interaction is found
between Asp at position 270 (EU numbering) in Fc CH2 domain B, and
Arg at position 131 in Fc.gamma.RIIb in the crystal structure of
the Fc(P238D)/Fc.gamma.RIIb extracellular region complex.
[0124] FIG. 29 shows a graph in which the horizontal axis shows the
relative value of Fc.gamma.RIIb-binding activity of each 2B
variant, and the vertical axis shows the relative value of
Fc.gamma.RIIa type R-binding activity of each 2B variant. The value
for the amount of binding of each 2B variant to each Fc.gamma.R was
divided by the value for the amount of binding of a control
antibody prior to alteration (altered Fc with substitution of Pro
at position 238 (EU numbering) with Asp) to each Fc.gamma.R; and
then the obtained value was multiplied by 100, and used as the
value of relative binding activity of each 2B variant towards each
Fc.gamma.R.
[0125] FIG. 30 shows Glu at position 233 (EU numbering) in Fc Chain
A and the surrounding residues in the extracellular region of
Fc.gamma.RIIb in the crystal structure of the
Fc(P238D)/Fc.gamma.RIIb extracellular region complex.
[0126] FIG. 31 shows Ala at position 330 (EU numbering) in Fc Chain
A and the surrounding residues in the extracellular region of
Fc.gamma.RIIb in the crystal structure of the
Fc(P238D)/Fc.gamma.RIIb extracellular region complex.
[0127] FIG. 32 shows the structures of Pro at position 271 (EU
numbering) of Fc Chain B after superimposing the crystal structures
of the Fc(P238D)/Fc.gamma.RIIb extracellular region complex and the
Fc(WT)/Fc.gamma.RIIIa extracellular region complex by least square
fitting based on Ca atom pair distances with respect to Fc Chain
B.
[0128] FIG. 33 shows the change in plasma concentrations of the
administered antigen-binding molecules of human FcgRIIb transgenic
mice when Fv4-IgG1 or Fv4-P587 was administered to the mice.
[0129] FIG. 34 shows the change in plasma concentrations of the
administered human IL-6R of human FcgRIIb transgenic mice when
Fv4-IgG1 or Fv4-P587 was administered to the mice.
[0130] FIG. 35 shows a non-limiting action mechanism for the
elimination of soluble antigens from plasma by administration of
antibodies that bind to antigens in an ion-concentration-dependent
manner, which are antibodies with enhanced Fc.gamma.R-binding at
neutral pH as compared to that of existing neutralizing
antibodies.
[0131] FIG. 36 shows the change in plasma concentrations of the
administered antigen-binding molecules of human FcgRIIb and human
FcRn transgenic mice when Fv4-IgG1, Fv4-P587, or Fv4-P587_LS was
administered to the mice.
[0132] FIG. 37 shows the change in plasma concentrations of the
administered human IL-6R of human FcgRIIb and human FcRn transgenic
mice when Fv4-IgG1, Fv4-P587, or Fv4-P587_LS was administered to
the mice.
MODE FOR CARRYING OUT THE INVENTION
[0133] The present invention provides an Fc region variant with
enhanced Fc.gamma.RIIb-binding activity, and/or enhanced binding
selectivity to Fc.gamma.RIIb compared to Fc.gamma.RIIa (type R), as
compared to an Fc region to which an amino acid alteration(s) has
not been introduced; and a polypeptide comprising the Fc region
variant.
[0134] More specifically, the invention provides an Fc region
variant containing an amino acid sequence that has a combination of
an amino acid alteration at position 238 (EU numbering) and other
specific amino acid alteration(s); and a polypeptide comprising the
Fc region variant. Furthermore, the present invention provides a
method of enhancing Fc.gamma.RIIb-binding activity, and/or
enhancing binding selectivity to Fc.gamma.RIIb, compared to
Fc.gamma.RIIa (type R) as compared to those of an Fc region to
which an amino acid alteration(s) has not been introduced, by
introducing amino acid alteration(s) into the Fc region. The
present invention also provides a method of suppressing production
of antibodies against an Fc region by introducing an amino acid
alteration(s) into the Fc region when the Fc region variant is
administered to an organism, as compared to when the Fc region
without introduction of amino acid alteration(s) is
administered.
[0135] "Polypeptides" of the present invention generally refers to
peptides or proteins approximately ten amino acids or more in
length. Furthermore, they are generally polypeptides derived from
organisms, but are not particularly limited, and for example, they
may be polypeptides comprising an artificially designed sequence.
Furthermore, they may be any of naturally-occurring polypeptides,
synthetic polypeptides, recombinant polypeptides, or such.
[0136] Preferred examples of the polypeptides of the present
invention include antibodies. More preferred examples include
naturally-occurring IgGs, particularly naturally-occurring human
IgGs. "Naturally-occurring (native) IgGs" refers to polypeptides
belonging to a class of antibodies practically encoded by
immunoglobulin gamma genes and comprising an amino acid sequence
identical to those of IgGs found in nature. For example, a
naturally-occurring human IgG means a naturally-occurring human
IgG1, naturally-occurring human IgG2, naturally-occurring human
IgG3, naturally-occurring human IgG4, or such. Naturally-occurring
IgGs also include mutants spontaneously produced from them.
[0137] While an IgK (Kappa, .kappa. chain), IgL1, IgL2, IgL3, IgL6,
and IgL7 (Lambda, .lamda. chain)-type constant region is present in
the antibody light chain constant region, it may be any light chain
constant region. For the human IgK (Kappa) constant region and
human IgL7 (Lambda) constant region, a plurality of allotype
sequences due to genetic polymorphism are described in "Sequences
of proteins of immunological interest", NIH Publication No.
91-3242, and any of them may be used in the present invention.
Furthermore, in the present invention, a light chain constant
region may be a light chain constant region that has been altered
with amino acid substitutions, additions, deletions, insertions,
and/or modifications or such. For the antibody Fc region, for
example, Fc regions of the IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3,
IgG4, and IgM types exist. For example, a human IgG antibody Fc
region can be used as the antibody Fc region of the present
invention, and human IgG1 antibody Fc regions are preferred. Fc
regions that can be used as an Fc region of the present invention
are, for example, those derived from naturally-occurring IgG
constant regions, or specifically, a constant region derived from
naturally-occurring human IgG1 (SEQ ID NO: 11), a constant region
derived from naturally-occurring human IgG2 (SEQ ID NO: 12), a
constant region derived from naturally-occurring human IgG3 (SEQ ID
NO: 13), and a constant region derived from naturally-occurring
human IgG4 (SEQ ID NO: 14). FIG. 21 shows the constant region
sequences of the naturally-occurring IgG1, IgG2, IgG3, and IgG4.
Constant regions of naturally-occurring IgGs also include mutants
spontaneously produced from them. For the constant regions of human
IgG1, human IgG2, human IgG3, and human IgG4 antibodies, a
plurality of allotype sequences due to genetic polymorphism are
described in "Sequences of proteins of immunological interest", NIH
Publication No. 91-3242, and any of them may be used in the present
invention. In particular, for the human IgG1 sequence, the amino
acid sequence at positions 356 to 358 (EU numbering) may be either
DEL or EEM.
[0138] "Fc.gamma. receptors" (herein, referred to as Fc.gamma.
receptors, Fc.gamma.R or FcgR) refers to receptors that may bind to
the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies,
and practically means any member of the family of proteins encoded
by the Fc.gamma. receptor genes. In humans, this 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 allotypes H131 (type H) and R131 (type
R)), Fc.gamma.RIIb (including Fc.gamma.RIIb-1 and Fc.gamma.RIIb-2),
and Fc.gamma.RIIc; and Fc.gamma.RIII (CD16) including isoforms
Fc.gamma.RIIIa (including allotypes V158 and F158), and
Fc.gamma.RIIIb (including allotypes Fc.gamma.RIIIb-NA1 and
Fc.gamma.RIIIb-NA2), and any human Fc.gamma.Rs, Fc.gamma.R isoforms
or allotypes yet to be discovered, but is not limited thereto.
Fc.gamma.RIIb1 and Fc.gamma.RIIb2 have been reported as splicing
variants of human Fc.gamma.RIIb. In addition, a splicing variant
named Fc.gamma.RIIb3 has been reported (J. Exp. Med, 1989, 170:
1369). In addition to these splicing variants, human Fc.gamma.RIIb
includes all splicing variants registered in NCBI, which are
NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and
NP_003992.3. Furthermore, human Fc.gamma.RIIb includes every
previously-reported genetic polymorphism, as well as Fc.gamma.RIIb
(Arthritis Rheum, 2003, 48: 3242-52; Hum Mol Genet, 2005, 14:
2881-92; and Arthritis Rheum. 2002 May; 46(5): 1242-54), and every
genetic polymorphism that will be reported in the future.
[0139] The Fc.gamma.R includes human, mouse, rat, rabbit, and
monkey-derived Fc.gamma.Rs but is not limited thereto, and may be
derived from any organism. Mouse Fc.gamma.Rs include Fc.gamma.RI
(CD64), Fc.gamma.RII (CD32), Fc.gamma.RIII (CD16), and
Fc.gamma.RIII-2 (CD16-2), and any mouse Fc.gamma.Rs, or Fc.gamma.R
isoforms or allotypes yet to be discovered, but are not limited
thereto. Favorable examples of such Fc.gamma. receptors include
human Fc.gamma.RI (CD64), Fc.gamma.RIIA (CD32), Fc.gamma.RIIB
(CD32), Fc.gamma.RIIIA (CD16), and/or Fc.gamma.RIIIB (CD16).
[0140] The polynucleotide sequence and amino acid sequence of
Fc.gamma.RI are set forth in SEQ ID NOs: 1 (NM_000566.3) and 2
(NP_000557.1), respectively;
the polynucleotide sequence and amino acid sequence of
Fc.gamma.RIIA are set forth in SEQ ID NOs: 3 (BC020823.1) and 4
(AAH20823.1), respectively; the polynucleotide sequence and amino
acid sequence of Fc.gamma.RIIB are set forth in SEQ ID NOs: 5
(BC146678.1) and 6 (AAI46679.1), respectively; the polynucleotide
sequence and amino acid sequence of Fc.gamma.RIIIA are set forth in
SEQ ID NOs: 7 (BC033678.1) and 8 (AAH33678.1), respectively; and
the polynucleotide sequence and amino acid sequence of
Fc.gamma.RIIIB are set forth in SEQ ID NOs 9 (BC128562.1) and 10
(AAI28563.1), respectively (the RefSeq Registration number is
indicated inside the parentheses).
[0141] In Fc.gamma.RIIa, there are two allotypes, one where the
amino acid at position 131 of Fc.gamma.RIIa is histidine (type H)
and the other where this amino acid is substituted with arginine
(type R) (J. Exp. Med, 172: 19-25, 1990).
[0142] Herein, an "Fc region to which an amino acid alteration(s)
has not been introduced", or a similar expression, refers to an Fc
region prior to introduction of amino acid alteration(s) of the
present invention. In the present invention, this may be, for
example, a native IgG Fc region, or an IgG Fc region produced by
adding an alteration other than the amino acid alteration(s) of the
present invention to a native IgG. Furthermore, in the present
invention, "Fc region variant" means an Fc region in which at least
one amino acid has been altered to another amino acid of the
present invention in the Fc region without introduction of amino
acid alteration(s) of the present invention. Herein, Fc region with
"at least one amino acid has been altered to another amino acid"
includes an Fc region introduced with this amino acid alteration,
and an Fc region consisting of the same amino acid sequence.
[0143] "Naturally-occurring IgGs" refers to polypeptides belonging
to a class of antibodies practically encoded by immunoglobulin
gamma genes and comprising an amino acid sequence identical to
those of IgGs found in nature. For example, a naturally-occurring
human IgG means a native human IgG1, native human IgG2, native
human IgG3, naturally-occurring human IgG4, or such.
Naturally-occurring IgGs also include mutants spontaneously
produced from them.
[0144] The Fc region of a native IgG means an Fc region comprising
an amino acid sequence identical to that of the Fc region derived
from an IgG found in nature. The heavy-chain constant region of a
native IgG is shown in FIG. 21 (SEQ ID NOs: 11-14), and for
example, it refers to, in FIG. 21, Fc regions in heavy-chain
constant region derived from native human IgG1, Fc regions in
heavy-chain constant region derived from native human IgG2, Fc
regions in heavy-chain constant region derived from native human
IgG3, and Fc regions in heavy-chain constant region derived from
native human IgG4. The Fc regions of native IgGs also include
mutants spontaneously produced from them.
[0145] In the present invention, whether or not the binding
activity towards each type of Fc.gamma.R is enhanced, or maintained
or decreased in a polypeptide comprising an Fc region variant or an
Fc region variant of the present invention can be determined, for
example, by observing whether there is a decrease or an increase in
the dissociation constant (KD) value obtained from the results of
sensorgram analysis, where various Fc.gamma.Rs are subjected to
interaction as an analyte with antibodies immobilized onto the
sensor chips or captured onto the sensor chips using Protein A,
Protein L, Protein A/G, Protein G, anti-lambda chain antibodies,
anti-kappa chain antibodies, antigenic peptides, antigenic
proteins, or such using a BIACORE.TM. system that is an interaction
analyzer that utilizes the surface plasmon resonance (SPR)
phenomena, as shown in the Examples. Alternatively, it can also be
determined by observing whether there is an increase or a decrease
in the value obtained by dividing the amount of change in the
resonance unit (RU) value on the sensorgram before and after
various types of Fc.gamma.Rs are subjected to interaction as an
analyte with antibodies immobilized onto the sensor chips or
captured onto the sensor chips using Protein A, Protein L, Protein
A/G, Protein G, anti-lambda chain antibodies, anti-kappa chain
antibodies, antigenic peptides, antigenic proteins, or such, by the
amount of change of resonance units (RU) before and after
antibodies are immobilized or captured onto the sensor chip.
Furthermore, it can be determined by observing an increase or a
decrease in the dissociation constant (KD) values obtained from
sensorgram analysis, where a sample such as an antibody to be
evaluated is subjected to interaction as an analyte using a sensor
chip onto which Fc.gamma.R is immobilized directly or via an
anti-tag antibody. Alternatively, it can be determined by observing
whether the amount of change in sensorgram values increases or
decreases before and after a sample such as an antibody to be
evaluated is subjected to interaction as an analyte with the sensor
chip onto which Fc.gamma.R is immobilized directly or via an
anti-tag antibody.
[0146] Specifically, the binding activity of an Fc region variant
towards an Fc.gamma. receptor can be measured by the Amplified
Luminescent Proximity Homogeneous Assay (ALPHA) screening, the
BIACORE.TM. method which utilizes the surface plasmon resonance
(SPR) phenomena, or such, in addition to ELISA or fluorescence
activated cell sorting (FACS) (Proc. Natl. Acad. Sci. USA (2006)
103 (11): 4005-4010).
[0147] ALPHA screening is performed by ALPHA technology which uses
two beads, a donor and an acceptor, based on the following
principles. Luminescent signals are detected only when molecules
bound to donor beads physically interact with molecules bound to
the acceptor beads, and the two beads are in close proximity to
each other. Laser-excited photosensitizer in the donor beads
converts ambient oxygen to excited-state singlet oxygen. Singlet
oxygen is dispersed around the donor beads, and when it reaches the
adjacent acceptor beads, chemiluminescent reaction is induced in
the beads, and light is ultimately emitted. When the molecules
bound to the donor beads do not interact with the molecules bound
to the acceptor beads, the chemiluminescent reaction does not take
place because singlet oxygen produced by the donor beads does not
reach the acceptor beads.
[0148] For example, a biotinylated polypeptide complex is bound to
the donor beads, and Fc.gamma. receptor tagged with glutathione S
transferase (GST) is linked to the acceptor beads. In the absence
of a competing polypeptide complex comprising an Fc region variant,
the polypeptide complex comprising a wild-type Fc region interacts
with the Fc.gamma. receptor and produces 520-620 nm signals. The
polypeptide complex comprising an untagged mutant Fc region
competes with the polypeptide complex comprising a wild-type Fc
region for interaction with the Fc.gamma. receptor. Relative
binding activities can be determined by quantifying the decrease in
fluorescence observed as a result of the competition. Biotinylation
of polypeptide complexes such as antibodies using Sulfo-NHS-biotin
and such is well known. The method of expressing the Fc.gamma.
receptor and GST in a cell carrying a fusion gene produced by
fusing a polynucleotide encoding the Fc.gamma. receptor in frame
with a polynucleotide encoding GST in an expressible vector, and
performing purification using a glutathione column is appropriately
adopted as a method for tagging an Fc.gamma. receptor with GST. The
obtained signals are preferably analyzed, for example, by fitting
them to a one-site competition model which uses a non-linear
regression analysis using software such as GRAPHPAD PRISM
(GraphPad, San Diego).
[0149] One of the substances (the ligand) in observation of an
interaction is immobilized onto a gold thin film on a sensor chip,
and by shining light from the reverse side of the sensor chip so
that total reflection takes place at the interface between the gold
thin film and glass, a portion of reduced reflection intensity is
formed in part of the reflected light (SPR signal). When the other
one of the substances (the analyte) in observation of an
interaction is made to flow on the sensor chip surface and the
ligand binds to the analyte, the mass of the immobilized ligand
molecule increases and the refractive index of the solvent on the
sensor chip surface changes. The position of the SPR signal shifts
as a result of this change in refractive index (on the other hand,
the signal position returns when this binding dissociates). The
Biacore.TM. system indicates the amount of shift mentioned above,
or more specifically the time variable of mass by plotting the
change in mass on the sensor chip surface on the ordinate as the
measurement data (sensorgram). The amount of analyte bound to the
ligand trapped on the sensor chip surface is determined from the
sensorgram. Kinetic parameters such as association rate constants
(ka) and dissociation rate constants (kd) are determined from the
curves of the sensorgram, and the dissociation constants (KD) are
determined from the ratio of these constants. In the BIACORE.TM.
method, a method for measuring inhibition is preferably used. An
example of the method for measuring inhibition is described in
Proc. Natl. Acad. Sci USA (2006) 103 (11): 4005-4010.
[0150] An Fc region with decreased Fc.gamma.R-binding activity or a
polypeptide comprising this Fc region refers to an Fc region
variant or a polypeptide comprising the Fc region variant which
binds to Fc.gamma.R with essentially weaker binding activity than a
polypeptide comprising the parent Fc region when assays are
performed by using substantially the same amount of a polypeptide
comprising an Fc region to which an amino acid alteration(s) has
not been introduced (also referred to as polypeptides comprising
parent Fc regions or parent polypeptides) and a polypeptide
comprising at least one amino acid alteration in the Fc region
(also referred to as a polypeptide comprising an Fc region variant
or an altered polypeptide).
[0151] Furthermore, an Fc region with enhanced Fc.gamma.R-binding
activity or a polypeptide comprising the Fc region refers to an Fc
region variant or a polypeptide comprising the Fc region variant
which binds to Fc.gamma.R with essentially stronger binding
activity than a polypeptide containing the parent Fc region when
assays are performed by using substantially the same amount of a
polypeptide comprising a parent Fc region and a polypeptide
comprising an Fc region variant.
[0152] A polypeptide with maintained Fc.gamma.R-binding activity
refers to a polypeptide that binds to Fc.gamma.R with binding
activity equivalent to or essentially not different from that of
the parent polypeptide when assays are performed by using
substantially the same amount of a polypeptide comprising a parent
Fc region and a polypeptide comprising the Fc region variant.
[0153] In the present invention, enhanced Fc.gamma.RIIb-binding
activity preferably means, for example, that the KD value ratio for
[KD value of a polypeptide comprising a parent Fc region for
Fc.gamma.RIIb]/[KD value of a polypeptide comprising an Fc region
variant for Fc.gamma.RIIb] in the KD values measured by the
above-mentioned measurement method preferably becomes 15.0 or
greater, 20.0 or greater, 25.0 or greater, 30.0 or greater, 35.0 or
greater, 40.0 or greater, 45.0 or greater, or even 50.0 or greater,
55.0 or greater, 60.0 or greater, 65.0 or greater, 70.0 or greater,
75.0 or greater, 80.0 or greater, 85.0 or greater, 90.0 or greater,
95.0 or greater, or 100.0 or greater.
[0154] Furthermore, "an Fc region variant of the present invention
shows enhanced binding selectivity to Fc.gamma.RIIb compared to
Fc.gamma.RIIa" means that:
(i) Fc.gamma.RIIb-binding activity is enhanced, and
Fc.gamma.RIIa-binding activity is maintained or decreased; (ii)
Fc.gamma.RIIb-binding activity is enhanced and
Fc.gamma.RIIa-binding activity is also enhanced, but the degree of
enhancement of Fc.gamma.RIIa-binding activity is lower than the
degree of enhancement of Fc.gamma.RIIb-binding activity; or (iii)
Fc.gamma.RIIb-binding activity is decreased, but the degree of
decrease of the binding activity is less than the degree of
decrease of Fc.gamma.RIIa-binding activity. Whether or not an Fc
region variant of the present invention is a variant with improved
binding selectivity for Fc.gamma.RIIb rather than for Fc.gamma.RIIa
can be determined, for example, by comparing the ratio of the KD
value for Fc.gamma.RIIa to the KD value for Fc.gamma.RIIb of the
polypeptide comprising an Fc region variant of the present
invention (KD value for Fc.gamma.RIIa/KD value for Fc.gamma.RIIb)
with the ratio of the KD value for Fc.gamma.RIIa to the KD value
for Fc.gamma.RIIb of the polypeptide comprising the parent Fc
region (KD value for Fc.gamma.RIIa/KD value for Fc.gamma.RIIb),
which were determined according to the above-mentioned examples.
Specifically, when the value of the KD ratio for the polypeptide
comprising the Fc region variant of the present invention is
greater than that of the polypeptide comprising the parent Fc
region, the polypeptide comprising the Fc region variant of the
present invention can be determined to have an improved binding
selectivity for Fc.gamma.RIIb rather than for Fc.gamma.RIIa in
comparison with the polypeptide comprising the parent Fc region
variant. In particular, since Fc.gamma.RIIa (type R)-binding
activity is likely to correlate with binding activity to
Fc.gamma.RIIb than to Fc.gamma.RIIa (type H), finding amino acid
alteration(s) that can enhance binding selectivity to Fc.gamma.RIIb
compared to Fc.gamma.RIIa (type R) is important for enhancing
binding selectivity to Fc.gamma.RIIb compared to other Fc.gamma.Rs
other than Fc.gamma.RIIb.
[0155] The binding selectivity between Fc.gamma.RIIa (type R) and
Fc.gamma.RIIb is, for example, a KD value ratio [KD value of the
polypeptide comprising the Fc region variant for Fc.gamma.RIIa
(type R)]/[KD value of the polypeptide comprising the Fc region
variant for Fc.gamma.RIIb] of preferably 10.0 or more for the KD
values measured by the measurement method described above, and more
preferably 20.0 or more.
[0156] The binding selectivity between Fc.gamma.RIIa (type H) and
Fc.gamma.RIIb is, for example, a KD value ratio [KD value of the
polypeptide comprising the Fc region variant for Fc.gamma.RIIa
(type H)]/[KD value of the polypeptide comprising the Fc region
variant for Fc.gamma.RIIb] of preferably 100.0 or more, 200 or
more, 300 or more, 400 or more, or 500 or more for the KD values
measured by the measurement method described above, and more
preferably 600 or more, 700 or more, 800 or more, or 900 or
more.
[0157] Furthermore, whether or not the binding activities of the
polypeptides of the present invention towards various Fc.gamma.Rs
were maintained, enhanced, or decreased can be determined from the
increase or decrease in the amount of binding of the various
Fc.gamma.Rs to the polypeptides of the present invention, which
were determined according to the examples described above. Here,
the amount of binding of the various Fc.gamma.Rs to the
polypeptides refers to values obtained by determining the
difference in the RU values of sensorgrams that changed before and
after interaction of various Fc.gamma.Rs as the analyte with each
polypeptide, and dividing them by differences in the RU values of
sensorgrams that changed before and after capturing polypeptides to
the sensor chips.
[0158] Fc region variants of the present invention are not
particularly limited in terms of their KD values (mol/L) for
Fc.gamma.RIIb, and for example, the values may be 9.times.10.sup.-7
or less, preferably 5.times.10.sup.-7 or less, more preferably
3.times.10.sup.-7 or less, even more preferably 1.times.10.sup.-7
or less, and yet even more preferably 5.times.10.sup.-8 or
less.
[0159] "Fc region" refers to the fragment consisting of a hinge
portion or a part thereof, CH2 domain, and CH3 domain in an
antibody molecule. According to EU numbering (herein, also called
the EU INDEX) (see FIG. 21), an IgG-class Fc region refers to, for
example, the region from cysteine at position 226 to the C
terminus, or from proline at position 230 to the C terminus, but is
not limited thereto.
[0160] The Fc region may be obtained preferably by re-eluting the
fraction adsorbed onto protein A column after partially digesting
IgG1, IgG2, IgG3, IgG4 monoclonal antibodies or such using a
protease such as pepsin. The protease is not particularly limited
as long as it can digest a full-length antibody so that Fab and
F(ab')2 will be produced in a restrictive manner by appropriately
setting the enzyme reaction conditions such as pH, and examples
include pepsin and papain.
[0161] The present invention provides Fc region variants having a
combination of alterations which includes alteration of amino acid
at position 238 (EU numbering) to another amino acid and alteration
of at least one amino acid selected from amino acids at positions
233, 234, 235, 237, 264, 265, 266, 267, 268, 269, 271, 272, 274,
296, 326, 327, 330, 331, 332, 333, 334, 355, 356, 358, 396, 409,
and 419 (EU numbering) to another amino acid in the Fc region of a
human IgG (IgG1, IgG2, IgG3, or IgG4). By combining alteration of
amino acid at position 238 (EU numbering) to another amino acid
with alteration of at least one amino acid selected from amino
acids at positions 233, 234, 237, 264, 265, 266, 267, 268, 269,
271, 272, 274, 296, 326, 327, 330, 331, 332, 333, 334, 355, 356,
358, 396, 409, and 419 (EU numbering) to another amino acid in the
human IgG Fc region, it is possible to provide a polypeptide
comprising an Fc region variant with enhanced Fc.gamma.RIIb-binding
activity, and/or enhanced binding selectivity to Fc.gamma.RIIb,
compared to Fc.gamma.RIIa, to Fc.gamma.RIIa (type R) in particular,
as compared to those of a polypeptide containing an Fc region to
which an amino acid alteration(s) has not been introduced. Other
amino acid alterations that are to be combined with the amino acid
alteration at position 238 (EU numbering) are preferably those at
positions 233, 237, 264, 267, 268, 271, 272, 296, 327, 330, 332,
333, and 396 (EU numbering), and particularly preferably those at
positions 233, 237, 264, 267, 268, 271, 296, 330, and 396 (EU
numbering). In particular, in terms of enhancement of
Fc.gamma.RIIb-binding activity, or enhancement of binding
selectivity to Fc.gamma.RIIb compared to Fc.gamma.RIIa, an example
of a preferred combination of amino acid alterations include
combination of alterations at amino acid positions 238, 268, and
271 (EU numbering) with at least one amino acid position selected
from 233, 237, 264, 267, 272, 296, 327, 330, 332, and 396 (EU
numbering).
[0162] Amino acids to be altered are not particularly limited as
long as they lead to enhancement of Fc.gamma.RIIb-binding activity
or enhancement of binding selectivity to Fc.gamma.RIIb compared to
Fc.gamma.RIIa as compared to before the alteration, but it is
preferred that the amino acid at position 238 is Asp, the amino
acid at position 233 is Asp, the amino acid at position 234 is Tyr,
the amino acid at position 235 is Phe, the amino acid at position
237 is Asp, the amino acid at position 264 is Ile, the amino acid
at position 265 is Glu, the amino acid at position 266 is Phe, Leu,
or Met, the amino acid at position 267 is Ala, Glu, Gly, or Gln,
the amino acid at position 268 is Asp, Gln, or Glu, the amino acid
at position 269 is Asp, the amino acid at position 271 is Gly, the
amino acid at position 272 is Asp, Phe, Ile, Met, Asn, Pro, or Gln,
the amino acid at position 274 is Gln, the amino acid at position
296 is Asp or Phe, the amino acid at position 326 is Ala or Asp,
the amino acid at position 327 is Gly, the amino acid at position
330 is Lys, Arg, or Ser, the amino acid at position 331 is Ser, the
amino acid at position 332 is Lys, Arg, Ser, or Thr, the amino acid
at position 333 is Lys, Arg, Ser, or Thr, the amino acid at
position 334 is Arg, Ser, or Thr, the amino acid at position 355 is
Ala or Gln, the amino acid at position 356 is Glu, the amino acid
at position 358 is Met, the amino acid at position 396 is Ala, Asp,
Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,
Val, Trp or Tyr, the amino acid at position 409 is Arg, and the
amino acid at position 419 is Glu, according to EU numbering. In
particular, when combining alterations at amino acid positions 238,
268, and 271 (EU numbering) with at least one amino acid position
selected from 233, 264, 267, 272, 296, 327, 330, 332, and 396 (EU
numbering), it is preferred that the amino acid at position 238 is
Asp, the amino acid at position 268 is Asp or Glu, the amino acid
at position 271 is Gly, the amino acid at position 233 is Asp, the
amino acid at position 237 is Asp, the amino acid at position 264
is Ile, the amino acid at position 267 is Ala or Gly, the amino
acid at position 272 is Asp or Pro, the amino acid at position 296
is Asp, the amino acid at position 327 is Gly, the amino acid at
position 330 is Arg, the amino acid at position 332 is Thr, and the
amino acid at position 396 is Leu or Met, according to EU
numbering.
[0163] In addition to these alterations, at least one different Fc
region alteration may be added in the present invention. The added
alteration is not particularly limited as long as
Fc.gamma.RIIb-binding activity is enhanced and/or binding
selectivity to Fc.gamma.RIIb compared to Fc.gamma.RIIa is enhanced.
Furthermore, alterations can be made by combining an alteration
where a portion of an Fc region is substituted with a corresponding
portion of Fc region of a different isotype. For example, it is
possible to enhance Fc.gamma.RIIb-binding activity and/or binding
selectivity to Fc.gamma.RIIb by combining the above-mentioned amino
acid alterations with substitution of the amino acid sequence from
Ala at position 118 to Thr at position 225 (EU numbering) in the
IgG1-derived Fc region with the amino acid sequence from Ala at
position 118 to Pro at position 222 (EU numbering) in the
IgG4-derived Fc region. A specific example includes a combination
of amino acid alterations introduced into IL6R-BP230, and the
alteration of substituting the amino acid sequence from Ala at
position 118 to Thr at position 225 (EU numbering) in G1d with the
amino acid sequence from Ala at position 118 to Pro at position 222
(EU numbering) in G4d, as of IL6R-BP478/IL6R-L described in Example
7.
[0164] Among them, alterations that lead to greater enhancement of
Fc.gamma.RIIb-binding activity, or lead to greater enhancement of
binding selectivity to Fc.gamma.RIIb compared to Fc.gamma.RIIa
(type R) are preferred. Examples of such a preferred combination of
amino acid alterations include the following (a) to (x):
(a) amino acid alterations at positions 238, 233, 237, 268, 271,
296, and 330 (EU numbering) of an Fc region; (b) amino acid
alterations at positions 238, 237, 268, 271, 296, and 330 (EU
numbering) of an Fc region; (c) amino acid alterations at positions
238, 233, 237, 268, 271, 296, 330, and 332 (EU numbering) of an Fc
region; (d) amino acid alterations at positions 238, 233, 237, 264,
267, 268, 271, and 330 (EU numbering) of an Fc region; (e) amino
acid alterations at positions 238, 233, 237, 267, 268, 271, 296,
330, and 332 (EU numbering) of an Fc region; (f) amino acid
alterations at positions 238, 237, 267, 268, 271, 296, 330, and 332
(EU numbering) of an Fc region; (g) amino acid alterations at
positions 238, 233, 237, 268, 271, 296, 327, and 330 (EU numbering)
of an Fc region; (h) amino acid alterations at positions 238, 233,
237, 264, 267, 268, and 271 (EU numbering) of an Fc region; (i)
amino acid alterations at positions 238, 233, 237, 264, 267, 268,
271, 296, and 330 (EU numbering) of an Fc region; (j) amino acid
alterations at positions 238, 233, 237, 264, 267, 268, 271, 296,
330, and 396 (EU numbering) of an Fc region; (k) amino acid
alterations at positions 238, 237, 264, 267, 268, 271, and 330 (EU
numbering) of an Fc region; (l) amino acid alterations at positions
238, 237, 264, 267, 268, 271, 296, and 330 (EU numbering) of an Fc
region; (m) amino acid alterations at positions 238, 264, 267, 268,
and 271 (EU numbering) of an Fc region; (n) amino acid alterations
at positions 238, 264, 267, 268, 271, and 296 (EU numbering) of an
Fc region; (o) amino acid alterations at positions 238, 237, 267,
268, 271, 296, and 330 (EU numbering) of an Fc region; (p) amino
acid alterations at positions 238, 233, 237, 264, 267, 268, 271,
330, and 396 (EU numbering) of the Fc region; (q) amino acid
alterations at positions 238, 233, 237, 264, 267, 268, 271, 296,
327, 330, and 396 (EU numbering) of an Fc region; (r) amino acid
alterations at positions 238, 233, 237, 264, 267, 268, 271, 272,
and 296 (EU numbering) of an Fc region; (s) amino acid alterations
at positions 238, 237, 264, 267, 268, 271, 272, and 330 (EU
numbering) of an Fc region; (t) amino acid alterations at positions
238, 237, 264, 267, 268, 271, 272, 296, and 330 (EU numbering) of
an Fc region; (u) amino acid alterations at positions 238, 233,
264, 267, 268, and 271 (EU numbering) of an Fc region; (v) amino
acid alterations at positions 238, 237, 267, 268, 271, 296, and 330
(EU numbering) of an Fc region; (w) amino acid alterations at
positions 238, 264, 267, 268, 271, 272, and 296 (EU numbering) of
an Fc region; and (x) amino acid alterations at positions 238, 233,
264, 267, 268, 271, and 296 (EU numbering) of an Fc region.
[0165] In addition, among these combinations, the following
combinations of amino acid alterations (a) to (x) below are more
preferred amino acid combinations:
(a) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 268 is Asp, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, and the amino acid at position 330 is Arg, according to EU
numbering, in an Fc region; (b) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 237
is Asp, the amino acid at position 268 is Asp or Glu, the amino
acid at position 271 is Gly, the amino acid at position 296 is Asp,
and the amino acid at position 330 is Arg, according to EU
numbering, in an Fc region; (c) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 233
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 268 is Asp, the amino acid at position 271 is Gly, the
amino acid at position 296 is Asp, the amino acid at position 330
is Arg, and the amino acid at position 332 is Thr, according to EU
numbering, in an Fc region; (d) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 233
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 264 is Ile, the amino acid at position 267 is Gly or Ala,
the amino acid at position 268 is Glu, the amino acid at position
271 is Gly, and the amino acid at position 330 is Arg, according to
EU numbering, in an Fc region; (e) an amino acid sequence in which
the amino acid at position 238 is Asp, the amino acid at position
233 is Asp, the amino acid at position 237 is Asp, the amino acid
at position 267 is Ala, the amino acid at position 268 is Glu, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, the amino acid at position 330 is Arg, and the amino acid
at position 332 is Thr, according to EU numbering, in an Fc region;
(f) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 267 is Ala, the amino acid at position 268 is Glu, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, the amino acid at position 330 is Arg, and the amino acid
at position 332 is Thr, according to EU numbering, in an Fc region;
(g) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 268 is Asp, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, the amino acid at position 327 is Gly, and the amino acid
at position 330 is Arg, according to EU numbering, in an Fc region;
(h) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, and the amino acid at position 271 is Gly, according to EU
numbering, in an Fc region; (i) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 233
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 264 is Ile, the amino acid at position 267 is Ala, the
amino acid at position 268 is Glu, the amino acid at position 271
is Gly, the amino acid at position 296 is Asp, and the amino acid
at position 330 is Arg, according to EU numbering, in an Fc region;
(j) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, the amino acid at position 271 is Gly, the amino acid at
position 296 is Asp, the amino acid at position 330 is Arg, and the
amino acid at position 396 is Met or Leu, according to EU
numbering, in the Fc region; (k) an amino acid sequence in which
the amino acid at position 238 is Asp, the amino acid at position
237 is Asp, the amino acid at position 264 is Ile, the amino acid
at position 267 is Ala, the amino acid at position 268 is Glu, the
amino acid at position 271 is Gly, and the amino acid at position
330 is Arg, according to EU numbering, in an Fc region; (l) an
amino acid sequence in which the amino acid at position 238 is Asp,
the amino acid at position 237 is Asp, the amino acid at position
264 is Ile, the amino acid at position 267 is Ala, the amino acid
at position 268 is Glu, the amino acid at position 271 is Gly, the
amino acid at position 296 is Asp, and the amino acid at position
330 is Arg, according to EU numbering, in an Fc region; (m) an
amino acid sequence in which the amino acid at position 238 is Asp,
the amino acid at position 264 is Ile, the amino acid at position
267 is Ala, the amino acid at position 268 is Glu, and the amino
acid at position 271 is Gly, according to EU numbering, in an Fc
region; (n) an amino acid sequence in which the amino acid at
position 238 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, the amino acid at position 271 is Gly, and the amino acid
at position 296 is Asp, according to EU numbering, in an Fc region;
(o) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 267 is Ala or Gly, the amino acid at position 268 is Glu,
the amino acid at position 271 is Gly, the amino acid at position
296 is Asp, and the amino acid at position 330 is Arg, according to
EU numbering, in an Fc region; (p) an amino acid sequence in which
the amino acid at position 238 is Asp, the amino acid at position
233 is Asp, the amino acid at position 237 is Asp, the amino acid
at position 264 is Ile, the amino acid at position 267 is Ala, the
amino acid at position 268 is Glu, the amino acid at position 271
is Gly, the amino acid at position 330 is Arg, and the amino acid
at position 396 is Met or Leu, according to EU numbering, in an Fc
region; (q) an amino acid sequence in which the amino acid at
position 238 is Asp, the amino acid at position 233 is Asp, the
amino acid at position 237 is Asp, the amino acid at position 264
is Ile, the amino acid at position 267 is Ala, the amino acid at
position 268 is Glu, the amino acid at position 271 is Gly, the
amino acid at position 296 is Asp, the amino acid at position 327
is Gly, the amino acid at position 330 is Arg, and the amino acid
at position 396 is Met, according to EU numbering, in an Fc region;
(r) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, the amino acid at position 271 is Gly, the amino acid at
position 272 is Asp, and the amino acid at position 296 is Asp,
according to EU numbering, in an Fc region; (s) an amino acid
sequence in which the amino acid at position 238 is Asp, the amino
acid at position 237 is Asp, the amino acid at position 264 is Ile,
the amino acid at position 267 is Ala, the amino acid at position
268 is Glu, the amino acid at position 271 is Gly, the amino acid
at position 272 is Pro, and the amino acid at position 330 is Arg,
according to EU numbering, in an Fc region; (t) an amino acid
sequence in which the amino acid at position 238 is Asp, the amino
acid at position 237 is Asp, the amino acid at position 264 is Ile,
the amino acid at position 267 is Ala, the amino acid at position
268 is Glu, the amino acid at position 271 is Gly, the amino acid
at position 272 is Pro, the amino acid at position 296 is Asp, and
the amino acid at position 330 is Arg, according to EU numbering,
in an Fc region; (u) an amino acid sequence in which the amino acid
at position 238 is Asp, the amino acid at position 233 is Asp, the
amino acid at position 264 is Ile, the amino acid at position 267
is Ala, the amino acid at position 268 is Glu, and the amino acid
at position 271 is Gly, according to EU numbering, in an Fc region;
(v) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 267 is Gly, the amino acid at position 268 is Asp, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, and the amino acid at position 330 is Arg, according to EU
numbering, in an Fc region; (w) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 264
is Ile, the amino acid at position 267 is Ala, the amino acid at
position 268 is Glu, the amino acid at position 271 is Gly, the
amino acid at position 272 is Asp, and the amino acid at position
296 is Asp, according to EU numbering, in an Fc region; and (x) an
amino acid sequence in which the amino acid at position 238 is Asp,
the amino acid at position 233 is Asp, the amino acid at position
264 is Ile, the amino acid at position 267 is Ala, the amino acid
at position 268 is Glu, the amino acid at position 271 is Gly, and
the amino acid at position 296 is Asp, according to EU numbering,
in an Fc region.
[0166] In addition, amino acid alterations performed for other
purpose(s) can be combined in polypeptides comprising an Fc region
variant of the present invention. For example, amino acid
substitutions that improve FcRn-binding activity (J. Immunol. 2006
Jan. 1; 176(1): 346-56; J Biol Chem. 2006 Aug. 18; 281(33):
23514-24; Int. Immunol. 2006 December; 18(12): 1759-69; Nat
Biotechnol. 2010 February; 28(2): 157-9; WO/2006/019447;
WO/2006/053301; and WO/2009/086320), and amino acid substitutions
for improving antibody heterogeneity or stability (WO/2009/041613)
may be added. Alternatively, polypeptides produced by conferring
polypeptides comprising an Fc region variant of the present
invention with the property of promoting disappearance of antigens,
which are described in WO 2011/122011 or PCT/JP2011/072550, and
polypeptides conferring the property for repeated binding to a
plurality of antigen molecules, which are described in WO
2009/125825, WO 2012/073992 or WO 2013/047752, are also included in
the present invention. Alternatively, with the objective of
increasing plasma retention, amino acid alterations that decrease
the pI of the constant region (WO/2012/016227) may be combined in a
polypeptide comprising an Fc region variant of the present
invention. Alternatively, with the objective of conferring binding
ability to other antigens, the amino acid alterations disclosed in
EP1752471 and EP1772465 may be combined in CH3 of a polypeptide
comprising an Fc region variant of the present invention.
[0167] When a polypeptide comprising an Fc region variant of the
present invention is an antigen-binding molecule such as an
antibody, amino acid alterations of enhancing human FcRn-binding
activity under an acidic pH range condition can be combined to
enhance the effect of the antigen-binding molecule to eliminate
antigens from plasma. More specifically, alterations used to
enhance human FcRn-binding activity under an acidic pH range
condition may be carried out on an IgG antibody, for example, by a
method of substituting Leu for Met at position 428, and
substituting Ser for Asn at position 434, according to EU numbering
(Nat Biotechnol, 2010 28: 157-159); a method of substituting Ala
for Asn at position 434 (Drug Metab Dispos. 2010 April; 38(4):
600-5); a method of substituting Tyr for Met at position 252,
substituting Thr for Ser at position 254, and substituting Glu for
Thr at position 256 (J Biol Chem, 2006, 281: 23514-23524); a method
for substituting Gln for Thr at position 250, and substituting Leu
for Met at position 428 (J Immunol. 2006, 176(1): 346-56); method
of substituting His for Asn at position 434 (Clinical Pharmacology
& Therapeutics (2011) 89(2): 283-290), or by using alterations
such as those described in WO2010106180, WO2010045193,
WO2009058492, WO2008022152, WO2006050166, WO2006053301,
WO2006031370, WO2005123780, WO2005047327, WO2005037867,
WO2004035752, WO2002060919, or such.
[0168] Furthermore, an antibody molecule produced by substituting
His for Asn at position 434 (EU numbering) in humanized anti-CD4
antibody to enhance human FcRn-binding activity under an acidic pH
range condition and to improve plasma retention properties was
recently reported to bind to rheumatoid factors (RF) (Clin
Pharmacol Ther. 2011 February; 89(2): 283-90). This antibody has a
human IgG1 Fc region, but by substituting His for Asn at position
434 which is positioned at the FcRn-binding site, it has been shown
to bind to rheumatoid factors that recognize this substituted
site.
[0169] As described above, various alterations have been reported
as alterations for enhancing human FcRn-binding activity under an
acidic pH range condition; however, by introducing these
alterations into the FcRn-binding site in an Fc region, affinity to
rheumatoid factors which recognize this site may become
enhanced.
[0170] However, by introducing alterations which do not reduce
FcRn-binding activity and reduce only binding activity to
rheumatoid factors into the site in the Fc region, antigen-binding
molecules with enhanced human FcRn-binding activity under an acidic
pH range condition and without affinity to rheumatoid factors can
be produced.
