U.S. patent application number 14/379825 was filed with the patent office on 2016-02-18 for antigen-binding molecule for promoting disappearance of antigen via fc gamma riib.
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 Kenta Haraya, Tomoyuki Igawa, Yuki Iwayanagi, Shojiro Kadono, Hitoshi Katada, Atsuhiko Maeda, Futa Mimoto.
Application Number | 20160046693 14/379825 |
Document ID | / |
Family ID | 49005844 |
Filed Date | 2016-02-18 |
United States Patent
Application |
20160046693 |
Kind Code |
A1 |
Igawa; Tomoyuki ; et
al. |
February 18, 2016 |
Antigen-Binding Molecule for Promoting Disappearance of Antigen via
Fc gamma RIIB
Abstract
The present invention provides antigen-binding molecules
containing (i) an antigen-binding domain whose antigen-binding
activity varies depending on ion concentration conditions, (ii) an
Fc.gamma.R-binding domain having Fc.gamma. RIIb-selective binding
activity, and (iii) an FcRn-binding domain having FcRn-binding
activity under an acidic pH range condition, and methods of
decreasing plasma antigen concentration as compared to before
administering the molecule, which include the step of administering
the molecule.
Inventors: |
Igawa; Tomoyuki; (Shizuoka,
JP) ; Maeda; Atsuhiko; (Shizuoka, JP) ;
Iwayanagi; Yuki; (Shizuoka, JP) ; Haraya; Kenta;
(Shizuoka, JP) ; Katada; Hitoshi; (Shizuoka,
JP) ; Kadono; Shojiro; (Kanagawa, JP) ;
Mimoto; Futa; (Shizuoka, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chugai Seiyaku Kabushiki Kaisha |
Tokyo |
|
JP |
|
|
Assignee: |
CHUGAI SEIYAKU KABUSHIKI
KAISHA
Tokyo
JP
|
Family ID: |
49005844 |
Appl. No.: |
14/379825 |
Filed: |
February 22, 2013 |
PCT Filed: |
February 22, 2013 |
PCT NO: |
PCT/JP2013/054461 |
371 Date: |
August 20, 2014 |
Current U.S.
Class: |
424/172.1 ;
435/69.6; 530/389.1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 2039/505 20130101; C07K 2317/526 20130101; C07K 2317/52
20130101; C07K 2317/76 20130101; C07K 2317/92 20130101; C07K
16/2866 20130101; C07K 16/00 20130101; C07K 2317/72 20130101; C07K
2317/94 20130101; C07K 2317/71 20130101; C07K 16/303 20130101; C07K
2317/524 20130101; C07K 2317/41 20130101; A61P 29/00 20180101 |
International
Class: |
C07K 16/00 20060101
C07K016/00; C07K 16/28 20060101 C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 24, 2012 |
JP |
PCT/JP2012/054624 |
Aug 24, 2012 |
JP |
2012-185866 |
Sep 28, 2012 |
JP |
PCT/JP2012/075092 |
Claims
1.-22. (canceled)
23. A method of removing an antigen from plasma, the method
comprising: (a) identifying a subject in need of having an antigen
removed from the subject's plasma; (b) providing an antigen-binding
molecule that specifically binds to the antigen, wherein the
antigen-binding molecule comprises: (i) an antigen-binding domain
that binds to the antigen in an ion concentration-dependent manner;
and (ii) an Fc region in which the amino acids at positions 238 and
271 (EU numbering) are aspartic acid and glycine, respectively, and
(c) administering the antigen-binding molecule to the subject.
24. The method of claim 23, wherein the Fc region is a modified Fc
region that differs from the amino acid sequence of a native Fc
region at sites including one or more of the following positions
(EU numbering): 233, 234, 237, 264, 265, 266, 267, 268, 269, 272,
274, 296, 326, 327, 330, 331, 332, 333, 355, 356, 358, 396, 409,
and 419.
25. The method of claim 24, wherein at least one of the following
positions in the modified Fc region is occupied by the indicated
amino acid (all positions indicated by EU numbering): aspartic acid
at position 233; tyrosine at position 234; aspartic acid at
position 237; isoleucine at position 264; glutamic acid at position
265; any one of phenylalanine, methionine, and leucine at position
266; any one of alanine, glutamic acid, glycine, and glutamine at
position 267; any one of aspartic acid, glutamic acid, and
glutamine at position 268; aspartic acid at position 269; any one
of aspartic acid, phenylalanine, isoleucine, methionine,
asparagine, proline, or glutamine at position 272; glutamine at
position 274; aspartic acid or phenylalanine at position 296;
alanine or aspartic acid at position 326; glycine at position 327;
lysine or arginine at position 330; serine at position 331;
threonine at position 332; any one of threonine, lysine, and
arginine at position 333; glutamine at position 355; glutamine at
position 356; methionine at position 358; any one of aspartic acid,
glutamic acid, phenylalanine, isoleucine, lysine, leucine,
methionine, glutamine, arginine, and tyrosine at position 396;
arginine at amino acid position 409; and glutamic acid at amino
acid position 419.
26. The method of claim 23, wherein the antigen-binding domain
binds the antigen in a calcium ion concentration-dependent
manner.
27. The method of claim 26, wherein the antigen-binding domain has
decreased binding to the antigen at a calcium ion concentration
between 0.1 .mu.M and 30 .mu.M compared to at a calcium ion
concentration between 100 .mu.M and 10 mM.
28. The method of claim 23, wherein the antigen-binding domain
binds the antigen in a pH-dependent manner.
29. The method of claim 28, wherein the antigen-binding domain has
decreased binding to the antigen at a pH of 4.0 to 6.5 compared to
at a pH of 6.7 to 10.
30. The method of claim 23, wherein the antigen-binding domain is
an antibody variable region.
31. The method of claim 23, wherein the Fc region is an Fc region
of any one of SEQ ID NOs: 14, 15, 16, or 17 with at least the
following two changes: the amino acids at positions 238 and 271 (EU
numbering) are substituted with aspartic acid and glycine,
respectively.
32. The method of claim 23, wherein, at a pH of 4.0 to 6.5, the
FcRn-binding activity of the Fc region is greater than the
FcRn-binding activity of an Fc region selected from the group of Fc
regions consisting of SEQ ID NOs: 14, 15, 16, and 17.