[0171] For alterations that reduce binding activity to rheumatoid
factors, alterations to positions 248-257, 305-314, 342-352,
380-386, 388, 414-421, 423, 425-437, 439, and 441-444 according to
EU numbering are used. Preferably, alterations to positions 387,
422, 424, 426, 433, 436, 438, and 440 are used. Particularly
preferably, alteration of substituting Glu or Ser for Val at
position 422, alteration of substituting Arg for Ser at position
424, alteration of substituting Asp for His at position 433,
alteration of substituting Thr for Tyr at position 436, alteration
of substituting Arg or Lys for Gln at position 438, and alteration
of substituting Glu or Asp for Ser at position 440 are used. These
alterations may be used alone or by combining alterations at
multiple positions.
[0172] Alternatively, to decrease binding activity to rheumatoid
factors, an N-type glycosylation sequence may be introduced into
this site. Specifically, Asn-Xxx-Ser/Thr (Xxx is any amino acid
other than Pro) is known as an N-type glycosylation sequence.
Adding an N-type sugar chain by introducing this sequence into the
site in the Fc region enables inhibition of binding to RF by steric
hindrance due to the N-type sugar chain. Alterations used to add an
N-type sugar chain are preferably alteration which substitutes Asn
for Lys at position 248, alteration which substitutes Asn for Ser
at position 424, alteration which substitutes Asn for Tyr at
position 436 and substitutes Thr for Gln at position 438, and
alteration which substitutes Asn for Gln at position 438.
Particularly preferably, the alteration which substitutes Asn for
Ser at position 424 is used.
[0173] Preferred example of a polypeptide comprising an Fc region
variant of the present invention includes a polypeptide comprising
at least two Fc region variants wherein the two Fc region variants
are associated, much like in an IgG antibody. When an IgG antibody
is used as a polypeptide of the present invention, the type of
constant region is not limited, and an IgG isotypes (subclasses)
such as IgG1, IgG2, IgG3, and IgG4 can be used. IgG antibodies of
the present invention are preferably human IgG, and more preferably
human IgG1 and human IgG4. The amino acid sequences of the
heavy-chain constant regions of human IgG1 and human IgG4 are
known. A plurality of allotype sequences due to genetic
polymorphisms have been described in Sequences of Proteins of
Immunological Interest, NIH Publication No. 91-3242 for the human
IgG1 constant region, and any of the sequences may be used in the
present invention.
[0174] The two associated Fc region variants included in the
aforementioned polypeptide may be Fc region variants introduced
with the same amino acid alteration(s) (hereinafter, referred to as
a polypeptide containing homologous Fc region variants) or Fc
region variants comprising different amino acid sequences where
each have been introduced with different amino acid alteration(s),
or alternatively Fc region variants comprising different amino acid
sequences where only one of the Fc regions has been introduced with
amino acid alteration(s) (hereinafter, referred as a polypeptide
containing heterologous Fc region variants). As the amino acid
alteration to be introduced into only one of the Fc regions,
alteration in the loop structure portion from positions 233 to 239
(EU numbering) in the Fc region CH2 domain involved in binding with
Fc.gamma.RIIb and Fc.gamma.RIIa is preferred; and preferably, an
alteration that enhances Fc.gamma.RIIb-binding activity and/or
enhances binding selectivity to Fc.gamma.RIIb compared to
Fc.gamma.RIIa (type R) of the loop structure of the CH2 region of
one of the Fc regions is introduced and an amino acid alteration
that destabilizes the loop structure of the CH2 region of the other
Fc region is introduced. Examples of amino acid alterations that
can destabilize the loop structure of the CH2 region may be
substitution of at least one amino acid selected from amino acids
at positions 235, 236, 237, 238, and 239 to another amino acid.
Specifically, the CH2 region loop structure can be destabilized,
for example, by altering the amino acid at position 235 to Asp,
Gln, Glu, or Thr, altering the amino acid at position 236 to Asn,
altering the amino acid at position 237 to Phe or Trp, altering the
amino acid at position 238 to Glu, Gly, or Asn, and altering the
amino acid at position 239 to Asp or Glu, according to EU
numbering.
[0175] To produce a polypeptide comprising heterologous Fc region
variants of the present invention, it is required that Fc region
variants having amino acids that differ from each other are
associated, or a polypeptide comprising heterologous Fc region
variants of interest is separated from other polypeptides
comprising homologous Fc region variants.
[0176] For association of polypeptides having different amino acids
from each other, a technique of suppressing unintended association
between H chains by introducing electrostatic repulsion into the
interface of the second constant region of the antibody H chain
(CH2) or the third constant region of the H chain (CH3) (WO
2006/106905) can be applied.
[0177] In the technology of suppressing unintended association
between H chains by introducing electrostatic repulsion into the
interface of CH2 or CH3, examples of amino acid residues in contact
at the interface of other constant regions of the H chain include
the residue at position 356 (EU numbering), the residue at position
439 (EU numbering), the region facing the residue at position 357
(EU numbering), the residue at position 370 (EU numbering), the
residue at position 399 (EU numbering), and the residue at position
409 (EU numbering) in the CH3 domain.
[0178] More specifically, for example, in an antibody containing
two types of H chain CH3 domains, the antibody in which one to
three pairs of amino acid residues selected from the amino acid
residues shown below in (1) to (3) in the first H chain CH3 domain
have the same type of charge can be produced: [0179] (1) amino acid
residues at positions 356 and 439 (EU numbering) which are amino
acid residues contained in the H chain CH3 domain; [0180] (2) amino
acid residues at positions 357 and 370 (EU numbering) which are
amino acid residues contained in the H chain CH3 domain; and [0181]
(3) amino acid residues at positions 399 and 409 (EU numbering)
which are amino acid residues contained in the H chain CH3
domain.
[0182] Furthermore, an antibody can be produced in which one to
three pairs of amino acid residues corresponding to the amino acid
residue pairs indicated above in (1) to (3) having the same type of
charge in the first H chain CH3 domain have charges opposite to the
corresponding amino acid residues in the aforementioned first H
chain CH3 domain, wherein the amino acid residue pairs are selected
from the amino acid residue pairs indicated above in (1) to (3) in
the second H chain CH3 domain which differs from the first H chain
CH3 domain.
[0183] The respective amino acid residues of (1) to (3) mentioned
above are positioned close to each other when associated. Those
skilled in the art can find sites that correspond to the
above-mentioned amino acid residues of (1) to (3) by homology
modeling and such using commercially available software for the
desired H chain CH3 domain or H chain constant region, and amino
acid residues of these sites can be altered when appropriate.
[0184] In the above-mentioned antibodies, for example, "charged
amino acid residues" are preferably selected from amino acid
residues included in either of groups (X) or (Y) below: (X)
glutamic acid (E) and aspartic acid (D); and (Y) lysine (K),
arginine (R), and histidine (H).
[0185] In the above-mentioned antibodies, the phrase "having the
same type of charge" means that, for example, all of the two or
more amino acid residues are amino acid residues included in either
of the above-mentioned groups (X) and (Y). The phrase "having the
opposite charge" means that, for example, when at least one of the
two or more amino acid residues is an amino acid residue included
in either one of the above-mentioned groups (X) and (Y), the
remaining amino acid residues are amino acid residues included in
the other group.
[0186] In a preferred embodiment of the above-mentioned antibody,
the first H chain CH3 domain and the second H chain CH3 domain may
be cross-linked by disulfide bonds.
[0187] In the present invention, the amino acid residues to be
altered are not limited to amino acid residues of the antibody
constant region or antibody variable region described above. Those
skilled in the art can find amino acid residues that form the
interface in polypeptide mutants or heteromultimers through
homology modeling and such using commercially available software,
and can alter the amino acid residues at those sites to regulate
association.
[0188] Other known techniques can be used additionally for
association of heterologous Fc region variants. Specifically, such
a technique is conducted by substituting an amino acid side chain
present in a variable region of one of the H chains in an antibody
with a larger side chain (knob; which means "bulge"), and
substituting an amino acid side chain present in a variable region
of the other H chain with a smaller side chain (hole; which means
"void"), to place the knob within the hole. This can promote
efficient association between Fc-region-containing polypeptides
having different amino acids (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).
[0189] In addition, other known techniques can also be used for
heterologous association of Fc region variants. Association of
polypeptides having different sequences can be induced efficiently
by complementary association of CH3 by using a strand-exchange
engineered domain CH3 produced by changing a portion of one of the
H chain CH3 of an antibody to an IgA-derived sequence corresponding
to that portion and introducing to the complementary portion of the
other H chain CH3, an IgA-derived sequence corresponding to that
portion (Protein Engineering Design & Selection, 23; 195-202,
2010). This known technique can also be used to efficiently induce
association between Fc region-containing polypeptides having
different amino acids from each other.
[0190] In addition, heterodimerized antibody production techniques
that use association of antibody CH1 and CL, and association of VH
and VL, which are described in WO2011/028952, can also be used.
[0191] As with the method described in WO2008/119353 and
WO2011/131746, it is also possible to use the technique of
producing heterodimerized antibodies by producing two types of
homodimerized antibodies in advance, incubating them under reducing
conditions to dissociate them, and allowing them to associate
again.
[0192] As with the method described in J. Mol. (2012) 420, 204-219,
it is also possible to use the technique of producing
heterodimerized antibodies by introducing charged residues such as
Lys, Arg, Glu, and Asp so that electrostatic repulsion is
introduced into CH3 of IgG1 and IgG2.
[0193] Furthermore, as with the method described in WO2012/058768,
it is also possible to use the technique of producing
heterodimerized antibodies by adding alterations to the CH2 and CH3
regions.
[0194] Furthermore, even in cases where polypeptides comprising
heterologous Fc region variants cannot be formed efficiently,
polypeptides comprising heterologous Fc region variants can be
obtained by separating and purifying them from polypeptides
comprising homologous Fc region variants. When producing a
polypeptide comprising heterologous Fc region variants consisting
of a first polypeptide and a second polypeptide which have
different sequences from each other, polypeptides comprising
homologous Fc region consisting of only two first polypeptides, and
polypeptide comprising homologous Fc region consisting of only two
second polypeptide are mixed in as impurities. Known technologies
can be used as a method for efficiently removing these two types of
polypeptides comprising homologous Fc region. A method has been
reported to be able to purify two types of homodimers and the
heterodimerized antibody of interest by ion exchange
chromatography, by creating a difference in isoelectric points by
introducing amino acid substitutions into the variable regions of
the two types of H chains (WO 2007114325). To date, as a method for
purifying heterodimerized antibodies, a method using Protein A has
been reported to purify a heterodimerized antibody comprising a
mouse IgG2a H chain that binds to Protein A and a rat IgG2b H chain
that does not bind to Protein A (WO 98050431 and WO 95033844).
[0195] Furthermore, a heterodimerized antibody alone can be
efficiently purified by using H chains in which amino acid residues
at the IgG-Protein A binding site, positions 435 and 436 (EU
numbering), are substituted with amino acids yielding different
Protein A affinities such as Tyr or His to change interaction of
each of the H chains with Protein A, and using a Protein A
column.
[0196] A plurality of these substitutions and technologies, for
example, two or more of them can be used in combination.
Furthermore, these alterations can be made separately to the first
polypeptide and the second polypeptide when necessary. Polypeptides
of the present invention may also be those produced based on the
products of the above-mentioned alterations.
[0197] In the present invention, amino acid alteration means any of
substitution, deletion, addition, insertion, and modification, or a
combination thereof. In the present invention, amino acid
alteration may be rephrased as amino acid mutation, and they are
used synonymously.
[0198] When substituting amino acid residues, substitution to a
different amino acid residue is carried out with the objective of
altering aspects such as (a)-(c) described below:
(a) polypeptide backbone structure in the sheet-structure or
helical-structure region; (b) electric charge or hydrophobicity at
the target site; or (c) size of the side chain.
[0199] Amino acid residues are classified into the following groups
based on their general side chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, and ile; (2)
neutral hydrophilic: cys, ser, thr, asn, and gln; (3) acidic: asp
and glu; (4) basic: his, lys, and arg; (5) residues that affect the
chain orientation: gly and pro; and (6) aromatic: trp, tyr, and
phe.
[0200] Substitution between amino acid residues within each of
these amino acid groups is referred to as conservative
substitution, and amino acid residue substitution between different
groups is referred to as non-conservative substitution.
Substitutions in the present invention may be conservative
substitutions or non-conservative substitutions, or a combination
of conservative substitutions and non-conservative
substitutions.
[0201] Amino acid sequence alterations are produced by various
methods known to those skilled in the art. Such methods include the
site-directed mutagenesis method (Hashimoto-Gotoh, T, Mizuno, T,
Ogasahara, Y, and Nakagawa, M. (1995) An
oligodeoxyribonucleotide-directed dual amber method for
site-directed mutagenesis. Gene 152: 271-275; Zoller, M J, and
Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA
fragments cloned into M13 vectors. Methods Enzymol. 100: 468-500;
Kramer, W, Drutsa, V, Jansen, H W, Kramer, B, Pflugfelder, M, and
Fritz, H J (1984) The gapped duplex DNA approach to
oligonucleotide-directed mutation construction. Nucleic Acids Res.
12: 9441-9456; Kramer W, and Fritz H J (1987)
Oligonucleotide-directed construction of mutations via gapped
duplex DNA Methods. Enzymol. 154, 350-367; and Kunkel, T A (1985)
Rapid and efficient site-specific mutagenesis without phenotypic
selection. Proc Natl Acad Sci USA. 82: 488-492), the PCR mutation
method, and the cassette mutation method, but are not limited
thereto.
[0202] Amino acid modification of the present invention includes
post-translational modification. A specific post-translational
modification may be addition or deletion of a sugar chain. For
example, in the IgG1 constant region consisting of the amino acid
sequence of SEQ ID NO: 11, the amino acid residue at position 297
(EU numbering) may be sugar chain-modified. The sugar-chain
structure for the modification is not limited. Generally,
antibodies expressed in eukaryotic cells comprise glycosylation in
the constant region. Therefore, antibodies expressed in cells such
as those below are normally modified by some type of sugar chain:
[0203] antibody-producing cells of mammals [0204] eukaryotic cells
transformed with an expression vector comprising a DNA encoding an
antibody
[0205] Eukaryotic cells shown here include yeast and animal cells.
For example, CHO cells and HEK293H cells are representative animal
cells used in transformation with an expression vector comprising
an antibody-encoding DNA. On the other hand, those without
glycosylation at this site are also included in the constant region
of the present invention. Antibodies whose constant region is not
glycosylated can be obtained by expressing an antibody-encoding
gene in prokaryotic cells such as Escherichia coli.
[0206] Specifically, for example, sialic acid may be added to the
sugar chain of an Fc region (MAbs. 2010 September-October; 2(5):
519-27).
[0207] Furthermore, the present invention provides antibodies
comprising any of Fc region variant described above.
[0208] The term "antibody/antibodies" in the present invention is
used in the broadest sense, and as long as the desired biological
activity is shown, it comprises any antibody such as monoclonal
antibodies (including full-length monoclonal antibodies),
polyclonal antibodies, antibody variants, antibody fragments,
polyspecific antibodies (multi-specific antibodies) (for example,
bispecific antibodies (diabodies)), chimeric antibodies, and
humanized antibodies.
[0209] Regarding the antibodies of the present invention, the
antigen type and antibody origin are not limited, and they may be
any type of antibodies. The origin of the antibodies is not
particularly limited, but examples include human antibodies, mouse
antibodies, rat antibodies, and rabbit antibodies.
[0210] Methods for producing the antibodies are well known to those
skilled in the art, and for example, monoclonal antibodies may be
produced by the hybridoma method (Kohler and Milstein, Nature 256:
495 (1975)), or the recombination method (U.S. Pat. No. 4,816,567).
Alternatively, they may be isolated from a phage antibody library
(Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol.
Biol. 222: 581-597 (1991)).
[0211] A humanized antibody is also called a reshaped human
antibody. Specifically, humanized antibodies prepared by grafting
the CDRs 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 mouse antibody CDRs to human FRs.
[0212] A vector for expressing a humanized antibody can be produced
by inserting a DNA encoding an antibody variable region in which
three CDRs and four FRs are ligated and a DNA encoding a human
antibody constant region into an expression vector so that these
DNAs are fused in frame. After this integration vector is
transfected into a host to establish recombinant cells, these cells
are cultured, and the DNA encoding the humanized antibody is
expressed to produce the humanized antibody in the culture of the
cells (see, European Patent Publication No. EP 239,400, and
International Patent Publication No. WO 1996/002576).
[0213] As necessary, an amino acid residue in an FR may be
substituted so that the CDRs of a reshaped human antibody form an
appropriate antigen-binding site. For example, a mutation can be
introduced into the amino acid sequence of an FR by applying the
PCR method used for grafting mouse CDRs to human FRs.
[0214] A desired human antibody can be obtained by DNA immunization
using a transgenic animal having the complete repertoire of human
antibody genes (see International Publication Nos. WO 1993/012227,
WO 1992/003918, WO 1994/002602, WO 1994/025585, WO 1996/034096, and
WO 1996/033735) as an animal for immunization.
[0215] Furthermore, technologies for obtaining a human antibody by
panning using a human antibody library are known. For example, a
human antibody V region is expressed on the surface of a phage as a
single-chain antibody (scFv) by the phage display method. The
scFv-expressing phage that binds to the antigen can be selected.
The DNA sequence that encodes the V region of the antigen-bound
human antibody can be determined by analyzing the genes of the
selected phage. After determining the DNA sequence of the scFv that
binds to the antigen, an expression vector can be prepared by
fusing the V-region sequence in-frame with the sequence of a
desired human antibody C region, and then inserting this into a
suitable expression vector. The expression vector is introduced
into suitable expression cells such as those described above, and
the human antibody can be obtained by expressing the human
antibody-encoding gene. These methods are already known (see,
International Publication Nos. WO 1992/001047, WO 1992/020791, WO
1993/006213, WO 1993/011236, WO 1993/019172, WO 1995/001438, and WO
1995/15388).
[0216] Variable regions constituting the antibodies of the present
invention can be variable regions that recognize any antigen.
[0217] Herein, there is no particular limitation on the antigen,
and it may be any antigens. Examples of such antigens preferably
include ligands (cytokines, chemokines, and such), receptors,
cancer antigens, MHC antigens, differentiation antigens,
immunoglobulins, and immune complexes partly containing
immunoglobulins.
[0218] Examples of cytokines include interleukins 1 to 18, colony
stimulating factors (G-CSF, M-CSF, GM-CSF, etc.), interferons
(IFN-.alpha., IFN-.beta., IFN-.gamma., etc.), growth factors (EGF,
FGF, IGF, NGF, PDGF, TGF, HGF, etc.), tumor necrosis factors
(TNF-.alpha. and TNF-.beta.), lymphotoxin, erythropoietin, leptin,
SCF, TPO, MCAF, and BMP.
[0219] Examples of chemokines include CC chemokines such as CCL1 to
CCL28, CXC chemokines such as CXCL1 to CXCL17, C chemokines such as
XCL1 to XCL2, and CX3C chemokines such as CX3CL1.
[0220] Examples of receptors include receptors belonging to
receptor families such as the hematopoietic growth factor receptor
family, cytokine receptor family, tyrosine kinase-type receptor
family, serine/threonine kinase-type receptor family, TNF receptor
family, G protein-coupled receptor family, GPI anchor-type receptor
family, tyrosine phosphatase-type receptor family, adhesion factor
family, and hormone receptor family. The receptors belonging to
these receptor families and their characteristics have been
described in many documents such as Cooke B A., King R J B., van
der Molen H J. ed. New Comprehesive Biochemistry Vol. 18B "Hormones
and their Actions Part II" pp. 1-46 (1988) Elsevier Science
Publishers BV; 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); and Flower D R. Flower (Biochim. Biophys.
Acta (1999) 1422 (3): 207-234).
[0221] Examples of specific receptors belonging to the
above-mentioned receptor families preferably include human or mouse
erythropoietin (EPO) receptors (Blood (1990) 76 (1): 31-35; and
Cell (1989) 57 (2): 277-285), human or 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 or mouse thrombopoietin (TPO) receptors (Proc Natl Acad Sci
USA. (1992) 89 (12): 5640-5644; EMBO J. (1993) 12(7): 2645-53),
human or mouse insulin receptors (Nature (1985) 313 (6005):
756-761), human or mouse Flt-3 ligand receptors (Proc. Natl. Acad.
Sci. USA. (1994) 91 (2): 459-463), human or mouse platelet-derived
growth factor (PDGF) receptors (Proc. Natl. Acad. Sci. USA. (1988)
85 (10): 3435-3439), human or mouse interferon (IFN)-.alpha. and
.beta. receptors (Cell (1990) 60 (2): 225-234; and Cell (1994) 77
(3): 391-400), human or mouse leptin receptors, human or mouse
growth hormone (GH) receptors, human or mouse interleukin (IL)-10
receptors, human or mouse insulin-like growth factor (IGF)-I
receptors, human or mouse leukemia inhibitory factor (LIF)
receptors, and human or mouse ciliary neurotrophic factor (CNTF)
receptors.
[0222] Cancer antigens are antigens that are expressed as cells
become malignant, and they are also called tumor-specific antigens.
Abnormal sugar chains that appear on cell surfaces or protein
molecules when cells become cancerous are also cancer antigens, and
they are also called sugar-chain cancer antigens. Examples of
cancer antigens preferably include GPC3 which is a receptor
belonging to the GPI anchor-type receptor family mentioned above,
and is also expressed in several cancers including liver cancer
(Int J Cancer. (2003) 103 (4): 455-65), as well as EpCAM which is
expressed in several cancers including lung cancer (Proc Natl Acad
Sci USA. (1989) 86 (1): 27-31), CA19-9, CA15-3, and sialyl SSEA-1
(SLX).
[0223] MHC antigens are roughly classified into MHC class I
antigens and MHC class II antigens. MHC class I antigens include
HLA-A, --B, --C, -E, --F, -G, and --H, and MHC class II antigens
include HLA-DR, -DQ, and -DP.
[0224] Differentiation antigens may 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.
[0225] Immunoglobulins include IgA, IgM, IgD, IgG, and IgE. Immune
complexes include a component of at least any of the
immunoglobulins.
[0226] Other antigens include, for example, the molecules below:
17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1
adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin
AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin
RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12,
ADAM15, ADAM17/TACE, ADAMS, ADAMS, ADAMTS, ADAMTS4, ADAMTS5,
addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7,alpha-1-antitrypsin,
alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL,
AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic
peptide, av/b3 integrin, Ax1, b2M, B7-1, B7-2, B7-H, B-lymphocyte
stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1,
BAK, Bax, BCA-1, BCAM, Bc1, BCMA, BDNF, b-ECGF, bFGF, BID, Bik,
BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4
BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2),
BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II
(BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic
factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5,
C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen
(CEA), cancer associated antigen, cathepsin A, cathepsin B,
cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin
L, cathepsin 0, cathepsin S, cathepsin V, cathepsin X/Z/P, CBL,
CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16,
CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24,
CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8,
CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6,
CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,
CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19,
CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32,
CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46,
CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80
(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147,
CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Botulinum toxin,
Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF,
CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1,
CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7,
CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15,
CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,cytokeratin
tumor associated antigen, DAN, DCC, DcR3, DC-SIGN, complement
regulatory factor (Decay accelerating factor), des (1-3)-IGF-I
(brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk,
EGAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN,
ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1,
EpCAM, ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa,
factor VII, factor VIIIc, factor IX, fibroblast activation protein
(FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8,
FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle stimulating
hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,
FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1,
GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2),
GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1),
GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR,
glucagon, Glut4, glycoprotein IIb/IIIa (GPIIb/IIIa), GM-CSF, gp130,
gp72, GRO, growth hormone releasing hormone, hapten (NP-cap or
NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH
envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF),
Hep B gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3),
Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD
glycoprotein, HGFA, high molecular weight melanoma-associated
antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR,
HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human
cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309,
IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE,
IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1,
IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8,
IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon
(INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain,
insulin B chain, insulin-like growth factor1, integrin alpha2,
integrin alpha3, integrin alpha4, integrin alpha4/beta1, integrin
alpha4/beta7, integrin alpha5 (alpha V), integrin alpha5/beta1,
integrin alpha5/beta3, integrin alpha6, integrin beta1, integrin
beta2,interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein
5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14,
kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3,
kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin
5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1 bp1, LBP,
LDGF, LECT2, lefty, Lewis-Y antigen, Lewis-Y associated antigen,
LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn,
L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing
hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC,
MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF
receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK,
MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15,
MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP,
mucin (Muc1), MUC18, Mullerian-inhibiting substance, Mug, MuSK,
NAIP, NAP, NCAD, N-C adherin, NCA 90, NCAM, NCAM, neprilysin,
neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF),
NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG,
OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone,
PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1,
PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental
alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin,
prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane
antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES,
RANTES, relaxin A chain, relaxin B chain, renin, respiratory
syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factor, RLIP76,
RPA2, RSK, 5100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh,
SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,
STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated
glycoprotein-72), TARC, TCA-3, T-cell receptor (for example, T-cell
receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT,
testis PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha,
TGF-beta, TGF-beta Pan Specific, TGF-betaRI (ALK-5), TGF-betaRII,
TGF-betaRIIb, TGF-betaRIII, TGF-beta1, TGF-beta2, TGF-beta3,
TGF-beta4, TGF-beta5, thrombin, thymus Ck-1, thyroid-stimulating
hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF,
TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI, TNF-RII,
TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER,
TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D
(TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),
TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B
(TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R,
TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR
AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF
RI CD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26
(TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35,
TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95),
TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9
(4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2),
TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3,
TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11
(TRANCE/RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand,
DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,
THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15
(TL1A/VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A
(TNF-.alpha. Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1),
TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5
(CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand
Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30
ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo,
TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor,
TRF, Trk, TROP-2, TSG, TSLP, tumor associated antigen CA125, tumor
associated antigen expressing Lewis-Y associated carbohydrates,
TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD,
VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3
(flt-4), VEGI, VIM, virus antigen, VLA, VLA-1, VLA-4, VNR integrin,
von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A,
WNT4, WNTSA, WNTSB, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A,
WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR,
XIAP, XPD, HMGB1, IgA, A.beta., CD81, CD97, CD98, DDR1, DKK1, EREG,
Hsp90, IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK9,
prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B,
tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1.1,
Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9,
EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b,
C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H,
properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin,
tissue factor, factor V, factor Va, factor VII, factor VIIa, factor
VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa,
factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor
XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA,
plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2,
Syndecan-3, Syndecan-4, LPA, and S1P; and receptors for hormone and
growth factors.
[0227] One or more amino acid residue alterations are allowed in
the amino acid sequences constituting the variable regions as long
as their antigen-binding activities are maintained. When altering a
variable region amino acid sequence, there is no particularly
limitation on the site of alteration and number of amino acids
altered. For example, amino acids present in CDR and/or FR can be
altered appropriately. When altering amino acids in a variable
region, the binding activity is preferably maintained without
particular limitation; and for example, as compared to before
alteration, the binding activity is 50% or more, preferably 80% or
more, and more preferably 100% or more. Furthermore, the binding
activity may be increased by amino acid alterations. For example,
the binding activity may be 2-, 5-, 10-times higher or such than
that before alteration. In the antibodies of the present invention,
alteration of amino acid sequence may be at least one of amino acid
residue substitution, addition, deletion, and modification.
[0228] For example, the modification of the N-terminal glutamine of
a variable region into pyroglutamic acid by pyroglutamylation is a
modification well known to those skilled in the art. Thus, when the
heavy-chain N terminus is glutamine, the antibodies of the present
invention comprise the variable regions in which the glutamine is
modified to pyroglutamic acid.
[0229] Antibody variable regions of the present invention may have
any sequences, and they may be antibody variable regions of any
origin, such as mouse antibodies, rat antibodies, rabbit
antibodies, goat antibodies, camel antibodies, humanized antibodies
produced by humanizing these non-human antibodies, and human
antibodies. "Humanized antibodies", also referred to as "reshaped
human antibodies", are antibodies in which the complementarity
determining regions (CDRs) of an antibody derived from a non-human
mammal, for example, a mouse antibody, are transplanted into the
CDRs of a human antibody. Methods for identifying CDRs are known
(Kabat et al., Sequence of Proteins of Immunological Interest
(1987), National Institute of Health, Bethesda, Md.; Chothia et
al., Nature (1989) 342: 877). Their common genetic recombination
techniques are also known (see, European Patent Application
Publication No. EP 125023 and WO 96/02576). Furthermore, these
antibodies may have various amino acid substitutions introduced
into their variable regions to improve their antigen binding,
pharmacokinetics, stability, and immunogenicity. Variable regions
of the antibodies of the present invention may be able to bind
antigens repeatedly due to their pH dependability in antigen
binding (WO 2009/125825).
[0230] .kappa. chain and .lamda. chain-type constant regions are
present in antibody light-chain constant regions, but either one of
the light chain constant regions is acceptable. Furthermore,
light-chain constant regions of the present invention may be
light-chain constant regions with amino acid alterations such as
substitutions, deletions, additions, and/or insertions.
[0231] For example, for the heavy chain constant regions of an
antibody of the present invention, heavy chain constant regions of
human IgG antibodies may be used and heavy chain constant regions
of human IgG1 antibodies and those of human IgG4 antibodies are
preferred.
[0232] Furthermore, Fc region variants of the present invention may
be made into Fc fusion protein molecules by linking to other
proteins, physiologically active peptides, and such. Herein, fusion
protein refers to a chimeric polypeptide comprising at least two
different polypeptides, which do not spontaneously link with each
other in natural. Examples of the other proteins and biologically
active peptides include receptors, adhesion molecules, ligands, and
enzymes, but are not limited thereto.
[0233] Preferred examples of Fc fusion protein molecules of the
present invention include proteins with Fc region fused to a
receptor protein that binds to a target, and such examples include
TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion
protein, and CTLA4-Fc fusion protein (Nat Med. 2003 January; 9(1):
47-52; BioDrugs. 2006; 20(3): 151-60). Furthermore, a protein to be
fused to a polypeptide of the present invention may be any molecule
as long as it binds to a target molecule, and examples include scFv
molecules (WO 2005/037989), single-domain antibody molecules (WO
2004/058821; WO 2003/002609), antibody-like molecules (Current
Opinion in Biotechnology 2006, 17: 653-658; Current Opinion in
Biotechnology 2007, 18: 1-10; Current Opinion in Structural Biology
1997, 7: 463-469; and Protein Science 2006, 15: 14-27) such as
DARPins (WO 2002/020565), Affibody (WO 1995/001937), Avimer (WO
2004/044011; WO 2005/040229), and Adnectin (WO 2002/032925).
Furthermore, antibodies and Fc fusion protein molecules may be
multispecific antibodies that bind to multiple types of target
molecules or epitopes.
[0234] Furthermore, the antibodies of the present invention include
antibody modification products. Such antibody modification products
include, for example, antibodies linked with various molecules such
as polyethylene glycol (PEG) and cytotoxic substances. Such
antibody modification products can be obtained by chemically
modifying antibodies of the present invention. Methods for
modifying antibodies are already established in this field.
[0235] The antibodies of the present invention may also be
bispecific antibodies. "Bispecific antibody" refers to an antibody
that has in a single molecule variable regions that recognize
different epitopes. The epitopes may be present in a single
molecule or in different molecules.
[0236] The polypeptides of the present invention can be prepared by
the methods known to those skilled in the art. For example, the
antibodies can be prepared by the methods described below, but the
methods are not limited thereto.
[0237] A DNA encoding an antibody heavy chain in which one or more
amino acid residues in the Fc region have been substituted with
other amino acids of interest and DNA encoding an antibody light
chain, are expressed. A DNA encoding a heavy chain in which one or
more amino acid residues in the Fc region are substituted with
other amino acids of interest can be prepared, for example, by
obtaining a DNA encoding the Fc region of a natural heavy chain,
and introducing an appropriate substitution so that a codon
encoding a particular amino acid in the Fc region encodes another
amino acid of interest.
[0238] Alternatively, a DNA encoding a heavy chain in which one or
more amino acid residues in the Fc region are substituted with
other amino acids of interest can also be prepared by designing and
then chemically synthesizing a DNA encoding a protein in which one
or more amino acid residues in the Fc region of the natural heavy
chain are substituted with other amino acids of interest. The
position and type of amino acid substitution are not particularly
limited. Furthermore, alteration is not limited to substitution,
and alteration may be any of deletion, addition, or insertion, or
combination thereof.
[0239] Alternatively, a DNA encoding a heavy chain in which one or
more amino acid residues in the Fc region are substituted with
other amino acids of interest can be prepared as a combination of
partial DNAs. Such combinations of partial DNAs include, for
example, the combination of a DNA encoding a variable region and a
DNA encoding a constant region, and the combination of a DNA
encoding an Fab region and a DNA encoding an Fc region, but are not
limited thereto. Furthermore, a DNA encoding a light chain can
similarly be prepared as a combination of partial DNAs.
[0240] Methods for expressing the above-described DNAs include the
methods described below. For example, a heavy chain expression
vector is constructed by inserting a DNA encoding a heavy chain
variable region into an expression vector along with a DNA encoding
a heavy chain constant region. Likewise, a light chain expression
vector is constructed by inserting a DNA encoding a light chain
variable region into an expression vector along with a DNA encoding
a light chain constant region. Alternatively, these heavy and light
chain genes may be inserted into a single vector.
[0241] When inserting a DNA encoding the antibody of interest into
an expression vector, the DNA is inserted so that the antibody is
expressed under the control of an expression-regulating region such
as an enhancer or promoter. Next, host cells are transformed with
this expression vector to express the antibody. In such cases, an
appropriate combination of host and expression vector may be
used.
[0242] Examples of the vectors include M13 vectors, pUC vectors,
pBR322, pBluescript, and pPCR-Script. Alternatively, when aiming to
subclone and excise cDNA, in addition to the vectors described
above, pGEM-T, pDIRECT, pT7, and such can be used.
[0243] Expression vectors are particularly useful when using
vectors for producing the polypeptides of the present invention.
For example, when a host cell is E. coli such as JM109, DH5.alpha.,
HB101, and XL1-Blue, the expression vectors must carry a promoter
that allows efficient expression in E. coli, for example, lacZ
promoter (Ward et al., Nature (1989) 341: 544-546; FASEB J. (1992)
6: 2422-2427; its entirety are incorporated herein by reference),
araB promoter (Better et al., Science (1988) 240: 1041-1043; its
entirety are incorporated herein by reference), T7 promoter, or
such. Such vectors include pGEX-5X-1 (Pharmacia), "QIAexpress
system" (QIAGEN), pEGFP, or pET (in this case, the host is
preferably BL21 that expresses T7 RNA polymerase) in addition to
the vectors described above.
[0244] The vectors may contain signal sequences for polypeptide
secretion. As a signal sequence for polypeptide secretion, a pelB
signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169: 4379;
its entirety are incorporated herein by reference) may be used when
a polypeptide is secreted into the E. coli periplasm. The vector
can be introduced into host cells by lipofectin method, calcium
phosphate method, and DEAE-Dextran method, for example.
[0245] In addition to E. coli expression vectors, the vectors for
producing the polypeptides of the present invention include
mammalian expression vectors (for example, pcDNA3 (Invitrogen),
pEGF-BOS (Nucleic Acids. Res. 1990, 18(17): p5322; its entirety are
incorporated herein by reference), pEF, and pCDM8), insect
cell-derived expression vectors (for example, the "Bac-to-BAC
baculovirus expression system" (GIBCO-BRL) and pBacPAK8),
plant-derived expression vectors (for example, pMH1 and pMH2),
animal virus-derived expression vectors (for example, pHSV, pMV,
and pAdexLcw), retroviral expression vectors (for example,
pZIPneo), yeast expression vectors (for example, "Pichia Expression
Kit" (Invitrogen), pNV11, and SP-Q01), and Bacillus subtilis
expression vectors (for example, pPL608 and pKTH50), for
example.
[0246] When aiming for expression in animal cells such as CHO, COS,
and NIH3T3 cells, the vectors must have a promoter essential for
expression in cells, for example, SV40 promoter (Mulligan et al.,
Nature (1979) 277: 108; its entirety are incorporated herein by
reference), MMTV-LTR promoter, EF1.alpha. promoter (Mizushima et
al., Nucleic Acids Res. (1990) 18: 5322; its entirety are
incorporated herein by reference), CAG promoter (Gene. (1990) 18:
5322; its entirety are incorporated herein by reference), and CMV
promoter, and more preferably they have a gene for selecting
transformed cells (for example, a drug resistance gene that allows
evaluation using an agent (neomycin, G418, or such)). Vectors with
such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV,
and pOP13, for example.
[0247] In addition, the following method can be used for stable
gene expression and gene copy number amplification in cells: CHO
cells deficient in a nucleic acid synthesis pathway are introduced
with a vector that carries a DHFR gene which compensates for the
deficiency (for example, pCHOI), and the vector is amplified using
methotrexate (MTX). Alternatively, the following method can be used
for transient gene expression: COS cells with a gene expressing
SV40 T antigen on their chromosome are transformed with a vector
with an SV40 replication origin (pcD and such). Replication origins
derived from polyoma virus, adenovirus, bovine papilloma virus
(BPV), and such can also be used. To amplify gene copy number in
host cells, the expression vectors may further carry selection
markers such as aminoglycoside transferase (APH) gene, thymidine
kinase (TK) gene, E. coli xanthine-guanine
phosphoribosyltransferase (Ecogpt) gene, and dihydrofolate
reductase (dhfr) gene.
[0248] Antibodies can be collected, for example, by culturing
transformed cells, and then separating the antibodies from the
inside of the transformed cells or from the culture media.
Antibodies can be separated and purified using an appropriate
combination of methods such as centrifugation, ammonium sulfate
fractionation, salting out, ultrafiltration, 1q, FcRn, protein A,
protein G column, affinity chromatography, ion exchange
chromatography, and gel filtration chromatography.
[0249] Furthermore, the present invention provides methods for
producing a polypeptide comprising an antibody Fc region variant
having enhanced Fc.gamma.RIIb-binding activity in comparison with a
polypeptide comprising a parent Fc region, which comprises adding
at least one amino acid alteration to the Fc region variant.
[0250] Examples include production methods comprising the following
steps:
(a) adding at least one amino acid alteration to an Fc region of
polypeptides comprising the Fc region; (b) measuring the
Fc.gamma.RIIb-binding activity of the polypeptides altered in step
(a); and (c) selecting polypeptides comprising an Fc region variant
having enhanced Fc.gamma.RIIb-binding activity in comparison with a
polypeptide comprising a parent Fc region.
[0251] A preferred embodiment is a method for producing a
polypeptide comprising an Fc region variant, which comprises the
steps of:
(a) altering a nucleic acid encoding the polypeptide so that the
Fc.gamma.RIIb-binding activity is enhanced in comparison with the
polypeptide comprising a parent Fc region; (b) introducing the
nucleic acid into host cells and culturing them to induce
expression; and (c) collecting the polypeptide from the host cell
culture.
[0252] Furthermore, antibodies and Fc fusion protein molecules
produced by this production method are also included in the present
invention.
[0253] The present invention also provides a method of producing a
polypeptide which comprises an Fc region variant with enhanced
binding selectivity to Fc.gamma.RIIb compared to Fc.gamma.RIIa
(type R) as compared to that of a polypeptide which comprises a
parent Fc region, wherein the method comprises adding at least one
amino acid alteration to an antibody Fc region variant in a
polypeptide comprising the Fc region variant.
[0254] An example is a production method comprising the steps
of:
(a) adding at least one amino acid alteration to an Fc region in a
polypeptide comprising the Fc region; (b) determining the
Fc.gamma.RIIa-binding activity and Fc.gamma.RIIb-binding activity
of the polypeptide altered in step (a); and (c) selecting a
polypeptide comprising an Fc region variant with enhanced binding
selectivity to Fc.gamma.RIIb compared to Fc.gamma.RIIa (type R) as
compared to that of a polypeptide comprising a parent Fc
region.
[0255] In a preferred embodiment, it is a method of producing
polypeptides comprising an Fc region variant, wherein the method
comprises the steps of:
(a) modifying a nucleic acid encoding a polypeptide comprising a
parent Fc region to achieve enhancement of binding selectivity to
Fc.gamma.RIIb compared to Fc.gamma.RIIa (type R) as compared to
that of the polypeptide; (b) transfecting the nucleic acid into a
host cell and culturing the cell for expression of the nucleic
acid; and (c) collecting the polypeptide from the host cell
culture.
[0256] Antibodies and Fc fusion protein molecules produced by the
production method are also included in the present invention.
[0257] Furthermore, the present invention provides a method of
producing a polypeptide comprising an Fc region variant with
enhanced Fc.gamma.RIIb-binding activity and enhanced binding
selectivity to Fc.gamma.RIIb compared to Fc.gamma.RIIa (type R) as
compared to those of a polypeptide comprising a parent Fc region,
wherein the method comprises adding at least one amino acid
alteration to an antibody Fc region variant in a polypeptide
comprising the antibody Fc region variant.
[0258] An example is a production method comprising the steps
of:
(a) adding at least one amino acid alteration to an Fc region in a
polypeptide comprising the Fc region; (b) determining the
Fc.gamma.RIIa-binding activity and Fc.gamma.RIIb-binding activity
of the polypeptide altered in step (a); and (c) selecting a
polypeptide comprising an Fc region variant with enhanced
Fc.gamma.RIIb-binding activity and enhanced binding selectivity to
Fc.gamma.RIIb compared to Fc.gamma.RIIa (type R) as compared to
those of a polypeptide comprising a parent Fc region.
[0259] In a preferred embodiment, it is a method of producing
polypeptides comprising an Fc region variant, wherein the method
comprises the steps of:
(a) modifying nucleic acid encoding a polypeptide comprising a
parent Fc region to achieve enhancement of Fc.gamma.RIIb-binding
activity and enhancement of binding selectivity to Fc.gamma.RIIb
compared to Fc.gamma.RIIa (type R) as compared to those of the
polypeptide; (b) transfecting the nucleic acid into a host cell and
culturing the cell for expression of the nucleic acid; and (c)
collecting the polypeptide from the host cell culture.
[0260] Antibodies and Fc fusion protein molecules produced by the
production method are also included in the present invention.
[0261] The present invention also provides methods for producing a
polypeptide in which antibody production against the polypeptide is
suppressed compared with a polypeptide comprising a parent Fc
region when administered in vivo, which comprise adding at least
one amino acid alteration in the Fc region of a polypeptide
comprising an antibody Fc region.
[0262] Examples include a production method comprising the
following steps:
(a) adding at least one amino acid alteration in the Fc region of a
polypeptide comprising an Fc region; and (b) confirming that
antibody production is suppressed when the polypeptide comprising
an Fc region altered in step (a) is administered in vivo in
comparison with a polypeptide comprising a parent Fc region.
[0263] Whether or not production of antibodies against the
polypeptide has been suppressed can be confirmed by methods of
administering the polypeptide to an animal and such. Alternatively,
suppression of antibody production can be determined by measuring
the binding activities towards Fc.gamma.RIIa and Fc.gamma.RIIb, and
observing an increase in the value obtained by dividing the KD
value for Fc.gamma.RIIa by the KD value for Fc.gamma.RIIb. Such
polypeptides are considered to be useful as pharmaceuticals since
they can suppress antibody production without activating activating
Fc.gamma.R.
[0264] In the above-mentioned production methods, it is preferred
that Fc.gamma.RIIb-binding activity is enhanced and binding
selectivity to Fc.gamma.RIIb compared to Fc.gamma.RIIa (type R) is
enhanced.
[0265] An example of a preferred embodiment of the above-mentioned
production method is altering an Fc region of human IgG so that
alteration of the amino acid at position 238 (EU numbering) to
another amino acid and alteration of at least one amino acid
selected from amino acids at positions 233, 234, 235, 237, 264,
265, 266, 267, 268, 269, 271, 272, 274, 296, 326, 327, 330, 331,
332, 333, 334, 355, 356, 358, 396, 409, and 419 according to EU
numbering to another amino acid are introduced into the Fc region.
Other amino acid alterations that are combined with the amino acid
alteration at position 238 (EU numbering) are preferably those at
amino acid positions 233, 237, 264, 267, 268, 271, 272, 296, 327,
330, 332, 333, and 396 according to EU numbering, and in particular
those at amino acid positions 233, 237, 264, 267, 268, 271, 296,
330, and 396. In particular, a preferred combination of amino acid
alterations in terms of enhancement of Fc.gamma.RIIb-binding
activity or enhancement of binding selectivity to Fc.gamma.RIIb
compared to Fc.gamma.RIIa includes, for example, the combination of
amino acid alterations at amino acid positions 238, 268, and 271
(EU numbering) with at least one amino acid alteration selected
from positions 233, 237, 264, 267, 272, 296, 327, 330, 332, and 396
(EU numbering).
[0266] The amino acids to be altered are not particularly limited
as long as those enhance Fc.gamma.RIIb-binding activity or enhance
binding selectivity to Fc.gamma.RIIb compared to Fc.gamma.RIIa as
compared to before the alteration, but preferably, the amino acid
at position 238 is Asp, the amino acid at position 233 is Asp, the
amino acid at position 234 is Tyr, the amino acid at position 237
is Asp, the amino acid at position 264 is Ile, the amino acid at
position 265 is Glu, the amino acid at position 266 is Phe, Leu or
Met, the amino acid at position 267 is Ala, Glu, Gly, or Gln, the
amino acid at position 268 is Asp, Gln, or Glu, the amino acid at
position 269 is Asp, the amino acid at position 271 is Gly, the
amino acid at position 272 is Asp, Phe, Ile, Met, Asn, Pro, or Gln,
the amino acid at position 274 is Gln, the amino acid at position
296 is Asp or Phe, the amino acid at position 326 is Ala or Asp,
the amino acid at position 327 is Gly, the amino acid at position
330 is Lys, Arg, or Ser, the amino acid at position 331 is Ser, the
amino acid at position 332 is Lys, Arg, Ser, or Thr, the amino acid
at position 333 is Lys, Arg, Ser, or Thr, the amino acid at
position 334 is Arg, Ser, or Thr, the amino acid at position 355 is
Ala or Gln, the amino acid at position 356 is Glu, the amino acid
at position 358 is Met, the amino acid at position 396 is Ala, Asp,
Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,
Val, Trp, or Tyr, the amino acid at position 409 is Arg, and the
amino acid at position 419 is Glu. The amino acids to be altered
are more preferably those that enhance Fc.gamma.RIIb-binding
activity and also enhance binding selectivity to Fc.gamma.RIIb
compared to Fc.gamma.RIIa as compared to before the alteration. In
particular, when combining alterations at amino acid positions 238,
268, and 271 (EU numbering) with at least one amino acid position
selected from 233, 264, 267, 272, 296, 327, 330, 332, and 396 (EU
numbering), it is preferred that the amino acid at position 238 is
Asp, the amino acid at position 268 is Asp or Glu, the amino acid
at position 271 is Gly, the amino acid at position 233 is Asp, the
amino acid at position 237 is Asp, the amino acid at position 264
is Ile, the amino acid at position 267 is Ala or Gly, the amino
acid at position 272 is Asp or Pro, the amino acid at position 296
is Asp, the amino acid at position 327 is Gly, the amino acid at
position 330 is Arg, the amino acid at position 332 is Thr, and the
amino acid at position 396 is Leu or Met, according to EU
numbering.
[0267] Among these combinations, introducing alterations that lead
to greater enhancement of Fc.gamma.RIIb-binding activity, or lead
to greater enhancement of binding selectivity to Fc.gamma.RIIb
compared to Fc.gamma.RIIa (type R) are preferred. Examples of such
a preferred combination of amino acid substitution as alterations
include the following combinations (a) to (x):
(a) amino acid alterations at positions 238, 233, 237, 268, 271,
296, and 330 (EU numbering) of an Fc region; (b) amino acid
alterations at positions 238, 237, 268, 271, 296, and 330 (EU
numbering) of an Fc region; (c) amino acid alterations at positions
238, 233, 237, 268, 271, 296, 330, and 332 (EU numbering) of an Fc
region; (d) amino acid alterations at positions 238, 233, 237, 264,
267, 268, 271, and 330 (EU numbering) of an Fc region; (e) amino
acid alterations at positions 238, 233, 237, 267, 268, 271, 296,
330, and 332 (EU numbering) of an Fc region; (f) amino acid
alterations at positions 238, 237, 267, 268, 271, 296, 330, and 332
(EU numbering) of an Fc region; (g) amino acid alterations at
positions 238, 233, 237, 268, 271, 296, 327, and 330 (EU numbering)
of an Fc region; (h) amino acid alterations at positions 238, 233,
237, 264, 267, 268, and 271 (EU numbering) of an Fc region; (i)
amino acid alterations at positions 238, 233, 237, 264, 267, 268,
271, 296, and 330 (EU numbering) of an Fc region; (j) amino acid
alterations at positions 238, 233, 237, 264, 267, 268, 271, 296,
330, and 396 (EU numbering) of an Fc region; (k) amino acid
alterations at positions 238, 237, 264, 267, 268, 271, and 330 (EU
numbering) of an Fc region; (l) amino acid alterations at positions
238, 237, 264, 267, 268, 271, 296, and 330 (EU numbering) of an Fc
region; (m) amino acid alterations at positions 238, 264, 267, 268,
and 271 (EU numbering) of an Fc region; (n) amino acid alterations
at positions 238, 264, 267, 268, 271, and 296 (EU numbering) of an
Fc region; (o) amino acid alterations at positions 238, 237, 267,
268, 271, 296, and 330 (EU numbering) of an Fc region; (p) amino
acid alterations at positions 238, 233, 237, 264, 267, 268, 271,
330, and 396 (EU numbering) of the Fc region; (q) amino acid
alterations at positions 238, 233, 237, 264, 267, 268, 271, 296,
327, 330, and 396 (EU numbering) of an Fc region; (r) amino acid
alterations at positions 238, 233, 237, 264, 267, 268, 271, 272,
and 296 (EU numbering) of an Fc region; (s) amino acid alterations
at positions 238, 237, 264, 267, 268, 271, 272, and 330 (EU
numbering) of an Fc region; (t) amino acid alterations at positions
238, 237, 264, 267, 268, 271, 272, 296, and 330 (EU numbering) of
an Fc region; (u) amino acid alterations at positions 238, 233,
264, 267, 268, and 271 (EU numbering) of an Fc region; (v) amino
acid alterations at positions 238, 237, 267, 268, 271, 296, and 330
(EU numbering) of an Fc region; (w) amino acid alterations at
positions 238, 264, 267, 268, 271, 272, and 296 (EU numbering) of
an Fc region; and (x) amino acid alterations at positions 238, 233,
264, 267, 268, 271, and 296 (EU numbering) of an Fc region.
[0268] In addition, among these combinations, the following
combinations of amino acid alterations (a) to (x) below are more
preferred amino acid combinations:
(a) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 268 is Asp, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, and the amino acid at position 330 is Arg, according to EU
numbering, in an Fc region; (b) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 237
is Asp, the amino acid at position 268 is Asp or Glu, the amino
acid at position 271 is Gly, the amino acid at position 296 is Asp,
and the amino acid at position 330 is Arg, according to EU
numbering, in an Fc region; (c) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 233
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 268 is Asp, the amino acid at position 271 is Gly, the
amino acid at position 296 is Asp, the amino acid at position 330
is Arg, and the amino acid at position 332 is Thr, according to EU
numbering, in an Fc region; (d) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 233
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 264 is Ile, the amino acid at position 267 is Gly or Ala,
the amino acid at position 268 is Glu, the amino acid at position
271 is Gly, and the amino acid at position 330 is Arg, according to
EU numbering, in an Fc region; (e) an amino acid sequence in which
the amino acid at position 238 is Asp, the amino acid at position
233 is Asp, the amino acid at position 237 is Asp, the amino acid
at position 267 is Ala, the amino acid at position 268 is Glu, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, the amino acid at position 330 is Arg, and the amino acid
at position 332 is Thr, according to EU numbering, in an Fc region;
(f) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 267 is Ala, the amino acid at position 268 is Glu, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, the amino acid at position 330 is Arg, and the amino acid
at position 332 is Thr, according to EU numbering, in an Fc region;
(g) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 268 is Asp, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, the amino acid at position 327 is Gly, and the amino acid
at position 330 is Arg, according to EU numbering, in an Fc region;
(h) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, and the amino acid at position 271 is Gly, according to EU
numbering, in an Fc region; (i) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 233
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 264 is Ile, the amino acid at position 267 is Ala, the
amino acid at position 268 is Glu, the amino acid at position 271
is Gly, the amino acid at position 296 is Asp, and the amino acid
at position 330 is Arg, according to EU numbering, in an Fc region;
(j) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, the amino acid at position 271 is Gly, the amino acid at
position 296 is Asp, the amino acid at position 330 is Arg, and the
amino acid at position 396 is Met or Leu, according to EU
numbering, in the Fc region; (k) an amino acid sequence in which
the amino acid at position 238 is Asp, the amino acid at position
237 is Asp, the amino acid at position 264 is Ile, the amino acid
at position 267 is Ala, the amino acid at position 268 is Glu, the
amino acid at position 271 is Gly, and the amino acid at position
330 is Arg, according to EU numbering, in an Fc region; (l) an
amino acid sequence in which the amino acid at position 238 is Asp,
the amino acid at position 237 is Asp, the amino acid at position
264 is Ile, the amino acid at position 267 is Ala, the amino acid
at position 268 is Glu, the amino acid at position 271 is Gly, the
amino acid at position 296 is Asp, and the amino acid at position
330 is Arg, according to EU numbering, in an Fc region; (m) an
amino acid sequence in which the amino acid at position 238 is Asp,
the amino acid at position 264 is Ile, the amino acid at position
267 is Ala, the amino acid at position 268 is Glu, and the amino
acid at position 271 is Gly, according to EU numbering, in an Fc
region; (n) an amino acid sequence in which the amino acid at
position 238 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, the amino acid at position 271 is Gly, and the amino acid
at position 296 is Asp, according to EU numbering, in an Fc region;
(o) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 267 is Ala or Gly, the amino acid at position 268 is Glu,
the amino acid at position 271 is Gly, the amino acid at position
296 is Asp, and the amino acid at position 330 is Arg, according to
EU numbering, in an Fc region; (p) an amino acid sequence in which
the amino acid at position 238 is Asp, the amino acid at position
233 is Asp, the amino acid at position 237 is Asp, the amino acid
at position 264 is Ile, the amino acid at position 267 is Ala, the
amino acid at position 268 is Glu, the amino acid at position 271
is Gly, the amino acid at position 330 is Arg, and the amino acid
at position 396 is Met or Leu, according to EU numbering, in an Fc
region; (q) an amino acid sequence in which the amino acid at
position 238 is Asp, the amino acid at position 233 is Asp, the
amino acid at position 237 is Asp, the amino acid at position 264
is Ile, the amino acid at position 267 is Ala, the amino acid at
position 268 is Glu, the amino acid at position 271 is Gly, the
amino acid at position 296 is Asp, the amino acid at position 327
is Gly, the amino acid at position 330 is Arg, and the amino acid
at position 396 is Met, according to EU numbering, in an Fc region;
(r) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 233 is Asp, the amino acid at
position 237 is Asp, the amino acid at position 264 is Ile, the
amino acid at position 267 is Ala, the amino acid at position 268
is Glu, the amino acid at position 271 is Gly, the amino acid at
position 272 is Asp, and the amino acid at position 296 is Asp,
according to EU numbering, in an Fc region; (s) an amino acid
sequence in which the amino acid at position 238 is Asp, the amino
acid at position 237 is Asp, the amino acid at position 264 is Ile,
the amino acid at position 267 is Ala, the amino acid at position
268 is Glu, the amino acid at position 271 is Gly, the amino acid
at position 272 is Pro, and the amino acid at position 330 is Arg,
according to EU numbering, in an Fc region; (t) an amino acid
sequence in which the amino acid at position 238 is Asp, the amino
acid at position 237 is Asp, the amino acid at position 264 is Ile,
the amino acid at position 267 is Ala, the amino acid at position
268 is Glu, the amino acid at position 271 is Gly, the amino acid
at position 272 is Pro, the amino acid at position 296 is Asp, and
the amino acid at position 330 is Arg, according to EU numbering,
in an Fc region; (u) an amino acid sequence in which the amino acid
at position 238 is Asp, the amino acid at position 233 is Asp, the
amino acid at position 264 is Ile, the amino acid at position 267
is Ala, the amino acid at position 268 is Glu, and the amino acid
at position 271 is Gly, according to EU numbering, in an Fc region;
(v) an amino acid sequence in which the amino acid at position 238
is Asp, the amino acid at position 237 is Asp, the amino acid at
position 267 is Gly, the amino acid at position 268 is Asp, the
amino acid at position 271 is Gly, the amino acid at position 296
is Asp, and the amino acid at position 330 is Arg, according to EU
numbering, in an Fc region; (w) an amino acid sequence in which the
amino acid at position 238 is Asp, the amino acid at position 264
is Ile, the amino acid at position 267 is Ala, the amino acid at
position 268 is Glu, the amino acid at position 271 is Gly, the
amino acid at position 272 is Asp, and the amino acid at position
296 is Asp, according to EU numbering, in an Fc region; and (x) an
amino acid sequence in which the amino acid at position 238 is Asp,
the amino acid at position 233 is Asp, the amino acid at position
264 is Ile, the amino acid at position 267 is Ala, the amino acid
at position 268 is Glu, the amino acid at position 271 is Gly, and
the amino acid at position 296 is Asp, according to EU numbering,
in an Fc region.
[0269] The present invention further provides a method of altering
a polypeptide to produce a polypeptide with enhanced
Fc.gamma.RIIb-binding activity or enhanced binding selectivity to
Fc.gamma.RIIb compared to Fc.gamma.RIIa (type R) as compared to
those of a polypeptide containing a parent Fc region. The present
invention also provides a method of altering a polypeptide to
produce a polypeptide with enhanced Fc.gamma.RIIb-binding activity
and enhanced binding selectivity to Fc.gamma.RIIb compared to
Fc.gamma.RIIa (type R) as compared to those of a polypeptide
containing a parent Fc region.
[0270] The present invention also provides methods for altering a
polypeptide for the production of a polypeptide whose antibody
production is suppressed compared with that of a polypeptide
comprising a parent Fc region when it is administered in vivo.
[0271] An example of a preferred embodiment includes the
combination of amino acid alterations described in the method of
producing polypeptides comprising Fc region variants with enhanced
Fc.gamma.RIIb-binding activity or enhanced binding selectivity to
Fc.gamma.RIIb compared to Fc.gamma.RIIa (type R). An example of a
more preferred embodiment includes the above-described combination
of amino acid alterations described in the method of producing
polypeptides comprising Fc region variants with enhanced
Fc.gamma.RIIb-binding activity and enhanced binding selectivity to
Fc.gamma.RIIb compared to Fc.gamma.RIIa (type R).
[0272] Furthermore, the present invention provides a nucleic acid
encoding a polypeptide comprising an Fc region having at least one
amino acid alteration, wherein the polypeptide comprises an Fc
region variant with enhanced Fc.gamma.RIIb-binding activity or
enhanced binding selectivity to Fc.gamma.RIIb compared to
Fc.gamma.RIIa (type R) as compared to those of a polypeptide
comprising a parent Fc region. The present invention also provides
a nucleic acid encoding a polypeptide comprising an Fc region
having at least one amino acid alteration, wherein the polypeptide
comprises an Fc region variant with enhanced Fc.gamma.RIIb-binding
activity and enhanced binding selectivity to Fc.gamma.RIIb compared
to Fc.gamma.RIIa (type R)-binding activity, as compared to those of
a polypeptide comprising a parent Fc region. The nucleic acid of
the present invention may be in any form such as DNA or RNA.
[0273] The present invention also provides vectors carrying the
above-described nucleic acids of the present invention. The type of
vector can be appropriately selected by those skilled in the art
depending on the host cells to be introduced with the vector. The
vectors include, for example, those described above.
[0274] Furthermore, the present invention relates to host cells
transformed with the above-described vectors of the present
invention. Appropriate host cells can be selected by those skilled
in the art. The host cells include, for example, those described
above. Specific examples include the following host cells.
[0275] When eukaryotic cells are used as host cells, animal cells,
plant cells, or fungal cells can be appropriately used.
Specifically, the animal cells include, for example, the following
cells.
(1) mammalian cells: CHO (Chinese hamster ovary cell line), COS
(Monkey kidney cell line), myeloma (Sp2/O, NS0, and such), BHK
(baby hamster kidney cell line), Hela, Vero, HEK293 (human
embryonic kidney cell line with sheared adenovirus (Ad)5 DNA),
FreeStyle293.TM. cell line, PER.C6 cell (human embryonic retinal
cell line transformed with the Adenovirus Type 5 (Ad5) E1A and E1B
genes), and such (Current Protocols in Protein Science (May, 2001,
Unit 5.9, Table 5.9.1)); (2) amphibian cells: Xenopus oocytes, or
such; and (3) insect cells: sf9, sf21, Tn5, or such.
[0276] 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.
[0277] Furthermore, the following cells can be used as fungal
cells: [0278] yeasts: the Saccharomyces genus such as Saccharomyces
serevisiae, and the Pichia genus such as Pichia pastoris; and
[0279] filamentous fungi: the Aspergillus genus such as Aspergillus
niger.
[0280] Furthermore, the present invention provides a method of
enhancing Fc.gamma.RIIb-binding activity and/or enhancing binding
selectivity to Fc.gamma.RIIb compared to Fc.gamma.RIIa (type
R)-binding activity, as compared to those of a polypeptide
comprising a parent Fc region, wherein the method comprises adding
at least one amino acid alteration to the Fc region in an Fc
region-comprising polypeptide.
[0281] The present invention also provides methods for suppressing
production of antibodies against a polypeptide comprising an Fc
region, as compared with a polypeptide comprising a parent Fc
region, when the polypeptide is administered in vivo, wherein the
method comprises adding at least one amino acid alteration in the
Fc region of the polypeptide.
[0282] An example of a preferred embodiment is the combination of
amino acid alterations described in the method of producing
polypeptides comprising Fc region variants with enhanced
Fc.gamma.RIIb-binding activity and/or enhanced binding selectivity
to Fc.gamma.RIIb compared to Fc.gamma.RIIa (type R).
[0283] Polypeptides produced by any of the above-mentioned methods
are also included in the present invention.
[0284] The present invention provides pharmaceutical compositions
comprising the polypeptide comprising an Fc region variant of the
present invention.
[0285] The pharmaceutical compositions of the present invention can
be formulated, in addition to the antibody or Fc-fusion protein
molecules of the present invention described above, with
pharmaceutically acceptable carriers by known methods. For example,
the compositions can be used parenterally, when the antibodies are
formulated in a sterile solution or suspension for injection using
water or any other pharmaceutically acceptable liquid. For example,
the compositions can be formulated by appropriately combining the
antibodies or Fc-fusion protein molecules with pharmaceutically
acceptable carriers or media, specifically, sterile water or
physiological saline, vegetable oils, emulsifiers, suspending
agents, surfactants, stabilizers, flavoring agents, excipients,
vehicles, preservatives, binding agents, and such, by mixing them
at a unit dose and form required by generally accepted
pharmaceutical implementations. 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. The content of the active ingredient in such a
formulation is adjusted so that an appropriate dose within the
required range can be obtained.
[0286] Sterile compositions for injection can be formulated using
vehicles such as distilled water for injection, according to
standard protocols.
[0287] Aqueous solutions used for injection include, for example,
physiological saline and isotonic solutions containing glucose or
other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and
sodium chloride. These can be used in conjunction with suitable
solubilizers such as alcohol, specifically ethanol, polyalcohols
such as propylene glycol and polyethylene glycol, and non-ionic
surfactants such as Polysorbate 80.TM. and HCO-50.
[0288] Oils include sesame oils and soybean oils, and can be
combined with solubilizers such as benzyl benzoate or benzyl
alcohol. These may also be formulated with buffers, for example,
phosphate buffers or sodium acetate buffers; analgesics, for
example, procaine hydrochloride; stabilizers, for example, benzyl
alcohol or phenol; or antioxidants. The prepared injections are
typically aliquoted into appropriate ampules.
[0289] The administration is preferably carried out parenterally,
and specifically includes injection, intranasal administration,
intrapulmonary administration, and percutaneous administration. For
example, injections can be administered systemically or locally by
intravenous injection, intramuscular injection, intraperitoneal
injection, or subcutaneous injection.
[0290] Furthermore, the method of administration can be
appropriately selected according to the age and symptoms of the
patient. A single dosage of the pharmaceutical composition
containing an antibody or a polynucleotide encoding an antibody can
be selected, for example, from the range of 0.0001 mg to 1000 mg
per kg of body weight. Alternatively, the dosage may be, for
example, in the range of 0.001 to 100000 mg/patient. However, the
dosage is not limited to these values. The dosage and method of
administration vary depending on the patient's body weight, age,
and symptoms, and can be appropriately selected by those skilled in
the art.
[0291] The above-mentioned polypeptides comprising an Fc region
variant of the present invention are useful as active ingredients
of pharmaceutical agents that suppress the activation of B cells,
mast cells, dendritic cells, and/or basophils. Polypeptides
comprising an Fc region variant of the present invention can
suppress the activation of B cells, mast cells, dendritic cells,
and/or basophils, by selectively working on Fc.gamma.RIIb without
activating activating Fc.gamma.R. B cell activation includes
proliferation, IgE production, IgM production, and IgA production.
The above-mentioned polypeptides comprising an Fc region variant of
the present invention cross-link Fc.gamma.RIIb with IgE to suppress
IgE production of B cells, with IgM to suppress IgM production of B
cells, and with IgA to suppress IgA production. Other than the
above, suppressive effects similar to those mentioned above are
exhibited by directly or indirectly cross-linking Fc.gamma.RIIb
with molecules that are expressed on B cells and comprise the ITAM
domain inside the cell or interact with the ITAM domain such as
BCR, CD19, and CD79b. Furthermore, activation of mast cells
includes proliferation, activation by IgE and such, and
degranulation. In mast cells, the above-mentioned polypeptides
comprising an Fc region variant of the present invention can
suppress proliferation, activation by IgE and such, and
degranulation by directly or indirectly cross-linking Fc.gamma.RIIb
with IgE receptor molecules that are expressed on mast cells and
comprise the ITAM domain or interact with the ITAM domain such as
Fc.epsilon.RI, DAP12, and CD200R3. Activation of basophils includes
proliferation and degranulation of basophils. Also in basophils,
the above-mentioned polypeptides comprising an Fc region variant of
the present invention can suppress proliferation, activation, and
degranulation by directly or indirectly cross-linking Fc.gamma.RIIb
with molecules on the cell membrane, which comprise the ITAM domain
inside the cell or interact with the ITAM domain. Activation of
dendritic cells includes proliferation and degranulation of
dendritic cells. Also in dendritic cells, the above-mentioned
polypeptides comprising an Fc region variant of the present
invention can suppress activation, degranulation, and proliferation
by directly or indirectly cross-linking Fc.gamma.RIIb with
molecules on the cell membrane, which comprise the ITAM domain
inside the cell or interact with the ITAM domain.
[0292] In the present invention, the polypeptides comprising an Fc
region variant of the present invention mentioned above are useful
as an active ingredient of therapeutic agents or preventive agents
for immunological inflammatory diseases. As described above, since
polypeptides comprising an Fc region variant of the present
invention can suppress activation of B cells, mast cells, dendritic
cells and/or basophils, administration of the polypeptides
comprising an Fc region variant of the present invention as a
result can treat or prevent immunological inflammatory diseases.
Without being limited thereto, the term "immunological inflammatory
diseases" comprises, rheumatoid arthritis, autoimmune hepatitis,
autoimmune thyroiditis, autoimmune blistering diseases, autoimmune
adrenocortical disease, autoimmune hemolytic anemia, autoimmune
thrombocytopenic purpura, megalocytic anemia, autoimmune atrophic
gastritis, autoimmune neutropenia, autoimmune orchitis, autoimmune
encephalomyelitis, autoimmune receptor disease, autoimmune
infertility, chronic active hepatitis, glomerulonephritis,
interstitial pulmonary fibrosis, multiple sclerosis, Paget's
disease, osteoporosis, multiple myeloma, uveitis, acute and chronic
spondylitis, gouty arthritis, inflammatory bowel disease, adult
respiratory distress syndrome (ARDS), psoriasis, Crohn's disease,
Basedow's disease, juvenile diabetes, Addison's disease, myasthenia
gravis, lens-induced uveitis, systemic lupus erythematosus,
allergic rhinitis, allergic dermatitis, ulcerative colitis,
hypersensitivity, muscle degeneration, cachexia, systemic
scleroderma, localized scleroderma, Sjogren's syndrome, Behchet's
disease, Reiter's syndrome, type I and type II diabetes, bone
resorption disorder, graft-versus-host reaction,
ischemia-reperfusion injury, atherosclerosis, brain trauma,
cerebral malaria, sepsis, septic shock, toxic shock syndrome,
fever, malgias due to staining, aplastic anemia, hemolytic anemia,
idiopathic thrombocytopenia, Goodpasture's syndrome, Guillain-Barre
syndrome, Hashimoto's thyroiditis, pemphigus, IgA nephropathy,
pollinosis, antiphospholipid antibody syndrome, polymyositis,
Wegener's granulomatosis, arteritis nodosa, mixed connective tissue
disease, fibromyalgia, asthma, atopic dermatitis, chronic atrophic
gastritis, primary biliary cirrhosis, primary sclerosing
cholangitis, autoimmune pancreatitis, aortitis syndrome, rapidly
progressive glomerulonephritis, megaloblastic anemia, idiopathic
thrombocytopenic purpura, primary hypothyroidism, idiopathic
Addison's disease, insulin-dependent diabetes mellitus, chronic
discoid lupus erythematosus, pemphigoid, herpes gestationis, linear
IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia
areata, vitiligo vulgaris, leukoderma acquisitum centrifugum of
Sutton, Harada's disease, autoimmune optic neuropathy, idiopathic
azoospermia, habitual abortion, hypoglycemia, chronic urticaria,
ankylosing spondylitis, psoriatic arthritis, enteropathic
arthritis, reactive arthritis, spondyloarthropathy, enthesopathy,
irritable bowel syndrome, chronic fatigue syndrome,
dermatomyositis, inclusion body myositis, Schmidt's syndrome,
Graves' disease, pernicious anemia, lupoid hepatitis, presenile
dementia, Alzheimer's disease, demyelinating disorder, amyotrophic
lateral sclerosis, hypoparathyroidism, Dressler's syndrome,
Eaton-Lambert syndrome, dermatitis herpetiformis, alopecia,
progressive systemic sclerosis, CREST syndrome (calcinosis,
Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and
telangiectasia), sarcoidosis, rheumatic fever, erythema multiforme,
Cushing's syndrome, transfusion reaction, Hansen's disease,
Takayasu arteritis, polymyalgia rheumatica, temporal arteritis,
giant cell arthritis, eczema, lymphomatoid granulomatosis, Kawasaki
disease, endocarditis, endomyocardial fibrosis, endophthalmitis,
fetal erythroblastosis, eosinophilic fasciitis, Felty syndrome,
Henoch-Schonlein purpura, transplant rejection, mumps,
cardiomyopathy, purulent arthritis, familial Mediterranean fever,
Muckle-Wells syndrome, and hyper-IgD syndrome.
[0293] Furthermore, in autoimmune diseases which may be caused by
production of antibodies against autoantigens (autoantibodies), the
polypeptides comprising an Fc region variant of the present
invention mentioned above are useful as an active ingredient of
pharmaceutical agents for treating or preventing the autoimmune
diseases by suppressing production of those autoantibodies. Use of
a molecule produced by fusing an antibody Fc portion with AchR (an
autoantigen of myasthenia gravis) has been reported to suppress
proliferation of B cells which express AchR-recognizing BCR, and
induce apoptosis (J. Neuroimmunol, 227: 35-43, 2010). Use of a
fusion protein formed between an antigen recognized by an
autoantibody and an antibody Fc region of the present invention
enables crosslinking of Fc.gamma.RIIb with BCR of a B cell
expressing BCR for that autoantigen, suppression of proliferation
of B cells expressing BCR for the autoantigen, and induction of
apoptosis. Such autoimmune diseases include Guillain-Barre
syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune
hepatitis, primary biliary cirrhosis, primary sclerosing
cholangitis, autoimmune pancreatitis, aortitis syndrome,
Goodpasture's syndrome, rapidly progressive glomerulonephritis,
megaloblastic anemia, autoimmune hemolytic anemia, autoimmune
neutropenia, idiopathic thrombocytopenic purpura, Basedow's
disease, Hashimoto's thyroiditis, primary hypothyroidism,
idiopathic Addison's disease, insulin-dependent diabetes mellitus,
chronic discoid lupus erythematosus, localized scleroderma,
pemphigus, pemphigoid, herpes gestationis, linear IgA bullous
dermatosis, epidermolysis bullosa acquisita, alopecia areata,
vitiligo vulgaris, leukoderma acquisitum centrifugum of Sutton,
Harada's disease, autoimmune optic neuropathy, idiopathic
azoospermia, habitual abortion, type II diabetes, hypoglycemia, and
chronic urticaria; but are not limited thereto.
[0294] Furthermore, the above-mentioned polypeptides comprising an
Fc Region variant of the present invention are useful as an active
ingredient in therapeutic agents for diseases with deficiency of a
biologically essential protein. For diseases with deficiency of a
biologically essential protein, therapeutic methods that administer
and supplement the protein as a pharmaceutical agent are used.
However, since the patient lacks the protein from the beginning,
the externally supplemented protein is recognized as a foreign
substance and antibodies against that protein are produced. As a
result, the protein becomes easily removed, and the effect as a
pharmaceutical is reduced. Use of a fusion protein comprising such
a protein and an antibody Fc region of the present invention
enables crosslinking between Fc.gamma.RIIb and BCR on B cells that
recognize the protein, and enables suppression of antibody
production against the protein. The proteins to be supplemented
include Factor VIII, Factor IX, TPO, EPO, .alpha.-iduronidase,
iduronate sulfatase, A-type heparan N-sulfatase, B type
.alpha.-N-acetylglucosaminidase, C type acetyl CoA:
.alpha.-glucosaminidase acetyltransferase, D type
N-acetylglucosamine 6-sulfatase, galactose 6-sulfatase,
N-acetylgalactosamine 4-sulfatase, .beta.-glucuronidase,
.alpha.-galactosidase, acidic .alpha.-galactosidase, and
glucocerebrosidase. These proteins may be supplemented for diseases
such as hemophilia, idiopathic thrombocytopenic purpura, renal
anemia, and lysosomal disease (mucopolysaccharidosis, Fabry's
disease, Pompe disease, and Gaucher's disease), without being
limited thereto.
[0295] Furthermore, the above-mentioned polypeptides comprising an
Fc region variant of the present invention are useful as an active
ingredient for antiviral agents. Antibodies that comprise an Fc
region of the present invention and are anti-virus antibodies can
suppress antibody-dependent enhancement observed with anti-virus
antibodies. Antibody-dependent enhancement is a phenomenon where a
virus uses neutralizing antibodies against the virus to become
phagocytosed via activating Fc.gamma.Rs, and infects
Fc.gamma.R-expressing cells so that the infection spreads. Binding
of anti-dengue-virus neutralizing antibodies to Fc.gamma.RIIb has
been reported to play an important role in suppressing
antibody-dependent enhancement (Proc. Natl. Acad. Sci. USA, 108:
12479-12484, 2011). Crosslinking Fc.gamma.RIIb with an immune
complex with dengue virus, which is formed by the anti-dengue-virus
neutralizing antibodies, inhibits Fc.gamma.R-mediated phagocytosis,
resulting in the suppression of antibody-dependent enhancement.
Examples of such viruses include dengue virus (DENV1, DENV2, and
DENV4) and HIV, but are not limited thereto.
[0296] Furthermore, polypeptides comprising an Fc region variant of
the present invention described above are useful as an active
ingredient in preventive agents or therapeutic agents for
arteriosclerosis. Antibodies against oxidized LDL, i.e., a cause
for arteriosclerosis, which are antibodies comprising an Fc region
of the present invention, can prevent Fc.gamma.RIIa-dependent
adhesion of inflammatory cells. It has been reported that while
anti-oxidized LDL antibodies inhibit the interaction between
oxidized LDL and CD36, anti-oxidized LDL antibodies bind to
endothelial cells, and monocytes recognize their Fc portion in an
Fc.gamma.RIIa-dependent or Fc.gamma.RI-dependent manner; and this
leads to adhesion (Immunol. Lett., 108: 52-61, 2007). Using
antibodies comprising an Fc region of the present invention for
such antibodies may inhibit Fc.gamma.RIIa-dependent binding and
suppress monocyte adhesion by Fc.gamma.RIIb-mediated inhibitory
signals.
[0297] In the present invention, polypeptides comprising an Fc
region variant of the present invention described above are useful
as an active ingredient in therapeutic agents or preventive agents
for cancer. As described above, it is known that enhancing the
Fc.gamma.RIIb binding enhances the agonistic activity of an agonist
antibody, and enhances the antitumor effect of the antibody.
Therefore, agonist antibodies using the Fc region variant of the
present invention are useful for treatment or prevention of cancer.
Specifically, the Fc region variant of the present invention
enhances the agonistic activity of agonist antibodies against, for
example, receptors of the TNF receptor family such as Aliases,
CD120a, CD120b, Lymphotoxin .beta. receptor, CD134, CD40, FAS,
TNFRSF6B, CD27, CD30, CD137, TNFRSF10A, TNFRSF10B, TNFRSF10C,
TNFRSF10D, RANK, Osteoprotegerin, TNFRSF12A, TNFRSF13B, TNFRSF13C,
TNFRSF14, Nerve growth factor receptor, TNFRSF17, TNFRSF18,
TNFRSF19, TNFRSF21, TNFRSF25, and Ectodysplasin A2 receptor and can
be used for treating or preventing cancer. Furthermore, in addition
to the above, agonistic activity is also enhanced for agonist
antibodies against molecules which need to interact with
Fc.gamma.RIIb for exhibiting its agonistic activity. In addition,
by incorporating the Fc region variant of the present invention
into a polypeptide having binding activity to a molecule such as
Kit, a type of receptor tyrosine kinase (RTK), which suppresses
cell proliferation upon crosslinking with Fc.gamma.RIIb, inhibitory
effect against cells expressing such molecule may be enhanced.
Without being limited thereto, cancer includes lung cancer
(including small cell lung cancer, non-small cell lung cancer,
pulmonary adenocarcinoma, and squamous cell carcinoma of the lung),
large intestine cancer, rectal cancer, colon cancer, breast cancer,
liver cancer, gastric cancer, pancreatic cancer, renal cancer,
prostate cancer, ovarian cancer, thyroid cancer,
cholangiocarcinoma, peritoneal cancer, mesothelioma, squamous cell
carcinoma, cervical cancer, endometrial cancer, bladder cancer,
esophageal cancer, head and neck cancer, nasopharyngeal cancer,
salivary gland tumor, thymoma, skin cancer, basal cell tumor,
malignant melanoma, anal cancer, penile cancer, testicular cancer,
Wilms' tumor, acute myeloid leukemia (including acute
myeloleukemia, acute myeloblastic leukemia, acute promyelocytic
leukemia, acute myelomonocytic leukemia, and acute monocytic
leukemia), chronic myelogenous leukemia, acute lymphoblastic
leukemia, chronic lymphatic leukemia, Hodgkin's lymphoma,
non-Hodgkin's lymphoma (Burkitt's lymphoma, chronic lymphocytic
leukemia, mycosis fungoides, mantle cell lymphoma, follicular
lymphoma, diffuse large-cell lymphoma, marginal zone lymphoma,
pilocytic leukemia plasmacytoma, peripheral T-cell lymphoma, and
adult T cell leukemia/lymphoma), Langerhans cell histiocytosis,
multiple myeloma, myelodysplastic syndrome, brain tumor (including
glioma, astroglioma, glioblastoma, meningioma, and ependymoma),
neuroblastoma, retinoblastoma, osteosarcoma, Kaposi's sarcoma,
Ewing's sarcoma, angiosarcoma, and hemangiopericytoma.
[0298] Furthermore, the present invention relates to methods for
treating or preventing immunological inflammatory diseases, which
comprise the step of administering to a subject (patient) a
polypeptide comprising an Fc region variant of the present
invention or a polypeptide comprising an Fc region variant produced
by production methods of the present invention.
[0299] The present invention also provides kits for use in the
therapeutic methods or preventive methods of the present invention,
which comprises at least a polypeptide comprising an Fc region
variant of the present invention or a polypeptide comprising an Fc
region variant produced by production methods of the present
invention, or a pharmaceutical composition of the present
invention. In addition, pharmaceutically acceptable carriers,
media, instructions on the method of use, and such may be included
in the kit. Furthermore, the present invention relates to use of a
polypeptide comprising an Fc region variant of the present
invention or a polypeptide comprising an Fc region variant produced
by production methods of the present invention in the production of
agents for treating or preventing immunological inflammatory
diseases. The present invention also relates to polypeptides
comprising an Fc region variant of the present invention or
polypeptides comprising an Fc region variant produced by production
methods of the present invention for use in the therapeutic methods
or preventive methods of the present invention.
[0300] As used herein, the three-letter and single-letter codes for
respective amino acids are as follows:
Alanine: Ala (A)
Arginine: Arg (R)
Asparagine: Asn (N)
[0301] Aspartic acid: Asp (D)
Cysteine: Cys (C)
Glutamine: Gln (Q)
[0302] Glutamic acid: Glu (E)
Glycine: Gly (G)
Histidine: His (H)
Isoleucine: Ile (I)
Leucine: Leu (L)
Lysine: Lys (K)
Methionine: Met (M)
Phenylalanine: Phe (F)
Proline: Pro (P)
Serine: Ser (S)
Threonine: Thr (T)
Tryptophan: Trp (W)
Tyrosine: Tyr (Y)
Valine: Val (V)
[0303] All prior art documents cited herein are incorporated by
reference in their entirety.
EXAMPLES
[0304] Herein below, the present invention will be specifically
described further with reference to the Examples, but it is not to
be construed as being limited thereto.
[Example 1] Assessment of Platelet Aggregation Ability of Existing
Antibodies Comprising an Fc with Enhanced FcgRIIb-Binding
[0305] As shown in Table 16 in Reference Example 4, an existing
FcgRIIb enhancement technique which introduces alterations
involving substitution of Glu for Ser at position 267 and
substitution of Phe for Leu at position 328 (EU numbering) into
native human IgG1 (Non-patent Document 28) shows 408-fold enhanced
binding to FcgRIIb and 0.51-fold decreased binding to FcgRIIaH,
while showing 522-fold enhanced binding to FcgRIIaR, as compared to
those of IgG1. As described in "Background Art", even if
FcgRIIb-binding is enhanced, when it comes to cells such as
platelets which only express FcgRIIa, only enhancement effects on
FcgRIIa may be affected. That is, existing techniques which enhance
binding to FcgRIIaR have the danger of enhancing
platelet-aggregating activity and increasing the risk for
developing thrombosis. To confirm this, it was assessed whether
platelet-aggregating activity is actually enhanced when
FcgRIIa-binding of an antibody is enhanced.
[0306] Using the method of Reference Example 1, omalizumab_VH-G1d
(SEQ ID NO: 25) was produced as the heavy chain and
omalizumab_VL-CK (SEQ ID NO: 26) was produced as the light chain of
a human IgG1 antibody that binds to IgE. Furthermore, to enhance
human Fc.gamma.RIIb-binding activity of omalizumab_VH-G1d,
omalizumab_VH-G1d-v3 was produced by substituting Glu for Ser at
position 267 and Phe for Leu at position 328 according to EU
numbering in omalizumab_VH-G1d. Using the method of Reference
Example 1, omalizumab-G1d-v3 which contains omalizumab_VH-G1d-v3 as
the heavy chain and omalizumab_VL-CK as the light chain was
produced. Platelet-aggregating ability was assessed using this
antibody.
[0307] Platelet aggregation was assayed using the platelet
aggregometer HEMA TRACER 712 (LMS Co.). First, about 50 ml of whole
blood was collected at a fixed amount into 4.5-ml evacuated blood
collection tubes containing 0.5 ml of 3.8% sodium citrate, and this
was centrifuged at 200 g for 15 minutes. The resultant supernatant
was collected and used as platelet-rich plasma (PRP). After PRP was
washed with buffer 1 (137 mM NaCl, 2.7 mM KCl, 12 mM NaHCO.sub.3,
0.42 mM NaH.sub.2PO.sub.4, 2 mM MgCl.sub.2, 5 mM HEPES, 5.55 mM
dextrose, 1.5 U/ml apyrase, 0.35% BSA), the buffer was replaced
with buffer 2 (137 mM NaCl, 2.7 mM KCl, 12 mM NaHCO.sub.3, 0.42 mM
NaH.sub.2PO.sub.4, 2 mM MgCl.sub.2, 5 mM HEPES, 5.55 mM dextrose, 2
mM CaCl.sub.2, 0.35% BSA) to prepare about 300,000/.mu.l washed
platelets. 156 .mu.l of the washed platelets was aliquoted into
assay cuvettes containing a stir bar set in the platelet
aggregometer. The platelets were stirred at 1000 rpm with the stir
bar in the cuvettes maintained at 37.0.degree. C. in the platelet
aggregometer. 44 .mu.l of the immune complex of omalizumab-G1d-v3
and IgE at a molar ratio of 1:1 (prepared at final concentrations
of 600 .mu.g/ml and 686 .mu.g/ml, respectively) was added to the
cuvettes. The platelets were reacted with the immune complex for
five minutes. Then, at a concentration that does not allow
secondary platelet aggregation, adenosine diphosphate (ADP, SIGMA)
was added to the reaction mixture to test whether the aggregation
is enhanced.
[0308] The result for each donor with an Fc.gamma.RIIa polymorphic
form (H/H or R/H) obtained from the above assay is shown in FIGS. 1
and 2. From the result in FIG. 1, it is shown that platelet
aggregation was enhanced with the Fc.gamma.RIIa polymorphic form
(R/H) when the immune complex is added. Meanwhile, as shown in FIG.
2, platelet aggregation was not enhanced with the Fc.gamma.RIIa
polymorphic form (H/H).
[0309] Next, platelet activation was assessed using activation
markers. Platelet activation can be measured based on the increased
expression of an activation marker such as CD62p (p-selectin) or
active integrin on the platelet membrane surface. 2.3 .mu.l of the
immune complex was added to 7.7 .mu.l of the washed platelets
prepared by the method described above. After five minutes of
reaction at room temperature, activation was induced by adding ADP
at a final concentration of 30 .mu.M, and whether the immune
complex enhances the ADP-dependent activation was assessed. A
sample added with phosphate buffer (pH 7.4; Gibco), instead of the
immune complex, was used as a negative control. Staining was
performed by adding, to each post-reaction sample, PE-labeled
anti-CD62 antibody (BECTON DICKINSON), PerCP-labeled anti-CD61
antibody, and FITC-labeled PAC-1 antibody (BD bioscience). Each of
the fluorescence intensities was measured using a flow cytometer
(FACS CantoII.TM. flow cytometer, BD bioscience).
[0310] The result on CD62p expression, obtained by the above assay
method, is shown in FIG. 3. The result on the activated integrin
expression is shown in FIG. 4. The washed platelets used were
obtained from a healthy person with the Fc.gamma.RIIa polymorphic
form R/H. Both CD62p and active integrin expressed on platelet
membrane surface, which is induced by ADP stimulation, was enhanced
in the presence of the immune complex.
[0311] From these results, in existing antibodies having an Fc with
enhanced human Fc.gamma.RIIb-binding, which have an Fc produced by
introducing substitution of Ser at position 267 with Glu and Leu at
position 328 with Phe (EU numbering) into an IgG1 Fc, the genetic
polymorphs of Fc.gamma.RIIa whose amino acid at position 131 is R
showed enhanced platelet-aggregating activity compared to when the
amino acid at position 131 is H. That is, existing antibodies
having an Fc with enhanced human Fc.gamma.RIIb binding was
suggested to have the danger of increasing the risk for developing
thrombosis due to platelet aggregation in humans carrying
Fc.gamma.RIIa type R, elucidating the value of an Fc with enhanced
selective binding to Fc.gamma.RIIb that overcomes this problem.
[Example 2] Production of Variants with Enhanced Binding to
FcgRIIb
[0312] As shown in Example 1, when enhancing binding to FcgRIIb, it
is necessary to enhance FcgRIIb-binding while also suppressing
binding to other activating FcgRs as much as possible. Therefore,
production of variants with enhanced binding or selectivity to
FcgRIIb was examined by combining alterations having the effect of
enhancing binding or improving selectivity to FcgRIIb.
Specifically, using as a base the P238D alteration, which shows
excellent effects in both enhancement of binding and improvement of
selectivity to FcgRIIb, alterations found to be effective upon
combination with P238D in Reference Examples 6, 8, and 9 were
further combined.