33. The method of claim 32, wherein the Fc region in which the
amino acids at positions 238 and 271 (EU numbering) are aspartic
acid and glycine is an Fc region having an amino acid substitution
at at least one or more of the following positions (indicated by EU
numbering): 244, 245, 249, 250, 251, 252, 253, 254, 255, 256, 257,
258, 260, 262, 265, 270, 272, 279, 283, 285, 286, 288, 293, 303,
305, 307, 308, 309, 311, 312, 314, 316, 317, 318, 332, 339, 340,
341, 343, 356, 360, 362, 375, 376, 377, 378, 380, 382, 385, 386,
387, 388, 389, 400, 413, 415, 423, 424, 427, 428, 430, 431, 433,
434, 435, 436, 438, 439, 440, 442, and 447 in the amino acid
sequence of the Fc region of any one of SEQ ID NOs: 14, 15, 16, or
17.
34. The method of claim 33, wherein the Fc region in which the
amino acids at positions 238 and 271 (EU numbering) are aspartic
acid and glycine has at least one of the following positions
occupied by the indicated amino acid (all positions indicated by EU
numbering): leucine at position 244; arginine at position 245;
proline at position 249; glutamine or glutamic acid at position
250; any one of arginine, aspartic acid, glutamic acid, and leucine
at position 251; any one of phenylalanine, serine, threonine, and
tyrosine at position 252; serine or threonine at position 254; any
one of arginine, glycine, isoleucine, and leucine at position 255;
any one of alanine, arginine, asparagine, aspartic acid, glutamine,
glutamic acid, proline, and threonine at position 256; any one of
alanine, isoleucine, methionine, asparagine, serine, and valine at
position 257; aspartic acid at position 258; serine at position
260; leucine at position 262; lysine at position 270; leucine or
arginine at position 272; any one of alanine, aspartic acid,
glycine, histidine, methionine, asparagine, glutamine, arginine,
serine, threonine, tryptophan, and tyrosine at position 279; any
one of alanine, aspartic acid, phenylalanine, glycine, histidine,
isoleucine, lysine, leucine, asparagine, proline, glutamine,
arginine, serine, threonine, tryptophan, and tyrosine at position
283; asparagine at position 285; phenylalanine at position 286;
asparagine or proline at position 288; valine at position 293; any
one of alanine, glutamic acid, glutamine, and methionine at
position 307; any one of isoleucine, proline, and threonine at
position 308; proline at position 309; any one of alanine, glutamic
acid, isoleucine, lysine, leucine, methionine, serine, valine, and
tryptophan at position 311; any one of alanine, aspartic acid, and
proline at position 312; alanine or leucine at position 314; lysine
at position 316; proline at position 317; asparagine or threonine
at position 318; any one of phenylalanine, histidine, lysine,
leucine, methionine, arginine, serine, and tryptophan at position
332; any one of asparagine, threonine, and tryptophan at position
339; proline at position 341; any one of glutamic acid, histidine,
lysine, glutamine, arginine, threonine, or tyrosine at position
343; arginine at position 375; any one of glycine, isoleucine,
methionine, proline, threonine, and valine at position 376; lysine
at position 377; any one of aspartic acid, asparagine, and valine
at position 378; any one of alanine, asparagine, serine, and
threonine at position 380; any one of phenylalanine, histidine,
isoleucine, lysine, leucine, methionine, asparagine, glutamine,
arginine, serine, threonine, valine, tryptophan, and tyrosine at
position 382; any one of alanine, arginine, aspartic acid, glycine,
His, lysine, serine, and threonine at position 385; any one of
arginine, aspartic acid, isoleucine, lysine, methionine, proline,
serine, and threonine at position 386; any one of alanine,
arginine, His, proline, serine, and threonine at amino acid
position 387; any one of asparagine, proline, and serine at
position 389; asparagine at position 423; asparagine at position
427; any one of leucine, methionine, phenylalanine, serine, and
threonine at position 428; any one of alanine, phenylalanine,
glycine, histidine, isoleucine, lysine, leucine, methionine,
asparagine, glutamine, arginine, serine, threonine, valine, and
tyrosine at position 430; histidine or asparagine at position 431;
any one of arginine, glutamine, histidine, isoleucine, lysine,
proline, and serine at position 433; any one of alanine, glycine,
histidine, phenylalanine, serine, tryptophan, and tyrosine at
position 434; any one of arginine, asparagine, histidine,
isoleucine, leucine, lysine, methionine, and threonine at position
436; any one of lysine, leucine, threonine, and tryptophan at
position 438; lysine at position 440; and lysine at position 442 in
the amino acid sequence of the Fc region of any one of SEQ ID NOs:
14, 15, 16, or 17.
35. The method of claim 23, wherein the antigen-binding molecule is
an antibody.
36. A pharmaceutical composition comprising an antigen-binding
molecule that specifically binds to an antigen, wherein the
antigen-binding molecule comprises: (i) an antigen-binding domain
that binds to the antigen in an ion concentration-dependent manner;
and (ii) an Fc region in which the amino acids at positions 238 and
271 (EU numbering) are aspartic acid and glycine, respectively.
37. The pharmaceutical composition of claim 36, wherein the Fc
region is a modified Fc region which differs from the amino acid
sequence of a native Fc region at sites including one or more of
the following positions (EU numbering): 233, 234, 237, 264, 265,
266, 267, 268, 269, 272, 274, 296, 326, 327, 330, 331, 332, 333,
355, 356, 358, 396, 409, and 419.
38. The pharmaceutical composition of claim 37, wherein at least
one of the following positions in the modified Fc region is
occupied by the indicated amino acid (all positions indicated by EU
numbering): aspartic acid at position 233; tyrosine at position
234; aspartic acid at position 237; isoleucine at position 264;
glutamic acid at position 265; any one of phenylalanine,
methionine, and leucine at position 266; any one of alanine,
glutamic acid, glycine, and glutamine at position 267; any one of
aspartic acid, glutamic acid, and glutamine at position 268;
aspartic acid at position 269; any one of aspartic acid,
phenylalanine, isoleucine, methionine, asparagine, proline, or
glutamine at position 272; glutamine at position 274; aspartic acid
or phenylalanine at position 296; alanine or aspartic acid at
position 326; glycine at position 327; lysine or arginine at
position 330; serine at position 331; threonine at position 332;
any one of threonine, lysine, and arginine at position 333;
glutamine at position 355; glutamine at position 356; methionine at
position 358; any one of aspartic acid, glutamic acid,
phenylalanine, isoleucine, lysine, leucine, methionine, glutamine,
arginine, and tyrosine at position 396; arginine at position 409;
and glutamic acid at position 419.
39. A method of producing an antigen-binding molecule that
specifically binds to an antigen, the method comprising: (a)
identifying a first coding sequence encoding an antigen-binding
domain of an antibody heavy chain, wherein the heavy chain, when
associated with an antibody light chain, forms an antigen-binding
molecule that binds to the antigen in an ion-dependent manner; (b)
providing a host cell containing a nucleic acid or nucleic acids
comprising (i) the first coding sequence linked in-frame to a
second coding sequence encoding an Fc region in which the amino
acids at positions 238 and 271 (indicated by EU numbering) are
aspartic acid and glycine, respectively, and (ii) a third coding
sequence encoding the light chain; (c) culturing the host cell to
express the nucleic acid or nucleic acids and thereby produce the
antigen-binding molecule; and (d) collecting the antigen-binding
molecule.
40. The method of claim 39, wherein the Fc region is a modified Fc
region which differs from the amino acid sequence of a native Fc
region at sites including one or more of the following positions
(EU numbering): 233, 234, 237, 264, 265, 266, 267, 268, 269, 272,
274, 296, 326, 327, 330, 331, 332, 333, 355, 356, 358, 396, 409,
and 419.
41. The method of claim 40, wherein at least one of the following
positions in the modified Fc region is occupied by the indicated
amino acid (all positions indicated by EU numbering): aspartic acid
at position 233; tyrosine at position 234; aspartic acid at
position 237; isoleucine at position 264; glutamic acid at position
265; any one of phenylalanine, methionine, and leucine at position
266; any one of alanine, glutamic acid, glycine, and glutamine at
position 267; any one of aspartic acid, glutamic acid, and
glutamine at position 268; aspartic acid at position 269; any one
of aspartic acid, phenylalanine, isoleucine, methionine,
asparagine, proline, or glutamine at position 272; glutamine at
position 274; aspartic acid or phenylalanine at position 296;
alanine or aspartic acid at position 326; glycine at position 327;
lysine or arginine at position 330; serine at position 331;
threonine at position 332; any one of threonine, lysine, and
arginine at position 333; glutamine at position 355; glutamine at
position 356; methionine at position 358; any one of aspartic acid,
glutamic acid, phenylalanine, isoleucine, lysine, leucine,
methionine, glutamine, arginine, and tyrosine at position 396;
arginine at position 409; and glutamic acid at position 419.
42. The method of claim 39, further comprising formulating the
antigen-binding molecule as a pharmaceutical composition.
43. The method of claim 40, further comprising formulating the
antigen-binding molecule as a pharmaceutical composition.
44. The method of claim 41, further comprising formulating the
antigen-binding molecule as a pharmaceutical composition.
Description
TECHNICAL FIELD
[0001] The present invention provides uses of antigen-binding
molecules for eliminating antigens from plasma; methods for
eliminating antigens from plasma, which comprise administering
antigen-binding molecules; pharmaceutical compositions comprising
antigen-binding molecules that are capable of eliminating antigens
from plasma; and methods for producing antigen-binding molecules
for eliminating antigens from plasma.
BACKGROUND ART
[0002] Antibodies are drawing attention as pharmaceuticals as they
are highly stable in plasma and have few side effects. At present,
a number of IgG-type therapeutic antibodies are available on the
market and many therapeutic antibodies are currently under
development (Non-patent Documents 1 and 2). Meanwhile, various
technologies applicable to second-generation therapeutic antibodies
have been reported, including those that enhance effector function,
antigen-binding ability, pharmacokinetics, and stability, and those
that reduce the risk of immunogenicity (Non-patent Document 3). In
general, the requisite dose of a therapeutic antibody is very high.
This, in turn, has led to problems, such as high production cost,
as well as the difficulty in producing subcutaneous formulations.
In theory, the dose of a therapeutic antibody may be reduced by
improving antibody pharmacokinetics or improving the affinity
between antibodies and antigens.
[0003] Literature has reported methods for improving antibody
pharmacokinetics using artificial substitution of amino acids in
constant regions (Non-patent Documents 4 and 5). Similarly,
affinity maturation has been reported as a technology for enhancing
antigen-binding activity and/or antigen-neutralizing activity of an
antibody (Non-patent Document 6). This technology enables
enhancement of antigen-binding activity by introduction of amino
acid mutations into the CDR region of a variable region or such.
The enhancement of antigen-binding ability enables improvement of
in vitro biological activity or reduction of dosage, and further
enables improvement of in vivo efficacy (Non-patent Document
7).
[0004] The antigen-neutralizing capacity of a single antibody
molecule having neutralizing activity depends on its affinity.
Therefore, the affinity of antibodies has been enhanced using
various methods in order to neutralize antigens with a small amount
of antibodies (Non-patent Document 6). Furthermore, if the affinity
of the antibody to the antigen could be made infinite by covalent
binding to the antigen, a single antibody molecule could neutralize
one antigen molecule (a divalent antibody can neutralize two
antigen molecules). However, the stoichiometric neutralization
reaction of one antibody molecule against one antigen molecule (one
divalent antibody against two antigens) is the limit of such
methods, and thus it is impossible to completely neutralize antigen
with an amount of antibody smaller than the amount of antigen. That
is, antigen-neutralizing effect by enhancing affinity has a limit
(Non-patent Document 8). To sustain the neutralization effect of a
neutralizing antibody for a certain period, the antibody must be
administered at a dose higher than the amount of antigens produced
in the body during the same period. With just the improvement of
antibody pharmacokinetics or affinity maturation technology
described above, there is thus a limitation in the reduction of the
required antibody dose. Accordingly, in order to sustain the
antigen-neutralizing effect for a target period with an amount of
antibody smaller than the amount of antigen, a single antibody must
neutralize multiple antigens. An antigen-binding molecule that
binds to an antigen in a pH- and/or metal ion
concentration-dependent manner has recently been reported as a
novel method for achieving the above objective (Patent Documents 1
and 2). The ion concentration-dependent antigen-binding molecules,
which strongly bind to an antigen under neutral pH and/or high
calcium ion concentration conditions in plasma and dissociate from
the antigen under acidic pH and/or low calcium ion concentration
conditions in the endosome, can dissociate from the antigen in the
endosome. When an ion concentration-dependent antigen-binding
molecule dissociates from the antigen is recycled to the plasma by
FcRn, it can bind to another antigen again. Thus, a single ion
concentration-dependent antigen-binding molecule can bind to a
number of antigens repeatedly.