[0313] The variable region of IL6R-H (SEQ ID NO: 18), which is
disclosed in WO2009/125825 and which is the variable region of an
antibody against the human interleukin 6 receptor was produced as
the antibody H chain variable region, and IL6R-G1d (SEQ ID NO: 19)
which has G1d produced by removing the C-terminal Gly and Lys of
human IgG1 was produced as the antibody H chain constant region.
Furthermore, IL6R-B3 (SEQ ID NO: 23) was produced by introducing
K439E into IL6R-G1d. Then, variants were produced from IL6R-B3 by
combining E233D, L234Y, G237D, S267Q, H268D, P271G, Y296D, K326D,
K326A, A330R, A330K, which are alterations found to be effective
upon combination with P238D in Reference Examples 6, 8, and 9.
IL6R-L (SEQ ID NO: 21) was commonly used as the antibody L chain.
These variants were used for antibody expression and purification
according to the method of Reference Example 1, and binding to each
FcgR (FcgRIa, FcgRIIa type H, FcgRIIa type R, FcgRIIb, and FcgRIIIa
type V) were assessed by the method of Reference Example 2.
[0314] The KD values of each variant for each FcgR are shown in
Table 1. "Alteration" in the Table refers to alterations introduced
into IL6R-B3 (SEQ ID NO: 23). Meanwhile, IL6R-B3/IL6R-L which is
used as a template for producing each of the variants is indicated
by an asterisk (*). "KD(IIaR)/KD(IIb)" in the Table shows the value
obtained by dividing the KD of each variant for FcgRIIaR by the KD
of each variant for FcgRIIb. The greater this value is, the higher
the selectivity to FcgRIIb is. "Parent polypeptide KD(IIb)/altered
polypeptide KD(IIb)" refers to a value obtained by dividing the KD
value of IL6R-B3/IL6R-L for FcgRIIb by the KD value of each variant
for FcgRIIb. Furthermore, "parent polypeptide KD(IIaR)/altered
polypeptide KD(IIaR)" refers to a value obtained by dividing the KD
value of IL6R-B3/IL6R-L for FcgR IIaR by the KD value of that
variant for FcgRIIaR. In Table 1, values shown in bold italicized
font were calculated using the following equation
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
described in Reference Example 2 since the binding of FcgR to IgG
was determined to be too weak to analyze accurately by kinetic
analysis.
[0315] Binding of IL6R-G1d/IL6R-L which carries the native human
IgG1 sequence was 1.3 fold for FcgRIa, 1.1 fold for FcgRIIaR, 1.1
fold for FcgRIIaH, 1.2 fold for FcgRIIb, and 0.9 fold for
FcgRIIIaV, when binding of IL6R-B3/IL6R-L produced by introducing
K439E into IL6R-G1d/IL6R-L to the respective FcgRs was defined as
1, and all of the binding to FcgRs were equivalent to that of
IL6R-G1d/IL6R-L. Therefore, comparing binding of each variant with
that of the IL6R-B3/IL6R-L prior to alteration can be considered to
be equivalent to comparing each variant with IL6R-G1d/IL6R-L which
carries the native human IgG1 sequence. Accordingly, in the
Examples hereafter, binding activity of each variant is compared
with that of the IL6R-B3/IL6R-L prior to alteration.
[0316] Table 1 shows that all variants showed improved affinity to
FcgRIIb in comparison with the IL6R-B3 prior to alteration, and
IL6R-BF648/IL6R-L showed 2.6 fold improved affinity which was the
lowest and IL6R-BP230/IL6R-L showed 147.6 fold improved affinity
which was the highest. Furthermore, the value of KD(IIaR)/KD(IIb),
which shows the degree of selectivity, is 10.0 for
IL6R-BP234/IL6R-L which showed the lowest value, and 32.2 for
IL6R-BP231/IL6R-L which showed the highest value, and all variants
improved their selectivity compared to the IL6R-B3/IL6R-L prior to
alteration, which showed a value of 0.3. All variants showed lower
binding to FcgRIa, FcgRIIaH, and FcgRIIIaV compared to the
IL6R-B3/IL6R-L prior to alteration.
[Example 3] X-Ray Structure Analysis of a Complex Formed Between an
Fc with Enhanced Fc.gamma.RIIb Binding and the Extracellular Region
of Fc.gamma.RIIb and a Complex Formed Between this Fc and the
Extracellular Region of Fc.gamma.RIIaR
[0317] The IL6R-BP230/IL6R-L variant showing the highest enhanced
FcgRIIb binding in Example 2 showed approximately 150-fold enhanced
binding to FcgRIIb and binding to FcgRIIa type R was suppressed to
about 1.9-fold increase when compared to the IL6R-B3/IL6R-L prior
to alteration. Therefore, IL6R-BP230/IL6R-L is an excellent variant
in terms of both binding and selectivity to FcgRIIb; however, to
produce more excellent variants, it is preferred that FcgRIIb
binding is further enhanced as well as binding to FcgRIIaR is
suppressed as much as possible.
[0318] As shown in FIG. 28 of Reference Example 7, in Fc having the
P238D alteration, formation of a strong electrostatic interaction
is observed between Asp at position 270 (EU numbering) of the CH2
domain B and Arg at position 131 of Fc.gamma.RIIb. While this
residue at position 131 is His in Fc.gamma.RIIIa and Fc.gamma.RIIa
type H, it is Arg in Fc.gamma.RIIa type R as in the case with
Fc.gamma.RIIb. As a result, there are no differences in the
interactions at this portion, and this is the reason that it is
difficult to bring about selectivity for Fc.gamma.RIIa type R.
[0319] Meanwhile, the extracellular regions of Fc.gamma.RIIa and
Fc.gamma.RIIb match 93% in amino acid sequence, that is, have very
high homology. When the crystal structure of the complex formed
between the native IgG1 Fc (hereinafter "Fc(WT)") and the
extracellular region of Fc.gamma.RIIa type R (J. Imunol. 2011, 187,
3208-3217) was analyzed, amino acid differences around an
interacting interface were found to be only three (G1n127, Leu132,
and Phe160) between Fc.gamma.RIIa type R and Fc.gamma.RIIb, and
improvement of selectivity over Fc.gamma.RIIa type R was expected
to be very difficult.
[0320] Therefore, to achieve further improvement of selectivity and
enhancement of Fc.gamma.RIIb-binding activity, the present
inventors considered that the amino acid mutations to be introduced
must be examined in detail by conformationally elucidating the
subtle differences in interactions caused by the difference in
receptors by obtaining not only the three-dimensional structure of
the complex formed between an Fc with enhanced Fc.gamma.RIIb
binding and the extracellular region of Fc.gamma.RIIb, but also
simultaneously, the three-dimensional structure of a complex formed
with the extracellular region of Fc.gamma.RIIa type R for which
improvement of selectivity is considered most difficult. Then,
X-ray structure analyses were performed on a complex formed between
the extracellular region of Fc.gamma.RIIb and Fc(P208), in which
the K439E alteration is removed from the Fc of IL6R-BP208/IL6R-L
(produced in Reference Example 9) which is a variant used as the
basis for production of IL6R-BP230/IL6R-L, and a complex formed
between the extracellular region of Fc.gamma.RIIa type R and
Fc(P208).
3-1. X-Ray Structure Analysis of a Complex Formed Between Fc(P208)
and the Extracellular Region of Fc.gamma.RIIb
[0321] The three-dimensional structure of the
Fc(P208)/Fc.gamma.RIIb extracellular region complex was determined
by X-ray structure analysis at 2.81 .ANG. resolution. The structure
obtained as a result of this analysis is shown in FIG. 5. The
extracellular region of Fc.gamma.RIIb is bound between two Fc CH2
domains, and this is similar to the three-dimensional structures of
complexes formed between Fc(WT) which is an Fc of a native IgG and
the respective extracellular region of Fc.gamma.RIIIa (Proc. Natl.
Acad. Sci. USA, 2011, 108, 12669-126674), Fc.gamma.RIIIb (Nature,
2000, 400, 267-273; J. Biol. Chem. 2011, 276, 16469-16477), or
Fc.gamma.RIIa analyzed so far.
[0322] However, detailed analysis revealed that in the
Fc(P208)/Fc.gamma.RIIb extracellular region complex, the loop
structure of positions 233 to 239 (EU numbering) continuing from
the hinge region of Fc CH2 domain A was changed compared to that of
the Fc(WT)/Fc.gamma.RIIa type R extracellular region complex due to
introduction of the G237D and P238D mutations (FIG. 6). As a
result, formation of a strong hydrogen bond was observed between
the main chain of Asp at position 237 (EU numbering) of Fc(P208)
and the side chain of Tyr at position 160 of Fc.gamma.RIIb (FIG.
7). As this Tyr160 is Phe in both the type H and type R of
Fc.gamma.RIIa, hydrogen bond formation is impossible. Therefore,
this hydrogen bond was considered to have an important contribution
to acquisition of selectivity which refers to enhancement of
Fc.gamma.RIIb-binding activity and decrease in
Fc.gamma.RIIa-binding activity.
[0323] On the other hand, the side chain of Asp at position 237 (EU
numbering) of Fc(P208) does not show remarkable interaction with
Fc.gamma.RIIb, and no interaction was observed with residues within
Fc. Ile at position 332 (EU numbering), Glu at position 333 (EU
numbering), and Lys at position 334 (EU numbering) in Fc were
positioned in close proximity around this Asp at position 237 (EU
numbering) (FIG. 8). If the loop structure can be stabilized by
substituting these positions with hydrophilic resides to form
interaction with the side chain of Asp at position 237 (EU
numbering), entropic energy loss accompanying formation of hydrogen
bond with Tyr at position 160 of Fc.gamma.RIIb can be decreased,
and this may lead to increase in the binding free energy, that is,
enhancement of binding activity.
[0324] Comparison of the X-ray crystal structure of the complex
formed between Fc(P238D) carrying the P238D alteration and the
extracellular region of Fc.gamma.RIIb, which is shown in Reference
Example 7, and the X-ray crystal structure of the complex formed
between Fc(P208) and the extracellular region of Fc.gamma.RIIb,
showed that compared to Fc(P238D), Fc(P208) contains five new
mutations, and most of them were only changes at the side-chain
level. However, by altering Pro at position 271 (EU numbering) to
Gly in the CH2 domain B of Fc, change in location was observed at
the main-chain-level, and structural changes had taken place at the
upstream loop formed by positions 266-270 (EU numbering) (FIG. 9).
As shown in Reference Example 8, in Fc(P238D), it has been
suggested that the Pro portion at position 271 (EU numbering) may
be stereochemically strained when Asp at position 270 (EU
numbering) forms a strong electrostatic interaction with Arg at
position 131 of Fc.gamma.RIIb. Structural changes observed this
time by introducing Gly at position 271 (EU numbering) may be the
result of releasing structural strain accumulated at the Pro
portion prior to alteration, and the amount of release of strain is
considered to have led to improvement of binding free energy with
Fc.gamma.RIIb, that is, the enhancement of binding activity.
[0325] Furthermore, Arg at position 292 (EU numbering) was
confirmed to undergo structural changes with taking two forms as a
result of structural changes of the loop at positions 266-271 (EU
numbering). In this case, Arg at position 292 (EU numbering) forms
electrostatic interaction with Asp at position 268 (EU numbering)
which is one of the other altered residues in Fc(P208) (FIG. 9),
and this may contribute to the stabilization of this loop
structure. The electrostatic interaction formed between Asp at
position 270 (EU numbering) in this loop and Arg at position 131 of
Fc.gamma.RIIb greatly contributes to Fc.gamma.RIIb-binding
activity; therefore, introduction of the H268D alteration may have
led to reduction of entropic energy loss accompanying binding and
increase in binding free energy, that is, enhancement of binding
activity by stabilizing the conformation of this loop structure to
its Fc.gamma.RIIb-bound form.
[0326] When further investigations for the possibility of
alterations aimed at further improving the activity were carried
out based on the structural analysis results, Ser at position 239
(EU numbering) was found as one of the candidate positions for
introducing alterations. As shown in FIG. 10, this Ser at position
239 (EU numbering) of CH2 domain B is positioned in the direction
where Lys at position 117 of Fc.gamma.RIIb extends in a most
structurally natural manner. However, as the electron density of
Lys at position 117 of Fc.gamma.RIIb was not detected in this
analysis, this Lys residue does not form a steady structure. Thus,
currently, involvement of this Lys residue in the interaction with
Fc(P208) is considered to be limited; however, if this Ser at
position 239 (EU numbering) of CH2 domain B is altered to
negatively-charged Asp or Glu, electrostatic interaction with the
positively-charged Lys at position 117 of Fc.gamma.RIIb can be
expected, and as a result, Fc.gamma.RIIb-binding activity can be
expected to become enhanced.
[0327] On the other hand, observing the structure of Ser at
position 239 (EU numbering) in CH2 domain A, it is considered that
the side chain of this amino acid forms hydrogen bonds with the
main chain of Gly at position 236 (EU numbering) and stabilizes the
loop structure from positions 233 to 239 which continues from the
hinge region and includes Asp at position 237 (EU numbering) which
forms a hydrogen bond with the Fc.gamma.RIIb Tyr160 side chain
(FIG. 7). Stabilizing this loop structure to the conformation taken
during binding may lead to reduction of entropic energy loss
accompanying binding, and as a result, enhancement of binding free
energy, that is, enhancement of binding activity. On the other
hand, if this Ser at position 239 (EU numbering) in CH2 domain A is
altered to Asp or Glu, the hydrogen bond with the main chain of Gly
at position 239 (EU numbering) may be lost, and may cause
electrostatic repulsion between Asp at position 265 (EU numbering)
which is present in close proximity, and thus may lead to large
destabilization of the loop structure. This destabilized energy
operates to decrease the binding free energy with Fc.gamma.R11b;
therefore, this may lead to decrease in binding activity.
[Expression and Purification of Fc(P208)]
[0328] Fc(P208) was prepared as follows. First, IL6R-P208 was
produced by altering Glu at position 439 (EU numbering) of
IL6R-BP208 (SEQ ID NO: 24) to Lys, which is the sequence of a
native human IgG1. Then, Cys at position 220 (EU numbering) of
IL6R-P208 was substituted with Ser. Then, genetic sequence of
Fc(P208) from Glu at position 216 (EU numbering) to its C terminal
was cloned by PCR. Using this cloned genetic sequence, production
of expression vectors, and expression and purification of Fc(P208)
were carried out according to the method of Reference Example 1.
Cys at position 220 (EU numbering) forms a disulfide bond with Cys
of the L chain in general IgG1. The L chain is not co-expressed
when Fc alone is prepared, and therefore, this residue was
substituted with Ser to avoid formation of unnecessary disulfide
bonds.
[Expression and Purification of the Fc.gamma.RIIb Extracellular
Region]
[0329] This was prepared according to the method of Reference
Example 2.
[Purification of the Fc(P208)/Fc.gamma.RIIb Extracellular Region
Complex]
[0330] To 1.5 mg of the Fc.gamma.RIIb extracellular region sample
obtained for crystallization, 0.15 mg of Endo F1 (Protein Science
1996, 5: 2617-2622) expressed and purified from Escherichia coli as
a glutathione S-transferase fusion protein was added. This was
allowed to remain at room temperature for three days in 0.1 M
Bis-Tris buffer at pH 6.5, and the N-linked oligosaccharide was
cleaved, leaving N-acetylglucosamine directly bound to Asn. Next,
this Fc.gamma.RIIb extracellular region sample subjected to
carbohydrate cleavage treatment was concentrated by ultrafiltration
with 5000 MWCO, and purified by gel filtration chromatography
(Superdex.RTM. 200 10/300 chromatography) using a column
equilibrated in 20 mM HEPES at pH 7.5 containing 0.1 M NaCl.
Furthermore, to the obtained carbohydrate-cleaved Fc.gamma.RIIb
extracellular region fraction, Fc(P208) was added so that the molar
ratio of the Fc.gamma.RIIb extracellular region would be present in
slight excess, and after concentration by ultrafiltration with
10000 MWCO, a sample of the Fc(P208)/Fc.gamma.RIIb extracellular
region complex was obtained through purification by gel filtration
chromatography (Superdex.RTM. 200 10/300 chromatography) using a
column equilibrated in 25 mM HEPES at pH 7.5 containing 0.1 M
NaCl.
[Crystallization of the Fc(P208)/Fc.gamma.RIIb Complex
Extracellular Region Complex]
[0331] A sample of Fc(P208)/Fc.gamma.RIIb extracellular region
complex were concentrated to about 10 mg/ml using 10000MWCO
ultrafiltration filter, and crystallized using the hanging drop
vapor diffusion method in combination with the seeding method. VDXm
plate (Hampton Research) was used for crystallization. Using a
reservoir solution containing 0.1 M Bis-Tris (pH 6.5), 19% (w/v)
PEG3350, and 0.2 M potassium phosphate dibasic, crystallization
drops were prepared at a mixing ratio of reservoir
solution:crystallization sample=0.85 .mu.l:0.85 .mu.l. Crystals of
the complex obtained under the same condition were crushed with
Seed Bead (Hampton Research) to prepare a seed crystal solution.
0.15 .mu.l of a diluted solution produced from the seed crystal
solution was added to the crystallization drops, which were sealed
in the wells containing reservoirs, and allowed to stand at
20.degree. C. This successfully yielded plate-like crystals.
[Measurement of X-Ray Diffraction Data from an
Fc(P208)/Fc.gamma.RIIb Extracellular Region Complex Crystal]
[0332] One of the obtained single crystals of the
Fc(P208)/Fc.gamma.RIIb extracellular region complex was soaked into
a solution of 0.1 M Bis-Tris pH 6.5, 24% (w/v) PEG3350, 0.2 M
potassium phosphate dibasic, 20% (v/v) ethylene glycol. The crystal
was fished out of the solution using a pin with attached tiny nylon
loop, and frozen in liquid nitrogen; and then X-ray diffraction
data was measured by BL32XU at Spring-8. During the measurement,
the crystal was constantly placed in a nitrogen stream at
-178.degree. C. to maintain in a frozen state, and a total of 300 X
ray diffraction images were collected using MX-225HE CCD detector
(RAYONIX) attached to a beam line with rotating the crystal
0.6.degree. at a time. Determination of cell parameters, indexing
of diffraction spots, and diffraction data processing from the
obtained diffraction images were performed using the Xia2 program
(J. Appl. Cryst. 2010, 43: 186-190), XDS Package (Acta. Cryst.
2010, D66: 125-132) and Scala (Acta. Cryst. 2006, D62: 72-82); and
finally, diffraction intensity data up to 2.81 .ANG. resolution was
obtained. The crystal belongs to the space group C222.sub.1, and
has the following cell parameters; a=156.69 .ANG., b=260.17 .ANG.,
c=56.85 .ANG., .alpha.=90.degree., .beta.=90.degree.,
.gamma.=90.degree..
[X Ray Structure Analysis of the Fc(P208)/Fc.gamma.RIIb
Extracellular Region Complex]
[0333] Crystal structure of the Fc(P208)/Fc.gamma.RIIb
extracellular region complex was determined by the molecular
replacement method using the program Phaser (J. Appl. Cryst. 2007,
40: 658-674). From the size of the obtained crystal lattice and the
molecular weight of the Fc(P208)/Fc.gamma.RIIb extracellular region
complex, the number of complexes in the asymmetric unit was
predicted to be one. From the structural coordinates of PDB code:
3SGJ which is the crystal structure of the Fc(WT)/Fc.gamma.RIIIa
extracellular region complex, the amino acid residue portions of
the A chain positions 239-340 and the B chain positions 239-340
were taken out as separate coordinates, and they were used
respectively as models for searching the Fc CH2 domains. The amino
acid residue portions of the A chain positions 341-444 and the B
chain positions 341-443 were taken out as a single set of
coordinates from the same structural coordinates of PDB code: 3SGJ;
and this was used as a model for searching the Fc CH3 domains.
Finally, from the structural coordinates of PDB code: 2FCB which is
a crystal structure of the Fc.gamma.RIIb extracellular region, the
amino acid residue portions of the A chain positions 6-178 was
taken out and used as a model for searching the Fc(P208). The
present inventors tried to determine the orientations and positions
of each search model of Fc CH3 domains, Fc.gamma.RIIb extracellular
region, and Fc CH2 domain in the crystal lattices using rotation
function and translation function, but failed to determine the
position of one of the CH2 domains. Then, with reference to the
crystal structure of the complex of Fc(WT)/Fc.gamma.RIIIa
extracellular region, the position of the last CH2 domain was
determined from an electron density map that was calculated based
on the phase determined from the remaining three parts. Thus, the
present inventors obtained an initial model for the crystal
structure of the Fc(P208)/Fc.gamma.RIIb extracellular region
complex. When rigid body refinement which moves the two Fc CH2
domains, the two Fc CH3 domains, and the Fc.gamma.RIIb
extracellular region was performed on the obtained initial model,
the crystallographic reliability factor, R value became 42.6%, and
the Free R value became 43.7% to diffraction intensity data from 25
.ANG. to 3.0 .ANG. at this point. Furthermore, structural
refinement using the program REFMAC5 (Acta Cryst. 2011, D67,
355-367), and revision of the model to observe the electron density
maps whose coefficient have 2Fo-Fc or Fo-Fc, which are calculated
based on the experimentally determined structural factor Fo, the
calculated structural factor Fc and the calculated phase using the
model, was carried out by the Coot program (Acta Cryst. 2010, D66:
486-501), and model refinement was carried out by repeating these
steps. Finally, as a result of incorporation of water molecules
into the model based on the electron density maps which use 2Fo-Fc
or Fo-Fc as the coefficient, and the following refinement, the
crystallographic reliability factor, R values and the Free R value
of the model containing 4786 non-hydrogen atoms became 24.5% and
28.2% to 27259 diffraction intensity data from 25 .ANG. to 2.81
.ANG. resolution, respectively.
3-2. X-Ray Structure Analysis of a Complex Formed Between Fc(P208)
and the Extracellular Region of Fc.gamma.RIIa Type R
[0334] As a result of structural analysis, the crystal structure of
the Fc(P208)/Fc.gamma.RIIa type R extracellular region complex was
determined at 2.87 .ANG. resolution. The crystal structure of the
Fc(P208)/Fc.gamma.RIIa type R extracellular region complex was
compared with the crystal structure of the Fc(P208)/Fc.gamma.RIIb
extracellular region complex shown in Example 3-1, and reflecting
the very high amino acid homology of the two receptors, hardly any
differences were observed for the overall structures (FIG. 11).
However, when the structure was examined in detail at the electron
density level, differences that may be used for improving
selectivity were found. In Fc.gamma.RIIa type R, the residue at
position 160 was Phe instead of Tyr, and as shown in FIG. 12, this
Phe cannot form a hydrogen bond with the main chain of amino acid
residue at position 237 (EU numbering) of the Fc CH2 domain A, that
was present in binding between FcgRIIb and Fc containing the P238D
alteration. While this may be the major factor for improvement of
selectivity for Fc.gamma.RIIa type R by introduction of the P238D
alteration, further comparison at the electron density level showed
that in the complex formed with Fc.gamma.RIIb, the electron density
of the side chains of Leu at position 235 (EU numbering) and Leu at
position 234 (EU numbering) in the Fc CH2 domain A can be
confirmed, whereas the electron densities of these side chains were
not clear in the complex formed with Fc.gamma.RIIa type R, and the
loop around position 237 (EU numbering) seems to be fluctuating as
a result of decrease in interaction with FcgRIIa type R around this
region. On the other hand, when structures of the same region are
compared for CH2 domain B (FIG. 13), the electron density to Asp at
position 237 (EU numbering) can be confirmed in the structure of
the complex formed with Fc.gamma.RIIb, whereas the electron density
to about three residues upstream of Asp at position 237 (EU
numbering) can be confirmed for the complex formed with
Fc.gamma.RIIa type R, and compared to binding with FcgRIIb, a wider
region seems to be used for the interaction. The above suggested
that in the region of positions 234 to 238 (EU numbering) of
Fc(P208), the CH2 domain A side may largely contribute to binding
with Fc.gamma.RIIb, and the CH2 domain B side may largely
contribute to binding with Fc.gamma.RIIaR.
[Expression and Purification of Fc.gamma.RIIa Type R Extracellular
Region]
[0335] This was prepared according to the method of Reference
Example 2.
[Purification of the Fc(P208)/Fc.gamma.RIIa Type R Extracellular
Region Complex]
[0336] To 1.5 mg of the purified Fc.gamma.RIIa type R extracellular
region sample, 0.15 mg of Endo F1 (Protein Science 1996, 5,
2617-2622) expressed and purified from Escherichia coli as a fusion
protein with glutathione S-transferase and 20 .mu.L of 5 U/mL Endo
F2 (QA-bio) and 20 .mu.L of 5 U/mL Endo F3 (QA-bio) were added.
This was left to stand at room temperature for nine days in 0.1 M
sodium acetate buffer (pH 4.5) condition, and then 0.07 mg of Endo
F1 (Protein Science 1996, 5, 2617-2622) expressed and purified from
Escherichia coli as a fusion protein with glutathione S-transferase
and 7.5 .mu.L of 5 U/mL Endo F2 (QA-bio) and 7.5 .mu.L of 5 U/mL
Endo F3 (QA-bio) were added. This was left to stand for three more
days, and the N-linked oligosaccharide was cleaved, while leaving
N-acetylglucosamine directly bound to Asn. Next, this Fc.gamma.RIIa
type R extracellular domain sample subjected to sugar chain
cleavage treatment was concentrated using 10000 MWCO
ultrafiltration filter, and purified by gel filtration
chromatography (Superdex.RTM. 200 10/300 chromatography) using a
column equilibrated with 25 mM HEPES (pH 7), 0.1 M NaCl.
Furthermore, to the obtained sugar chain-cleaved Fc.gamma.RIIa type
R extracellular region fraction, Fc(P208) was added so that the
molar ratio of Fc.gamma.RIIa type R extracellular region would be
present in slight excess, and after concentration using 10000 MWCO
ultrafiltration filter, a sample of the Fc(P208)/Fc.gamma.RIIa type
R extracellular region complex was obtained through purification by
gel filtration chromatography (Superdex.RTM. 200 10/300
chromatography) using a column equilibrated with 25 mM HEPES (pH
7), 0.1 M NaCl.
[Crystallization of the Complex of Fc(P208)/Fc.gamma.RIIaR Type
Extracellular Region]
[0337] A sample of Fc(P208)/Fc.gamma.RIIa R type extracellular
region complex concentrated to about 10 mg/ml with a 10000 MWCO
ultrafiltration filter was crystallized using the sitting drop
vapor diffusion method. Using a reservoir solution of 0.1 M
Bis-Tris (pH 7.5), 26% (w/v) PEG3350, 0.2 M ammonium sulfate,
crystallization drops were prepared at a mixing ratio of reservoir
solution:crystallization sample=0.8 .mu.l:1.0 .mu.l. The drops were
then tightly sealed and allowed to stand at 20.degree. C. This
succeeded in yielding plate-like crystals.
[X-Ray Diffraction Data Measurement from Fc(P208)/Fc.gamma.RIIa
Type R Extracellular Region Complex Crystal]
[0338] A single crystal of Fc(P208)/Fc.gamma.RIIa type R
extracellular region complex prepared was soaked into a solution of
0.1 M Bis-Tris (pH 7.5)), 27.5% (w/v) PEG3350, 0.2 M ammonium
sulfate, 20% (v/v) glycerol. Then, the crystal was fished out of
the solution using a pin with attached tiny nylon loop, and frozen
in liquid nitrogen. X-ray diffraction data of the single crystal
was measured at synchrotron radiation facility Photon Factory
BL-17A in the High Energy Accelerator Research Organization. The
crystal was constantly placed in a nitrogen stream at -178.degree.
C. to maintain in a frozen state during the measurement. A total of
225 X-ray diffraction images from the single crystal were collected
using CCD detector Quantum 315r (ADSC) equipped to the beam line
with rotating the single crystal at 0.6.degree. at a time. Based on
the obtained diffraction images, lattice constant determination,
diffraction spot indexing, and diffraction data processing were
performed using programs Xia2 (J. Appl. Cryst. (2010) 43, 186-190),
XDS Package (Acta Cryst. (2010) D66, 125-132), and Scala (Acta
Cryst. (2006) D62, 72-82). Finally, diffraction intensity data up
to 2.87 .ANG. resolution was obtained. The crystal belongs to the
space group C222.sub.1 with lattice constant a=154.31 .ANG.,
b=257.61 .ANG., c=56.19 .ANG., .alpha.=90.degree.,
.beta.=90.degree., and .gamma.=90.degree..
[X-Ray Crystal Structure Analysis of Fc(P208)/Fc.gamma.RIIa Type R
Extracellular Region Complex]
[0339] The structure was determined by a molecular replacement
method using program Phaser (J. Appl. Cryst. (2007) 40, 658-674).
The number of complexes in an asymmetrical unit was estimated to be
one from the size of the obtained crystal lattice and the molecular
weight of Fc(P208)/Fc.gamma.RIIa type R extracellular region
complex. Using, as a search model, the crystallographic structure
of Fc(P208)/Fc.gamma.RIIb extracellular region complex obtained as
described in Example 3-1, the orientation and position in the
crystal lattices were determined based on the rotation function and
translation function. The crystallographic reliability factor R
value for the data of diffracted intensity at 25 to 3.0 .ANG. was
38.4% and Free R value was 38.0% after rigid body refinement of the
obtained initial model which moves the two CH2 domains and two CH3
domains of the Fc, and the extracellular region of Fc.gamma.RIIa
type R. Then, structural model refinement was achieved by repeating
structural refinement using program REFMAC5 (Acta Cryst. (2011)
D67, 355-367) followed by revision of the model performed using
program Coot (Acta Cryst. (2010) D66, 486-501) with reference to
the electron density maps where the coefficients 2Fo-Fc and Fo-Fc
were calculated using experimentally determined structural factor
Fo, structural factor Fc calculated according to the model, and the
phases calculated according to the model. Finally, as a result of
incorporation of water molecules into the model based on the
electron density maps which use 2Fo-Fc or Fo-Fc as the coefficient,
and the following refinement, the crystallographic reliability
factor, R values and the Free R value of the model containing 4758
non-hydrogen atoms became 26.3% and 29.8% to 24838 diffraction
intensity data from 25 .ANG. to 2.87 .ANG. resolution,
respectively.
[Example 4] Fc Variants Whose Alteration Sites were Determined
Based on Crystal Structures
[0340] As shown in Example 3, it was suggested that electrostatic
interaction between Asp at position 268 (EU numbering) and Arg at
position 292 (EU numbering) is formed as a result of structural
changes nearby which accompany introduction of the P271G alteration
into the CH2 domain B of the FcgRIIb-binding-enhanced variant
Fc(P208) (FIG. 9). This interaction functions to stabilize the loop
structure of positions 266 to 271 (EU numbering), and as a result
it may have contributed to the enhancement of Fc.gamma.RIIb
binding. Accordingly, it was examined whether strengthening the
electrostatic interaction with Arg at position 292 (EU numbering)
to stabilize this loop structure by altering Asp at position 268
(EU numbering) to Glu leads to enhancement of interaction with
FcgRIIb. Furthermore, as shown in FIG. 8, Tyr at position 160 (EU
numbering) of FcgRIIb forms hydrogen bonds with the main chain of
Asp at position 237 (EU numbering) of the Fc(P208) CH2 domain A,
and plays an important role in binding with FcgRIIb. While the side
chain portion of Asp at position 237 (EU numbering) does not form a
particular interaction, Ile at position 332 (EU numbering), Glu at
position 333 (EU numbering), and Lys at position 334 (EU numbering)
are positioned nearby in the molecule. Examination of whether
substituting these sites with hydrophilic residues to strengthen
the interaction with Asp at position 237 (EU numbering) and
stabilize the loop structure near this residue will enhance
interaction with Tyr at position 160 of Fc.gamma.RIIb were carried
out.
[0341] Variants were produced by introducing H268E, I332T, I332S,
1332E, I332K, E333K, E333R, E333S, E333T, K334S, K334T, and K334E
individually into IL6R-BP230/IL6R-L (SEQ ID NO: 27/SEQ ID NO: 21)
which was produced in Example 2. IL6R-L (SEQ ID NO: 21) was
commonly used as the antibody L chain. These variants were used for
antibody expression and purification according to the method of
Reference Example 1, and binding to each FcgR (FcgRIa, FcgRIIa type
H, FcgRIIa type R, FcgRIIb, and FcgRIIIa type V) was assessed using
the method of Reference Example 2.
[0342] The KD of each variant to each FcgR is shown in Table 2. In
the table, "alteration" refers to an alteration introduced into
IL6R-BP3 (SEQ ID NO: 23). IL6R-B3/IL6R-L which is used as the
template to produce IL6R-BP230 is indicated by an asterisk (*). "KD
(IIb) of parent polypeptide/KD (IIb) of altered polypeptide" in the
table shows the value obtained by dividing the KD value of
IL6R-B3/IL6R-L for FcgRIIb by the KD value of each variant for
FcgRIIb. Meanwhile, "KD (IIaR) of parent polypeptide/KD (IIaR) of
altered polypeptide" shows the value obtained by dividing the KD
value of IL6R-B3/IL6R-L for FcgR IIaR by the KD value of each
variant for FcgR IIaR. "KD (IIaR)/KD (IIb)" shows the value
obtained by dividing the KD of each variant for FcgRIIaR by the KD
of the variant for FcgRIIb. The greater the value, the higher the
selectivity to FcgRIIb is. In Table 2, values shown in bold
italicized font are ones for which the binding of FcgR to IgG was
concluded to be too weak to analyze correctly by kinetic analysis
and thus was calculated using:
KD=CR.sub.max/(R.sub.eg-RI)-C [Equation 2]
described in Reference Example 2.
[0343] Both FcgRIIb binding and FcgRIIb selectivity of
IL6R-BP264/IL6R-L, IL6R-BP465/IL6R-L, IL6R-BP466/IL6R-L, and
IL6R-BP470, resulting from introducing H268E, E333K, E333R, and
E333T, respectively, into IL6R-BP230/IL6R-L were increased as
compared to those of IL6R-BP230/IL6R-L. The FcgRIIb selectivity of
IL6R-BP391/IL6R-L introduced with the I332T was reduced while its
FcgRIIb binding was increased as compared to IL6R-BP230/IL6R-L.
[Example 5] Introduction of Comprehensive Alterations into the Area
Around Position 271 (EU Numbering)
[0344] When the X-ray crystal structure of the complex formed
between Fc(P238D) carrying the P238D alteration and the
Fc.gamma.RIIb extracellular region and the X-ray crystal structure
of the complex formed between Fc(P208) and the Fc.gamma.RIIb
extracellular region were compared, the structure near position 271
(EU numbering) showed the greatest structural change (FIG. 9). As
shown in Reference Example 8, in Fc(P238D), it was suggested that
when Asp at position 270 (EU numbering) forms a strong
electrostatic interaction with Arg at position 131 of
Fc.gamma.RIIb, the Pro at position 271 (EU numbering) portion may
be stereochemically strained. In the structure of
Fc(P208)/Fc.gamma.RIIb, introduction of the P271G alteration causes
positional changes at the main-chain level to remove this
structural strain. As a result, the structure near position 271 (EU
numbering) may have been largely changed. If alterations that
further stabilize this changed structure can be efficiently
introduced, entropic energy loss accompanying formation of
electrostatic interaction with Arg at position 131 of Fc.gamma.RIIb
can be reduced, and this may lead to enhancement of binding
activity. Accordingly, comprehensive alterations were introduced
into the area around position 271 (EU numbering) to screen for
alterations that show effects of enhancing binding to FcgRIIb or
improving selectivity to FcgRIIb.
[0345] IL6R-BP267 (SEQ ID NO: 29) produced by introducing E233D,
G237D, P238D, H268E, and P271G into IL6R-B3 (SEQ ID NO: 23) was
used as the template for introducing comprehensive alterations.
Amino acids at positions 264, 265, 266, 267, 269, and 272 (EU
numbering)) in IL6R-BP267 were individually substituted with any of
the 18 amino acids other than the original amino acids and Cys.
IL6R-L (SEQ ID NO: 21) was commonly used as the antibody L chain.
These variants were used for antibody expression and purification
according to the method of Reference Example 1, and binding to each
FcgR (FcgRIa, FcgRIIa type H, FcgRIIa type R, FcgRIIb, and FcgRIIIa
type V) was assessed using the method of Reference Example 2. From
among the obtained variants, those that enhanced FcgRIIb-binding or
improved selectivity to FcgRIIb compared to those of the
IL6R-BP267/IL6R-L prior to alteration introduction are summarized
in Table 3.
[0346] The KD value of each variant to each FcgR is shown in Table
3. In the table, "alteration added to IL6R-BP267" refers to an
alteration introduced into IL6R-BP267 (SEQ ID NO: 29), which was
used as a template. IL6R-B3/IL6R-L which is used as the parent to
produce IL6R-B3 is indicated by asterisk (*). In the table, "KD
(IIb) of parent polypeptide/KD (IIb) of altered polypeptide" shows
the value obtained by dividing the KD value of IL6R-B3/IL6R-L for
FcgRIIb by the KD value of each variant for FcgRIIb. Meanwhile, "KD
(IIaR) of parent polypeptide/KD (IIaR) of altered polypeptide"
shows the value obtained by dividing the KD of IL6R-B3/IL6R-L for
FcgRIIaR by the KD of each variant for FcgR IIaR. "KD (IIaR)/KD
(11b)" shows the value obtained by dividing the KD value of each
variant for FcgRIIaR by the KD value of the variant for FcgRIIb.
The greater the value is, the higher the selectivity to FcgRIIb is.
In Table 3, the values shown in bold italicized font are ones for
which the binding of FcgR to IgG was concluded to be too weak to
analyze correctly by kinetic analysis and thus was calculated
using:
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
described in Reference Example 2.
[0347] All the binding of variants shown in Table 3 to FcgRIa,
FcgRIIaH, and FcgRIIIaV were comparable or reduced as compared to
that of IL6R-B3/IL6R-L. Meanwhile, the FcgRIIb binding of variants
resulting from adding the S267A, V264I, E269D, S267E, V266F, S267G,
and V266M, respectively, to IL6R-BP267/IL6R-L was increased as
compared to that of IL6R-BP267/IL6R-L prior to addition of
alteration. Meanwhile, the KD (IIaR)/KD (IIb) values of variants
resulting from adding the S267A, S267G, E272M, E272Q, D265E, E272D,
E272N, V266L, E272I, and E272F alterations, respectively, to
IL6R-BP267/IL6R-L were increased as compared to that of
IL6R-BP267/IL6R-L prior to addition of alteration, demonstrating
the effect to improve the FcgRIIb selectivity.
[Example 6] Enhancement of the FcgRIIb Binding by Introducing
Alterations into CH3 Region
[0348] An alteration substituting Leu for Pro at position 396 (EU
numbering) has been reported to enhance the FcgRIIb binding (Cancer
Res. (2007) 67, 8882-8890). Position 396 (EU numbering) is present
at a position which is not directly involved in the interaction
with FcgR. However, the amino acid can be assumed to have an effect
on the interaction with FcgR by changing the antibody structure.
Thus, the present inventors assessed whether the FcgRIIb binding is
enhanced or its FcgRIIb selectivity is increased by comprehensive
introduction of amino acid alterations at position 396 (EU
numbering).
[0349] IL6R-BP423 (SEQ ID NO: 33) produced by introducing E233D,
G237D, P238D, S267A, H268E, P271G, and A330R into IL6R-B3 (SEQ ID
NO: 23) was used as the template. Variants were produced by
substituting the amino acid at position 396 (EU numbering)) in
IL6R-BP423 with any of the 18 amino acids other than the original
amino acid and cysteine. IL6R-L (SEQ ID NO: 21) was commonly used
as the antibody L chain. These variants were used for antibody
expression and purification according to the method of Reference
Example 1, and binding to each FcgR (FcgRIa, FcgRIIa type H,
FcgRIIa type R, FcgRIIb, and FcgRIIIa type V) was assessed using
the method of Reference Example 2. Binding of the obtained variants
to each FcgR are summarized in Table 4.
[0350] In the Table, "alteration added to IL6R-BP423" refers to an
alteration introduced into IL6R-BP423. IL6R-B3/IL6R-L which was
used as the template to produce IL6R-BP423 is indicated by asterisk
(*). In the table, "KD (IIb) of parent polypeptide/KD (IIb) of
altered polypeptide" shows the value obtained by dividing the KD
value of IL6R-B3/IL6R-L for FcgRIIb by the KD value of each variant
for FcgRIIb. Meanwhile, "KD (IIaR) of parent polypeptide/KD (IIaR)
of altered polypeptide" shows the value obtained by dividing the KD
value of IL6R-B3/IL6R-L for FcgR IIaR by the KD value of each
variant for FcgR IIaR. "KD (IIaR)/KD (IIb)" shows the value
obtained by dividing the KD of each variant for FcgRIIaR by the KD
of the variant for FcgRIIb. The greater the value, the higher the
selectivity to FcgRIIb is. In Table 4, the values shown in bold
italicized font are ones for which the binding of FcgR to IgG was
concluded to be too weak to analyze correctly by kinetic analysis
and thus was calculated using:
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
described in Reference Example 2.
[0351] The result shown in Table 4 demonstrates that: the
FcgRIIb-binding activity of IL6R-BP456/IL6R-L resulting from
introducing P396M into IL6R-BP423/IL6R-L, IL6R-BP455/1L6R-L
resulting from introducing P396L into IL6R-BP423/IL6R-L,
IL6R-BP464/IL6R-L resulting from introducing P396Y into
IL6R-BP423/IL6R-L, IL6R-BP450/1L6R-L resulting from introducing
P396F into IL6R-BP423/IL6R-L, IL6R-BP448/IL6R-L resulting from
introducing P396D into IL6R-BP423/IL6R-L, IL6R-BP458/IL6R-L
resulting from introducing P396Q into IL6R-BP423/IL6R-L,
IL6R-BP453/IL6R-L resulting from introducing P3961 into
IL6R-BP423/IL6R-L, IL6R-BP449/IL6R-L resulting from introducing
P396E into IL6R-BP423/IL6R-L, IL6R-BP454/IL6R-L resulting from
introducing P396K into IL6R-BP423/IL6R-L, and IL6R-BP459/IL6R-L
resulting from introducing P396R into IL6R-BP423/IL6R-L was all
increased as compared to that of IL6R-BP423/IL6R-L prior to
introduction of the alterations. Meanwhile, the KD (IIaR)/KD (IIb)
value of IL6R-BP456/IL6R-L resulting from introducing P396M into
IL6R-BP423/IL6R-L was larger as compared to that of
IL6R-BP423/IL6R-L prior to introduction of the alteration,
demonstrating the improved FcgRIIb selectivity. As seen in Table 4,
the affinity of the prepared variants to FcgRIa, FcgRIIaH, and
FcgRIIIaV was all lower than that of IL6R-B3/IL6R-L, which was the
parent polypeptide.
[Example 7] Preparation of Variants with Enhanced FcgRIIb Binding
Using Subclass Sequences
[0352] Subclass exists in human IgG and its FcgR binding profile
varies. The present inventors assessed whether the difference in
the affinity to each FcgR between IgG1 and IgG4 could be utilized
to increase the FcgRIIb binding and/or improve the selectivity.
[0353] First, IgG1 and IgG4 were analyzed for their affinity to
each FcgR. IL6R-G4d (SEQ ID NO: 30) containing G4d was constructed
as the antibody H chain. G4d lacks the C-terminal Gly and Lys and
contains a substitution of Pro for Ser at position 228 (EU
numbering) in human IgG4. IL6R-L (SEQ ID NO: 21) was commonly used
as the antibody L chain. IL6R-G1d/IL6R-L and IL6R-G4d/IL6R-L were
expressed and purified according to the method described in
Reference Example 1. These were assessed for their binding to each
FcgR (FcgRIa, FcgRIIa type H, FcgRIIa type R, FcgRIIb, or FcgRIIIa
type V) by the method described in Reference Example 2. The binding
of the resulting variants to each FcgR is summarized in Table
5.
TABLE-US-00001 TABLE 5 KD FOR KD FOR KD FOR KD FOR KD FOR FcgRIa
FcgRIIaR FcgRIIaH FcgRIIb FcgRIIIaV VARIANT NAME (mol/L) (mol/L)
(mol/L) (mol/L) (mol/L) IL6R-G1d/IL6R-L 1.2E-10 9.7E-07 6.5E-07
3.9E-06 4.2E-07 IL6R-G4d/IL6R-L 6.6E-10 2.1E-06 3.4E-06 2.6E-06
3.4E-06
[0354] Compared to IL6R-G1d/IL6R-L, IL6R-G4d/IL6R-L was found to
have 1.5-fold stronger binding to FcgRIIb and 2.2-fold weaker
binding to FcgRIIaR. Furthermore, compared to IL6R-G1d/IL6R-L,
IL6R-G4d/IL6R-L had weaker affinity to FcgRIa, FcgRIIaH, and
FcgRIIIaV. The above-mentioned results revealed that compared to
IL6R-G1d, IL6R-G4d has excellent selectivity and binding to
FcgRIIb.