[0005] On the other hand, the plasma retention of an antigen is
very short compared to antibodies recycled via FcRn binding.
However, even though the plasma retention of the antigen itself is
short, when a typical antibody with such a long plasma retention
binds to the antigen, the plasma retention of the antigen-antibody
complex is prolonged similar to the antibody. Thus, normally, when
an antibody is administered, the antigen bound by the antibody
exists in the form of an antigen-antibody complex, which prolongs
plasma retention of the antigen (antigen is not easily eliminated
from plasma), and causes an increase of plasma antigen
concentration. On the other hand, an ion concentration-dependent
antigen-binding molecule can suppress increase in plasma antigen
concentration by dissociating from the antigen in the endosome.
However, this suppression of increase in plasma antigen
concentration is affected by the balance with the amount of the
antigens produced in vivo. Therefore, the possibility that
administration of such ion concentration-dependent antigen-binding
molecules may elevate the plasma antigen concentration as compared
to before administration was considered (Patent Document 3).
[0006] Recently, antigen-binding molecules that bind to FcRn under
neutral conditions were produced. Administration of an
antigen-binding molecule that binds to an antigen in an ion
concentration-dependent manner and binds to FcRn under neutral
conditions revealed that the molecule can decrease the plasma
antigen concentration as compared to before administration (Patent
Document 3). While typical antibodies increase the plasma antigen
concentration when administered, antigen-binding molecules having
FcRn-binding activity under a neutral pH condition and
antigen-binding molecules that bind to an antigen in an ion
concentration-dependent manner and having FcRn-binding activity
under a neutral pH condition can decrease the plasma antigen
concentration when they are administered. Since such
antigen-binding molecules can actively eliminate antigens from
plasma via endocytosis that takes place as a result of binding to
FcRn, these molecules are highly useful as pharmaceuticals.
[0007] On the other hand, besides FcRn, several Fc.gamma. receptors
(Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIIb, and Fc.gamma.RIIIa)
exist as receptors for IgG (Non-patent Document 9). Since binding
activity of antibodies to activating Fc.gamma. receptors play an
important role in an antibody's cytotoxicity, antibodies targeting
membrane antigens, whose cytotoxicities have been enhanced by
enhancing their binding activity to activating Fc.gamma. receptors
have been developed to date (Non-patent Documents 10 and 11).
Similarly, since binding activity to inhibitory Fc.gamma. receptor
(Fc.gamma.RIIb) plays an important role in immunosuppression
activity (Non-patent Documents 12, 13, and 14), agonistic activity
(Non-Patent Documents 15 and 16), and such, antibodies targeting
membrane antigens, which have enhanced binding activity to
inhibitory Fc.gamma. receptors, are being studied (Non-Patent
Documents 17 and 18).
[0008] Effects of antibodies that bind to soluble antigens on
Fc.gamma.R binding have been examined mainly from the viewpoint of
side effects. For example, it is known that the risk for
thromboembolism increased in a group of patients who were
administered bevacizumab, an antibody against VEGF (Non-patent
Document 19). Similarly, thromboembolism has been observed in
clinical development tests of antibodies against the CD40 ligand,
and the clinical study was discontinued (Non-patent Document 20).
Fc.gamma.RIIa, an activating Fc.gamma. receptor, is expressed on
platelet cells, while an inhibitory Fc.gamma. receptor
Fc.gamma.RIIb is not (Non-patent Document 21), and later studies
using animal models and such have suggested that both of the
administered antibodies aggregate platelets via binding to
Fc.gamma.RIIa on the platelets, and form blood clots as a result
(Non-patent Documents 22 and 23). In patients with 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 24). Furthermore, there are reports
that when an antibody with enhanced Fc.gamma.RIIb-binding is used
as a pharmaceutical, a decrease in risk of antibody production can
be expected (Non-Patent Document 25), and an antibody that binds to
a membrane antigen, whose Fc.gamma.RIIa-binding has been enhanced,
enhances antibody-dependent cellular phagocytosis (ADCP) via
macrophages and dendritic cells (Non-patent Document 26). However,
the binding activity towards activating and/or inhibitory Fc.gamma.
receptors of antibodies targeting soluble antigens had not been
known to have an effect on plasma kinetics of antibodies or
antigens bound by the antibodies in the organisms administered with
the antibodies.
PRIOR ART DOCUMENTS
Patent Documents
[0009] [Patent Document 1] WO2009/125825 [0010] [Patent Document 2]
WO2012/073992 [0011] [Patent Document 3] WO2011/122011
Non-Patent Documents
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development of therapeutic antibodies., Mol. Cells, (2005) 20 (1),
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antibody with longer serum half-life., J. Immunol. (2006) 176 (1),
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[0018] [Non-patent Document 7] Wu H, Pfarr D S, Johnson S, Brewah Y
A, Woods R M, Patel N K, White W I, Young J F, Kiener P A.,
Development of Motavizumab, an Ultra-potent Antibody for the
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[0019] [Non-patent Document 8] Hanson C V, Nishiyama Y, Paul S.,
Catalytic antibodies and their applications., Curr. Opin.
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FcgammaR: current models., Immunol. Lett. (2002) 82, 57-65 [0021]
[Non-patent Document 10] Clynes, R., Yoshizumi, T., Moroi, Y.,
Houghton, A. N., and Ravetch, J. V., Fc Receptors are required for
passive and active immunity to melanoma., Proc. Natl. Acad. Sci.
U.S.A (1998) 95, 652-656 [0022] [Non-patent Document 11] Clynes R
A, Towers T L, Presta L G, Ravetch J V, Inhibitory Fc receptors
modulate in vivo cytoxicity against tumor targets., Nat. Med.