[0355] FIG. 14 shows the comparison of the sequences of G1d and G4d
from CH1 to the C terminus (positions 118 to 445 (EU numbering)).
The boxed amino acids in FIG. 14 show residues that are different
between G1d and G4d. Several sites predicted to be involved in
interaction with FcgR were selected from among these different
amino acids, and whether further improvement of selectivity and
binding is possible was examined by transferring the sequence of
G4d having excellent selectivity and binding to FcgRIIb into
FcgRIIb-enhanced variants.
[0356] Specifically, IL6R-BP473 was produced by introducing A327G
into IL6R-BP230, IL6R-BP472 was produced by introducing A330S into
IL6R-BP230, IL6R-BP471 was produced by introducing P331S into
IL6R-BP230, IL6R-BP474 was produced by introducing A330S and P331S
into IL6R-BP230, IL6R-BP475 was produced by introducing A327G and
A330S into IL6R-BP230, IL6R-BP476 was produced by introducing
A327G, A330S, and P331S into IL6R-BP230, and IL6R-BP477 was
produced by introducing A327G and P331S into IL6R-BP230.
Furthermore IL6R-BP478 (SEQ ID NO: 31) was produced by substituting
Ala at position 118 to Thr at position 225 (EU numbering) in
IL6R-BP230 with a G4d sequence (Ala at position 118 to Pro at
position 222 (EU numbering)). IL6R-L (SEQ ID NO: 21) was commonly
used as the antibody L chain. These variants were used for antibody
expression and purification according to the method of Reference
Example 1, and binding to each FcgR (FcgRIa, FcgRIIa type H,
FcgRIIa type R, FcgRIIb, and FcgRIIIa type V) was assessed using
the method of Reference Example 2.
[0357] The KD value of each variant to each FcgR is shown in Table
6. "KD (IIb) of parent polypeptide/KD (IIb) of altered polypeptide"
in the table shows the value obtained by dividing the KD value of
IL6R-B3/IL6R-L for FcgRIIb by the KD value of each variant for
FcgRIIb. "Alteration(s) added to IL6R-BP230" refers to an
alteration introduced into IL6R-BP230. IL6R-B3/IL6R-L used as the
template to produce IL6R-BP230 is indicated by *1. Meanwhile,
IL6R-BP478 (SEQ ID NO: 31), in which the segment from Ala at
position 118 up to Thr at position 225 (EU numbering) in IL6R-BP230
is substituted with the sequence of G4d (Ala at position 118 up to
Pro at position 222 (EU numbering)), is indicated by *2. "KD (IIaR)
of parent polypeptide/KD (IIaR) of altered polypeptide" shows the
value obtained by dividing the KD value of IL6R-B3/IL6R-L for FcgR
IIaR by the KD value of the variant for FcgR IIaR. "KD (IIaR)/KD
(IIb)" shows the value obtained by dividing the KD of each variant
for FcgRIIaR by the KD of the variant for FcgRIIb. The greater the
value, the higher the selectivity to FcgRIIb is. In Table 6, the
values shown in bold italicized font are ones for which the binding
of FcgR to IgG was concluded to be too weak to analyze correctly by
kinetic analysis and thus was calculated using:
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
described in Reference Example 2.
[0358] Of the variants shown in Table 6, IL6R-BP473/IL6R-L
introduced with A327G showed FcgRIIb binding increased by 1.2 times
compared to that of IL6R-BP230/IL6R-L. Compared to
IL6R-BP230/IL6R-L, IL6R-BP478/IL6R-L produced by substituting Ala
at position 118 to Thr at position 225 (EU numbering) in IL6R-BP230
with a G4d sequence (Ala at position 118 to Pro at position 222 (EU
numbering)), showed 1.1-fold increase in both FcgRIIb-binding and
FcgRIIaR-binding. Compared to IL6R-B3/IL6R-L which is the parent
peptide, all variants had lower affinity to FcgRIa, FcgRIIaH, and
FcgRIIIaV.
[0359] Furthermore, as shown in FIG. 14, the other sites where the
amino acids are different between G1d and G4d include positions
268, 274, 296, 355, 356, 358, 409, 419, and 445 (EU numbering).
Therefore, by substituting these sites with IgG4-derived amino
acids, selectivity and binding to FcgRIIb may be enhanced.
[0360] In the examination carried out so far, transferring A327G,
which is in the human IgG4 sequence, to the variant
IL6R-BP230/IL6R-L was shown to enhance Fc.gamma.RIIb-binding
activity. A further examination was performed for portions that do
not match between the IgG4 and IgG1 sequences.
[0361] Specifically, variants were produced by introducing the
following alterations into IL6R-BP230 as the antibody H chain:
K274Q was introduced to produce IL6R-BP541; Y296F was introduced to
produce IL6R-BP542; H268Q was introduced to produce IL6R-BP543;
R355Q was introduced to produce IL6R-BP544; D356E was introduced to
produce IL6R-BP545; L358M was introduced to produce IL6R-BP546;
K409R was introduced to produce IL6R-BP547; and Q419E was
introduced to produce IL6R-BP548. Meanwhile, IL6R-L was used as the
common antibody L chain. Antibodies that contain the above heavy
chain variant and the light chain IL6R-L were purified according to
the methods described in Reference Example 1. The purified
antibodies were assessed for their binding to each Fc.gamma.R
(Fc.gamma.RIa, Fc.gamma.RIIaH, Fc.gamma.RIIaR, Fc.gamma.RIIb, or
Fc.gamma.RIIIaV) by the method of Reference Example 2.
[0362] The KD of each variant for each Fc.gamma.R is shown in Table
7. In this Table, "parent polypeptide KD(IIb)/altered polypeptide
KD(IIb)" refers to a value obtained by dividing the KD value of
IL6R-B3/IL6R-L for Fc.gamma.RIIb by the KD value of each variant
for Fc.gamma.RIIb. In the Table, "alterations added to IL6R-BP230"
indicate alterations introduced into IL6R-BP230. Meanwhile,
IL6R-B3/IL6R-L used as a template when producing IL6R-BP230 was
indicated as *1. "Parent polypeptide KD(IIaR)/altered polypeptide
KD(IIaR)" refers to a value obtained by dividing the KD value of
IL6R-B3/IL6R-L for Fc.gamma.RIIaR by the KD value of each variant
for Fc.gamma.RIIaR. "KD(IIaR)/KD(IIb)" is a value obtained by
dividing the KD of each variant for Fc.gamma.RIIaR by the KD of
this variant for Fc.gamma.RIIb. The larger this value is, the
higher the selectivity to Fc.gamma.RIIb is. The Table 7 values
shown in bold italicized font were calculated using the following
equation
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
shown in Reference Example 2 since the binding of Fc.gamma.R to IgG
was determined to be too weak to analyze correctly by kinetic
analysis.
[0363] As shown in Table 7, compared to the IL6R-BP230/IL6R-L prior
to alteration, IL6R-BP541/IL6R-L produced by introducing K274Q into
IL6R-BP230/IL6R-L, IL6R-BP544/IL6R-L produced by introducing R355Q
into IL6R-BP230/IL6R-L, IL6R-BP545/1L6R-L produced by introducing
D356E into IL6R-BP230/IL6R-L, and IL6R-BP546/IL6R-L produced by
introducing L358M into IL6R-BP230/IL6R-L, had enhanced
Fc.gamma.RIIb-binding. Among them, IL6R-BP544/IL6R-L produced by
introducing R355Q into IL6R-BP230/IL6R-L, IL6R-BP545/1L6R-L
produced by introducing D356E into IL6R-BP230/IL6R-L, and
IL6R-BP546/IL6R-L produced by introducing L358M into
IL6R-BP230/IL6R-L show increased KD(IIaR)/KD(IIb) values when
compared to that of the IL6R-BP230/IL6R-L prior to alteration,
showing that these alterations enhance selectivity to Fc.gamma.RIIb
as well.
[Example 8] Examination of Combining Alterations that Bring about
Improvement of Selectivity and Enhancement of Binding to
FcgRIIb
[0364] The combinations of alterations that were found in the
examinations carried out so far to improve selectivity or binding
activity to Fc.gamma.RIIb were examined to achieve further
optimization.
[0365] Combination of alterations that achieves improvement of
selectivity and/or enhancement of binding to Fc.gamma.RIIb in the
examinations carried out so far was introduced into IL6R-B3. As a
comparative control, IL6R-BP253 was produced by introducing the
S267E and L328F alterations, which are known to enhance binding to
Fc.gamma.RIIb (Seung et al., Mol. Immunol. (2008) 45, 3926-3933),
into IL6R-B3. IL6R-L was used for the antibody L chain. An antibody
containing the above-mentioned heavy-chain variant and the light
chain of IL6R-L, which was expressed according to the method of
Reference Example 1, was purified. Binding of the purified antibody
to each Fc.gamma.R (Fc.gamma.RIa, Fc.gamma.RIIaH, Fc.gamma.RIIaR,
Fc.gamma.RIIb, and Fc.gamma.RIIIaV) was assessed using the method
of Reference Example 2.
[0366] The KD of each variant for each Fc.gamma.R is shown in Table
8. "Alteration" in the Table refers to alterations introduced into
IL6R-B3. Meanwhile, IL6R-B3/IL6R-L used as a template for producing
each of the variants is indicated by an asterisk (*). "Parent
polypeptide KD(IIb)/altered polypeptide KD(IIb)" refers to a value
obtained by dividing the KD value of IL6R-B3/IL6R-L for
Fc.gamma.RIIb by the KD value of each variant for Fc.gamma.RIIb.
Furthermore, "parent polypeptide KD(IIaR)/altered polypeptide
KD(IIaR)" refers to a value obtained by dividing the KD value of
IL6R-B3/IL6R-L for Fc.gamma.R IIaR by the KD of the respective
variant for Fc.gamma.RIIaR. "KD(IIaR)/KD(IIb)" shows a value
obtained by dividing the KD of each variant for Fc.gamma.RIIaR by
the KD of the respective variant for Fc.gamma.RIIb. The larger this
value is, the higher the selectivity to Fc.gamma.RIIb compared to
Fc.gamma.RIIaR is. "KD(IIaH)/KD(IIb)" shows a value obtained by
dividing the KD of each variant for Fc.gamma.RIIaH by the KD of the
respective variant for Fc.gamma.RIIb. The larger this value is, the
higher the selectivity to Fc.gamma.RIIb compared to Fc.gamma.RIIaH
is. The values shown in bold italicized font in Table 8 were
calculated using the following equation
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
shown in Reference Example 2 since the binding of Fc.gamma.R to IgG
was determined to be too weak to correctly analyze by kinetic
analysis.
TABLE-US-00002 TABLE 8 VARIANT KD (mol/L) KD(IIaR)/ KD(IIaH)/
KD(IIaR) OF THE PARENT POLYPEPTIDE/ KD(IIb) OF THE PARENT
POLYPEPTIDE/ NAME ALTERATION Fc.gamma.RIa Fc.gamma.RIIaR
Fc.gamma.RIIaH Fc.gamma.RIIb Fc.gamma.RIIIaV KD(IIb) KD(IIb)
KD(IIaR) OF THE ALTERED POLYPEPTIDE KD(IIb) OF THE ALTERED
POLYPEPTIDE G1d G1d 3.2E-10 1.0E-06 6.7E-07 2.6E-06 3.5E-07 0.4 0.3
1.1 1.2 B3 B3 4.2E-10 1.1E-06 7.7E-07 3.1E-06 3.3E-07 0.3 0.2 1.0
1.0 BP253 S267E/L328F 6.7E-11 2.1E-09 1.2E-06 1.1E-08 3.6E-06 0.2
107.1 528.8 276.8 BP262 G237D/P238D/H268E/P271G 1.0E-08 2.0E-06
1.2E-07 17.0 375.0 0.5 25.8 BP264 E233D/G237D/P238D/H268E/ 7.4E-09
3.5E-07 1.2E-08 28.3 227.6 3.2 252.0 P271G/Y296D/A330R BP265
G237D/P238D/H268E/P271G/ 2.3E-08 6.3E-07 1.5E-08 41.2 789.5 1.8
203.9 Y296D/A330R BP266 E233D/G237D/P238D/H268E/ 1.4E-08 3.2E-07
1.8E-08 18.0 621.5 3.4 175.1 P271G/A330R BP268
E233D/G237D/P238D/H268E/ 4.5E-09 1.8E-06 9.2E-08 19.6 228.3 0.6
33.7 P271G/Y296D BP269 G237D/P238D/H268E/P271G/ 1.4E-08 2.2E-06
1.1E-07 19.6 637.2 0.5 27.4 Y296D BP243 E233D/G237D/P238D/S267A/
7.7E-10 1.8E-07 5.1E-09 34.2 390.6 6.3 605.5 H268E/P271G/A330R
BP425 E233D/G237D/P238D/V266L/ 4.1E-09 2.2E-07 9.1E-09 23.6 1644.7
5.1 339.9 S267A/H268E/P271G/A330R BP426 E233D/G237D/P238D/S267A/
1.0E-09 1.6E-07 5.9E-09 27.6 8361.8 6.8 529.0
H268E/E269D/P271G/A330R BP428 E233D/G237D/P238D/S267G/ 4.9E-09
3.9E-07 1.4E-08 28.0 3000.0 2.8 221.4 H268E/P271G/A330R BP429
E233D/G237D/P238D/V264I/ 6.2E-09 1.7E-07 5.4E-09 31.5 648.1 6.5
574.1 S267G/H268E/P271G/A330R BP430 E233D/G237D/P238D/V266L/
1.7E-08 2.2E-07 1.2E-08 18.5 909.1 4.9 256.2
S267G/H268E/P271G/A330R BP431 E233D/G237D/P238D/S267G/ 3.6E-09
4.1E-07 1.2E-08 34.6 649.6 2.7 265.0 H268E/E269D/P271G/A330R BP433
E233D/G237D/P238D/H268D/ 7.5E-10 6.8E-07 3.4E-08 20.0 216.0 1.6
91.7 P271G/Y296D/A330K/I332T BP434 E233D/G237D/P238D/H268D/ 5.5E-10
3.4E-07 1.2E-08 27.2 333.3 3.3 252.0 P271G/Y296D/K326D/A330R/ I332T
BP435 E233D/G237D/P238D/H268D/ 1.0E-09 4.2E-07 1.6E-08 27.1 217.9
2.6 198.7 P271G/Y296D/K326A/A330R/ I332T BP436
E233D/G237D/P238D/S267A/ 2.6E-10 2.2E-07 2.1E-06 5.1E-09 43.8 411.0
4.9 606.7 H268E/P271G/Y296D/A330R/ I332T BP437
G237D/P238D/S267A/H268E/ 7.5E-10 2.2E-07 5.9E-09 37.7 236.5 4.9
523.6 P271G/Y296D/A330R/I332T BP438 E233D/G237D/P238D/S267A/
2.1E-10 1.8E-07 1.6E-06 5.5E-09 32.7 293.6 6.2 568.8
H268E/P271G/A330R/I332T BP439 E233D/G237D/P238D/V264I/ 8.7E-09
1.3E-07 6.1E-09 20.9 460.5 8.7 509.9 V266L/S267A/H268E/P271G/ A330R
BP440 E233D/G237D/P238D/V264I/ 8.7E-09 1.3E-07 5.2E-09 24.0 307.1
8.8 595.0 H268E/P271G/A330R BP441 E233D/G237D/P238D/V266L/ 1.7E-08
3.6E-07 1.5E-08 24.0 582.8 3.0 205.3 H268E/P271G/A330R BP442
E233D/G237D/P238D/H268E/ 4.5E-09 3.8E-07 1.2E-08 30.6 379.0 2.9
250.0 E269D/P271G/A330R BP443 E233D/G237D/P238D/V266L/ 1.8E-08
5.1E-07 2.3E-08 21.7 406.0 2.2 132.5 H268E/E269D/P271G/A330R BP445
E233D/G237D/P238D/V264I/ 2.0E-09 8.0E-08 2.6E-09 31.0 581.4 13.8
1201.6 S267A/H268E/P271G/A330R BP479 E233D/G237D/P238D/V264I/
5.3E-09 9.0E-07 5.6E-08 16.1 268.3 1.2 55.5 V266L/S267A/H268E/P271G
BP480 E233D/G237D/P238D/V266L/ 1.3E-08 6.3E-06 2.0E-07 32.1 107.7
0.2 15.9 H268E/E269D/P271G BP481 E233D/G237D/P238D/V264I/ 1.0E-09
4.0E-07 1.9E-08 20.5 350.5 2.8 159.8 S267A/H268E/P271G BP483
E233D/G237D/P238D/V266L/ 1.3E-09 1.3E-06 7.8E-08 16.8 230.8 0.8
39.7 S267A/H268E/P271G BP484 E233D/G237D/P238D/S267A/ 8.2E-10
7.8E-07 4.6E-08 17.1 240.7 1.4 67.8 H268E/E269D/P271G BP487
E233D/G237D/P238D/V264I/ 1.2E-09 3.9E-08 8.4E-07 1.2E-09 33.8 730.4
28.3 2695.7 S267A/H268E/P271G/A330R/ P396M BP488
E233D/G237D/P238D/V264I/ 2.2E-09 7.4E-08 1.6E-06 1.9E-09 40.1 864.9
14.8 1675.7 S267A/H268E/P271G/Y296D/ A330R BP489
E233D/G237D/P238D/V264I/ 1.3E-09 4.3E-08 8.7E-07 1.0E-09 42.8 870.0
25.7 3100.0 S267A/H268E/P271G/Y296D/ A330R/P396M BP490
G237D/P238D/V264I/S267A/ 4.5E-09 1.1E-07 2.4E-06 2.4E-09 46.7
1000.0 9.8 1291.7 H268E/P271G/A330R BP491 G237D/P238D/V264I/S267A/
5.3E-09 1.2E-07 2.2E-06 3.0E-09 38.8 723.7 9.3 1019.7
H268E/P271G/Y296D/A330R BP492 P238D/V264I/S267A/H268E/ 7.9E-10
9.2E-07 2.4E-08 38.8 678.0 1.2 131.4 P271G BP493
P238D/V264I/S267A/H268E/ 8.2E-10 1.1E-06 2.1E-08 52.1 900.5 1.0
146.9 P271G/Y296D BP494 G237D/P238D/S267A/H268E/ 3.9E-09 2.5E-07
6.6E-09 38.6 820.7 4.3 471.1 P271G/Y296D/A330R BP495
G237D/P238D/S267G/H268E/ 8.3E-09 4.9E-07 9.7E-09 50.9 1243.5 2.2
321.2 P271G/Y296D/A330R BP496 E233D/G237D/P238D/V264I/ 1.2E-09
4.7E-07 3.7E-06 1.8E-08 25.5 201.1 2.3 168.5
S267A/H268E/P271G/Y296D BP497 E233D/G237D/P238D/V264I/ 2.1E-09
8.5E-08 9.6E-07 4.1E-09 21.0 236.5 12.9 763.5
S267A/H268E/P271G/A327G/ A330R BP498 E233D/G237D/P238D/V264I/
1.3E-09 5.1E-08 9.3E-07 1.7E-09 30.8 563.6 21.7 1878.8
S267A/H268E/P271G/A330R/ P396L BP499 E233D/G237D/P238D/V264I/
1.2E-09 4.9E-08 1.0E-06 1.5E-09 33.8 684.9 22.3 2123.3
S267A/H268E/P271G/Y296D/ A330R/P396L BP500 G237D/P238D/V264I/S267A/
2.3E-09 7.2E-07 2.4E-08 29.9 1033.1 1.5 128.1 H268E/P271G/Y296D
BP501 G237D/P238D/V264I/S267A/ 2.1E-09 6.3E-07 2.5E-08 25.1 555.6
1.7 123.0 H268E/P271G BP502 E233D/G237D/P238D/V264I/ 2.1E-09
1.1E-07 1.3E-06 3.7E-09 29.5 352.3 10.1 840.1
S267A/H268E/P271G/Y296D/ A327G/A330R BP503 E233D/G237D/P238D/V264I/
1.2E-09 5.7E-08 8.6E-07 1.7E-09 33.2 502.9 19.4 1812.9
S267A/H268E/P271G/Y296D/ A327G/A330R/P396M BP504
E233D/G237D/P238D/V264I/ 1.4E-09 4.5E-07 2.4E-08 18.5 658.4 2.4
127.6 S267A/H268E/P271G/E272P BP505 E233D/G237D/P238D/V264I/
1.1E-09 4.3E-07 2.1E-08 20.0 514.0 2.6 144.9
S267A/H268E/P271G/E272D BP506 E233D/G237D/P238D/V264I/ 3.1E-09
1.2E-07 2.5E-06 3.4E-09 35.1 731.0 9.2 906.4
S267A/H268E/P271G/E272P/ Y296D/A330R BP507 E233D/G237D/P238D/V264I/
2.6E-09 1.0E-07 1.8E-06 2.9E-09 34.2 618.6 11.1 1065.3
S267A/H268E/P271G/E272P/ A330R BP508 E233D/G237D/P238D/V264I/
1.4E-09 5.4E-07 2.1E-08 26.0 961.5 2.0 149.0
S267A/H268E/P271G/E272P/ Y296D BP509 E233D/G237D/P238D/V264I/
1.1E-09 5.2E-07 1.8E-08 29.2 443.8 2.1 174.2
S267A/H268E/P271G/E272D/ Y296D BP510 G237D/P238D/V264I/S267A/
6.0E-09 1.7E-07 4.0E-06 3.8E-09 43.5 1041.7 6.6 807.3
H268E/P271G/E272P/A330R BP511 G237D/P238D/V264I/S267A/ 6.0E-09
1.8E-07 4.3E-06 3.5E-09 50.6 1235.6 6.3 890.8
H268E/P271G/E272P/Y296D/ A330R BP531 E233D/G237D/P238D/V264I/
9.4E-09 1.2E-07 3.5E-06 3.8E-09 33.1 933.3 8.9 826.7
S267G/H268E/P271G/Y296D/ A330R/P396M BP532 E233D/G237D/P238D/V264I/
1.2E-08 9.4E-08 1.9E-06 3.2E-09 29.3 593.8 11.7 968.8
H268E/P271G/Y296D/A330R/ P396M BP533 E233D/G237D/P238D/V264I/
7.7E-09 1.2E-07 2.6E-06 4.1E-09 29.3 634.1 9.2 756.1
S267G/H268E/P271G/Y296D/ A330R/P396L BP534 E233D/G237D/P238D/V264I/
9.3E-09 9.1E-08 1.8E-06 3.0E-09 30.7 606.1 12.1 1043.8
H268E/P271G/Y296D/A330R/ P396L BP535 E233D/G237D/P238D/V264I/
1.1E-08 9.2E-08 3.2E-06 4.0E-09 23.2 806.0 11.9 780.9
S267G/H268E/P271G/Y296D/ A327G/A330R/P396M BP536
E233D/G237D/P238D/V264I/ 8.9E-09 7.9E-08 1.3E-06 3.0E-09 26.6 437.7
13.9 1043.8 H268E/P271G/Y296D/A327G/ A330R/P396M BP537
G237D/P238D/V264I/S267G/ 2.9E-08 2.7E-07 3.1E-06 6.9E-09 39.1 447.3
4.1 447.3 H268E/P271G/A330R BP538 G237D/P238D/V264I/H268E/ 5.5E-08
2.0E-07 3.0E-06 5.3E-09 38.6 568.2 5.4 587.1 P271G/A330R BP539
G237D/P238D/V264I/S267G/ 6.4E-08 3.3E-07 5.6E-06 8.4E-09 39.0 666.7
3.4 369.0 H268E/P271G/E272P/Y296D/ A330R BP540
G237D/P238D/V264I/H268E/ 9.6E-08 2.1E-07 4.6E-06 5.7E-09 36.6 802.8
5.2 541.0 P271G/E272P/Y296D/A330R BP549 G237D/P238D/S267G/H268E/
1.8E-08 5.7E-07 1.6E-08 35.9 696.2 1.9 196.2 P271G/A330R BP550
G237D/P238D/V264I/S267G/ 2.5E-08 3.4E-07 5.0E-06 7.6E-09 44.2 655.3
3.3 406.3 H268E/P271G/E272D/Y296D/ A330R BP551
G237D/P238D/V264I/H268E/ 3.2E-08 2.5E-07 2.8E-06 6.4E-09 38.1 435.5
4.5 482.1 P271G/E272D/Y296D/A330R BP552 E233D/G237D/P238D/V264I/
3.2E-09 9.7E-08 1.9E-06 2.6E-09 37.3 733.6 11.4 1196.9
S267A/H268E/P271G/E272D/ Y296D/A330R BP553 E233D/G237D/P238D/V264I/
3.4E-09 8.6E-08 1.4E-06 3.1E-09 27.8 453.1 12.8 1003.2
S267A/H268E/P271G/E272D/ A330R BP554 G237D/P238D/V264I/S267A/
8.0E-09 1.5E-07 2.3E-06 4.4E-09 32.7 518.0 7.6 698.2
H268E/P271G/E272D/A330R BP555 G237D/P238D/V264I/S267A/ 9.4E-09
1.6E-07 3.2E-06 4.1E-09 39.7 778.6 6.7 754.3
H268E/P271G/E272D/Y296D/ A330R BP556 G237D/P238D/V264I/S267G/
4.3E-08 3.0E-07 5.8E-06 8.4E-09 35.4 692.1 3.7 369.9
H268E/P271G/Y296D/A330R BP557 G237D/P238D/S267G/H268D/ 1.3E-08
8.5E-07 2.0E-08 42.0 746.3 1.3 154.2 P271G/Y296D/A330R BP558
G237D/P238D/V264I/S267G/ 1.3E-08 3.3E-07 4.9E-06 9.0E-09 36.4 543.2
3.4 343.7 H268E/P271G/E272D/A330R BP559 P238D/V264I/S267A/H268E/
1.1E-09 1.6E-06 2.8E-08 58.4 711.7 0.7 110.3 P271G/E272D/Y296D
BP560 P238D/S267G/H268E/P271G/ 5.6E-09 4.2E-06 1.8E-07 22.8 168.5
0.3 16.8 Y296D/A330R BP561 E233D/G237D/P238D/H268D/ 9.4E-09 5.1E-07
5.3E-06 1.8E-08 28.0 291.2 2.2 170.3 P271G/E272D/Y296D/A330R BP562
G237D/P238D/H268D/P271G/ 2.5E-08 6.8E-07 2.4E-08 29.0 466.1 1.6
131.4 E272D/Y296D/A330R BP563 E233D/G237D/P238D/H268E/ 1.2E-08
4.6E-07 8.3E-06 1.6E-08 29.1 525.3 2.4 196.2
P271G/E272D/Y296D/A330R BP564 G237D/P238D/H268E/P271G/ 3.1E-08
5.8E-07 2.2E-08 26.2 454.5 1.9 140.9 E272D/Y296D/A330R BP565
E233D/G237D/P238D/S267A/ 2.4E-09 2.3E-07 4.7E-06 5.5E-09 41.5 856.1
4.8 564.7 H268E/P271G/Y296D/A330R BP567 E233D/P238D/V264I/S267A/
2.1E-10 8.9E-07 1.4E-08 64.4 1231.9 1.2 224.6 H268E/P271G/Y296D
BP568 E233D/P238D/V264I/S267A/ 1.9E-10 6.8E-07 1.5E-08 46.1 748.3
1.6 210.9 H268E/P271G
Among the variants described in Table 8, IL6R-BP253/IL6R-L produced
by adding known alterations that enhance Fc.gamma.RIIb binding
showed 277-fold and 529-fold enhanced binding activities to
Fc.gamma.RIIb and Fc.gamma.RIIaR, respectively, compared to those
of the IL6R-B3/IL6R-L prior to alteration. Furthermore,
Fc.gamma.RIa-binding activity of IL6R-BP253/IL6R-L was also
enhanced compared to that of IL6R-B3/IL6R-L. On the other hand,
binding of IL6R-BP253/IL6R-L to Fc.gamma.RIIaH and Fc.gamma.RIIIaV
was decreased compared to those of IL6R-B3/IL6R-L. Among the other
variants, binding to Fc.gamma.RIa was slightly enhanced for
IL6R-BP436/IL6R-L, IL6R-BP438/IL6R-L, IL6R-BP567/IL6R-L, and
IL6R-BP568/IL6R-L, compared to that of the IL6R-B3/IL6R-L prior to
alteration but Fc.gamma.RIa binding was decreased in all of the
other variants. Furthermore, binding to Fc.gamma.RIIaH and
Fc.gamma.RIIIaV were decreased in all variants when compared to
those of IL6R-B3/IL6R-L.
[0367] Comparison of variants produced in this examination with the
existing variant IL6R-BP253/IL6R-L having enhanced Fc.gamma.RIIb
binding showed that the value of KD (IIaH)/KD (IIb) is 107.7 for
IL6R-BP480/IL6R-L which showed the lowest value and is 8362 for
IL6R-BP426/IL6R-L which showed the highest value, and the values
for all variants were higher than 107.1 for IL6R-BP253/IL6R-L.
Furthermore, the value of KD (IIaR)/KD (IIb) is 16.1 for
IL6R-BP479/IL6R-L which showed the lowest value and is 64.4 for
IL6R-BP567/IL6R-L which showed the highest value, and the values
for all variants were higher than 0.2 for IL6R-BP253/IL6R-L. From
these results, all of the variants shown in Table 8 have been shown
to be variants with improved selectivity to Fc.gamma.RIIb as
compared to the existing variant into which alteration(s) to
enhance Fc.gamma.RIIb binding is introduced. In particular,
IL6R-BP559/IL6R-L, IL6R-BP493/IL6R-L, IL6R-BP557/IL6R-L,
IL6R-BP492/IL6R-L, IL6R-BP500/IL6R-L, and IL6R-BP567/IL6R-L all
have Fc.gamma.RIIaR binding maintained at not more than 1.5 times
that of IL6R-B3/IL6R-L, and at the same time Fc.gamma.RIIb-binding
activity enhanced by 100 times or more; therefore, these variants
were expected to show effects yielded by enhanced binding to
Fc.gamma.RIIb while avoiding side effects caused by enhanced
binding to Fc.gamma.RIIaR.
[0368] In addition, regarding IL6R-BP489/IL6R-L, IL6R-BP487/IL6R-L,
IL6R-BP499/IL6R-L, IL6R-BP498/IL6R-L, IL6R-BP503/IL6R-L,
IL6R-BP488/IL6R-L, IL6R-BP490/IL6R-L, IL6R-BP445/IL6R-L,
IL6R-BP552/IL6R-L, IL6R-BP507/IL6R-L, IL6R-BP536/IL6R-L,
IL6R-BP534/IL6R-L, IL6R-BP491/IL6R-L, IL6R-BP553/IL6R-L,
IL6R-BP532/IL6R-L, IL6R-BP506/IL6R-L, IL6R-BP511/IL6R-L,
IL6R-BP502/IL6R-L, IL6R-BP531/IL6R-L, IL6R-BP510/IL6R-L,
IL6R-BP535/IL6R-L, IL6R-BP497/IL6R-L, IL6R-BP533/IL6R-L,
IL6R-BP555/IL6R-L, IL6R-BP554/IL6R-L, IL6R-BP436/IL6R-L,
IL6R-BP423/IL6R-L, IL6R-BP440/IL6R-L, IL6R-BP538/IL6R-L,
IL6R-BP429/IL6R-L, IL6R-BP438/IL6R-L, IL6R-BP565/IL6R-L,
IL6R-BP540/IL6R-L, IL6R-BP426/IL6R-L, IL6R-BP437/IL6R-L,
IL6R-BP439/IL6R-L, IL6R-BP551/IL6R-L, IL6R-BP494/IL6R-L,
IL6R-BP537/IL6R-L, IL6R-BP550/IL6R-L, IL6R-BP556/IL6R-L,
IL6R-BP539/IL6R-L, IL6R-BP558/IL6R-L, IL6R-BP425/IL6R-L, and
IL6R-BP495/IL6R-L, their Fc.gamma.RIIb binding was higher than that
of IL6R-BP253/IL6R-L added with the existing alteration that
enhances the Fc.gamma.RIIb binding. Further, the enhancement of the
Fc.gamma.RIIb binding ranges from 321 times (lowest) to 3100 times
(highest), compared to the binding of IL6R-B3/IL6R-L (which is
defined to be 1), from IL6R-BP495/IL6R-L to IL6R-BP489/IL6R-L,
respectively. Therefore, one may say that these variants are
superior to existing technology with regard to both selectivity and
binding to Fc.gamma.RIIb.
[0369] Here, variants related to IL6R-BP567/IL6R-L which is
considered to be the best in terms of selectivity for Fc.gamma.RIIb
were studied from the perspective of immunogenicity. The Y296D
alteration has been introduced into IL6R-BP567/IL6R-L which showed
the highest selectivity and into IL6R-BP493/IL6R-L which showed
Fc.gamma.RIIaR binding that is completely equivalent to that of the
native form and showed 147-fold enhanced binding to Fc.gamma.RIIb.
Y296 has been reported to be included in a Tregitope sequence (De
Groot et al., Blood (2008) 112, 3303-3311), and introducing
alterations into this site may lead to loss of immunosuppressive
functions that a native IgG1 normally have. Therefore, from the
perspective of immunogenicity, variants that do not include the
Y296D alteration are more preferred. IL6R-BP568/IL6R-L and
IL6R-BP492/IL6R-L were produced by removing the Y296D alteration
from IL6R-BP567/IL6R-L and IL6R-BP493/IL6R-L, respectively.
Considering the selectivity and binding activity to Fc.gamma.RIIb,
removing the Y296D alteration from IL6R-BP492/IL6R-L and
IL6R-BP568/IL6R-L decreased both selectivity and binding activity
compared to when Y296D was included. However, compared to the
native form, binding to Fc.gamma.RIIaR is 1.6-fold and binding to
Fc.gamma.RIIb is 211-fold for IL6R-BP568/IL6R-L, and binding to
Fc.gamma.RIIaR is 1.2-fold and binding to Fc.gamma.RIIb is 131-fold
for IL6R-BP492/IL6R-L, and therefore high selectivity and binding
activity were still maintained. These results allow one to say that
IL6R-BP568/IL6R-L and IL6R-BP492/IL6R-L are excellent variants not
only in terms of selectivity and binding activity to Fc.gamma.RIIb
but also in terms of immunogenicity.
[Example 9] Enhancement of Binding to FcgRIIb by a Heterodimerized
Antibody
[0370] 9-1. Examination of Introducing P238D into Only One of the
Chains
[0371] As shown in FIG. 26 in Reference Example 7, the reason why
Fc(P238D) acquired high binding to FcgRIIb is by introducing the
P238D alteration, the region that had formed a hydrophobic core
with the surrounding residues in the case of Pro could no longer
exist in the hydrophobic core upon change to Asp and directed to
the solvent side, and resulting in the great change in the loop
structure of domain A. However, whether it is necessary to
introduce the P238D alteration into both chains or whether it is
acceptable to introduce P238D into one of the chains and introduce
other alterations into the other chain still has to be verified.
Accordingly, heterodimerized antibodies with different alterations
introduced into each of the antibody H chains were used for this
verification.
[0372] The variable region (SEQ ID NO: 15) of a glypican 3 antibody
comprising the CDRs of GpH7 which is an anti-glypican 3 antibody
with improved plasma kinetics disclosed in WO 2009/041062 was used
as the antibody H chain. GpH7-A5 (SEQ ID NO: 35) produced by
introducing the D356K and H435R alterations into GpH7-G1d (SEQ ID
NO: 34) in which Gly and Lys are removed from the C terminus of
IgG1 carrying GpH7 as the variable region, and GpH7-B3 (SEQ ID NO:
17) produced by introducing the K439E alteration into GpH7-G1d were
used. The D356K and K439E alterations introduced into the
respective H chains were introduced to efficiently form the
heterodimers for each H chain when producing heterodimerized
antibodies comprising two H chains (WO2006/106905). H435R is an
alteration that inhibits binding to Protein A, and was introduced
to efficiently separate the dimeric heteromer comprising two H
chains each introduced with different alterations from the dimeric
homomer comprising two H chains each introduced with the same
alterations. Variants in which the amino acids at positions 236,
237, and 238 (EU numbering) in GpH7-B3 (SEQ ID NO: 17) produced in
Reference Example 3 were substituted with any of the 18 amino acids
other than the original amino acid and Cys were used as one of the
H chains. GpH7-AP001 produced by introducing P238D into GpH7-A5
(SEQ ID NO: 35) was used as the other chain. GpL16-k0 (SEQ ID NO:
16) of glypican 3 antibody with improved plasma kinetics disclosed
in WO 2009/041062 was commonly used as the antibody L chain. These
variants were expressed and purified according to the method of
Reference Example 1, and binding to each of the FcgRIIa type R and
FcgRIIb was assessed using the method of Reference Example 2. The
amount binding to FcgR of each variant is shown in FIG. 15.
[0373] The G237W, G237F, G236N, P238G, P238N, P238E, and P238D
alterations shown in FIG. 15 refer to alterations introduced into
GpH7-B3. A5/B3 refers to GpH7-A5/GpH7-B3/GpL16-k0 which has no
alterations introduced into both chains, and a variant containing
P238D in only one of the chains refers to
GpH7-A5/GpH7-BF648/GpL16-k0. Their results are shown in Table
9.
TABLE-US-00003 TABLE 9 BINDING BINDING AMOUNT OF AMOUNT OF ALTERED
ALTERED POLYPEPTIDE TO POLYPEPTIDE TO FcgRIIaR WHEN FcgRIIb WHEN
AMOUNT THAT OF THAT OF BINDING GpH7-AP001/ GpH7-AP001/ TO FcgRIIb/
ALTERATION ALTERATION GpH7-BF-648 GpH7-BF648 AMOUNT INTRODUCED
INTRODUCED IS DEFINED IS DEFINED BINDING VARIANT NAME INTO GpH7-A5
INTO GpH7-B3 AS 100 AS 100 TO FcgRIIaR GpH7-G1d/GpL16-k0 * * 515 65
0.5 GpH7-A5/GpH7-B3/GpL16-k0 570 77 0.5 GpH7-A5/GpH7-BF648/GpL16-k0
P238D 677 131 0.8 GpH7-AP001/GpH7-BF648/GpL16-k0 P238D P238D 100
100 4.0 GpH7-AP001/GpH7-BP032/GpL16-k0 P238D G236N 538 129 1.0
GpH7-AP001/GpH7-BP044/GpL16-k0 P238D G237F 609 127 0.8
GpH7-AP001/GpH7-BP057/GpL16-k0 P238D G237W 561 121 0.9
GpH7-AP001/GpH7-BP061/GpL16-k0 P238D P238E 111 80 2.9
GpH7-AP001/GpH7-BP063/GpL16-k0 P238D P238G 161 69 1.7
GpH7-AP001/GpH7-BP069/GpL16-k0 P238D P238N 131 72 2.2
[0374] In Table 9, "amount binding to FcgRIIb/amount binding to
FcgRIIaR" are values obtained by dividing the amount binding to
FcgRIIb of each variant by the amount binding to FcgRIIaR of each
variant, and shows that the higher the value is, the higher
selectivity to FcgRIIb is. Furthermore, while the phrases
"alteration introduced into GpH7-A5" and "alteration introduced
into GpH7-B3" indicate alterations introduced into GpH7-A5 and
GpH7-B3, respectively; and GpH7-G1d which was used as the template
when producing GpH7-A5 and GpH7-B3 is indicated by an asterisk (*).
From the results of Table 9, GpH7-AP001/GpH7-BF648/GpL16-k0 having
the P238D alteration in both chains had the highest selectivity to
FcgRIIb. GpH7-AP001/GpH7-BP061/GpL16-k0,
GpH7-AP001/GpH7-BP069/GpL16-k0, and GpH7-AP001/GpH7-BP063/GpL16-k0
which have P238E, P238N, and P238G in the other chain show the
values of "amount binding to FcgRIIb/amount binding to FcgRIIaR"
that are 2.9, 2.2, and 1.7, respectively, and even when compared to
GpH7-AP001/GpH7-BF648/GpL16-k0 which has the P238D alteration in
both chains, high selectivity to FcgRIIb was maintained.
Furthermore, since GpH7-AP001/GpH7-BF648/GpL16-k0 having the P238D
alteration in both chains also maintained 69% or greater affinity
for FcgRIIb, one can say that if the P238D alteration is present in
one of the chains, the other chain may have the P238E, P238N, or
P238G alteration. In addition, focusing on FcgRIIb-binding,
compared to GpH7-AP001/GpH7-BF648/GpL16-k0 having P238D in both
chains, GpH7-A5/GpH7-BF648/GpL16-k0 which has P238D only in one of
the chains and does not have alteration in the other chain showed
stronger binding to FcgRIIb; and GpH7-AP001/GpH7-BP032/GpL16-k0,
GpH7-AP001/GpH7-BP044/GpL16-k0, and GpH7-AP001/GpH7-BP057/GpL16-k0
which have P238D in one of their chains and have G236N, G237F, and
G237W, respectively, in the other chain were found to bind more
strongly to FcgRIIb.
9-2. Verification of Alterations Based on the Structural
Information of Fc(P208)/FcgRIIb
[0375] As shown in FIG. 10, in the crystal structure of
Fc(P208)/FcgRIIb, electron density of Lys at position 117 of
FcgRIIb was not observed, and this residue was considered not to be
largely involved in binding with Fc(P208); but by substituting Asp
or Glu for Ser at position 239 (EU numbering) of the CH2 domain B,
which is positioned nearby, an electrostatic interaction may be
formed with this Lys at position 117 of FcgRIIb. On the other hand,
as shown in FIG. 7, in CH2 domain A, Ser at position 239 (EU
numbering) forms a hydrogen bond with Gly at position 236 (EU
numbering), and stabilization of the loop structure from positions
233 to 239 (EU numbering) may contribute to strengthen the binding
with Tyr at position 160 (EU numbering), and substitutions in this
portion is predicted to cause decrease in binding activity
accompanying destabilization of the loop structure in CH2 domain A,
and these effects were predicted to cancel each other in homologous
alterations. Accordingly, in this examination, the S239D alteration
or the S239E alteration was introduced only into one of the chains
by heterodimerization, and effects of enhancement of binding to
FcgRIIb were examined.
[0376] As one of the antibody H chains, IL6R-BP256 and IL6R-BP257
were produced by introducing S239D and S239E, respectively, into
IL6R-BP208 (SEQ ID NO: 24). Similarly, S239D was introduced into
IL6R-BP230 (SEQ ID NO: 27) to produce IL6R-BP259, and S239E was
introduced into IL6R-BP230 to produce IL6R-BP260. IL6R-AP002 was
produced by introducing the same alterations as those included in
CH2 of IL6R-BP208, which are E233D, G237D, P238D, H268D, P271G, and
A330R, into IL6R-A5 (SEQ ID NO: 69), and IL6R-AP009 was produced by
introducing the same alterations as those included in CH2 of
IL6R-BP230, which are E233D, G237D, P238D, H268D, P271G, Y296D, and
A330R, into IL6R-A5, and they were used as the other antibody H
chain. As a comparison, IL6R-BP253 (SEQ ID NO: 32) was produced by
introducing S267E and L328F, which is a known FcgRIIb enhancement
technique (Non-patent Document 28), into IL6R-B3. IL6R-L (SEQ ID
NO: 21) was commonly used as the antibody L chain. Antibodies were
expressed using these variants and then purified according to the
method of Reference Example 1, and binding to each FcgR (FcgRIa,
FcgRIIa type H, FcgRIIa type R, FcgRIIb, and FcgRIIIa type V) was
assessed using the method of Reference Example 2.
[0377] The KD values of each variant for each FcgR are shown in
Table 10. "Parent polypeptide KD(IIb)/altered polypeptide KD(IIb)"
refers to a value obtained by dividing the KD value of
IL6R-B3/IL6R-L for FcgRIIb by the KD value of each variant for
FcgRIIb. Furthermore, "parent polypeptide KD(IIaR)/altered
polypeptide KD(IIaR)" refers to a value obtained by dividing the KD
value of IL6R-B3/IL6R-L for FcgRIIaR by the KD value of each
variant for FcgRIIaR. "KD(IIaR)/KD(IIb)" is a value obtained by
dividing the KD of each variant for FcgRIIaR by the KD of each
variant for FcgRIIb. The larger this value is, the higher the
selectivity to FcgRIIb is. The Table 10 values shown in bold
italicized font were calculated using the following equation
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
shown in Reference Example 2 since the binding of FcgR to IgG was
too weak to accurately analyze by kinetic analysis.