(2000) 6, 443-446 [0023] [Non-patent Document 12] Wernersson S,
Karlsson M C, Dahlstrom J, Mattsson R, Verbeek J S, Heyman B.,
IgG-mediated enhancement of antibody responses is low in Fc
receptor gamma chain-deficient mice and increased in Fc gamma
RII-deficient mice., J. Immunol. (1999) 163 (2), 618-622 [0024]
[Non-patent Document 13] Yuasa T, Kubo S, Yoshino T, Ujike A,
Matsumura K, Ono M, Ravetch J V, Takai T., Deletion of fcgamma
receptor IIB renders H-2(b) mice susceptible to collagen-induced
arthritis., J. Exp. Med. (1999) 189 (1), 187-194 [0025] [Non-patent
Document 14] Nakamura A, Yuasa T, Ujike A, Ono M, Nukiwa T, Ravetch
J V, Takai T., Fcgamma receptor IIB-deficient mice develop
Goodpasture's syndrome upon immunization with type IV collagen: a
novel murine model for autoimmune glomerular basement membrane
disease., J. Exp. Med. (2000) 191 (5), 899-906 [0026] [Non-patent
Document 15] Li F, Ravetch J V., Inhibitory Fc.gamma. receptor
engagement drives adjuvant and anti-tumor activities of agonistic
CD40 antibodies., Science (2011) 333 (6045), 1030-1034 [0027]
[Non-patent Document 16] Wilson N S, Yang B, Yang A, Loeser S,
Marsters S, Lawrence D, Li Y, Pitti R, Totpal K, Yee S, Ross S,
Vernes J M, Lu Y, Adams C, Offringa R, Kelley B, Hymowitz S, Daniel
D, Meng G, Ashkenazi A., An Fc.gamma. receptor-dependent mechanism
drives antibody-mediated target-receptor signaling in cancer
cells., Cancer Cell (2011) 19 (1), 101-113 [0028] [Non-patent
Document 17] Moore G L, Chen H, Karki S, Lazar G A., Engineered Fc
variant antibodies with enhanced ability to recruit complement and
mediate effector functions., Mol. Immunol. (2008) 45, 3926-3933
[0029] [Non-patent Document 18] Li F, Ravetch J V., Apoptotic and
antitumor activity of death receptor antibodies require inhibitory
Fc.gamma. receptor engagement., Proc. Natl. Acad. Sci. USA. (2012)
109 (27), 10966-10971 [0030] [Non-patent Document 19] Scappaticci F
A, Skillings J R, Holden S N, Gerber H P, Miller K, Kabbinavar F,
Bergsland E, Ngai J, Holmgren E, Wang J, Hurwitz H., Arterial
thromboembolic events in patients with metastatic carcinoma treated
with chemotherapy and bevacizumab., J. Natl. Cancer Inst. (2007) 99
(16), 1232-1239 [0031] [Non-patent Document 20] Boumpas D T, Furie
R, Manzi S, Illei G G, Wallace D J, Balow J E, Vaishnaw A, A short
course of BG9588 (anti-CD40 ligand antibody) improves serologic
activity and decreases hematuria in patients with proliferative
lupus glomerulonephritis., Arthritis. Rheum. (2003) 48 (3),
719-727. [0032] [Non-patent Document 21] Mackay M, Stanevsky A,
Wang T, Aranow C, Li M, Koenig S, Ravetch J V, Diamond B.,
Selective dysregulation of the FcgammaIIB receptor on memory B
cells in SLE., J. Exp. Med. (2006) 203 (9), 2157-2164 [0033]
[Non-patent Document 22] Meyer T, Robles-Carrillo L, Robson T,
Langer F, Desai H, Davila M, Amaya M, Francis J L, Amirkhosravi A.,
Bevacizumab immune complexes activate platelets and induce
thrombosis in FCGR2A transgenic mice., J. Thromb. Haemost. (2009) 7
(1), 171-181 [0034] [Non-patent Document 23] Robles-Carrillo L,
Meyer T, Hatfield M, Desai H, Davila M, Langer F, Amaya M, Garber
E, Francis J L, Hsu Y M, Amirkhosravi A., Anti-CD40L immune
complexes potently activate platelets in vitro and cause thrombosis
in FCGR2A transgenic mice., J. Immunol. (2010) 185 (3), 1577-1583
[0035] [Non-patent Document 24] Duffau P, Seneschal J, Nicco C,
Richez C, Lazaro E, Douchet I, Bordes C, Viallard J F, Goulvestre
C, Pellegrin J L, Weil B, Moreau J F, Batteux F, Blanco P.,
Platelet CD154 potentiates interferon-alpha secretion by
plasmacytoid dendritic cells in systemic lupus erythematosus., Sci.
Transl. Med. (2010) 2 (47), 47-63 [0036] [Non-patent Document 25]
Desai D D, Harbers S O, Flores M, Colonna L, Downie M P, Bergtold
A, Jung S, Clynes R., Fc gamma receptor IIB on dendritic cells
enforces peripheral tolerance by inhibiting effector T cell
responses., J. Immunol. (2007) 178 (10), 6217-6226 [0037]
[Non-patent Document 26] Richards J O, Karki S, Lazar G A, Chen H,
Dang W, Desjarlais J R., Optimization of antibody binding to
FcgammaRIIa enhances macrophage phagocytosis of tumor cells., Mol.
Cancer Ther. (2008) 7 (8) 2517-2527
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0038] The present invention was achieved in view of the above
circumstances. As mentioned above, the binding activity towards
activating and/or inhibitory Fc.gamma. receptors of antibodies
targeting soluble antigens had not been known to have an effect on
plasma kinetics of antibodies or antigens bound by the antibodies
in the organisms administered with the antibodies. More
specifically, an objective of the present invention is to suppress
increase in plasma concentration of an antigen bound by an
antigen-binding molecule by administering the antigen-binding
molecule that has a binding activity towards a pathogenic antigen
present in a soluble form in plasma, and also has a desired binding
activity towards activating and/or inhibitory Fc.gamma. receptors.
Another objective of the present invention is to optimize the
binding activity towards activating and/or inhibitory Fc.gamma.
receptors of antigen-binding molecules against the disease-causing
antigens present in a soluble form in plasma, and thereby optimize
the suppression of the increase in plasma concentration of antigens
bound by the antigen-binding molecules.
Means for Solving the Problems
[0039] Specifically, the present invention provides antigen-binding
molecules comprising (i) an antigen-binding domain whose
antigen-binding activity varies depending on ion concentration
conditions, (ii) an Fc.gamma.-binding domain having
Fc.gamma.RIIb-selective binding activity, and (iii) an FcRn-binding
domain having FcRn-binding activity under an acidic pH range
condition, and methods for decreasing plasma concentration of the
antigen as compared to before administering the antigen-binding
molecule, which comprises the step of administering the molecule.
Furthermore, the present invention provides agents for decreasing
plasma concentration of the antigen, which comprise an
antigen-binding molecule comprising (i) an antigen-binding domain
whose antigen-binding activity varies depending on ion
concentration conditions, (ii) an Fc.gamma.-binding domain having
Fc.gamma.RIIb-selective binding activity, and (iii) an FcRn-binding
domain having FcRn-binding activity under an acidic pH range
condition. The present invention also provides pharmaceutical
compositions which comprise an antigen-binding molecule comprising
(i) an antigen-binding domain whose antigen-binding activity varies
depending on ion concentration conditions, (ii) an
Fc.gamma.-binding domain having Fc.gamma.RIIb-selective binding
activity, and (iii) an FcRn-binding domain having FcRn-binding
activity under an acidic pH range condition. The present invention
also provides uses of the antigen-binding molecule for decreasing
plasma concentration of the antigen, wherein the molecule comprises
(i) an antigen-binding domain whose antigen-binding activity varies
depending on ion concentration conditions, (ii) an
Fc.gamma.-binding domain having Fc.gamma.RIIb-selective binding
activity, and (iii) an FcRn-binding domain having FcRn-binding
activity under an acidic pH range condition. In addition to the
above, the present invention provides methods of producing and/or
methods of screening for the antigen-binding molecules. Although it
is not particularly intended to limit the invention, specifically,
the following is provided as a non-limiting embodiment:
[1] use of an antigen-binding molecule for eliminating antigen from
plasma, wherein the antigen-binding molecule comprises an
antigen-binding domain whose antigen-binding activity varies
depending on ion concentration conditions, and an Fc region in
which the amino acid at position 238 is Asp and the amino acid at
position 271 is Gly as indicated by EU numbering; [2] the use of
[1], wherein the Fc region has an amino acid substitution at at
least one or more positions selected from the group consisting of
233, 234, 237, 264, 265, 266, 267, 268, 269, 272, 274, 296, 326,
327, 330, 331, 332, 333, 355, 356, 358, 396, 409, and 419 as
indicated by EU numbering; [3] the use of [2], wherein the amino
acids of the Fc region include any one or more of the following
amino acids indicated by EU numbering: Asp at amino acid position
233; Tyr at amino acid position 234; Asp at amino acid position
237; Ile at amino acid position 264; Glu at amino acid position
265; any one of Phe, Met, and Leu at amino acid position 266; any
one of Ala, Glu, Gly, and Gln at amino acid position 267; any one
of Asp, Glu, and Gln at amino acid position 268; Asp at amino acid
position 269; any one of Asp, Phe, Ile, Met, Asn, Pro, and 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 or Arg at amino acid position
330; Ser at amino acid position 331; Thr at amino acid position
332; any one of Thr, Lys, and Arg at amino acid position 333; Gln
at amino acid position 355; Glu at amino acid position 356; Met at
amino acid position 358; any one of Asp, Glu, Phe, Ile, Lys, Leu,
Met, Gln, Arg, and Tyr at amino acid position 396; Arg at amino
acid position 409; and Glu at amino acid position 419; [4] the use
of any one of [1] to [3], wherein the antigen-binding domain is an
antigen-binding domain whose antigen-binding activity varies
depending on calcium ion concentration conditions; [5] the use of
[4], wherein the antigen-binding domain is an antigen-binding
domain whose antigen-binding activity varies such that the
antigen-binding activity under a low calcium ion concentration
condition is lower than an antigen-binding activity under a high
calcium ion concentration condition; [6] the use of any one of [1]
to [3], wherein the antigen-binding domain is an antigen-binding
domain whose antigen-binding activity varies depending on pH
conditions; [7] the use of [6], wherein the antigen-binding domain
is an antigen-binding domain whose antigen-binding activity varies
such that the antigen-binding activity under an acidic pH range
condition is lower than an antigen-binding activity under a neutral
pH range condition; [8] the use of any one of [1] to [7], wherein
the antigen-binding domain is an antibody variable region; [9] the
use of any one of [1] to [8], wherein the Fc region is an Fc region
in which the amino acid at position 238 is Asp and the amino acid
at position 271 is Gly as indicated by EU numbering in the Fc
region included in any one of SEQ ID NOs: 14, 15, 16, or 17; [10]
the use of any one of [1] to [8], wherein the FcRn-binding activity
of the Fc region under an acidic pH range condition is enhanced
compared to the FcRn-binding activity of the Fc region included in
any one of SEQ ID NO: 14, 15, 16, or 17; [11] the use of [10],
wherein the Fc region with enhanced binding is an Fc region having
an amino acid substitution at least one or more positions selected
from the group consisting of 244, 245, 249, 250, 251, 252, 253,
254, 255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283, 285,
286, 288, 293, 303, 305, 307, 308, 309, 311, 312, 314, 316, 317,
318, 332, 339, 340, 341, 343, 356, 360, 362, 375, 376, 377, 378,
380, 382, 385, 386, 387, 388, 389, 400, 413, 415, 423, 424, 427,
428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447, as
indicated by EU numbering, in the amino acid sequence of the Fc
region included in any one of SEQ ID NO: 14, 15, 16, or 17; [12]
the use of [11], wherein the Fc region with enhanced binding
comprises at least one or more amino acids selected from the group
consisting of: Leu at amino acid position 244; Arg at amino acid
position 245; Pro at amino acid position 249; Gln or Glu at amino
acid position 250; any one of Arg, Asp, Glu, and Leu at amino acid
position 251; any one of Phe, Ser, Thr, and Tyr at amino acid
position 252; Ser or Thr at amino acid position 254; any one of
Arg, Gly, Ile, and Leu at amino acid position 255; any one of Ala,
Arg, Asn, Asp, Gln, Glu, Pro, and Thr at amino acid position 256;
any one of Ala, Ile, Met, Asn, Ser, and Val at amino acid position
257; Asp at amino acid position 258; Ser at amino acid position
260; Leu at amino acid position 262; Lys at amino acid position
270; Leu or Arg at amino acid position 272; any one of Ala, Asp,
Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, and Tyr at amino acid
position 279; any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu,
Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at amino acid position
283; Asn at amino acid position 285; Phe at amino acid position
286; Asn or Pro at amino acid position 288; Val at amino acid
position 293; any one of Ala, Glu, Gln, and Met at amino acid
position 307; any one of Ile, Pro, and Thr at amino acid position
308; Pro at amino acid position 309; any one of Ala, Glu, Ile, Lys,
Leu, Met, Ser, Val, and Trp at amino acid position 311; any one of
Ala, Asp, and Pro at amino acid position 312; Ala or Leu at amino
acid position 314; Lys at amino acid position 316; Pro at amino
acid position 317; Asn or Thr at amino acid position 318; any one
of Phe, His, Lys, Leu, Met, Arg, Ser, and Trp at amino acid
position 332; any one of Asn, Thr, and Trp at amino acid position
339; Pro at amino acid position 341; any one of Glu, His, Lys, Gln,
Arg, Thr, or Tyr at amino acid position 343; Arg at amino acid
position 375; any one of Gly, Ile, Met, Pro, Thr, and Val at amino
acid position 376; Lys at amino acid position 377; any one of Asp,
Asn, and Val at amino acid position 378; any one of Ala, Asn, Ser,
and Thr at amino acid position 380; any one of Phe, His, Ile, Lys,
Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid
position 382; any one of Ala, Arg, Asp, Gly, His, Lys, Ser, and Thr
at amino acid position 385; any one of Arg, Asp, Ile, Lys, Met,
Pro, Ser, and Thr at amino acid position 386; any one of Ala, Arg,
His, Pro, Ser, and Thr at amino acid position 387; any one of Asn,
Pro, and Ser at amino acid position 389; Asn at amino acid position
423; Asn at amino acid position 427; any one of Leu, Met, Phe, Ser,
and Thr at amino acid position 428; any one of Ala, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, and Tyr at amino
acid position 430; His or Asn at amino acid position 431; any one
of Arg, Gln, His, Ile, Lys, Pro, and Ser at amino acid position
433; any one of Ala, Gly, His, Phe, Ser, Trp, and Tyr at amino acid
position 434; any one of Arg, Asn, His, Ile, Leu, Lys, Met, and Thr
at amino acid position 436; any one of Lys, Leu, Thr, and Trp at
amino acid position 438; Lys at amino acid position 440; and Lys at
amino acid position 442 as indicated by EU numbering, in the amino
acid sequence of the Fc region included in any one of SEQ ID NO:
14, 15, 16, or 17; [13] the use of any one of [1] to [12], wherein
the antigen-binding molecule is an antibody; [14] a pharmaceutical
composition comprising an antigen-binding molecule which comprises
an antigen-binding domain whose antigen-binding activity varies
depending on ion concentration conditions, and an Fc region in
which the amino acid at position 238 is Asp and the amino acid at
position 271 is Gly as indicated by EU numbering; [15] the
pharmaceutical composition of [14], wherein the Fc region has an
amino acid substitution at at least one or more positions selected
from the group consisting of 233, 234, 237, 264, 265, 266, 267,
268, 269, 272, 274, 296, 326, 327, 330, 331, 332, 333, 355, 356,
358, 396, 409, and 419 as indicated by EU numbering; [16] the
pharmaceutical composition of [15], wherein the amino acids of the
Fc region include any one or more of the following amino acids
indicated by EU numbering: Asp at amino acid position 233; Tyr at
amino acid position 234; Asp at amino acid position 237; Ile at
amino acid position 264; Glu at amino acid position 265; any one of
Phe, Met, and Leu at amino acid position 266; any one of Ala, Glu,
Gly, and Gln at amino acid position 267; any one of Asp, Glu, and
Gln at amino acid position 268; Asp at amino acid position 269; any
one of Asp, Phe, Ile, Met, Asn, Pro, and 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 or Arg at amino acid position 330; Ser at
amino acid position 331; Thr at amino acid position 332; any one of
Thr, Lys, and Arg at amino acid position 333; Gln at amino acid
position 355; Glu at amino acid position 356; Met at amino acid
position 358; any one of Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln,
Arg, and Tyr at amino acid position 396; Arg at amino acid position
409; and Glu at amino acid position 419; [17] a method of producing
an antigen-binding molecule, comprising the steps of (a) to (e)
below: (a) obtaining an antigen-binding domain whose
antigen-binding activity varies depending on ion concentration
conditions; (b) obtaining a gene encoding the antigen-binding
domain selected in step (a); (c) operably linking the gene obtained
in step (b) with a gene encoding an Fc region in which the amino
acid at position 238 is Asp and the amino acid at position 271 is
Gly as indicated by EU numbering; (d) culturing host cells
containing the gene operably linked in step (c); and (e) isolating
an antigen-binding molecule from the culture solution obtained in
step (d); [18] the production method of [17], wherein the Fc region
has an amino acid substitution at at least one or more positions
selected from the group consisting of 233, 234, 237, 264, 265, 266,
267, 268, 269, 272, 274, 296, 326, 327, 330, 331, 332, 333, 355,
356, 358, 396, 409, and 419 as indicated by EU numbering; [19] the
production method of [18], wherein the amino acids of the Fc region
include any one or more of the following amino acids indicated by
EU numbering: Asp at amino acid position 233; Tyr at amino acid
position 234; Asp at amino acid position 237; Ile at amino acid
position 264; Glu at amino acid position 265; any one of Phe, Met,
and Leu at amino acid position 266; any one of Ala, Glu, Gly, and
Gln at amino acid position 267; any one of Asp, Glu, and Gln at
amino acid position 268; Asp at amino acid position 269; any one of
Asp, Phe, Ile, Met, Asn, Pro, and 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 or Arg at amino acid position 330; Ser at amino
acid position 331; Thr at amino acid position 332; any one of Thr,
Lys, and Arg at amino acid position 333; Gln at amino acid position
355; Met at amino acid position 356; Met at amino acid position
358; any one of Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln, Arg, and
Tyr at amino acid position 396; Arg at amino acid position 409; and
Glu at amino acid position 419; [20] a method of producing a
pharmaceutical composition comprising an antigen-binding molecule,
which comprises the steps of: (a) obtaining an antigen-binding
domain whose antigen-binding activity varies depending on ion
concentration conditions; (b) obtaining a gene encoding the
antigen-binding domain selected in step (a); (c) operably linking
the gene obtained in step (b) with a gene encoding an Fc region in