[0378] As shown in Table 10, compared to IL6R-BP208/IL6R-L, both
IL6R-AP002/IL6R-BP256/IL6R-L and IL6R-AP002/IL6R-BP257/IL6R-L
produced by introducing S239D and S239E, respectively, into one of
the chains of IL6R-BP208/IL6R-L showed enhanced binding to FcgRIIb.
Furthermore, the value of KD(IIaR)/KD(IIb) was greater than that of
IL6R-BP256/IL6R-L, and selectivity to FcgRIIb was also improved. On
the other hand, compared to IL6R-BP208/IL6R-L, both selectivity and
binding to FcgRIIb of IL6R-BP256/IL6R-L produced by introducing
S239D into both chains of IL6R-BP208/IL6R-L and IL6R-BP257/IL6R-L
produced by introducing S239E into both chains of IL6R-BP208/IL6R-L
had significantly decreased. This way, when S239D or S239E was
introduced into only one of the chains, effects of enhancing
binding to FcgRIIb was observed, whereas when S239D or S239E was
introduced into both chains, binding to FcgRIIb significantly
decreased. The main reason why this happened may be, as described
previously, the destabilization of loop structure in CH2 domain A.
Similar results were observed when S239D and S239E were introduced
using IL6R-BP230/IL6R-L as the template.
IL6R-AP009/IL6R-BP259/IL6R-L and IL6R-AP009/IL6R-BP260/IL6R-L
produced by introducing S239D and S239E, respectively, into one of
the chains of IL6R-BP230/IL6R-L showed both higher selectivity and
binding to FcgRIIb than those of IL6R-BP230/IL6R-L. On the other
hand, IL6R-BP259/IL6R-L and IL6R-BP260/IL6R-L produced by
introducing S239D and S239E respectively into both chains showed
greatly decreased selectivity and binding to FcgRIIb as compared to
those of IL6R-BP230/IL6R-L. Furthermore, variants produced by
introducing S239D or S239E into one of the chains of
IL6R-BP208/IL6R-L and IL6R-BP230/IL6R-L all showed greater
selectivity as well as binding to FcgRIIb as compared to those of
IL6R-BP253/IL6R-L utilizing a known FcgRIIb-enhancement
technique.
9-3. Verification of Alterations Based on Structural Information on
Fc(P208)/FcgRIIaR
[0379] Comparison of the crystal structures of Fc(P208) with
FcgRIIb and with FcgRIIaR in Example 3 showed that there is a
difference in electron density around position 237 (EU numbering)
where a hydrogen bond is formed with Tyr at position 160 of
FcgRIIb, and the CH2 domain A side was suggested to have a larger
contribution to binding with FcgRIIb, and the CH2 domain B side was
suggested to have a larger contribution to binding with FcgRIIaR
(FIGS. 12 and 13). For example, from how the electron density
looks, in the binding with the FcgRIIa type R, Leu at position 234
and Leu at position 235 (EU numbering) in CH2 domain B are
considered to be involved in binding with the receptor, whereas
these residues may only have little involvement in the binding to
FcgRIIb. Then, by substituting these two residues with residues
other than hydrophobic residues, interaction with FcgRIIa type R
may be reduced by a greater degree. However, at the CH2 domain A
side, the residues of Leu at position 234 and Leu at position 235
(EU numbering) are considered to contribute to stabilization of the
loop structure around position 237 (EU numbering), and in
particular, it is highly likely that they are more greatly involved
in binding with FcgRIIb. Therefore, substituting these residues
with residues other than hydrophobic residues may decrease
interaction of CH2 domain A with FcgRIIb. In particular, Leu at
position 235 (EU numbering) forms a favorable hydrophobic
interaction in CH2 domain A of a complex structure formed with
FcgRIIb, and since it is considered to have a large contribution to
stabilization of the loop structure around position 237 (EU
numbering), this residue was examined by substituting a residue in
only one of the chains with a non-hydrophobic residue. If the
hydrophobic interaction at the CH2 domain A in particular can
further be strengthened to further stabilize the loop structure
around position 237 (EU numbering) by substituting Leu at position
235 (EU numbering) with hydrophobic amino acids other than Leu in
both chains, that may lead to reduction of entropic energy loss
accompanying hydrogen bond formation with Tyr at position 160 of
FcgRIIb and may cause enhancement of selectivity and binding to
FcgRIIb; therefore, these were examined as well.
[0380] As the antibody H chain, IL6R-BP264 (SEQ ID NO: 28) was
produced by introducing E233D, G237D, P238D, H268E, P271G, Y296D,
and A330R into IL6R-B3 (SEQ ID NO: 23) was used as the template.
Variants in which Leu at position 234 (EU numbering) of IL6R-BP264
individually was substituted with Asn, Ser, Asp, Gln, Glu, Thr,
Arg, His, Gly, Lys, and Tyr were produced. Variants in which the
amino acid at position 235 (EU numbering) in IL6R-BP264 was
substituted with any of the 18 amino acids other than the original
amino acid and Cys were also produced. For the other antibody H
chain, IL6R-AP029 (SEQ ID NO: 42) was produced by introducing
E233D, G237D, P238D, H268E, P271G, Y296D, and A330R into IL6R-A5
(SEQ ID NO: 69). IL6R-L (SEQ ID NO: 21) was commonly used as the
antibody L chain. Variants produced by introducing L234N, L234S,
L234D, L234Q, L234E, L234T, L234R, L234H, L234G, L234K, and L234Y
into IL6R-BP264 and variants produced by introducing L235W, L235M,
L235P, L235F, L235A, L235V, and L235I, respectively, into
IL6R-BP264 were prepared as homologous antibodies containing the
same alteration in both chains and variants produced by introducing
L235N, L235S, L235D, L235Q, L235E, L235T, L235R, L235H, L235G,
L235K, and L235Y, respectively, were combined with IL6R-AP029 to
prepare heterodimeric antibodies, and those were then subjected to
examination.
[0381] These variants were used for antibody expression and
purification according to the method of Reference Example 1, and
binding to each FcgR (FcgRIa, FcgRIIa type H, FcgRIIa type R,
FcgRIIb, and FcgRIIIa type V) was assessed using the method of
Reference Example 2. FIG. 16 shows a graph in which KD values of
each variant for FcgRIIb are shown on the horizontal axis and the
KD values of each variant for FcgRIIaR are shown on the vertical
axis.
[0382] As shown in FIG. 16, IL6R-BP404/IL6R-L produced by
introducing L234Y into both chains of IL6R-BP264/IL6R-L showed
slightly enhanced binding to FcgRIIb as compared to that of the
IL6R-BP264/IL6R-L prior to alteration.
[0383] From among these variants, IL6R-BP404/IL6R-L with enhanced
FcgRIIb binding and variants with improved selectivity to FcgRIIb
are summarized in Table 11. In this Table, "parent polypeptide KD
(IIb)/altered polypeptide KD (IIb)" refers to a value obtained by
dividing the KD value of IL6R-B3/IL6R-L for FcgRIIb by the KD value
of each variant for FcgRIIb. "Parent polypeptide KD (IIaR)/altered
polypeptide KD (IIaR)" refers to a value obtained by dividing the
KD value of IL6R-B3/IL6R-L for FcgR IIaR by the KD value of each
variant for FcgRIIaR. "KD (IIaR)/KD (IIb)" is a value obtained by
dividing the KD of each variant for FcgRIIaR by the KD of each
variant for FcgRIIb. The larger this value is, the higher the
selectivity to FcgRIIb is. The Table 11 values shown in bold
italicized font were calculated using the following equation
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
shown in Reference Example 2 since the binding of FcgR to IgG was
too weak to accurately analyze by kinetic analysis.
[0384] As shown in Table 11, IL6R-BP404/IL6R-L produced by
introducing L234Y into both chains of IL6R-BP264/IL6R-L showed
1.1-fold increase in FcgRIIb binding as compared to that of the
IL6R-BP264/IL6R-L prior to introduction of alterations.
IL6R-BP408/IL6R-L produced by introducing L235Q into both chains of
IL6R-BP264/IL6R-L, IL6R-BP419/IL6R-L produced by introducing L235F
into both chains of IL6R-BP264/IL6R-L, IL6R-AP029/IL6R-BP407/IL6R-L
produced by introducing L235D into one of the chains of
IL6R-BP264/IL6R-L, IL6R-AP029/IL6R-BP408/IL6R-L produced by
introducing L235Q into one of the chains of IL6R-BP264/IL6R-L,
IL6R-AP029/IL6R-BP409/IL6R-L produced by introducing L235E into one
of the chains of IL6R-BP264/IL6R-L, and
IL6R-AP029/IL6R-BP410/IL6R-L produced by introducing L235T into one
of the chains of IL6R-BP264/IL6R-L all showed KD(IIaR)/KD(IIb)
values that were larger compared to that of the IL6R-BP264/IL6R-L
prior to introduction of alteration, and they were variants with
improved selectivity to FcgRIIb.
[Example 10] Assessment of Immunogenicity of Fc Variants with
Enhanced FcgRIIb Binding Using an In Silico Immunogenicity
Prediction Tool
[0385] When using the Fc variants described in this Example as
therapeutic antibodies, it is preferred that production of
anti-drug antibodies that weaken their pharmacological effect is
not induced. Since antibodies with high immunogenicity tend to
induce production of anti-drug antibodies, immunogenicity of
therapeutic antibodies is preferably as low as possible. As efforts
to avoid increase in immunogenicity of the variants as much as
possible, one can use in silico immunogenicity prediction tools
that predict T-cell epitopes such as Epibase.TM. and EpiMatrix
prediction tools. Epibase.TM. Light (Lonza) is an in silico
immunogenicity prediction tool to calculate the binding ability of
9-mer peptide to MHC class II which contains major DRB1 alleles
using the FASTER algorithm (Expert Opin Biol Ther. 2007 March;
7(3): 405-18). This tool can identify T-cell epitopes with strong
binding (strong epitopes) and medium binding (medium epitopes) to
MHC class II.
[0386] DRB1 allotype population frequency is reflected in the
calculation, and for this, Caucasian population frequency shown in
Table 12 below can be used.
TABLE-US-00004 TABLE 12 DRB1*1501 24.5% DRB1*0301 23.7% DRB1*0701
23.3% DRB1*0401 16.2% DRB1*0101 15.0% DRB1*1101 11.6% DRB1*1301
10.9% DRB1*1302 8.2% DRB1*0404 5.9% DRB1*1104 5.8% DRB1*1601 5.0%
DRB1*1401/1454 4.9% DRB1*0801 4.9% DRB1*0102 3.8%
DRB1*1201/1206/1210 3.3% DRB1*0407 2.7% DRB1*0901 2.3% DRB1*1303
2.0% DRB1*1001 1.9% DRB1*0405 1.5% DRB1*0403 1.0% DRB1*1102 0.7%
DRB1*0802 0.7% DRB1*1502 0.5% DRB1*0804 0.4% DRB1*1404 0.4%
DRB1*0803 0.3% DRB1*0406 0.2% DRB1*1402 0.2% DRB1*1602 0.2%
DRB1*1202 0.1% DRB1*0304 0.1% OR LESS DRB1*1405 0.1% OR LESS
DRB1*0410 0.1% OR LESS DRB1*1503 0.1% OR LESS DRB1*1106 0.1% OR
LESS DRB1*1504 0.1% OR LESS DRB1*1304 0.1% OR LESS DRB1*1110 0.1%
OR LESS DRB1*1406 0.1% OR LESS DRB1*0411 0.1% OR LESS DRB1*0302
0.1% OR LESS DRB1*1312 0.1% OR LESS
This tool was used to compare the total number of T-cell epitopes
with strong binding and medium binding that are included in the
sequences (the sequence from position 118 to the C terminus (EU
numbering)) of various Fc variants reported so far and Fc variants
with selectively enhanced binding to FcgRIIb described in the
Example. Specifically, the following antibodies were produced as
comparison controls for evaluating pre-existing techniques: Fc(DLE)
(SEQ ID NO: 78) which is an antibody Fc region introduced with the
S239D, A330L, and I332E alterations, which has been previously
reported to enhance FcgRIIIa-binding (Proc Natl Acad Sci USA. 2006,
103: 4005-10); Fc(YTE) (SEQ ID NO: 79) which is an antibody Fc
region introduced with the M252Y, S254T, and T256E alterations,
which has been previously reported to enhance FcRn-binding (J Biol
Chem. 2006, 281: 23514-24); Fc(EF) (SEQ ID NO: 80) which is an
antibody Fc region introduced with the S267E and L328F alterations,
which has been reported to enhance FcgRIIb-binding (Mol Immunol.
2008, 45: 3926-33); and Fc(P208) (SEQ ID NO: 81) which is an Fc
region of an antibody introduced with the E233D, G237D, P238D,
H268D, P271G, and A330R alterations, which has been reported to
enhance FcgRIIb-binding and is described in WO2012/115241.
Furthermore, Fc(P587) (SEQ ID NO: 70), which is an antibody Fc
region introduced with the E233D, P238D, 52641, S267A, H268E, and
P271G alterations in a similar manner to the BP568 variant, which
enhances FcgRIIb-binding and is described in the Examples, and
Fc(P588) (SEQ ID NO: 71), which is an antibody Fc region introduced
with the P238D, S264I, S267A, H268E, and P271G alterations in a
similar manner to BP492, were produced. The total number of strong
and medium binding epitopes in these Fc variants was compared using
the Epibase.TM. prediction tool. The results are shown in Table
13.
TABLE-US-00005 TABLE 13 NUMBER OF T-cell epitope Fc (DLE) 2 Fc
(YTE) 5 Fc (EF) 4 Fc (P208) 5 Fc (P587) 2 Fc (P588) 2
[0387] These results indicate that among the existing Fc variants,
Fc(P587) and Fc(P588) which are Fc regions of variants described in
the Examples have small number of T-cell epitopes and low
immunogenicity risk. When using the variants as pharmaceuticals,
this property indicates that the possibility of inducing anti-drug
antibodies is lowered and that the variants have excellent
properties.
[Example 11] Assessment of Blood Kinetics of Fc Variants with
Enhanced Human FcgRIIb Binding Using Human FcgRIIb Transgenic Mice
(11-1) Outline of the Examination
[0388] As indicated in WO2013/047752, compared to a native human
IgG, the plasma concentration of a target soluble antigen can be
reduced significantly in a living organism by administering an
antigen-binding molecule having human-FcRn-binding activity under
an acidic pH range condition and comprising an antigen-binding
domain whose antigen binding activity of the antigen-binding
molecule changes depending on an ion concentration condition, and
an FcgR-binding domain with higher FcgR-binding activity than the
FcgR-binding domain of a native human IgG Fc region, wherein the
sugar chain linked to position 297 (EU numbering) is a
fucose-containing sugar chain. It has also been reported that when
an antigen-binding molecule that has enhanced binding activity
particularly to FcgRIIb among the FcgRs is administered in vivo,
elimination of soluble antigens in plasma is accelerated, and the
concentration of soluble antigens in plasma can be reduced
effectively. In this Example, an Fc variant with enhanced binding
to human FcgRIIb was administered to genetically-modified
transgenic mice introduced with human FcgRIIb to test whether the
elimination rate of the target soluble antigens can be accelerated
by the Fc variant with actually enhanced binding to human FcgRIIb
described herein.
(11-2) Preparation of Antibodies with Enhanced Binding to
FcgRIIb
[0389] The following antibodies were used as the Fc variants with
enhanced human FcgRIIb-binding:
IL6R-P587 was produced by introducing the E233D, P238D, 52641,
S267A, H268E, and P271G alterations in a similar manner to BP568
into IL6R-G1d (SEQ ID NO: 19) consisting of a constant region of
G1d which has the C-terminal Gly and Lys removed from human IgG1,
and a variable region of an antibody against human interleukin-6
receptor (human IL-6R) disclosed in WO2009/125825. Fv4-P587
comprising IL-6R-P587 as the antibody H chain and IL6R-L2 (SEQ ID
NO: 74) which is the L chain of an anti-human IL-6R antibody
disclosed in WO2009/125825 as the antibody L chain was prepared
according to the method of Reference Example 1. As a comparison
control, Fv4-IgG1 comprising IL6R-G1d (SEQ ID NO: 19) and IL6R-L2
(SEQ ID NO: 74) as the antibody H chain and L chain, respectively,
was prepared similarly according to the method of Reference Example
1. As described in WO2009/125825, Fv4-G1d and Fv4-P587 prepared
herein comprises an antigen-binding domain whose antigen-binding
activity of the antigen-binding molecule changes depending on the
condition of proton ion concentration, that is, the antigen-binding
activity of the antigen-binding domain binds to a human IL-6R
(antigen) under acidic pH conditions weaker than under neutral pH
conditions.
(11-3) Production of Human FcgRIIb Transgenic Mice
[0390] Human FcgRIIb transgenic mice were produced by the following
method.
[0391] Transgenic mice were produced by introducing the human
FcgRIIb gene into C57BL/6(B6) mice. Production of transgenic mice
was carried out in accordance with the procedure described in "Nagy
et al., (Manipulating the mouse embryo, CSHL press. (2003)
399-506)" and in "Ueda et al. (Latest Technology for Gene
Targeting", Yodosha. (2000) 190-207)". More specifically,
transgenic mice were produced by microinjecting into pronuclear
fertilized eggs of B6 mice a bacterial artificial chromosome into
which the genomic region of the human FcgRIIb gene (GeneBank
#NW_004077999: 18,307,411-18,381,603) was cloned. Mice transferred
with the human FcgRIIb gene were selected from the obtained mice by
Southern blotting using a probe that specifically hybridizes with
the human FcgRIIb gene and by performing PCR. Blood and liver were
collected from the human FcgRIIb transgenic mice, and expression of
the human FcgRIIb gene was confirmed by Reverse Transcription
Polymerase Chain Reaction (RT-PCR) using primers that specifically
amplify the human FcgRIIb gene. As a result, expression of the
human FcgRIIb gene was detected. Furthermore, mouse peripheral
blood mononuclear cells (PBMC) were isolated from the blood of the
human FcgRIIb transgenic mice, and the expression of human FcgRIIb
in PBMC was confirmed by fluorescence activated cell sorting (FACS)
analyses. As a result, expression of human FcgRIIb was detected.
The above confirmed that human FcgRIIb transgenic mice which
express human FcgRIIb were established.
(11-4) In Vivo Test of Simultaneous Administration of Antigens and
Antibodies Using the Human FcgRIIb Transgenic Mice
[0392] Using the human FcgRIIb transgenic mice produced in (11-3),
soluble human IL-6R which is the antigen and the anti-human IL-6R
antibody prepared in (11-2) were administered simultaneously, and
the plasma concentrations of soluble human IL-6R and anti-human
IL-6R antibody after the administration were evaluated.
[0393] A mixed solution of soluble human IL-6R and anti-human IL-6R
antibody (5 .mu.g/mL and 0.1 mg/mL, respectively) was administered
in a single dose at 10 mL/kg to the tail vein. In this case, since
anti-human IL-6R antibody is present sufficiently in excess with
respect to soluble human IL-6R, almost all of the soluble human
IL-6R is considered to be bound to the antibody. Blood was
collected five minutes, 1 hour, 4 hours, 7 hours, 1 day, 3 days, 7
days, 14 days, 21 days, and 28 days after administration. The
collected blood was immediately centrifuged at 4.degree. C. and
15,000 rpm for 15 minutes to obtain the plasma. The separated
plasma was stored in a freezer set at -20.degree. C. or lower until
the time of measurement. The above-described Fv4-P587 and Fv4-IgG1
were used for the anti-human IL-6R antibody.
(11-5) Measurement of Plasma Anti-Human IL-6R Antibody
Concentration by ELISA
[0394] Concentration of anti-human IL-6R antibody in mouse plasma
was measured by ELISA. First, anti-human IgG (.gamma.-chain
specific) antibody F(ab')2 fragment (Sigma) was aliquoted into a
Nunc-Immuno.TM. MaxiSorp.TM. plate (Nalge Nunc International),
followed by allowing to stand overnight at 4.degree. C. to prepare
an anti-human IgG-immobilized plate. Calibration curve samples of
plasma concentration at 0.8, 0.4, 0.2, 0.1, 0.05, 0.025 and 0.0125
.mu.g/mL and mouse plasma assay samples diluted to 100-fold or more
were prepared. Mixtures obtained by adding 200 .mu.L of 20 ng/mL
soluble human IL-6R to 100 .mu.l of the calibration curve samples
or plasma assay samples were then stirred for 1 hour at room
temperature. Subsequently, the anti-human IgG-immobilized plate in
which the mixtures had been dispensed was further stirred for one
hour at room temperature. Then, a biotinylated anti-human IL-6R
antibody (R&D) was reacted with the samples at room temperature
for one hour and Streptavidin-PolyHRP80 (Stereospecific Detection
Technologies) was reacted with the samples at room temperature for
one hour. The chromogenic reaction was carried out using TMB One
Component HRP Microwell Substrate (BioFX Laboratories) as
substrate. After the reaction was stopped by adding 1N sulfuric
acid (Showa Chemical), absorbance at 450 nm was measured with a
microplate reader. Antibody concentrations in the mouse plasma were
calculated from absorbance values of the calibration curve using
the SOFTmax.TM. PRO analysis software (Molecular Devices). The time
course of plasma antibody concentration in human FcgRIIb transgenic
mice after intravenous administration measured by this method is
shown in FIG. 33.
(11-5) Measurement of Plasma Human IL-6R Concentration by
Electrochemiluminescence
[0395] The human IL-6R concentration in mouse plasma was measured
by electrochemiluminescence. Calibration curve samples of human
IL-6R were prepared at plasma concentrations of 12.5, 6.25, 3.13,
1.56, 0.781, 0.391, and 0.195 ng/mL, and mouse plasma assay samples
were prepared by diluting 50 fold or more. Monoclonal Anti-human
IL-6R Antibody (R&D) which has been ruthenium-labeled using
SULFO-TAG NHS Ester (Meso Scale Discovery), Biotinylated Anti-human
IL-6 R Antibody (R&D), and tocilizumab solution were mixed in
and allowed to react overnight at 37.degree. C. Then, the mixed
solution was aliquoted into the Streptavidin Gold Multi-ARRAY Plate
(Meso Scale Discovery) subjected to blocking using a TBS-Tween
solution containing 0.5% BSA (w/v) overnight at 5.degree. C. After
allowing to react for two more hours at room temperature, the plate
was washed. Immediately after the Read Buffer T (.times.2) (Meso
Scale Discovery) was aliquoted into the plate, and measurements
were carried out using the SECTOR.RTM. Imager 2400 (Meso Scale
Discovery). The hSIL-6R concentrations were calculated based on the
response from the calibration curve using the analytical software
SOFTmax.TM. PRO (Molecular Devices). Time course of the soluble
human IL-6R concentration in plasma of human FcgRIIb transgenic
mice after intravenous administration, which was measured by this
method, is shown in FIG. 34.
(11-6) Effects of Enhancing Human FcgRIIb Binding
[0396] The in vivo test results were compared for Fv4-P587 whose
human FcgRIIb-binding has been enhanced and Fv4-IgG1. As shown in
FIG. 33, the plasma retention of both antibodies were nearly equal;
however, as shown in FIG. 34, elimination of human IL-6R was
confirmed to be accelerated when human IL-6R was administered
simultaneously with Fv4-P587 having enhanced human FcgRIIb-binding
as compared to when human IL-6R was administered simultaneously
with Fv4-IgG1. More specifically, an antibody that binds to human
IL-6R in a pH-dependent manner was found to be able to decrease the
concentration of soluble human IL-6R by enhancing its binding
ability to human FcgRIIb.
[0397] Without being bound by a particular theory, one may consider
from this result that according to the mechanism indicated in FIG.
35, the soluble antigens in plasma that bind to this antibody
disappear as a result of being incorporated into FcgRIIb-expressing
cells via human Fc.gamma.RIIb.
[0398] Soluble human IL-6R bound to an antibody that binds soluble
human IL-6R is recycled into plasma by FcRn along with the
antibody. In contrast, Fv4-IgG1 which is an antibody that binds to
soluble human IL-6R in a pH-dependent manner dissociates the
antibody-bound soluble human IL-6R under acidic conditions in the
endosome. Since the dissociated soluble human IL-6R is degraded by
the lysosome, elimination of soluble human IL-6R can be
significantly accelerated, and Fv4-IgG1 which is an antibody that
binds to soluble human IL-6R in a pH-dependent manner binds to FcRn
in the endosome and is then recycled into plasma. This recycled
antibody can bind again to soluble human IL-6R; therefore, antigen
(soluble human IL-6R)-binding and recycling into plasma by FcRn are
repeated. It is considered that as a result, single antibody
molecule can bind to the soluble human IL-6R several times
repeatedly. Furthermore, it is considered that by enhancing the
FcgRIIb-binding activity of Fv4-IgG1 which shows pH-dependent
antigen binding, a complex formed between an antibody that binds to
the soluble human IL-6R and a soluble human IL-6R is quickly
incorporated into cells via FcgRIIb to enable decrease in the
soluble human IL-6R concentration more efficiently (FIG. 35).
[Example 12] Evaluation of Blood Kinetics of Fc Variants with
Enhanced Binding to Human FcgRIIb Using Human FcgRIIb and Human
FcRn Transgenic Mice
(12-1) Outline of the Examination
[0399] As indicated in WO2013/047752, use of an antigen-binding
molecule having higher Fc.gamma.R-binding activity than that of the
native IgG Fc region and whose human FcRn-binding activity under an
acidic pH range condition is enhanced confirmed that plasma
retention properties are improved compared to antigen-binding
molecules whose human FcRn-binding activity under an acidic pH
range condition is not enhanced. On the other hand, there have been
reports that an antigen-binding molecule having higher
Fc.gamma.R-binding activity than that of the native human IgG Fc
region and whose human FcRn-binding activity under an acidic pH
range condition is enhanced showed decreased plasma concentration
of target antigen as compared to an antigen-binding molecule having
higher Fc.gamma.R-binding activity than that of the native human
IgG Fc region and whose human FcRn-binding activity under an acidic
pH range condition is not enhanced. Accordingly, whether the
antigen-binding molecules carrying the Fc region variants with
enhanced human FcgRIIb binding described in the Examples have
similar properties was investigated.
(12-2) Production of an Antigen-Binding Molecule Having Higher
Fc.gamma.R-Binding Activity than that of a Native Human IgG Fc
Region and Whose Human FcRn-Binding Activity Under an Acidic pH
Range Condition is Enhanced
[0400] In addition to Fv4-IgG1 and Fv4-P587 described in Example
11-2, Fv4-P587-LS was prepared according to the method of Reference
Example 1, where Fv4-P587-LS contains IL6R-L2 as the antibody L
chain and IL6R-P587-LS (SEQ ID NO: 73) as the antibody H chain.
IL6R-P587-LS was produced by introducing into IL6R-P587, the H
chain of Fv4-P587, alterations consisting of substitution of Met at
position 428 with Leu and substitution of Asn at position 434 with
Ser, according to EU numbering, which are substitutions that have
been previously reported to improve blood kinetics of antibodies
(Nat. Biotechnol. 2010. 28; 157-159).
(12-3) Analysis of Interaction with Human FcRn
[0401] Analysis of interaction between a prepared antibody and the
human FcRn was carried out using a Biacore.TM. T200 surface plasmon
resonance system. An appropriate amount of protein L (BioVision)
was immobilized onto Sensor Chip CM4 (GE Healthcare) by the amine
coupling method and the antibodies of interest were captured onto
it. Next, a diluted FcRn and a running buffer (used as a control
solution) were injected to allow interaction of the antibodies
captured onto this sensor chip with human FcRn. 50 mmol/L sodium
phosphate, 150 mmol/L NaCl, and 0.05% (w/v) Tween20 (pH 6.0) was
used as the running buffer, and this running buffer was also used
to dilute FcRn. 10 mmol/L glycine-HCl (pH 1.5) was used for chip
regeneration. All measurements were performed at 25.degree. C.
Kinetic parameters such as association rate constants ka (1/Ms) and
dissociation rate constants kd (1/s) were determined from the
sensorgram obtained from the measurements, and the KD (M) of each
antibody for human FcRn were determined from the values of these
constants. The Biacore.TM. T200 Evaluation Software (GE Healthcare)
was used to calculate each parameter. KD values of the antibodies
prepared this time for human FcRn as measured by this method are
shown in Table 14. As shown in Table 14, compared to Fv4-P587,
Fv4-P587-LS was confirmed to have enhanced FcRn binding under
acidic conditions.
TABLE-US-00006 TABLE 14 KD FOR HUMAN FcRn AT pH 6.0 (.mu.mol/L)
IgG1 1.4 P587 1.5 P587-LS 0.12
(12-4) Production of Human FcgRIIb and Human FcRn Transgenic
Mice
[0402] Human FcgRIIb and human FcRn transgenic mice, and mouse FcRn
knockout mice were produced by the following method.
[0403] First, mouse FcRn knockout mice were produced. Production of
knockout mice was carried out according to the procedure described
in "Nagy et al., (Manipulating the mouse embryo, CSHL press. (2003)
399-506)". More specifically, a targeting vector to destroy the
mouse FcRn gene is prepared and introduced into ES cells (derived
from C57BL/6 mice) to destroy mouse FcRn gene by homologous
recombination. RNA was extracted from the liver of the established
mouse FcRn knockout mice, and by using cDNA synthesized from this
RNA as a template, RT-PCR was carried out using primers that
specifically amplify mouse FcRn. As a result, the mouse FcRn gene
was not detected from mouse FcRn knockout mice. Next, transgenic
mice were produced by introducing the human FcgRIIb and human FcRn
genes into the mouse FcRn knockout mice. Transgenic mice were
produced according to the procedures described in "Nagy et al.,
(Manipulating the mouse embryo, CSHL press. (2003) 399-506)" and in
"Ueda et al. (Latest Technology for Gene Targeting, Yodosha. (2000)
190-207)". More specifically, the mice were produced by
microinjecting into pronuclear fertilized eggs of mouse FcRn
knockout mice a bacterial artificial chromosome into which the
genomic regions of the human FcRn gene (GeneBank
#NC_000019.9:50,000,108-50,039,865) and the human FcgRIIb gene
(GeneBank #NW_004077999:18,307,411-18,381,603) were cloned. Mice
introduced with the human FcRn gene and the human FcgRIIb gene and
made to be homozygous for the mouse FcRn knockout allele were
selected from the obtained mice by Southern blotting using a probe
that specifically hybridizes with each gene and by PCR. Blood was
collected from the human FcgRIIb and human FcRn transgenic mice and
mouse FcRn knockout mice, and expression of the human FcRn gene and
the human FcgRIIb gene were confirmed by RT-PCR using primers that
specifically amplify the human FcRn gene and the human FcgRIIb
gene. As a result, expressions of the human FcRn gene and the human
FcgRIIb gene were detected. The above confirmed that human FcgRIIb
and human FcRn transgenic mice and mouse FcRn knockout mice which
express human FcRn and human FcgRIIb and do not express mouse FcRn
were established.
(12-5) Improvement of Pharmacokinetics by Enhancing Human
FcRn-Binding Activity Under an Acidic pH Range Condition
[0404] In vivo examination was carried out in a manner similar to
the method of Example 11 by administering Fv4-IgG1, Fv4-P587, and
Fv4-P587-LS individually to human FcgRIIb and human FcRn transgenic
mice, and plasma concentrations of soluble IL-6R and anti-human
IL-6R antibody were measured for the mouse groups. The results of
measuring the plasma concentrations of anti-human IL-6R antibody
and soluble IL-6R are shown in FIG. 36 and FIG. 37,
respectively.
[0405] In the group of mice administered with Fv4-P587-LS in which
the binding activity of Fv4-P587 to human FcRn under an acidic pH
range condition has been enhanced, plasma retention of antibodies
was found to be improved compared to that in the group of mice
administered with Fv4-P587. In addition, Fv4-P587-LS showed
improved plasma retention than Fv4-IgG1. On the other hand, the
plasma concentration of soluble IL-6R in the group of
Fv4-P587-LS-administered mice was equivalent to that in the group
of Fv4-P587-administered mice. In the group of Fv4-P587-LS- or
Fv4-P587-administered mice, plasma concentration of soluble IL-6R
was decreased compared to in the group of Fv4-IgG1-administered
mice.
[0406] Accordingly, administration of antibody in which the human
FcRn-binding activity of an antigen-binding molecule under an
acidic pH range condition has been enhanced, wherein human
Fc.gamma.RIIb-binding activity of the antigen-binding molecule is
higher than that of a native human IgG Fc region, showed that
plasma retention of the administered antigen molecule can be
improved in a living organism receiving the administration.
Furthermore, even if the plasma retention is improved in a living
organism administered with the antigen-binding molecule, the
antigen-eliminating effect of the living organism was shown not to
decrease, but rather can be maintained.
[0407] Alterations to enhance the human FcRn-binding activity under
an acidic pH range condition are not particularly limited; and
include: the method for substituting Leu for Met at position 428
and Ser for Asn at position 434 (EU numbering) in an IgG antibody
(Nat. Biotechnol, (2010) 28, 157-159); the method for substituting
Ala for Asn at position 434 (Drug. Metab. Dispos. (2010) 38 (4),
600-605); the method for substituting Tyr for Met at position 252,
Thr for Ser at position 254, and Glu for Thr at position 256 (J.
Biol. Chem. (2006) 281, 23514-23524); the method for substituting
Gln for Thr at position 250 and Leu for Met at position 428 (J.
Immunol. (2006) 176 (1) 346-356); the method for substituting His
for Asn at position 434 (Clin. Pharm. & Ther. (2011) 89 (2)
283-290); and alterations disclosed in WO2010/106180,
WO2010/045193, WO2009/058492, WO2008/022152, WO2006/050166,
WO2006/053301, WO2006/031370, WO2005/123780, WO2005/047327,
WO2005/037867, WO2004/035752, WO2002/060919, and such.
[Reference Example 1] Construction of Antibody Expression Vectors;
and Expression and Purification of Antibodies
[0408] Synthesis of full-length genes encoding the nucleotide
sequences of the H chain and L chain of the antibody variable
regions was carried out by production methods known to those
skilled in the art using Assemble PCR and such. Introduction of
amino acid substitutions was carried out by methods known to those
skilled in the art using PCR or such. The obtained plasmid fragment
was inserted into an animal cell expression vector, and the H-chain
expression vector and L-chain expression vector were produced. The
nucleotide sequence of the obtained expression vector was
determined by methods known to those skilled in the art. The
produced plasmids were introduced transiently into the HEK293H cell
line derived from human embryonic kidney cancer cells (Invitrogen)
or into FreeStyle293.TM. cells (Invitrogen) for antibody
expression. The obtained culture supernatant was collected, and
then passed through a 0.22 .mu.m MILLEX.RTM.-GV filter (Millipore),
or through a 0.45 .mu.m MILLEX.RTM.-GV filter (Millipore) to obtain
the culture supernatant. Antibodies were purified from the obtained
culture supernatant by methods known to those skilled in the art
using a Protein A Sepharose.RTM. 4 Fast Flow gel filtration medium
(GE Healthcare) or a Protein G Sepharose.RTM. 4 Fast Flow gel
filtration medium (GE Healthcare). For the concentration of the
purified antibodies, their absorbance at 280 nm was measured using
a spectrophotometer. From the obtained value, the extinction
coefficient calculated by the methods such as PACE was used to
calculate the antibody concentration (Protein Science 1995; 4:
2411-2423).
[Reference Example 2] Method for Preparing Fc.gamma.R and Method
for Analyzing the Interaction Between an Altered Antibody and
Fc.gamma.R
[0409] Extracellular domains of Fc.gamma.Rs were prepared by the
following method. First, a gene of the extracellular domain of
Fc.gamma.R was synthesized by a method well known to those skilled
in the art. At that time, the sequence of each Fc.gamma.R was
produced based on the information registered at NCBI. Specifically,
Fc.gamma.RI was produced based on the sequence of NCBI Accession
No. NM_000566.3, Fc.gamma.RIIa was produced based on the sequence
of NCBI Accession No. NM_001136219.1, Fc.gamma.RIIb was produced
based on the sequence of NCBI Accession No. NM_004001.3,
Fc.gamma.RIIIa was produced based on the sequence of NCBI Accession
No. NM_001127593.1, and Fc.gamma.RIIIb was produced based on the
sequence of NCBI Accession No. NM_000570.3, and a His tag was
attached to the C terminus. Furthermore, polymorphism is known for
Fc.gamma.RIIa, Fc.gamma.RIIIa, and Fc.gamma.RIIIb, and the
polymorphic sites were produced by referring to J. Exp. Med., 1990,
172: 19-25 for Fc.gamma.RIIa; J. Clin. Invest., 1997, 100 (5):
1059-1070 for Fc.gamma.RIIIa; and J. Clin. Invest., 1989, 84,
1688-1691 for Fc.gamma.RIIIb.
[0410] The obtained gene fragments were inserted into an animal
cell expression vector, and expression vectors were produced. The
produced expression vectors were introduced transiently into human
embryonic kidney cancer cell line-derived FreeStyle293.TM. cells
(Invitrogen) to express the proteins of interest. Regarding
Fc.gamma.RIIb used for crystallographic analysis, the protein of
interest was expressed in the presence of Kifunensine at a final
concentration of 10 .mu.g/mL, so that the sugar chain added to
Fc.gamma.RIIb will be the high-mannose type. Cells were cultured,
and after collection of the obtained culture supernatant, this was
passed through a 0.22 .mu.m filter to obtain the culture
supernatant. In principle, the obtained culture supernatants were
purified in the following four steps. The steps carried out were,
cation exchange column chromatography (SP Sepharose.RTM. 4 FF gel
filtration medium) in step 1, affinity column chromatography
(HisTrap.TM. HP affinity column) for His tag in step 2, gel
filtration column chromatography (Superdex.RTM. 200 gel filtration
column) in step 3, and aseptic chromatography in step 4. However,
for Fc.gamma.RI, anion exchange column chromatography using Q
Sepharose.RTM. 4 FF chromatography medium was performed as step 1.
The purified proteins were subjected to absorbance measurements at
280 nm using a spectrophotometer; and from the obtained values, the
concentrations of the purified proteins were calculated using the
absorption coefficient calculated using methods such as PACE
(Protein Science 1995; 4: 2411-2423).
[0411] Analysis of interaction between each altered antibody and
the Fc.gamma. receptor prepared as mentioned above was carried out
using a Biacore.TM. T100 surface plasmon resonance system (GE
Healthcare), a Biacore.TM. T200 surface plasmon resonance system
(GE Healthcare), a Biacore.TM. A100 surface plasmon resonance
system, and Biacore.TM. 4000 surface plasmon resonance system.
HBS-EP+ (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM ethylene diamine
tetraacetic acid (EDTA), 0.05% polysorbate 20) (GE Healthcare) was
used as the running buffer, and the measurement temperature was set
to 25.degree. C. Chips produced by immobilizing the antigen
peptide, Protein A (Thermo Scientific), Protein A/G (Thermo
Scientific), and Protein L (ACTIGEN or BioVision) by the amine
coupling method to a Series S sensor Chip CM5 (GE Healthcare) or
Series S sensor Chip CM4 (GE Healthcare), or alternatively, chips
produced by allowing preliminarily biotinylated antigen peptides to
interact with and immobilize onto a Series S Sensor Chip SA
(certified) (GE Healthcare) were used.
[0412] After capturing of antibodies of interest onto these sensor
chips, an Fc.gamma. receptor diluted with the running buffer was
allowed to interact, the amount bound to an antibody was measured,
and the antibodies were compared. However, since the amount of
Fc.gamma. receptor bound depends on the amount of the captured
antibodies, the amount of Fc.gamma. receptor bound was divided by
the amount of each antibody captured to obtain corrected values,
and these values were compared. Furthermore, antibodies captured
onto the chips were washed by reaction with 10 mM glycine-HCl, pH
1.5, and the chips were regenerated and used repeatedly.
[0413] Kinetic analyses for calculating the KD values of each
altered antibody for Fc.gamma.R were performed according to the
following method. First, antibodies of interest were captured onto
the above-mentioned sensor chips, and an Fc.gamma. receptor diluted
with the running buffer was allowed to interact. The Biacore.TM.
Evaluation Software was used to globally fit the measured results
to the obtained sensorgram using the 1:1 Langmuir binding model,
and the association rate constant ka (L/mol/s) and the dissociation
rate constant kd (1/s) were calculated; and from those values the
dissociation constants KD (mol/L) were calculated.
[0414] When the interaction between each of the altered antibodies
and Fc.gamma.R was weak, and correct analysis was determined to be
impossible by the above-mentioned kinetic analysis, the KD for such
interactions were calculated using the following 1:1 binding model
equation described in the Biacore.TM. T100 Software Handbook
BR1006-48 Edition AE.
[0415] The behavior of interacting molecules according to the 1:1
binding model on a Biacore.TM. surface plasmon resonance system can
be described by Equation 1 shown below.
R.sub.eq=CR.sub.max/(KD+C)+RI [Equation 1]
R.sub.eq: a plot of steady-state binding levels against analyte
concentration C: concentration RI: bulk refractive index
contribution in the sample R.sub.max: analyte binding capacity of
the surface
[0416] When this equation is rearranged, KD can be expressed as
Equation 2 shown below.
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
[0417] KD can be calculated by substituting the values of
R.sub.max, RI, and C into this equation. The values of RI and C can
be determined from the sensorgram of the measurement results and
measurement conditions. R.sub.max was calculated according to the
following method. As a target of comparison, for antibodies that
had sufficiently strong interactions as evaluated simultaneously in
the same round of measurement, the R.sub.max value was obtained
through global fitting using the 1:1 Langmuir binding model, and
then it was divided by the amount of the comparison antibody
captured onto the sensor chip, and multiplied by the captured
amount of an altered antibody to be evaluated.
[Reference Example 3] Comprehensive Analysis of the Binding of Fc
Variants to Fc.gamma.R
[0418] Mutations were introduced into IgG1 antibodies to generate
antibodies that have decreased Fc-mediated binding towards
activating Fc.gamma.R, specifically both allotypes of
Fc.gamma.RIIa, types H and R, as well as enhanced Fc.gamma.RIIb
binding relative to IgG1; and binding to each Fc.gamma.R was
analyzed comprehensively.
[0419] The variable region (SEQ ID NO: 15) of a glypican 3 antibody
comprising the CDR of GpH7 which is an anti-glypican 3 antibody
with improved plasma kinetics disclosed in WO 2009/041062 was used
as the common antibody H chain. Similarly, for the common antibody
L chain, GpL16-k0 (SEQ ID NO: 16) of the glypican 3 antibody with
improved plasma kinetics disclosed in WO 2009/041062 was used.
Furthermore, B3 in which a K439E mutation has been introduced into
G1d produced by removing the C terminal Gly and Lys of IgG1 was
used as the antibody H chain constant region. This H chain is
referred to as GpH7-B3 (SEQ ID NO: 17), and the L chain is referred
to as GpL16-k0 (SEQ ID NO: 16).
[0420] With respect to GpH7-B3, the amino acids that are considered
to be involved in Fc.gamma.R binding and the surrounding amino
acids (positions 234 to 239, 265 to 271, 295, 296, 298, 300, and
324 to 337, according to EU numbering) were substituted
respectively with 18 types of amino acids excluding the original
amino acids and Cys. These Fc variants are referred to as B3
variants. B3 variants were expressed and purified using the method
of Reference Example 1, and the binding to each Fc.gamma.R
(Fc.gamma.RIa, Fc.gamma.RIIa type H, Fc.gamma.RIIa type R,
Fc.gamma.RIIb, and Fc.gamma.RIIIa) was comprehensively evaluated
using the method of Reference Example 2.
[0421] Figures were produced based on the results of interaction
analysis with each Fc.gamma.R by the method below. The value of the
amount of Fc.gamma.R binding of each B3 variant-derived antibody
was divided by the value of the amount of Fc.gamma.R binding of the
antibody used for comparison which does not have mutations
introduced into B3 (an antibody having the sequence of a
naturally-occurring human IgG1 at positions 234 to 239, 265 to 271,
295, 296, 298, 300, and 324 to 337, according to EU numbering). The
value obtained by multiplying this value by 100 was used as an
indicator of the relative Fc.gamma.R-binding activity of each
variant. The horizontal axis shows the value of the relative
Fc.gamma.RIIb-binding activity of each variant, and the vertical
axis shows the value of the respective relative binding activity of
each variant towards activating Fc.gamma.Rs: Fc.gamma.RIa,
Fc.gamma.RIIa type H, Fc.gamma.RIIa type R, and Fc.gamma.RIIIa
(FIGS. 17, 18, 19, and 20).
[0422] As shown by labels in FIGS. 17-20, the results show that of
all alterations, when only mutations called mutation A (alteration
produced by substituting Pro at position 238 (EU numbering) with
Asp) and mutation B (alteration produced by substituting Leu at
position 328 (EU numbering) with Glu) were introduced, there were
remarkable enhancement of binding to Fc.gamma.RIIb and remarkable
suppression of binding to both types of Fc.gamma.RIIa compared with
before the introduction.
[Reference Example 4] SPR Analysis of Variants that Selectively
Bind to Fc.gamma.RIIb
[0423] With regard to the alteration identified in Example 1 where
Pro at position 238 (EU numbering) is substituted with Asp, the
binding to each Fc.gamma.R was analyzed in detail.
[0424] The variable region of IL6R-H (SEQ ID NO: 18), which is the
variable region of the antibody against the human interleukin 6
receptor disclosed in WO 2009/125825, was used as the antibody H
chain variable region, and IL6R-G1d (SEQ ID NO: 19) which comprises
G1d with deletion of C-terminal Gly and Lys of human IgG1 was used
as the antibody H chain constant region in the IgG1 H chain. Pro at
position 238 (EU numbering) in IL6R-G1d was substituted with Asp to
produce IL6R-G1d-v1 (SEQ ID NO: 20). Next, Leu at position 328 (EU
numbering) in IL6R-G1d was substituted with Glu to produce
IL6R-G1d-v2. Furthermore, for comparison, Ser at position 267 (EU
numbering) was substituted with Glu, and Leu at position 328 (EU
numbering) was substituted with Phe in IL6R-G1d to produce
IL6R-G1d-v3 as described in Non-patent Document 28. IL6R-L (SEQ ID
NO: 21), which is the L chain of tocilizumab, was utilized as a
mutual antibody L chain; and together with each H chain, the
antibodies were expressed and purified according to the method of
Reference Example 1. The obtained antibodies which comprise an
amino acid sequence derived from IL6R-G1d, IL6R-G1d-v1,
IL6R-G1d-v2, or IL6R-G1d-v3 as the antibody H chain are referred to
as IgG1, IgG1-v1, IgG1-v2, and IgG1-v3, respectively.
[0425] Next, kinetic analysis of interactions between these
antibodies and Fc.gamma.R was carried out using a Biacore.TM. T100
surface plasmon resonance system (GE Healthcare). HBS-EP+(GE
Healthcare) was used as the running buffer, and the measurement
temperature was set to 25.degree. C. A chip produced by
immobilizing Protein A onto a Series S Sensor Chip CM5 (GE
Healthcare) by the amine-coupling method was used. An antibody of
interest was captured onto this chip to interact with each
Fc.gamma.R that had been diluted with the running buffer, and
binding to the antibody was measured. After the measurement, the
antibody captured on the chip was washed off by allowing reaction
with 10 mM glycine-HCl, pH 1.5, and the chip was regenerated and
used repeatedly. The sensorgrams obtained as measurement results
were analyzed by the 1:1 Langmuir binding model using the
Biacore.TM. Evaluation Software to calculate the binding rate
constant ka (L/mol/s) and dissociation rate constant kd (1/s), and
the dissociation constant KD (mol/L) was calculated from these
values.
[0426] This time, since the binding of IgG1-v1 and IgG1-v2 to
Fc.gamma.RIIa type H and to Fc.gamma.RIIIa was weak, kinetic
parameters such as KD could not be calculated from the
above-mentioned analytical method. Regarding such interactions, KD
values were calculated using the following 1:1 binding model
described in the Biacore.TM. T100 Software Handbook BR1006-48
Edition AE.
[0427] The behavior of interacting molecules according to the 1:1
binding model on a Biacore.TM. surface plasmon resonance system can
be described by Equation 1 shown below.
R.sub.eq=CR.sub.max/(KD+C)+RI [Equation 1]
R.sub.eq: a plot of steady-state binding levels against analyte
concentration C: concentration RI: bulk refractive index
contribution in the sample R.sub.max: analyte binding capacity of
the surface
[0428] When this equation is rearranged, KD can be expressed as
Equation 2 shown below.
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
[0429] KD can be calculated by substituting the values of
R.sub.max, RI, and C into this equation. From the current
measurement conditions, RI=0, C=2 .mu.mol/L can be used.
Furthermore, the R.sub.max value obtained when globally fitting the
sensorgram obtained as a result of analyzing the interaction of
each Fc.gamma.R with IgG1 using the 1:1 Langmuir binding model was
divided by the amount of IgG1 captured, this was multiplied by the
amount of IgG1-v1 and IgG1-v2 captured, and the resulting value was
used as R.sub.max. This calculation is based on the hypothesis that
the limit quantity of each Fc.gamma.R that can be bound by IgG1
remains unchanged for all variants produced by introducing
mutations into IgG1, and the R.sub.max at the time of measurement
is proportional to the amount of antibody bound on the chip at the
time of measurement. R.sub.eq was defined as the amount of binding
of each Fc.gamma.R to each variant on the sensor chip observed at
the time of measurement.
[0430] Under these measurement conditions, the amount of binding
(R.sub.eq) of IgG1-v1 and IgG1-v2 to Fc.gamma.RIIa type H was
approximately 2.5 RU and 10 RU, respectively, and the amount of
binding (R.sub.eq) of IgG1-v1 and IgG1-v2 to Fc.gamma.RIIIa was
approximately 2.5 RU and 5 RU, respectively. The amount of IgG1,
IgG1-v1, and IgG1-v2 captured in the analysis of interactions with
H-type Fc.gamma.RIIa was 452 RU, 469.2 RU, and 444.2 RU,
respectively, and the amount of IgG1, IgG1-v1, and IgG1-v2 captured
in the analysis of interactions with Fc.gamma.RIIIa was 454.5 RU,
470.8 RU, and 447.1 RU, respectively. The R.sub.max values obtained
from global fitting of sensorgrams obtained as a result of
analyzing the interaction of IgG1 with H-type Fc.gamma.RIIa and
Fc.gamma.RIIIa using the 1:1 Langmuir binding model were 69.8 RU
and 63.8 RU, respectively. When these values were used, the
calculated R.sub.max values of IgG1-v1 and IgG1-v2 to Fc.gamma.RIIa
type H were 72.5 RU and 68.6 RU, respectively, and the calculated
R.sub.max values of IgG1-v1 and IgG1-v2 to Fc.gamma.RIIIa were 66.0
RU and 62.7 RU, respectively. These values were substituted into
Equation 2 to calculate the KD of IgG1-v1 and IgG1-v2 for
Fc.gamma.RIIa type H and Fc.gamma.RIIIa.
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
[0431] The KD values of IgG1, IgG1-v1, IgG1-v2, and IgG1-v3 for
each Fc.gamma.R (the KD values of each antibody for each
Fc.gamma.R) are shown in Table 15, and the relative KD values of
IgG1-v1, IgG1-v2, and IgG1-v3 obtained by taking the KD values of
IgG1 for each Fc.gamma.R and dividing them by the KD values of
IgG1-v1, IgG1-v2, and IgG1-v3 for each Fc.gamma.R (the relative KD
values of each antibody for each Fc.gamma.R) are shown in Table
16.
TABLE-US-00007 TABLE 15 IgG1 IgG1-v1 IgG1-v2 IgG1-v3 Fc.gamma.RIa
3.4E-10 7.3E-09 4.6E-10 1.9E-10 Fc.gamma.RIIa R 1.2E-06 1.2E-05
2.9E-06 2.3E-09 Fc.gamma.RIIa H 7.7E-07 5.6E-05* 1.2E-05* 1.5E-06
Fc.gamma.RIIb 5.3E-06 1.1E-06 2.3E-06 1.3E-08 Fc.gamma.RIIIa
3.1E-06 5.1E-05* 2.3E-05* 8.8E-06 (mol/L)
[0432] In Table 15 shown above, "*" means that the KD value was
calculated using Equation 2 because binding of Fc.gamma.R to IgG
was not sufficiently observed.
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
TABLE-US-00008 TABLE 16 IgG1-v1 IgG1-v2 IgG1-v3 Fc.gamma.RIa 0.047
0.74 1.8 Fc.gamma.RIIa R 0.10 0.41 522 Fc.gamma.RIIa H 0.014 0.064
0.51 Fc.gamma.RIIb 4.8 2.3 408 Fc.gamma.RIIIa 0.061 0.14 0.35 (THE
VALUE OBTAINED BY DIVIDING THE KD VALUE OF IgG1 FOR EACH Fc.gamma.R
BY THE KD VALUE OF EACH ANTIBODY IgG1 FOR EACH Fc.gamma.R)
[0433] According to Table 16, when compared with that of IgG1, the
binding activity of IgG1-v1 was decreased to 0.047-fold for
Fc.gamma.RIa, decreased to 0.10-fold for Fc.gamma.RIIa type R,
decreased to 0.014-fold for Fc.gamma.RIIa type H, decreased to
0.061-fold for Fc.gamma.RIIIa, and increased to 4.8-fold for
Fc.gamma.RIIb.
[0434] Furthermore, according to Table 16, when compared with that
of IgG1, the binding activity of IgG1-v2 was decreased to 0.74-fold
for Fc.gamma.RIa, decreased to 0.41-fold for Fc.gamma.RIIa type R,
decreased to 0.064-fold for Fc.gamma.RIIa type H, decreased to
0.14-fold for Fc.gamma.RIIIa, and increased to 2.3-fold for
Fc.gamma.RIIb.
[0435] More specifically, these results demonstrated that IgG1-v1
having an alteration of substituting Pro at position 238 (EU
numbering) with Asp and IgG1-v2 having an alteration of
substituting Leu at position 328 (EU numbering) with Glu have the
properties of weakening the binding to all activating Fc.gamma.Rs
including both allotypes of Fc.gamma.RIIa, while enhancing the
binding to Fc.gamma.RIIb which is an inhibitory Fc.gamma.R.
[0436] Next, selectivity of the obtained variant to Fc.gamma.RIIb
was evaluated by using the ratio of Fc.gamma.RIIb-binding activity
to the binding activity towards type R or type H of Fc.gamma.RIIa
as the indicator. Specifically, I/A(R) or I/A(H), which is a value
obtained by dividing the KD value for Fc.gamma.RIIa type R or type
H by the KD value for Fc.gamma.RIIb, was used as an indicator for
the selectivity of Fc.gamma.RIIb with respect to each
Fc.gamma.RIIa. This indicator has a greater value when the KD value
for Fc.gamma.RIIb becomes smaller or when the KD value for
Fc.gamma.RIIa becomes larger. That is, a variant that shows a
larger value shows an increased binding activity for Fc.gamma.RIIb
relative to Fc.gamma.RIIa. These indicators are summarized in Table
17 for each variant.
TABLE-US-00009 TABLE 17 IgG1 IgG1-v1 IgG1-v2 IgG1-v3 I/A (R) 0.23
11 1.3 0.18 I/A (H) 0.15 51 5.2 115
[0437] According to the results of Table 17, in comparison with
IgG1, IgG1-v3 which was produced by applying the existing
technology showed a greater I/A(H) value than that of IgG1 and a
greater selectivity for Fc.gamma.RIIb, but a smaller I/A(R) value
than that of IgG1 and an improved selectivity for Fc.gamma.RIIb. On
the other hand, IgG1-v1 and IgG1-v2 found in the Examples have
larger I/A(R) and I/A(H) values than those of IgG1, and improved
selectivity for Fc.gamma.RIIb over both allotypes of
Fc.gamma.RIIa.
[0438] So far, alterations having such properties have not been
reported, and they are in fact very rare as shown in FIGS. 17, 18,
19, and 20. Alterations produced by substituting Pro at position
238 (EU numbering) with Asp or substituting Leu at position 328 (EU
numbering) with Glu are very useful for the development of
therapeutic agents for immunological inflammatory diseases and
such
[0439] Furthermore, Table 16 shows that IgG1-v3 described in
Non-patent Document 28 certainly shows a 408-fold enhanced binding
to Fc.gamma.RIIb, while the binding to Fc.gamma.RIIa type H is
decreased to 0.51 fold, and the binding to Fc.gamma.RIIa type R is
enhanced to 522 fold. According to these results, since IgG1-v1 and
IgG1-v2 suppress their binding to both Fc.gamma.RIIa types R and H,
and enhance their binding to Fc.gamma.RIIb, they are considered to
be variants that bind with a greater Fc.gamma.RIIb selectivity
compared with IgG1-v3. Specifically, alterations produced by
substituting Pro at position 238 (EU numbering) with Asp or
substituting Leu at position 328 (EU numbering) with Glu are very
useful for the development of therapeutic agents for immunological
inflammatory diseases and such.
[Reference Example 5] Effects of Combining Fc.gamma.RIIb-Selective
Binding Alterations with Other Fc Region Amino Acid
Substitutions
[0440] Further enhancement of the selectivity for Fc.gamma.RIIb was
attempted based on the variant which has improved selectivity for
Fc.gamma.RIIb and has a substitution of Pro at position 238 (EU
numbering) with Asp found in Reference Examples 3 and 4.
[0441] First, into IL6R-G1d_v1 (SEQ ID NO: 20) produced by
introducing into IL6R-G1d the alteration produced by substituting
Pro at position 238 (EU numbering) with Asp, the substitution of
Leu at position 328 (EU numbering) with Glu as described in
Reference Example 4 which enhances selectivity for Fc.gamma.RIIb
was introduced to produce the IL6R-G1d-v4 variant. This was
combined with IL6R-L (SEQ ID NO: 21) and prepared according to the
method of Reference Example 1. The obtained antibody having the
amino acid sequence derived from IL6R-G1d-v4 as the antibody H
chain has been named IgG1-v4. The binding activities of IgG1,
IgG1-v1, IgG1-v2, and IgG1-v4 to Fc.gamma.RIIb were evaluated
according to the method of Reference Example 2, and those results
are shown in Table 18.
TABLE-US-00010 TABLE 18 Relative KD KD for for Fc.gamma.RIIb
Fc.gamma.RIIb (KD of IgG1/KD Variant Alteration (mol/L) of each
variant) IgG1 -- 5.30E-06 1 IgG1-v1 Substitution of Pro at position
1.10E-06 4.8 238 (EU numbering) with Asp IgG1-v2 Substitution of
Leu at position 2.30E-06 2.3 328 (EU numbering) with Glu IgG1-v4
Substitution of Pro at position 1.10E-05 0.47 238 (EU numbering)
with Asp and substitution of Leu at position 328 (EU numbering)
with Glu
[0442] From the results of Table 18, since L328E improves the
Fc.gamma.RIIb-binding activity by 2.3 fold compared with IgG1,
combining it with P238D which similarly improves the
Fc.gamma.RIIb-binding activity by 4.8 fold compared with IgG1 was
anticipated to further increase the degree of improvement of
Fc.gamma.RIIb-binding activity; however, in reality, the
Fc.gamma.RIIb-binding activity of the variant containing a
combination of these alterations was decreased to 0.47 fold
compared with that of IgG1. This result is an effect that could not
have been predicted from the respective alterations.
[0443] Similarly, into IL6R-G1d-v1 (SEQ ID NO: 20) produced by
introducing into IL6R-G1d the alteration produced by substituting
Pro at position 238 (EU numbering) with Asp, the substitutions of
Ser at position 267 (EU numbering) with Glu and of Leu at position
328 (EU numbering) with Phe as described in Reference Example 4
which improve Fc.gamma.RIIb-binding activity were introduced, and
the IL6R-G1d-v5 variant was prepared according to the method of
Reference Example 1. The obtained antibody having the amino acid
sequence derived from IL6R-G1d-v5 as the antibody H chain has been
named IgG1-v5. The Fc.gamma.RIIb-binding activities of IgG1,
IgG1-v1, IgG1-v3, and IgG1-v5 were evaluated according to the
method of Reference Example 2, and those results are shown in Table
19.
[0444] S267E/L328F which had an enhancing effect on Fc.gamma.RIIb
in Reference Example 4 was introduced into the P238D variant and
its Fc.gamma.RIIb-binding activities before and after introducing
this alteration were evaluated. The results are shown in Table
19.
TABLE-US-00011 TABLE 19 Relative KD KD for for Fc.gamma.RIIb
Fc.gamma.RIIb (KD of IgG1/KD Variant Alteration (mol/L) of each
variant) IgG1 -- 5.30E-06 1 IgG1-v1 Substitution of Pro at position
1.10E-06 4.8 238 (EU numbering) with Asp IgG1-v3 Substitution of
Ser at position 1.30E-08 408 267 (EU numbering) with Glu and
substitution of Leu at position 328 (EU numbering) with Phe IgG1-v5
Substitution of Pro at position 4.50E-07 12 238 (EU numbering) with
Asp, substitution of Ser at position 267 (EU numbering) with Glu,
and substitution of Leu at position 328 (EU numbering) with Phe
[0445] From the results of Table 19, since S267E/L328F improves the
Fc.gamma.RIIb-binding activity by 408 fold compared with IgG1,
combining it with P238D which similarly improves the
Fc.gamma.RIIb-binding activity by 4.8 fold as compared with IgG1
was anticipated to further increase the degree of improvement of
Fc.gamma.RIIb-binding activity; however, in reality, in a similar
manner to the former example, the Fc.gamma.RIIb-binding activity of
the variant containing a combination of these alterations was
improved only 12 fold or so as compared with that of IgG1. This
result is also an effect that could not have been predicted from
the effects of the respective alterations.
[0446] These results showed that while the substitution of Pro at
position 238 (EU numbering) with Asp alone improves
Fc.gamma.RIIb-binding activity, the effect is not exhibited when it
is combined with other alterations that improve the
Fc.gamma.RIIb-binding activity. A reason for this may be that the
structure at the interacting interface between Fc and Fc.gamma.R is
changed by introducing the substitution of Pro at position 238 (EU
numbering) with Asp and the effects of alterations observed in the
naturally-occurring antibody are no longer reflected in the
results. Accordingly, it was considered to be extremely difficult
to create an Fc with excellent selectivity for Fc.gamma.RIIb using
an Fc comprising substitution of Pro at position 238 (EU numbering)
with Asp as a template, since the information on effects of
alterations obtained with naturally-occurring antibodies could not
be applied.
[Reference Example 6] Comprehensive Analysis of Fc.gamma.RIIb
Binding of Variants Introduced with an Alteration at the Hinge
Portion in Addition to the P238D Alteration
[0447] As shown in Reference Example 5, in an Fc produced by
substituting Pro at position 238 (EU numbering) with Asp in a
naturally-occurring human IgG1, an anticipated combinatorial effect
could not be obtained even by combining it with another alteration
predicted to further increase Fc.gamma.RIIb binding. Therefore,
based on the Fc variant produced by substituting Pro at position
238 (EU numbering) with Asp, examination was carried out by
comprehensively introducing alterations into the Fc to find
variants that further enhance Fc.gamma.RIIb binding. For the
antibody H chains, IL6R-F11 (SEQ ID NO: 22) was produced by
introducing an alteration of substituting Met at position 252 (EU
numbering) with Tyr and an alteration of substituting Asn at
position 434 (EU numbering) with Tyr into IL6R-G1d (SEQ ID NO: 19),
and IL6R-F652 was prepared by introducing an additional alteration
of substituting Pro at position 238 (EU numbering) with Asp.
Expression plasmids containing an antibody H chain sequence were
prepared for each of the antibody H chain sequences produced by
substituting the region near the residue at position 238 (EU
numbering) (positions 234 to 237, and 239 (EU numbering)) in
IL6R-F652 each with 18 amino acids excluding the original amino
acids and cysteine. IL6R-L (SEQ ID NO: 21) was utilized as a common
antibody L chain for all of the antibodies. These variants were
expressed, purified, and expressed by the method of Reference
Example 1. These Fc variants are called PD variants. Interactions
of each PD variant with Fc.gamma.RIIa type R and Fc.gamma.RIIb were
comprehensively evaluated by the method of Reference Example 2.
[0448] With regard to the results of analyzing the interaction with
the respective Fc.gamma.Rs, a figure was produced according to the
following method. The value obtained by dividing the value for the
amount of binding of each PD variant to each Fc.gamma.R by the
value for the amount of Fc.gamma.R binding of the pre-altered
antibody which is used as the control (IL6R-F652/IL6R-L, which has
an alteration of substituting Pro at position 238 (EU numbering)
with Asp) and then multiplying the result by 100, was used as the
relative binding activity value of each PD variant to each
Fc.gamma.R. The horizontal axis shows relative values of the
Fc.gamma.RIIb-binding activity of each PD variant, and the vertical
axis shows relative values of the Fc.gamma.RIIa type R-binding
activity values of each PD variant (FIG. 22).
[0449] As a result, eleven types of alterations were found to have
the effects of enhancing Fc.gamma.RIIb binding and maintaining or
enhancing Fc.gamma.RIIa type R-binding in comparison with the
antibody before introducing alterations. The activities of these
eleven variants to bind Fc.gamma.RIIb and Fc.gamma.RIIa R are
summarized in Table 20. In the table, "alteration" refers to the
alteration introduced into IL6R-F11 (SEQ ID NO: 22).
TABLE-US-00012 TABLE 20 RELATIVE RELATIVE BINDING BINDING ACTIVITY
ACTIVITY TO TO VARIANT NAME ALTERATION Fc.gamma.RIIb Fc.gamma.RIIaR
IL6R-F652/IL6R-L P238D 100 100 IL6R-PD042/IL6R-L P238D/L234W 106
240 IL6R-PD043/IL6R-L P238D/L234Y 112 175 IL6R-PD079/IL6R-L
P238D/G237A 101 138 IL6R-PD080/IL6R-L P238D/G237D 127 222
IL6R-PD081/IL6R-L P238D/G237E 101 117 IL6R-PD082/IL6R-L P238D/G237F
108 380 IL6R-PD086/IL6R-L P238D/G237L 112 268 IL6R-PD087/IL6R-L
P238D/G237M 109 196 IL6R-PD094/IL6R-L P238D/G237W 122 593
IL6R-PD095/IL6R-L P238D/G237Y 124 543 IL6R-PD097/IL6R-L P238D/S239D
139 844
[0450] FIG. 23 shows relative values for the Fc.gamma.RIIb-binding
activity obtained by additionally introducing these eleven
alterations into a variant carrying the P238D alteration, and
relative values for the Fc.gamma.RIIb-binding activity obtained by
introducing these alterations into an Fc that does not contain the
P238D alteration in Reference Example 3. These eleven alterations
enhanced the amount of Fc.gamma.RIIb binding compared with before
introduction when they were further introduced into the P238D
variant, but on the contrary, the effect of lowering Fc.gamma.RIIb
binding was observed for eight of those alterations except G237F,
G237W, and S239D, when they were introduced into the variant that
does not contain P238D (GpH7-B3/GpL16-k0) used in Reference Example
3.
[0451] Reference Example 5 and these results showed that from the
effects of introducing alterations into a naturally-occurring IgG1,
it is difficult to predict the effects of introducing the same
alterations into the variant containing an Fc with the P238D
alteration. In other words, it would not have been possible to
discover these eight alterations identified this time without this
investigation.
[0452] The results of measuring KD values of the variants indicated
in Table 20 for Fc.gamma.RIa, Fc.gamma.RIIaR, Fc.gamma.RIIaH,
Fc.gamma.RIIb, and Fc.gamma.RIIIaV by the method of Reference
Example 2 are summarized in Table 21. In the table, "alteration"
refers to the alteration introduced into IL6R-F11 (SEQ ID NO: 22).
The template used for producing IL6R-F11, IL6R-G1d/IL6R-L, is
indicated with an asterisk (*). Furthermore, "KD(IIaR)/KD(IIb)" and
"KD(IIaH)/KD(IIb)" in the table respectively show the value
obtained by dividing the KD value of each variant for
Fc.gamma.RIIaR by the KD value of each variant for Fc.gamma.RIIb,
and the value obtained by dividing the KD value of each variant for
Fc.gamma.RIIaH by the KD value of each variant for Fc.gamma.RIIb.
KD(IIb) of the parent polypeptide/KD(IIb) of the altered
polypeptide refers to a value obtained by dividing the KD value of
the parent polypeptide for Fc.gamma.RIIb by the KD value of each
variant for Fc.gamma.RIIb. In addition, Table 21 shows KD values
for the stronger of the Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding
activities of each variant/KD values for the stronger of the
Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding activities of the parent
polypeptide. Here, parent polypeptide refers to a variant which has
IL6R-F11 (SEQ ID NO: 22) as the H chain. It was determined that due
to weak binding of Fc.gamma.R to IgG, it was impossible to
accurately analyze by kinetic analysis, and thus the values shown
in bold italicized font in Table 21 were calculated by using
Equation 2 of Reference Example 2.
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
[0453] Table 21 shows that all variants improved their affinity for
Fc.gamma.RIIb in comparison with IL6R-F11, and the range of
improvement was 1.9 fold to 5.0 fold. The ratio of KD value of each
variant for Fc.gamma.RIIaR/KD value of each variant for
Fc.gamma.RIIb, and the ratio of KD value of each variant for
Fc.gamma.RIIaH/KD value of each variant for Fc.gamma.RIIb represent
an Fc.gamma.RIIb-binding activity relative to the
Fc.gamma.RIIaR-binding activity and Fc.gamma.RIIaH-binding
activity, respectively. That is, these values show the degree of
binding selectivity of each variant for Fc.gamma.RIIb, and a larger
value indicates a higher binding selectivity for Fc.gamma.RIIb. For
the parent polypeptide IL6R-F11/IL6R-L, the ratio of KD value for
Fc.gamma.RIIaR/KD value for Fc.gamma.RIIb and the ratio of KD value
for Fc.gamma.RIIaH/KD value for Fc.gamma.RIIb are both 0.7, and
accordingly all variants in Table 21 showed improvement of binding
selectivity for Fc.gamma.RIIb in comparison with the parent
polypeptide. When the KD value for the stronger of the
Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding activities of a
variant/KD value for the stronger of the Fc.gamma.RIIaR- and
Fc.gamma.RIIaH-binding activities of the parent polypeptide is 1 or
more, this means that the stronger of the Fc.gamma.RIIaR- and
Fc.gamma.RIIaH-binding activities of a variant has equivalent or
reduced binding compared with the binding by the stronger of the
Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding activities of the parent
polypeptide. Since this value was 0.7 to 5.0 for the variants
obtained this time, one may say that binding by the stronger of the
Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding activities of the
variants obtained this time was nearly the same or decreased in
comparison with the parent polypeptide. These results showed that
compared with the parent polypeptide, the variants obtained this
time have enhanced binding activity to Fc.gamma.RIIb while having
maintained or decreased binding activities to Fc.gamma.RIIa type R
and type H, and thus have improved selectivity for Fc.gamma.RIIb.
Furthermore, compared with IL6R-F11, all variants had lower
affinity to Fc.gamma.RIa and Fc.gamma.RIIIaV.
[Reference Example 7] X-Ray Structure Analysis of a Complex Formed
Between an Fc Containing P238D and an Extracellular Region of
Fc.gamma.RIIb
[0454] As indicated earlier in Reference Example 5, even though an
alteration that improves Fc.gamma.RIIb-binding activity or
selectivity for Fc.gamma.RIIb is introduced into an Fc containing
P238D, the Fc.gamma.RIIb-binding activity was found to decrease,
and the reason for this may be that the structure of the
interacting interface between Fc and Fc.gamma.RIIb is changed due
to introduction of P238D. Therefore, to pursue the reason for this
phenomena, the three-dimensional structure of the complex formed
between an IgG1 Fc containing the P238D mutation (hereinafter,
Fc(P238D)) and the extracellular region of Fc.gamma.RIIb was
elucidated by X-ray crystal structure analysis, and the
three-dimensional structure and binding mode were compared to those
of the complex formed between the Fc of a naturally-occurring IgG1
(hereinafter, Fc(WT)) and the extracellular region of
Fc.gamma.RIIb. Many reports have been made on the three-dimensional
structure of a complex formed between an Fc and an Fc.gamma.R
extracellular region; and the three-dimensional structures of the
Fc(WT)/Fc.gamma.RIIIb extracellular region complex (Nature, 2000,
400: 267-273; J. Biol. Chem. 2011, 276: 16469-16477), the
Fc(WT)/Fc.gamma.RIIIa extracellular region complex (Proc. Natl.
Acad. Sci. USA, 2011, 108: 12669-126674), and the
Fc(WT)/Fc.gamma.RIIa extracellular region complex (J. Imunol. 2011,
187: 3208-3217) have been analyzed. While the three-dimensional
structure of the Fc(WT)/Fc.gamma.RIIb extracellular region complex
has not been analyzed, the three-dimensional structure of a complex
formed with Fc(WT) is known for Fc.gamma.RIIa, and the
extracellular regions of Fc.gamma.RIIa and Fc.gamma.RIIb match 93%
in amino acid sequence and have very high homology. Thus, the
three-dimensional structure of the Fc(WT)/Fc.gamma.RIIb
extracellular region complex was predicted by modeling using the
crystal structure of the Fc(WT)/Fc.gamma.RIIa extracellular region
complex.
[0455] The three-dimensional structure of the
Fc(P238D)/Fc.gamma.RIIb extracellular region complex was determined
by X-ray crystal structure analysis at 2.6 .ANG. resolution. The
structure obtained as a result of this analysis is shown in FIG.
24. The Fc.gamma.RIIb extracellular region is bound between two Fc
CH2 domains, and this is similar to the three-dimensional
structures of complexes formed between Fc(WT) and the respective
extracellular region of Fc.gamma.RIIIa, Fc.gamma.RIIIb, or
Fc.gamma.RIIa analyzed so far.
[0456] Next, for detailed comparison, the crystal structure of the
Fc(P238D)/Fc.gamma.RIIb extracellular region complex and the model
structure of the Fc(WT)/Fc.gamma.RIIb extracellular region complex
were superimposed by least square fitting based on Ca atom pair
distances with respect to the Fc.gamma.RIIb extracellular region
and the Fc CH2 domain A (FIG. 25). In that case, the degree of
overlap between Fc CH2 domains B was not satisfactory, and
conformational differences were found in this portion. Furthermore,
using the crystal structure of the Fc(P238D)/Fc.gamma.RIIb
extracellular region complex and the model structure of the
Fc(WT)/Fc.gamma.RIIb extracellular region complex, pairs of atoms
that have a distance of 3.7 .ANG. or less between the Fc.gamma.RIIb
extracellular region and Fc CH2 domain B were extracted and
compared in order to observe the differences in interatomic
interactions between Fc.gamma.RIIb and Fc CH2 domain B in Fc(WT)
and Fc(P238D). As shown in Table 22, the interatomic interactions
between Fc CH2 domain B and Fc.gamma.RIIb in Fc(P238D) and Fc(WT)
do not match.
TABLE-US-00013 TABLE 22 Fc (P238D) CH2 DOMAIN B INTERACTION Fc (WT)
CH2 DOMAIN B INTERACTION FcgRIIb ATOM PARTNER (DISTANCE BETWEEN
ATOMS, A) PARTNER (DISTANCE BETWEEN ATOMS, A) Val 116 CG2 Asp 265
OD2 (3.47) Gly 237 O (3.65) Ser 126 OG Ser 298 N (3.31) Ser 298 CB
(3.32) Tyr 296 O (3.05) Lys 128 CA Ser 298 OG (3.50) Phe 129 CB Ser
298 O (3.36) Phe 129 CD2 Asn 297 CB (3.50) Asn 297 CG (3.43) Lys
128 C Ser 298 OG (3.47) Phe 129 N Ser 298 OG (3.30) Phe 129 G Ser
267 OG (3.54) Arg 131 CB Val 266 O (3.02) Arg 131 CG Val 266 O
(3.22) Arg 131 CD Val 266 CG1 (3.45) Val 266 C (3.55) Val 266 O
(3.10) Arg 131 NE Ala 327 O (3.60) Val 266 C (3.66) Val 266 O
(3.01) Val 266 N (3.49) Arg 131 CZ Asp 270 CG (3.64) Val 266 N
(3.13) Asp 270 OD2 (3.22) Asp 270 OD1 (3.27) Asp 327 CB (3.63) Arg
131 NN1 Asp 270 CG (3.19) Val 266 CG1 (3.47) Asp 270 OD2 (2.83) Val
266 N (3.43) Asp 270 OD1 (2.99) Thr 299 OG1 (3.66) Ser 267 CB
(3.56) Ser 298 O (3.11)
[0457] Furthermore, the X-ray crystal structure of the
Fc(P238D)/Fc.gamma.RIIb extracellular region complex and the model
structure of the Fc(WT)/Fc.gamma.RIIb extracellular region complex
were superimposed by least square fitting based on Ca atom pair
distances with respect to the only Fc CH2 domain A or the only Fc
CH2 domain B, and the detailed structures near P238D were compared.
The location of the amino acid residue at position 238 (EU
numbering), which is the mutation introduction position, is changed
between Fc(P238D) and Fc(WT), one can see that along with this
change, the nearby loop structure continuing from the hinge region
is changed between Fc(P238D) and Fc(WT) (FIG. 26). Originally in
Fc(WT), Pro at position 238 (EU numbering) is present on the inner
side of the protein, and forms a hydrophobic core with the
surrounding residues. However, when this residue is changed to a
charged and very hydrophilic Asp, the presence in the same
hydrophobic core would cause energetical disadvantage in terms of
desolvation. Therefore, in Fc(P238D), to cancel this energetically
disadvantageous situation, the amino acid residue at position 238
(EU numbering) changes its orientation to face the solvent side,
and this may have caused this change in the nearby loop structure.
Furthermore, since this loop continues from the hinge region
crosslinked by an S--S bond, its structural change will not be
limited to a local change, and will affect the relative positioning
of the FcCH2 domain A and domain B. As a result, the interatomic
interactions between Fc.gamma.RIIb and Fc CH2 domain B have been
changed. Therefore, predicted effects could not be observed when
alterations that improve selectivity and binding activity towards
Fc.gamma.RIIb in a naturally-occurring IgG were combined with an Fc
containing the P238D alteration.
[0458] Furthermore, as a result of structural changes due to
introduction of P238D in Fc CH2 domain A, a hydrogen bond has been
found between the main chain of Gly at adjacent position 237 (EU
numbering) and Tyr at position 160 in Fc.gamma.RIIb (FIG. 27). The
residue in Fc.gamma.RIIa that corresponds to this Tyr 160 is Phe;
and when the binding is to Fc.gamma.RIIa, this hydrogen bond is not
formed. The amino acid at position 160 is one of the few
differences between Fc.gamma.RIIa and Fc.gamma.RIIb at the
interface of interaction with Fc, the presence of this hydrogen
bond which is specific to Fc.gamma.RIIb is presumed to have led to
improvement of Fc.gamma.RIIb-binding activity and decrease of
Fc.gamma.RIIa-binding activity in Fc(P238D), and improvement of its
selectivity. Furthermore, in Fc CH2 domain B, an electrostatic
interaction is observed between Asp at position 270 (EU numbering)
and Arg at position 131 in Fc.gamma.RIIb (FIG. 28). In
Fc.gamma.RIIa type H, which is one of the allotypes of
Fc.gamma.RIIa, the corresponding residue is His, and therefore
cannot form this electrostatic interaction. This can explain why
the Fc(P238D)-binding activity is lowered in Fc.gamma.RIIa type H
compared with Fc.gamma.RIIa type R. Observations based on such
results of X-ray crystal structure analysis showed that the change
of the loop structure beside P238D due to P238D introduction and
the accompanying change in the relative domain positioning causes
formation of new interactions not found in the naturally-occurring
IgG, and this led to a selective binding profile of P238D variants
for Fc.gamma.RIIb.
[Expression and Purification of Fc(P238D)]
[0459] An Fc containing the P238D alteration was prepared as
follows. First, Cys at position 220 (EU numbering) of hIL6R-IgG1-v1
(SEQ ID NO: 20) was substituted with Ser. Then, genetic sequence of
Fc(P238D) from Glu at position 216 (EU numbering) to its C terminus
was cloned by PCR. Using this cloned genetic sequence, production
of expression vectors, and expression and purification of Fc(P238D)
were carried out according to the method of Reference Example 1.
Cys at position 220 (EU numbering) forms a disulfide bond with Cys
of the L chain in general IgG1. The L chain is not co-expressed
when Fc alone is prepared, and therefore, this residue was
substituted with Ser to avoid formation of unnecessary disulfide
bonds.
[Expression and Purification of the Fc.gamma.RIIb Extracellular
Region]
[0460] This was prepared according to the method of Reference
Example 2.
[Purification of the Fc(P238D)/Fc.gamma.RIIb Extracellular Region
Complex]
[0461] To 2 mg of the Fc.gamma.RIIb extracellular region sample
obtained for crystallization, 0.29 mg of Endo F1 (Protein Science
1996, 5: 2617-2622) expressed and purified from Escherichia coli as
a glutathione S-transferase fusion protein was added. This was
allowed to remain at room temperature for three days in 0.1 M
Bis-Tris buffer at pH 6.5, and the N-linked oligosaccharide was
cleaved, leaving N-acetylglucosamine directly bound to Asn. Next,
this Fc.gamma.RIIb extracellular domain sample subjected to
carbohydrate cleavage treatment was concentrated by ultrafiltration
with 5000 MWCO, and purified by gel filtration chromatography
(Superdex.RTM. 200 10/300 chromatography) using a column
equilibrated in 20 mM HEPS at pH 7.5 containing 0.05 M NaCl.
Furthermore, to the obtained carbohydrate-cleaved Fc.gamma.RIIb
extracellular region fraction, Fc(P238D) was added so that the
molar ratio of the Fc.gamma.RIIb extracellular region would be
present in slight excess, and after concentration by
ultrafiltration with 10000 MWCO, a sample of the
Fc(P238D)/Fc.gamma.RIIb extracellular region complex was obtained
through purification by gel filtration chromatography
(Superdex.RTM. 200 10/300 chromatography) using a column
equilibrated in 20 mM HEPS at pH 7.5 containing 0.05 M NaCl.
[Crystallization of the Fc(P238D)/Fc.gamma.RIIb Extracellular
Region Complex]
[0462] A sample of the Fc(P238D)/Fc.gamma.RIIb extracellular region
complex was concentrated to approximately 10 mg/mL by
ultrafiltration with 10000 MWCO, and crystallization was carried
out by the sitting drop vapor diffusion method. A Hydra.RTM. II
Plus One pipetting robot (MATRIX) was used for crystallization; and
for a reservoir solution containing 100 mM Bis-Tris pH 6.5, 17%
PEG3350, 0.2 M ammonium acetate, and 2.7% (w/v) D-Galactose, a
crystallization drop was produced by mixing at a ratio of reservoir
solution:crystallization sample=0.2 .mu.L:0.2 .mu.L, and after
sealing, this was allowed to remain at 20.degree. C., and thin
plate-like crystals were successfully obtained.
[Measurement of X-Ray Diffraction Data from an
Fc(P238D)/Fc.gamma.RIIb Extracellular Region Complex Crystal]
[0463] One of the obtained single crystals of the
Fc(P238D)/Fc.gamma.RIIb extracellular region complex was soaked
into a solution of 100 mM Bis-Tris pH 6.5, 20% PEG3350, ammonium
acetate, 2.7% (w/v) D-Galactose, 22.5% (v/v) ethylene glycol. The
crystal was fished out of the solution using a pin with attached
tiny nylon loop, and frozen in liquid nitrogen; and then X-ray
diffraction data was measured at synchrotron radiation facility
Photon Factory BL-1A in High Energy Accelerator Research
Organization. During the measurement, the crystal was constantly
placed in a nitrogen stream at -178.degree. C. to maintain in a
frozen state, and a total of 225 X ray diffraction images were
collected using Quantum 270 CCD detector (ADSC) attached to a beam
line with rotating the crystal 0.8.degree. at a time. Determination
of cell parameters, indexing of diffraction spots, and diffraction
data processing from the obtained diffraction images were performed
using the Xia2 program (CCP4 Software Suite), XDS Package (Walfgang
Kabsch) and Scala (CCP4 Software Suite); and finally, diffraction
intensity data up to 2.46 .ANG. resolution was obtained. The
crystal belongs to the space group P21, and has the following cell
parameters; a=48.85 .ANG., b=76.01 .ANG., c=115.09 .ANG.,
.alpha.=90.degree., .beta.=100.70.degree., .gamma.=90.degree..
[X Ray Structure Analysis of the Fc(P238D)/Fc.gamma.RIIb
Extracellular Region Complex]
[0464] Crystal structure of the Fc(P238D)/Fc.gamma.RIIb
extracellular region complex was determined by the molecular
replacement method using the program Phaser (CCP4 Software Suite).
From the size of the obtained crystal lattice and the molecular
weight of the Fc(P238D)/Fc.gamma.RIIb extracellular region complex,
the number of complexes in the asymmetric unit was predicted to be
one. From the structural coordinates of PDB code: 3SGJ which is the
crystal structure of the Fc(WT)/Fc.gamma.RIIIa extracellular region
complex, the amino acid residue portions of the A chain positions
239-340 and the B chain positions 239-340 were taken out as
separate coordinates, and they were used respectively as models for
searching the Fc CH2 domains. The amino acid residue portions of
the A chain positions 341-444 and the B chain positions 341-443
were taken out as a single set of coordinates from the same
structural coordinates of PDB code: 3SGJ; and this was used as a
model for searching the Fc CH3 domains. Finally, from the
structural coordinates of PDB code: 2FCB which is a crystal
structure of the Fc.gamma.RIIb extracellular region, the amino acid
residue portions of the A chain positions 6-178 was taken out and
used as a model for searching the Fc.gamma.RIIb extracellular
region. The orientation and position of each search model in the
crystal lattice were determined in the order of Fc CH3 domain,
Fc.gamma.RIIb extracellular region, and Fc CH2 domain, based on the
rotation function and translation function to obtain the initial
model for the crystal structure of the Fc(P238D)/Fc.gamma.RIIb
extracellular region complex. When rigid body refinement which
moves the two Fc CH2 domains, the two Fc CH3 domains, and the
Fc.gamma.RIIb extracellular region was performed on the obtained
initial model, the crystallographic reliability factor, R value
became 40.4%, and the Free R value became 41.9% to diffraction
intensity data from 25 .ANG. to 3.0 .ANG. at this point.
Furthermore, structural refinement using the program Refmac5 (CCP4
Software Suite), and revision of the model to observe the electron
density maps whose coefficient have 2Fo-Fc or Fo-Fc, which are
calculated based on the experimentally determined structural factor
Fo, the calculated structural factor Fc and the calculated phase
using the model, was carried out by the Coot program (Paul Emsley),
and model refinement was carried out by repeating these steps.
Finally, as a result of incorporation of water molecules into the
model based on the electron density maps which use 2Fo-Fc or Fo-Fc
as the coefficient, and the following refinement, the
crystallographic reliability factor, R values and the Free R value
of the model containing 4846 non-hydrogen atoms became 23.7% and
27.6% to 24291 diffraction intensity data from 25 .ANG. to 2.6
.ANG. resolution, respectively.
[Production of a Model Structure of the Fc(WT)/Fc.gamma.RIIb
Extracellular Region Complex]
[0465] Based on the structural coordinates of PDB code: 3RY6 which
is a crystal structure of the Fc(WT)/Fc.gamma.RIIa extracellular
region complex, the Build Mutants function of the Discovery Studio
3.1 program (Accelrys) was used to introduce mutations to match the
amino acid sequence of Fc.gamma.RIIb into Fc.gamma.RIIa in this
structural coordinates. In that case, the Optimization Level was
set to High, Cut Radius was set to 4.5, five models were generated,
and the one with the best energy score among them was employed as
the model structure for the Fc(WT)/Fc.gamma.RIIb extracellular
region complex.
[Reference Example 8] Analysis of Fc.gamma.R Binding of Fc Variants
Whose Alteration Sites were Determined Based on Crystal
Structures
[0466] Based on the results of X-ray structure analysis on the
complex formed between Fc(P238D) and the Fc.gamma.RIIb
extracellular region obtained in Reference Example 7, comprehensive
alterations were introduced into sites on the Fc variant having
substitution of Pro at position 238 (EU numbering) with Asp that
were predicted to affect interaction with Fc.gamma.RIIb, (residues
of positions 233, 240, 241, 263, 265, 266, 267, 268, 271, 273, 295,
296, 298, 300, 323, 325, 326, 327, 328, 330, 332, and 334 (EU
numbering)) and variants with a combination of alterations that
enhance Fc.gamma.RIIb binding were examined.
[0467] IL6R-B3 (SEQ ID NO: 23) was produced by introducing into
IL6R-G1d (SEQ ID NO: 19) produced in Reference Example 4, the
alteration produced by substituting Lys at position 439 (EU
numbering) with Glu. Next, IL6R-BF648 was produced by introducing
into IL6R-B3, the alteration produced by substituting Pro at
position 238 (EU numbering) with Asp. IL6R-L (SEQ ID NO: 21) was
utilized as the common antibody L chain for all of the antibodies.
These antibody variants were expressed and purified according to
the method of Reference Example 1, and binding to each of the
Fc.gamma.Rs (Fc.gamma.RIa, Fc.gamma.RIIa type H, Fc.gamma.RIIa type
R, Fc.gamma.RIIb, and Fc.gamma.RIIIa type V) was comprehensively
evaluated by the method of Reference Example 2.
[0468] A figure was produced according to the following method for
the results of analyzing the interactions with the respective
Fc.gamma.Rs. The value for the amount of binding of each variant to
each Fc.gamma.R was divided by the value for the amount of binding
of the pre-altered control antibody (IL6R-BF648/IL6R-L with Pro at
position 238 (EU numbering) substituted with Asp) to each
Fc.gamma.R, and the obtained was then multiplied by 100 and used as
the relative binding activity value of each variant to each
Fc.gamma.R. The horizontal axis shows the relative binding activity
value of each variant to Fc.gamma.RIIb, and the vertical axis shows
the relative binding activity value of each variant to
Fc.gamma.RIIa type R (FIG. 29).
[0469] As shown in FIG. 29, the results show that of all the
alterations, 24 types of alterations were found to have an effect
of maintaining or enhancing Fc.gamma.RIIb binding in comparison
with the pre-altered antibody. The binding of these variants to
each of the Fc.gamma.Rs are shown in Table 23. In the table,
"alteration" refers to the alteration introduced into IL6R-B3 (SEQ
ID NO: 23; IL6R-2B999 in Table 23). The template used for producing
IL6R-B3, IL6R-G1d/IL6R-L, is indicated with an asterisk (*).
TABLE-US-00014 TABLE 23 RELATIVE RELATIVE RELATIVE RELATIVE
RELATIVE BINDING BINDING BINDING BINDING BINDING ACTIVITY ACTIIVTY
ACTIVITY ACTIVITY ACTIVITY VARIANT NAME ALTERNATION TO FcgRIa TO
FcgRIIaR TO FcgRIIaH TO FcgRIIb TO FcgRIIIaV IL6R-G1d/IL6R-L * 140
650 1670 62 3348 IL6R-2B999/IL6R-L 145 625 1601 58 3264
IL6R-BF648/IL6R-L P238D 100 100 100 100 100 IL6R-2B002/IL6R-L
P238D/E233D 118 103 147 116 147 IL6R-BP100/IL6R-L P238D/S267A 121
197 128 110 138 IL6R-BP102/IL6R-L P238D/S267Q 104 165 66 106 86
IL6R-BP103/IL6R-L P238D/S267V 56 163 69 107 77 IL6R-BP106/IL6R-L
P238D/H268D 127 150 110 116 127 IL6R-BP107/IL6R-L P238D/H268E 123
147 114 118 129 IL6R-BP110/IL6R-L P238D/H268N 105 128 127 101 127
IL6R-BP112/IL6R-L P238D/P271G 119 340 113 157 102 IL6R-2B128/IL6R-L
P238D/Y296D 95 87 37 103 96 IL6R-2B169/IL6R-L P238D/V323I 73 92 83
104 94 IL6R-2B171/IL6R-L P238D/V323L 116 117 115 113 122
IL6R-2B172/IL6R-L P238D/V323M 140 244 179 132 144 IL6R-BP136/IL6R-L
P238D/K326A 117 159 103 119 102 IL6R-BP117/IL6R-L P238D/K326D 124
166 96 118 105 IL6R-BP120/IL6R-L P238D/K326E 125 175 92 114 103
IL6R-BP126/IL6R-L P238D/K326L 113 167 132 103 146 IL6R-BP119/IL6R-L
P238D/K326M 117 181 133 110 145 IL6R-BP142/IL6R-L P238D/K326N 98
103 97 106 102 IL6R-BP121/IL6R-L P238D/K326Q 118 155 135 113 157
IL6R-BP118/IL6R-L P238D/K326S 101 132 128 104 144 IL6R-BP116/IL6R-L
P238D/K326T 110 126 110 108 114 IL6R-BP911/IL6R-L P238D/A330K 52
101 108 119 120 IL6R-BP078/IL6R-L P238D/A330M 106 101 89 105 91
IL6R-BP912/IL6R-L P238D/A330R 60 81 93 103 97
[0470] The results of measuring KD values of the variants shown in
Table 23 for Fc.gamma.RIa, Fc.gamma.RIIaR, Fc.gamma.RIIaH,
Fc.gamma.RIIb, and Fc.gamma.RIIIa type V by the method of Reference
Example 2 are summarized in Table 24. In the table, "alteration"
refers to the alteration introduced into IL6R-B3 (SEQ ID NO: 23).
The template used for producing IL6R-B3, IL6R-G1d/IL6R-L, is
indicated with an asterisk (*). Furthermore, "KD(IIaR)/KD(IIb)" and
"KD(IIaH)/KD(IIb)" in the table respectively represent the value
obtained by dividing the KD value of each variant for
Fc.gamma.RIIaR by the KD value of each variant for Fc.gamma.RIIb,
and the value obtained by dividing the KD value of each variant for
Fc.gamma.RIIaH by the KD value of each variant for Fc.gamma.RIIb.
"KD(IIb) of the parent polypeptide/KD(IIb) of the altered
polypeptide" refers to the value obtained by dividing the KD value
of the parent polypeptide for Fc.gamma.RIIb by the KD value of each
variant for Fc.gamma.RIIb. In addition, the "KD value for the
stronger of the Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding
activities of each variant/KD value for the stronger of the
Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding activities of the parent
polypeptide" are shown in Table 24. Here, parent polypeptide refers
to the variant which has IL6R-B3 (SEQ ID NO: 23) as the H chain. It
was determined that due to weak binding of Fc.gamma.R to IgG, it
was impossible to accurately analyze by kinetic analysis, and thus
the values shown in bold italicized font in Table 24 were
calculated by using Equation 2 of Reference Example 2.
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
[0471] Table 24 shows that in comparison with IL6R-B3 (IL6R-2B999
in Table 24), all variants showed improvement of affinity for
Fc.gamma.RIIb, and the range of improvement was 2.1 fold to 9.7
fold. The ratio of KD value of each variant for Fc.gamma.RIIaR/KD
value of each variant for Fc.gamma.RIIb, and the ratio of KD value
of each variant for Fc.gamma.RIIaH/KD value of each variant for
Fc.gamma.RIIb represent an Fc.gamma.RIIb-binding activity relative
to the Fc.gamma.RIIaR-binding activity and Fc.gamma.RIIaH-binding
activity, respectively. That is, these values show the degree of
binding selectivity of each variant for Fc.gamma.RIIb, and a
greater value indicates a higher binding selectivity for
Fc.gamma.RIIb. Since the ratio of KD value for Fc.gamma.RIIaR/KD
value for Fc.gamma.RIIb, and the ratio of KD value for
Fc.gamma.RIIaH/KD value for Fc.gamma.RIIb in the parent polypeptide
IL6R-B3/IL6R-L were 0.3 and 0.2, respectively, all variants in
Table 24 showed improvement of binding selectivity for
Fc.gamma.RIIb in comparison with the parent polypeptide. When the
KD value for the stronger of the Fc.gamma.RIIaR- and
Fc.gamma.RIIaH-binding activities of a variant/KD value for the
stronger of the Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding
activities of the parent polypeptide is 1 or more, this means that
the stronger of the Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding
activities of a variant has equivalent or decreased binding
compared with the binding by the stronger of the Fc.gamma.RIIaR-
and Fc.gamma.RIIaH-binding activities of the parent polypeptide.
Since this value was 4.6 to 34.0 for the variants obtained this
time, one may say that in comparison with the parent polypeptide,
the variants obtained this time had reduced binding by the stronger
of the Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding activities. These
results showed that compared with the parent polypeptide, the
variants obtained this time have maintained or decreased
Fc.gamma.RIIa type R- and type H-binding activities, enhanced
Fc.gamma.RIIb-binding activity, and improved selectivity for
Fc.gamma.RIIb. Furthermore, compared with IL6R-B3, all variants had
lower affinity to Fc.gamma.RIa and Fc.gamma.RIIIaV.
[0472] With regard to the promising variants among the obtained
combination variants, the factors leading to their effects were
studied using the crystal structure. FIG. 30 shows the crystal
structure of the Fc(P238D)/Fc.gamma.RIIb extracellular region
complex. In this figure, the H chain positioned on the left side is
Fc Chain A, and the H chain positioned on the right side is Fc
Chain B. Here, one can see that the site at position 233 (EU
numbering) in Fc Chain A is located near Lys at position 113 (EU
numbering) of Fc.gamma.RIIb. However, in this crystal structure,
the E233 side chain is in a condition of considerably high
mobility, and its electron density is not well observed. Therefore,
the alteration produced by substituting Glu at position 233 (EU
numbering) with Asp leads to decrease in the degree of freedom of
the side chain since the side chain becomes one carbon shorter. As
a result, the entropy loss when forming an interaction with Lys at
position 113 (EU numbering) of Fc.gamma.RIIb may be decreased, and
consequently this is speculated to contribute to improvement of
binding free energy.
[0473] Similarly, FIG. 31 shows the environment near the site at
position 330 (EU numbering) in the structure of the
Fc(P238D)/Fc.gamma.RIIb extracellular region complex. This figure
shows that the environment around the site at position 330 (EU
numbering) of Fc Chain A of Fc(P238D) is a hydrophilic environment
composed of Ser at position 85, Glu at position 86, Lys at position
163, and such (EU numbering) of Fc.gamma.RIIb. Therefore, the
alteration produced by substituting Ala at position 330 (EU
numbering) with Lys or Arg is speculated to contribute to
strengthening the interaction with Ser at position 85 (EU
numbering) or Glu at position 86 (EU numbering) in
Fc.gamma.RIIb.
[0474] FIG. 32 depicts the structures of Pro at position 271 (EU
numbering) of Fc Chain B after superimposing the crystal structures
of the Fc(P238D)/Fc.gamma.RIIb extracellular region complex and the
Fc(WT)/Fc.gamma.RIIIa extracellular region complex by least square
fitting based on Ca atom pair distances with respect to Fc Chain B.
These two structures match well, but have different
three-dimensional structures of Pro at position 271 (EU numbering).
When the weak electron density around this area in the crystal
structure of the Fc(P238D)/Fc.gamma.RIIb extracellular region
complex is also taken into consideration, it is suggested that
there is possibility that Pro at position 271 (EU numbering) in
Fc(P238D)/Fc.gamma.RIIb causes a large strain on the structure,
thus disturbing the loop structure to attain an optimal structure.
Therefore, one may consider that the alteration produced by
substituting Pro at position 271 (EU numbering) with Gly gives
flexibility to this loop structure and contributes to enhancement
of binding by reducing the energetic barrier when allowing to form
an optimum structure upon interaction with Fc.gamma.RIIb.
[Reference Example 9] Examination of the Combinatorial Effect of
Alterations that Enhance Fc.gamma.RIIb Binding when Combined with
P238D
[0475] Of the alterations obtained in Reference Examples 6 and 8,
those that enhanced Fc.gamma.RIIb binding or maintained
Fc.gamma.RIIb binding and showed effects of suppressing binding to
other Fc.gamma.Rs were combined with each other, and their effects
were examined.
[0476] Particularly good alterations were selected from Tables 19
and 22, and they were combined and introduced into the antibody H
chain IL6R-BF648 in a similar manner to the method of Reference
Example 8. IL6R-L was utilized as the common antibody L chain for
all of the antibodies, the antibodies were expressed and purified
according to the method of Reference Example 1, and binding to each
of the Fc.gamma.Rs (Fc.gamma.RIa, Fc.gamma.RIIa type H,
Fc.gamma.RIIa type R, Fc.gamma.RIIb, and Fc.gamma.RIIIa type V) was
comprehensively evaluated by the method of Reference Example 2.
[0477] Relative binding activities were calculated for the results
of analyzing interactions with the respective Fc.gamma.Rs according
to the following method. The value for the amount of binding of
each variant to each Fc.gamma.R was divided by the value for the
amount of binding of the pre-altered control antibody
(IL6R-BF648/IL6R-L with substitution of Pro at position 238 (EU
numbering) with Asp) to each Fc.gamma.R, and multiplied by 100; and
then the value was used as the relative binding activity value of
each variant to each Fc.gamma.R. The horizontal axis shows the
relative binding activity value of each variant to Fc.gamma.RIIb,
and the vertical axis shows the relative binding activity value of
each variant to Fc.gamma.RIIa type R (Table 25).
[0478] In the table, "alteration" refers to the alteration
introduced into IL6R-B3 (SEQ ID NO: 23). The template used for
producing IL6R-B3, IL6R-G1d/IL6R-L, is indicated with an asterisk
(*).
TABLE-US-00015 TABLE 25 RELATIVE RELATIVE RELATIVE RELATIVE
RELATIVE BINDING BINDING BINDING BINDING BINDING ACTIVITY ACTIVITY
ACTIVITY ACTIVITY ACTIVITY VARIANT NAME ALTERATION to FcgRIa to
FcgRIIaR to FcgRIIaH to FcgRIIb to FcgRIIIaV IL6R-G1d/IL6R-L * 140
650 1670 62 3348 IL6R-B3/IL6R-L 145 625 1601 58 3264
IL6R-BF648/IL6R-L P238D 100 100 100 100 100 IL6R-2B253/IL6R-L
E233D/P238D/V323M 155 288 207 156 126 IL6R-2B261/IL6R-L
E233D/P238D/Y296D 100 94 91 115 87 IL6R-BP082/IL6R-L
E233D/P238D/A330K 74 126 106 136 87 IL6R-BP083/IL6R-L
P238D/Y296D/A330K 50 87 91 122 107 IL6R-BP084/IL6R-L
P238D/V323M/A330K 109 203 162 141 106 IL6R-BP085/IL6R-L
G237D/P238D/A330K 19 279 158 152 104 IL6R-BP086/IL6R-L
P238D/K326A/A330K 72 155 116 137 123 IL6R-BP087/IL6R-L
L234Y/P238D/A330K 33 163 179 137 158 IL6R-BP088/IL6R-L
G237D/P238D/K326A/A330K 25 377 166 161 122 IL6R-BP089/IL6R-L
L234Y/P238D/K326A/A330K 43 222 186 147 136 IL6R-BP129/IL6R-L
E233D/P238D/Y296D/A330K 68 111 98 138 95 IL6R-BP130/IL6R-L
E233D/P238D/V323M/A330K 104 272 224 160 115 IL6R-BP131/IL6R-L
E233D/G237D/P238D/A330K 33 364 253 160 118 IL6R-BP132/IL6R-L
E233D/P238D/K326A/A330K 91 191 130 150 120 IL6R-BP133/IL6R-L
E233D/L234Y/P238D/A330K 41 174 151 137 114 IL6R-BP143/IL6R-L
L234Y/P238D/K326A 86 238 143 133 114 IL6R-BP144/IL6R-L
G237D/P238D/K326A 64 204 108 121 128 IL6R-BP145/IL6R-L
L234Y/G237D/P238D 41 350 224 152 153 IL6R-BP146/IL6R-L
L234Y/G237D/P238D/K326A 50 445 203 156 180 IL6R-BP147/IL6R-L
L234Y/G237D/P238D/K326A/A330K 24 650 582 177 209 IL6R-BP148/IL6R-L
E233D/L234Y/G237D/P238D/K326A/A330K 33 603 462 176 227
IL6R-BP149/IL6R-L E233D/L234Y/G237D/P238D/Y296D/K326A/A330K 29 539
401 173 186 IL6R-BP150/IL6R-L L234Y/G237D/P238D/K326A/A330R 30 757
770 183 204 IL6R-BP151/IL6R-L E233D/L234Y/G237D/P238D/K326A/A330R
39 705 621 180 221 IL6R-BP152/IL6R-L
E233D/L234Y/G237D/P238D/Y296D/K326A/A330R 34 638 548 178 146
IL6R-BP176/IL6R-L E233D/P238D/K326D/A330K 102 201 128 147 131
IL6R-BP177/IL6R-L E233D/L234Y/G237D/P238D/P271G/K326D/A330K 57 691
409 177 186 IL6R-BP178/IL6R-L E233D/G237D/P238D/P271G/A330K 51 653
259 179 110 IL6R-BP179/IL6R-L G237D/P238D/P271G/K326A/A330K 39 570
226 177 125 IL6R-BP180/IL6R-L G237D/P238D/P271G/A330K 29 602 203
179 100 IL6R-BP181/IL6R-L E233D/P238D/P271G/K326A/A330K 108 362 150
170 122 IL6R-BP182/IL6R-L E233D/P238D/P271G/Y296D/A330K 95 413 139
173 120 IL6R-BP183/IL6R-L E233D/L234Y/P238D/P271G/K326A/A330K 83
423 191 164 113 IL6R-BP184/IL6R-L E233D/P238D/P271G/A330K 96 436
131 171 106 IL6R-BP185/IL6R-L
E233D/L234Y/G237D/P238D/P271G/K326A/A330K 47 670 446 179 191
IL6R-BP186/IL6R-L E233D/L234Y/G237D/P238D/P271G/Y296D/K326A/ 43 614
368 175 143 A330K IL6R-BP187/IL6R-L L234Y/P238D/P271G/K326A/A330K
68 387 205 157 124 IL6R-BP188/IL6R-L
E233D/G237D/P238D/H268D/P271G/A330K 74 636 234 179 121
IL6R-BP189/IL6R-L G237D/P238D/H268D/P271G/K326A/A330K 56 557 183
177 141 IL6R-BP190/IL6R-L G237D/P238D/H268D/P271G/A330K 50 615 224
181 155 IL6R-BP191/IL6R-L E233D/P238D/H268D/P271G/K326A/A330K 125
382 145 170 142 IL6R-BP192/IL6R-L
E233D/P238D/H268D/P271G/Y296D/A330K 109 406 122 172 118
IL6R-BP193/IL6R-L E233D/P238D/H268D/P271G/A330K 113 449 154 173 135
IL6R-BP194/IL6R-L E233D/L234Y/G237D/P238D/H268D/P271G/K326A/ 69 672
395 178 249 A330K IL6R-BP195/IL6R-L
E233D/L234Y/G237D/P238D/H268D/P271G/Y296D/ 68 661 344 181 221
K326A/A330K IL6R-BP196/IL6R-L L234Y/P238D/H268D/P271G/K326A/A330K
89 402 195 157 137 IL6R-BP197/IL6R-L
E233D/L234Y/G237D/P238D/H268D/P271G/Y296D/ 71 642 294 179 206
K326D/A330K IL6R-BP198/IL6R-L
E233D/L234Y/P238D/H268D/P271G/K326A/A330K 104 449 188 164 157
IL6R-BP199/IL6R-L E233D/P238D/K326D/A330R 112 172 116 144 103
IL6R-BP200/IL6R-L E233D/L234Y/G237D/P238D/P271G/K326D/A330R 60 754
517 188 164 IL6R-BP201/IL6R-L E233D/G237D/P238D/P271G/A330R 57 696
359 186 121 IL6R-BP202/IL6R-L G237D/P238D/P271G/K326A/A330R 43 615
285 185 108 IL6R-BP203/IL6R-L G237D/P238D/P271G/A330R 35 637 255
185 88 IL6R-BP204/IL6R-L E233D/P238D/P271G/K326A/A330R 110 301 137
165 121 IL6R-BP205/IL6R-L E233D/P238D/P271G/Y296D/A330R 97 335 108
167 93 IL6R-BP206/IL6R-L E233D/P238D/P271G/A330R 101 362 123 168 92
IL6R-BP207/IL6R-L E233D/P238D/A330R 74 103 103 124 97
IL6R-BP208/IL6R-L E233D/G237D/P238D/H268D/P271G/A330R 81 690 310
188 118 IL6R-BP209/IL6R-L G237D/P238D/H268D/P271G/K326A/A330R 68
625 267 186 153 IL6R-BP210/IL6R-L G237D/P238D/H268D/P271G/A330R 57
661 279 187 135 IL6R-BP211/IL6R-L
E233D/P238D/H268D/P271G/K326A/A330R 128 312 111 165 87
IL6R-BP212/IL6R-L E233D/P238D/H268D/P271G/Y296D/A330R 117 363 135
173 122 IL6R-BP213/IL6R-L E233D/P238D/H268D/P271G/A330R 118 382 123
169 100 IL6R-BP214/IL6R-L E233D/L234Y/G237D/P238D/Y296D/K326D/A330K
36 498 285 174 165
[0479] The results of measuring KD values of the variants shown in
Table 25 for Fc.gamma.RIa, Fc.gamma.RIIaR, Fc.gamma.RIIaH,
Fc.gamma.RIIb, and Fc.gamma.RIIIa type V by the method of Reference
Example 2 are summarized in Table 26. In the table, "alteration"
refers to the alteration introduced into IL6R-B3 (SEQ ID NO: 23).
The template used for producing IL6R-B3, IL6R-G1d/IL6R-L, is
indicated with an asterisk (*). Furthermore, "KD (IIaR)/KD (IIb)"
and "KD (IIaH)/KD (IIb)" in the table respectively represent the
value obtained by dividing the KD value of each variant for
Fc.gamma.RIIaR by the KD value of each variant for Fc.gamma.RIIb,
and the value obtained by dividing the KD value of each variant for
Fc.gamma.RIIaH by the KD value of each variant for Fc.gamma.RIIb.
"KD (IIb) of the parent polypeptide/KD (IIb) of the altered
polypeptide" refers to the value obtained by dividing the KD value
of the parent polypeptide for Fc.gamma.RIIb by the KD value of each
variant for Fc.gamma.RIIb. In addition, the "KD value for the
stronger of the Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding
activities of each variant/KD value for the stronger of the
Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding activities of the parent
polypeptide" are shown in Table 26. Here, parent polypeptide refers
to the variant which has IL6R-B3 (SEQ ID NO: 23) as the H chain. It
was determined that due to weak binding of Fc.gamma.R to IgG, it
was impossible to accurately analyze by kinetic analysis, and thus
the values shown in bold italicized font in Table 26 were
calculated by using Equation 2 of Reference Example 2.
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
[0480] Table 26 shows that in comparison with IL6R-B3, all variants
showed improvement of affinity for Fc.gamma.RIIb, and the range of
improvement was 3.0 fold to 99.0 fold. The ratio of KD value of
each variant for Fc.gamma.RIIaR/KD value of each variant for
Fc.gamma.RIIb, and the ratio of KD value of each variant for
Fc.gamma.RIIaH/KD value of each variant for Fc.gamma.RIIb represent
an Fc.gamma.RIIb-binding activity relative to the
Fc.gamma.RIIaR-binding activity and Fc.gamma.RIIaH-binding
activity, respectively. That is, those values show the degree of
binding selectivity of each variant for Fc.gamma.RIIb, and a
greater value indicates a higher binding selectivity for
Fc.gamma.RIIb. Since the ratio of KD value for Fc.gamma.RIIaR/KD
value for Fc.gamma.RIIb, and the ratio of KD value for
Fc.gamma.RIIaH/KD value for Fc.gamma.RIIb of the parent polypeptide
IL6R-B3/IL6R-L were 0.3 and 0.2, respectively, all variants in
Table 26 showed improvement of binding selectivity for
Fc.gamma.RIIb in comparison with the parent polypeptide. When the
KD value for the stronger of the Fc.gamma.RIIaR- and
Fc.gamma.RIIaH-binding activities of a variant/KD value for the
stronger of the Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding
activities of the parent polypeptide is 1 or more, this means that
the stronger of the Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding
activities of a variant has equivalent or decreased binding
compared with the binding by the stronger of the Fc.gamma.RIIaR-
and Fc.gamma.RIIaH-binding activities of the parent polypeptide.
Since this value was 0.7 to 29.9 for the variants obtained this
time, one may say that binding by the stronger of the
Fc.gamma.RIIaR- and Fc.gamma.RIIaH-binding activities of the
variants obtained this time was nearly equivalent or decreased
compared with that of the parent polypeptide. These results showed
that compared with the parent polypeptide, the variants obtained
this time have maintained or decreased Fc.gamma.RIIa type R- and
type H-binding activities, enhanced Fc.gamma.RIIb-binding activity,
and improved selectivity for Fc.gamma.RIIb. Furthermore, compared
with IL6R-B3, all variants had lower affinity for Fc.gamma.RIa and
Fc.gamma.RIIIaV.
[0481] The variable region and constant region in the sequence of
the respective SEQ ID NOs are summarized in the following Table. In
the table, "B3" refers to "2B999(B3)", "omlizH" refers to
"omalizumab_VH", and "omlizL" refers to "omalizumab_VL".
TABLE-US-00016 TABLE 27 SEQ ID NO VARIABLE REGION CONSTANT REGION
15 GpH7 16 GpL16 k0 17 GpH7 B3 18 IL6R 19 IL6R G1d 20 IL6R IgG1-v1
21 IL6R-L k0 22 IL6R F11 23 IL6R B3 24 IL6R BP208 25 omlizH G1d 26
omlizL CK 27 IL6R BP230 28 IL6R BP264 29 IL6R BP267 30 IL6R G4d 31
IL6R BP478 32 IL6R BP253 33 IL6R BP423 34 GpH7 G1d 35 GpH7 A5 36
IL6R BP404 37 IL6R BP408 38 IL6R BP419 39 IL6R BP407 40 IL6R BP409
41 IL6R BP410 42 IL6R AP029 43 BP230 44 BP231 45 BP265 46 BP391 47
BP429 48 BP436 49 BP437 50 BP445 51 BP473 52 BP478 53 BP481 54
BP487 55 BP488 56 BP489 57 BP490 58 BP491 59 BP492 60 BP493 61
BP494 62 BP495 63 BP498 64 BP499 65 BP503 66 BP509 67 BP510 68
BP511 69 IL6R A5 70 Fc(P587) 71 Fc(P588) 72 IL6R P587 73 IL6R
P587-LS 74 IL6R-L2 k0 75 BP557 76 BP559 77 BP567 78 Fc(DLE) 79
Fc(YTE) 80 Fc(EF) 81 Fc(P208)
INDUSTRIAL APPLICABILITY
[0482] An Fc region variant with enhanced Fc.gamma.RIIb-binding
activity, and enhanced binding selectivity to Fc.gamma.RIIb
compared to Fc.gamma.RIIa (type R), as compared to those of a
polypeptide comprising an Fc region to which amino acid
alteration(s) have not been introduced; and a polypeptide which
comprises the Fc region variant. Use of the polypeptide enables
transmission of an inhibitory signal of inflammatory immune
response mediated by phosphorylation of ITIM of Fc.gamma.RIIb.
Furthermore, by conferring an antibody Fc with the property of
selective Fc.gamma.RIIb binding, anti-drug antibody production may
be suppressed through Fc.gamma.RIIb-mediated immunosuppressive
actions.
Sequence CWU 1
1
8111125DNAHomo sapiens 1atgtggttct 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 11252374PRTHomo sapiens 2Met 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
3703951DNAHomo sapiens 3atgactatgg 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
9514316PRTHomo sapiens 4Met 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 3155876DNAHomo sapiens 5atgggaatcc
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
8766291PRTHomo sapiens 6Met 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 2907765DNAHomo sapiens
7atgtggcagc 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 7658254PRTHomo
sapiens 8Met 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 2509702DNAHomo sapiens
9atgtggcagc 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 70210233PRTHomo sapiens 10Met
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
23011330PRTartificialAn artificially generated sequence 11Ala 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 33012326PRTartificialAn artificially
generated sequence 12Ala 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 32513377PRTartificialAn artificially generated sequence 13Ala
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 37514327PRTartificialAn artificially generated
sequence 14Ala 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
32515115PRTArtificialAn artificially generated sequence 15Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Thr 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 Glu Gly Leu Glu Trp Ile
35 40 45Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Glu Ser
Phe 50 55 60Gln Asp 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 11516219PRTArtificialAn
artificially generated sequence 16Asp Ile Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys
Gln Ala Ser Glu 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 Val Glu Ile Glu 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 21517443PRTArtificialAn
artificially generated sequence 17Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Thr 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 Glu Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Asp Pro
Lys Thr Gly Asp Thr Ala Tyr Ser Glu Ser Phe 50 55 60Gln Asp 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 Leu Leu 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 Asn 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 Glu
Ser Leu Ser Leu Ser Pro 435 44018119PRTArtificialAn artificially
generated sequence 18Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly
His Ser Ile Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro
Pro Gly Glu Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly
Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu
Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 11519447PRTArtificialAn artificially generated
sequence 19Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile
Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu
Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr
Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150
155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265
270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro 435 440 44520447PRTArtificialAn artificially generated
sequence 20Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile
Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu
Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr
Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150
155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Asp225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265
270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 44521214PRTArtificialAn artificially
generated sequence 21Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Asp Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Arg Leu His Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr
Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 21022447PRTArtificialAn artificially generated sequence 22Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp
20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu
Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro
Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala Met
Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Tyr Ile Ser 245 250 255Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410
415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430Ala Leu His Tyr His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 435 440 44523447PRTArtificialAn artificially generated sequence
23Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu
Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn
Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala
Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser
Leu Ser Pro 435 440 44524447PRTArtificialAn artificially generated
sequence 24Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile
Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu
Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr
Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150
155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Asp Leu Leu Gly Asp Asp225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Asp 260 265
270Gly Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Arg Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu
Ser Leu Ser Pro 435 440 44525449PRTArtificialAn artificially
generated sequence 25Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Val Ser Gly
Tyr Ser Ile Thr Ser Gly 20 25 30Tyr Ser Trp Asn Trp Ile Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp 35 40 45Val Ala Ser Ile Thr Tyr Asp Gly
Ser Thr Asn Tyr Asn Pro Ser Val 50 55 60Lys Gly Arg Ile Thr Ile Ser
Arg Asp Asp Ser Lys Asn Thr Phe Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
His Tyr Phe Gly His Trp His Phe Ala Val Trp Gly 100 105 110Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro26218PRTArtificialAn artificially generated
sequence 26Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val
Asp Tyr Asp 20 25 30Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu
Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95Glu Asp Pro Tyr Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150
155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 21527447PRTArtificialAn artificially generated sequence
27Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu
Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn
Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala
Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Asp Leu Leu Gly Asp Asp225 230 235 240Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Asp 260 265 270Gly
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Asp Asn Ser Thr Tyr Arg Val
290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Arg Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser
Leu Ser Pro 435 440 44528447PRTArtificialAn artificially generated
sequence 28Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile
Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu
Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr
Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150
155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Asp Leu Leu Gly Asp Asp225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Glu Glu Asp 260 265
270Gly Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Asp Asn Ser Thr Tyr
Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Arg Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu
Ser Leu Ser Pro 435 440 44529447PRTArtificialAn artificially
generated sequence 29Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly
His Ser Ile Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro
Pro Gly Glu Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly
Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu
Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Asp Leu Leu Gly Asp Asp225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Glu
Glu Asp 260 265 270Gly Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr
Gln Glu Ser Leu Ser Leu Ser Pro 435 440 44530444PRTArtificialAn
artificially generated sequence 30Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala
Val Ser Gly His Ser Ile Ser His Asp 20 25 30His Ala Trp Ser Trp Val
Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser
Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105
110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe225 230
235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345
350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Arg Leu
Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Leu 435 44031444PRTArtificialAn
artificially generated sequence 31Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala
Val Ser Gly His Ser Ile Ser His Asp 20 25 30His Ala Trp Ser Trp Val
Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser
Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105
110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys
Pro Pro Cys Pro Ala Pro Asp Leu Leu Gly Asp Asp Ser Val Phe225 230
235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Asp
Gly Glu Val 260 265 270Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Asp Asn Ser
Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn
Lys Ala Leu Pro Arg Pro Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345
350Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375
380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp 405 410 415Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Glu Ser Leu
Ser Leu Ser Pro 435 44032447PRTArtificialAn artificially generated
sequence 32Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile
Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu
Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr
Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150
155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Glu His Glu Asp 260 265
270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala
Phe Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu
Ser Leu Ser Pro 435 440 44533447PRTArtificialAn artificially
generated sequence 33Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly
His Ser Ile Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro
Pro Gly Glu Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly
Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu
Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Asp Leu Leu Gly Asp Asp225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ala Glu
Glu Asp 260 265 270Gly Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Arg Pro Ile Glu Lys 325 330 335Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr
Gln Glu Ser Leu Ser Leu Ser Pro 435 440 44534443PRTArtificialAn
artificially generated sequence 34Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Thr 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 Glu Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Asp Pro
Lys Thr Gly Asp Thr Ala Tyr Ser Glu Ser Phe 50 55 60Gln Asp 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 Leu Leu 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 Asn 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 44035443PRTArtificialAn artificially
generated sequence 35Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Thr 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 Glu Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Asp Pro Lys Thr Gly
Asp Thr Ala Tyr Ser Glu Ser Phe 50 55 60Gln Asp 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 Leu Leu 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 Asn 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
Lys 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 430Arg Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 44036447PRTArtificialAn artificially generated
sequence 36Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile
Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu
Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr
Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150
155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Asp Tyr Leu Gly Asp Asp225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Glu Glu Asp 260 265
270Gly Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Asp Asn Ser Thr Tyr
Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Arg Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu
Ser Leu Ser Pro 435 440 44537447PRTArtificialAn artificially
generated sequence 37Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly
His Ser Ile Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro
Pro Gly Glu Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly
Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu
Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Asp Leu Gln Gly Asp Asp225 230 235 240Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Glu Glu Asp 260 265 270Gly Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala
Lys Thr Lys Pro Arg Glu Glu Gln Asp Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Arg
Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410
415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser
Pro 435 440 44538447PRTArtificialAn artificially generated sequence
38Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu
Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn
Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala
Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Asp Leu Phe Gly Asp Asp225 230 235 240Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Glu Glu Asp 260 265 270Gly
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Asp Asn Ser Thr Tyr Arg Val
290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Arg Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser
Leu Ser Pro 435 440 44539447PRTArtificialAn artificially generated
sequence 39Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile
Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu
Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr
Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150
155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Asp Leu Asp Gly Asp Asp225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Glu Glu Asp 260 265
270Gly Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Asp Asn Ser Thr Tyr
Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Arg Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu
Ser Leu Ser Pro 435 440 44540447PRTArtificialAn artificially
generated sequence 40Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly
His Ser Ile Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro
Pro Gly Glu Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly
Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu
Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Asp Leu Glu Gly Asp Asp225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Glu
Glu Asp 260 265 270Gly Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Asp
Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Arg Pro Ile Glu Lys 325 330 335Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr
Gln Glu Ser Leu Ser Leu Ser Pro 435 440 44541447PRTArtificialAn
artificially generated sequence 41Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala
Val Ser Gly His Ser Ile Ser His Asp 20 25 30His Ala Trp Ser Trp Val
Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser
Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105
110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Asp Leu Thr Gly Asp Asp225 230
235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
Glu Glu Asp 260 265 270Gly Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln
Asp Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Arg Pro Ile Glu Lys 325 330 335Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345
350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His
Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440
44542447PRTArtificialAn artificially generated sequence 42Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr
Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25
30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp
35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser
Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp
Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Asp Leu Leu Gly Asp Asp225 230 235 240Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Glu Glu Asp 260 265 270Gly Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala
Lys Thr Lys Pro Arg Glu Glu Gln Asp Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Arg
Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro
Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn Arg Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 435 440 44543328PRTArtificialAn
artificially generated sequence 43Ala 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 Asp Leu Leu Gly Asp Asp 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 Asp Glu Asp Gly 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 Asp 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 Arg 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 Glu Ser Leu
Ser Leu Ser Pro 32544328PRTArtificialAn artificially generated
sequence 44Ala 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 Asp Asp 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 Asp Glu Asp Gly 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 Asp 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 Arg 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 Glu Ser Leu Ser Leu Ser Pro
32545328PRTArtificialAn artificially generated sequence 45Ala 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 Asp Asp
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 Glu Glu Asp Gly 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 Asp 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 Arg 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 Glu Ser Leu Ser Leu Ser Pro
32546328PRTArtificialAn artificially generated sequence 46Ala 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 Asp Leu Leu Gly Asp Asp
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 Asp Glu Asp Gly 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 Asp 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 Arg Pro Thr 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 Glu Ser Leu Ser Leu Ser Pro
32547328PRTArtificialAn artificially generated sequence 47Ala 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 Asp Leu Leu Gly Asp Asp
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 Ile Asp Val
Gly Glu Glu Asp Gly 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 Arg 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 Glu Ser Leu Ser Leu Ser Pro
32548328PRTArtificialAn artificially generated sequence 48Ala 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 Asp Leu Leu Gly Asp Asp
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
Ala Glu Glu Asp Gly 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 Asp 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 Arg Pro Thr 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 Glu Ser Leu Ser Leu Ser Pro
32549328PRTArtificialAn artificially generated sequence 49Ala 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 Asp Asp
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
Ala Glu Glu Asp Gly 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 Asp 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 Arg Pro Thr 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 Glu Ser Leu Ser Leu Ser Pro
32550328PRTArtificialAn artificially generated sequence 50Ala 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 Asp Leu Leu Gly
Asp Asp 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 Ile
Asp Val Ala Glu Glu Asp Gly 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 Arg 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 Glu Ser Leu Ser Leu Ser Pro
32551328PRTArtificialAn artificially generated sequence 51Ala 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 Asp Leu Leu Gly Asp Asp
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 Asp Glu Asp Gly 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 Asp 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 Gly Leu Pro Arg 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 Glu Ser Leu Ser Leu Ser Pro
32552325PRTArtificialAn artificially generated sequence 52Ala 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
Pro Cys Pro Ala Pro 100 105 110Asp Leu Leu Gly Asp Asp 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 Asp Glu
Asp Gly Glu Val Lys Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Asp 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
Ala Leu 195 200 205Pro Arg Pro 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
Arg Asp Glu Leu 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 285Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 290 295
300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu
Ser305 310 315 320Leu Ser Leu Ser Pro 32553328PRTArtificialAn
artificially generated sequence 53Ala 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 Asp Leu Leu Gly Asp Asp 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 Ile Asp Val Ala Glu Glu Asp Gly 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 Glu Ser Leu
Ser Leu Ser Pro 32554328PRTArtificialAn artificially generated
sequence 54Ala 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 Asp Leu
Leu Gly Asp Asp 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 Ile Asp Val Ala Glu Glu Asp Gly 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 Arg 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 Met 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 Glu Ser Leu Ser Leu Ser Pro
32555328PRTArtificialAn artificially generated sequence 55Ala 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 Asp Leu Leu Gly Asp Asp
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 Ile Asp Val
Ala Glu Glu Asp Gly 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 Asp 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 Arg 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 Glu Ser Leu Ser Leu Ser Pro
32556328PRTArtificialAn artificially generated sequence 56Ala 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 Asp Leu Leu Gly Asp Asp
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 Ile Asp Val
Ala Glu Glu Asp Gly 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 Asp 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 Arg 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 Met 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 Glu Ser Leu Ser Leu Ser Pro
32557328PRTArtificialAn artificially generated sequence 57Ala 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 Asp Asp
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 Ile
Asp Val Ala Glu Glu Asp Gly 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 Arg 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 Glu Ser Leu Ser Leu Ser Pro
32558328PRTArtificialAn artificially generated sequence 58Ala 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 Asp Asp
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 Ile Asp Val
Ala Glu Glu Asp Gly 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 Asp 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 Arg 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 Glu Ser Leu Ser Leu Ser Pro
32559328PRTArtificialAn artificially generated sequence 59Ala 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 Asp
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 Ile Asp Val
Ala Glu Glu Asp Gly 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 Glu Ser Leu Ser Leu Ser Pro
32560328PRTArtificialAn artificially generated sequence 60Ala 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 Asp
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 Ile Asp Val
Ala Glu Glu Asp Gly 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 Asp 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 Glu Ser Leu Ser Leu Ser Pro
32561328PRTArtificialAn artificially generated sequence 61Ala 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 Asp Asp
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
Ala Glu Glu Asp Gly 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 Asp 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 Arg 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 Glu Ser Leu Ser Leu Ser Pro
32562328PRTArtificialAn artificially generated sequence 62Ala 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 Asp Asp
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
Gly Glu Glu Asp Gly 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 Asp 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 Arg 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 Glu Ser Leu Ser Leu Ser Pro
32563328PRTArtificialAn artificially generated 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 Asp Leu Leu Gly Asp Asp
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 Ile Asp Val
Ala Glu Glu Asp Gly 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 Arg 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 Leu 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 Glu Ser Leu Ser Leu Ser Pro
32564328PRTArtificialAn artificially generated 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 Asp Leu Leu Gly Asp Asp
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 Ile Asp Val
Ala Glu Glu Asp Gly 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 Asp 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 Arg 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 Leu 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 Glu Ser Leu Ser Leu Ser Pro
32565328PRTArtificialAn artificially generated 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 Asp Leu Leu Gly Asp Asp
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 Ile Asp Val
Ala Glu Glu Asp Gly 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 Asp 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 Gly Leu Pro Arg 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 Met 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 Glu Ser Leu Ser Leu Ser Pro
32566328PRTArtificialAn artificially generated 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 Asp Leu Leu Gly Asp Asp
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 Ile Asp Val
Ala Glu Glu Asp Gly Asp 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 Asp 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 Glu Ser Leu Ser Leu Ser Pro
32567328PRTArtificialAn artificially generated sequence 67Ala 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 Asp Asp
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 Ile Asp Val
Ala Glu Glu Asp Gly Pro 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 Arg 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 Glu Ser Leu Ser Leu Ser Pro
32568328PRTArtificialAn artificially generated sequence 68Ala 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 Asp Asp
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 Ile Asp Val
Ala Glu Glu Asp Gly Pro 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 Asp 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 Arg 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 Glu Ser Leu Ser Leu Ser Pro
32569447PRTArtificialAn artificially generated sequence 69Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr
Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25
30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp
35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser
Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp
Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Lys Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410
415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 435 440 44570328PRTArtificialAn artificially generated sequence
70Ala 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 Asp Leu Leu Gly
Gly Asp 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 Ile
Asp Val Ala Glu Glu Asp Gly 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
32571328PRTArtificialAn artificially generated sequence 71Ala 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 Asp
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 Ile Asp Val
Ala Glu Glu Asp Gly 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 32572447PRTArtificialAn artificially generated sequence
72Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu
Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn
Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala
Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Asp Leu Leu Gly Gly Asp225 230 235 240Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Ile Asp Val Ala Glu Glu Asp 260 265 270Gly
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro 435 440 44573447PRTArtificialAn artificially generated
sequence 73Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile
Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu
Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr
Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150
155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Asp Leu Leu Gly Gly Asp225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Ile Asp Val Ala Glu Glu Asp 260 265
270Gly Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Leu His Glu 420 425 430Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 44574214PRTArtificialAn artificially
generated sequence 74Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Ser Val Thr Ile Thr Cys Gln Ala Ser
Thr Asp Ile Ser Ser His 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Glu Leu Leu Ile 35 40 45Tyr Tyr Gly Ser His Leu Leu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Phe Thr Ile Ser Ser Leu Glu Ala65 70 75 80Glu Asp Ala Ala Thr
Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 21075328PRTArtificialAn artificially generated sequence 75Ala
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 Asp
Asp 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 Gly Asp Glu Asp Gly 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 Asp 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 Arg 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 Glu Ser Leu Ser Leu Ser Pro
32576328PRTArtificialAn artificially generated sequence 76Ala 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 Asp
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 Ile Asp Val
Ala Glu Glu Asp Gly Asp 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 Asp 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 Glu Ser Leu Ser Leu Ser Pro
32577328PRTArtificialAn artificially generated sequence 77Ala 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 Asp Leu Leu Gly Gly Asp
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 Ile Asp Val
Ala Glu Glu Asp Gly 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 Asp 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 Glu Ser Leu Ser Leu Ser Pro
32578328PRTArtificialAn artificially generated sequence 78Ala 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
Asp 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 Leu Pro Glu 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
32579328PRTArtificialAn artificially generated sequence 79Ala 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 Tyr
Ile Thr Arg Glu 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
32580328PRTArtificialAn artificially generated sequence 80Ala 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
Glu 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 Phe 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
32581328PRTArtificialAn artificially generated sequence 81Ala 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 Asp Leu Leu Gly Asp Asp
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 Asp Glu Asp Gly 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 Arg 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 325
* * * * *