which the amino acid at position 238 (EU numbering) is Asp and the
amino acid at position 271 (EU numbering) is Gly; (d) culturing
host cells containing the gene operably linked in step (c); and (e)
isolating an antigen-binding molecule from the culture solution
obtained in step (d); [21] the production method of [20], wherein
the Fc region has an amino acid substitution at least one or more
positions selected from the group consisting of positions 233, 234,
237, 264, 265, 266, 267, 268, 269, 272, 274, 296, 326, 327, 330,
331, 332, 333, 355, 356, 358, 396, 409, and 419 (EU numbering);
[22] the production method of [21], wherein the amino acids of the
Fc region include any one or more of the following amino acids
indicated by EU numbering: Asp at amino acid position 233; Tyr at
amino acid position 234; Asp at amino acid position 237; Ile at
amino acid position 264; Glu at amino acid position 265; any one of
Phe, Met, and Leu at amino acid position 266; any one of Ala, Glu,
Gly, and Gln at amino acid position 267; any one of Asp, Glu, and
Gln at amino acid position 268; Asp at amino acid position 269; any
one of Asp, Phe, Ile, Met, Asn, Pro, and 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 or Arg at amino acid position 330; Ser at
amino acid position 331; Thr at amino acid position 332; any one of
Thr, Lys, and Arg at amino acid position 333; Gln at amino acid
position 355; Glu at amino acid position 356; Met at amino acid
position 358; any one of Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln,
Arg, and Tyr at amino acid position 396; Arg at amino acid position
409; and Glu at amino acid position 419; [23] a method of
eliminating an antigen from plasma, which comprises administering
an effective amount of an antigen-binding molecule comprising an
antigen-binding domain whose antigen-binding activity varies
depending on ion concentration conditions, and an Fc region in
which the amino acid at position 238 is Asp and the amino acid at
position 271 is Gly as indicated by EU numbering; [24] the method
of [23], wherein the Fc region has an amino acid substitution at
least one or more positions selected from the group consisting of
233, 234, 237, 264, 265, 266, 267, 268, 269, 272, 274, 296, 326,
327, 330, 331, 332, 333, 355, 356, 358, 396, 409, and 419 as
indicated by EU numbering; [25] the method of [24], wherein the
amino acids of the Fc region include any one or more of the
following amino acids indicated by EU numbering: Asp at amino acid
position 233; Tyr at amino acid position 234; Asp at amino acid
position 237; Ile at amino acid position 264; Glu at amino acid
position 265; any one of Phe, Met, and Leu at amino acid position
266; any one of Ala, Glu, Gly, and Gln at amino acid position 267;
any one of Asp, Glu, and Gln at amino acid position 268; Asp at
amino acid position 269; any one of Asp, Phe, Ile, Met, Asn, Pro,
and 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 or Arg at amino
acid position 330; Ser at amino acid position 331; Thr at amino
acid position 332; any one of Thr, Lys, and Arg at amino acid
position 333; Gln at amino acid position 355; Glu at amino acid
position 356; Met at amino acid position 358; any one of Asp, Glu,
Phe, Ile, Lys, Leu, Met, Gln, Arg, and Tyr at amino acid position
396; Arg at amino acid position 409; and Glu at amino acid position
419; [26] the method of any one of [23] to [25], wherein the
antigen-binding domain is an antigen-binding domain whose
antigen-binding activity varies depending on calcium ion
concentration conditions; [27] the method of [26], wherein the
antigen-binding domain is an antigen-binding domain whose
antigen-binding activity varies such that the antigen-binding
activity under a low calcium ion concentration condition is lower
than an antigen-binding activity under a high calcium ion
concentration condition; [28] the method of any one of [23] to
[25], wherein the antigen-binding domain is an antigen-binding
domain whose antigen-binding activity varies depending on pH
conditions; [29] the method of [28], wherein the antigen-binding
domain is an antigen-binding domain whose antigen-binding activity
varies such that the antigen-binding activity under an acidic pH
range condition is lower than an antigen-binding activity under a
neutral pH range condition; [30] the method of any one of [23] to
[29], wherein the antigen-binding domain is an antibody variable
region; [31] the method of any one of [23] to [30], wherein the
aforementioned Fc region is the Fc region contained in any one of
SEQ ID NOs: 14, 15, 16, or 17 in which the amino acid at position
238 is Asp and the amino acid at position 271 is Gly as indicated
by EU numbering; [32] the method of any one of [23] to [30],
wherein the FcRn-binding activity of the Fc region under an acidic
pH range condition is enhanced compared to the FcRn-binding
activity of the Fc region contained in any one of SEQ ID NO: 14,
15, 16, or 17; [33] the method of [32], wherein the Fc region with
enhanced binding is an Fc region having an amino acid substitution
at least one or more positions selected from the group consisting
of positions 244, 245, 249, 250, 251, 252, 253, 254, 255, 256, 257,
258, 260, 262, 265, 270, 272, 279, 283, 285, 286, 288, 293, 303,
305, 307, 308, 309, 311, 312, 314, 316, 317, 318, 332, 339, 340,
341, 343, 356, 360, 362, 375, 376, 377, 378, 380, 382, 385, 386,
387, 388, 389, 400, 413, 415, 423, 424, 427, 428, 430, 431, 433,
434, 435, 436, 438, 439, 440, 442, and 447, as indicated by EU
numbering, in the amino acid sequence of the Fc region contained in
any one of SEQ ID NO: 14, 15, 16, or 17; [34] the method of [33],
wherein the Fc region with enhanced binding comprises at least one
or more amino acids selected from the
group consisting of: Leu at amino acid position 244; Arg at amino
acid position 245; Pro at amino acid position 249; Gln or Glu at
amino acid position 250; any one of Arg, Asp, Glu, and Leu at amino
acid position 251; any one of Phe, Ser, Thr, and Tyr at amino acid
position 252; Ser or Thr at amino acid position 254; any one of
Arg, Gly, Ile, and Leu at amino acid position 255; any one of Ala,
Arg, Asn, Asp, Gln, Glu, Pro, and Thr at amino acid position 256;
any one of Ala, Ile, Met, Asn, Ser, and Val at amino acid position
257; Asp at amino acid position 258; Ser at amino acid position
260; Leu at amino acid position 262; Lys at amino acid position
270; Leu or Arg at amino acid position 272; any one of Ala, Asp,
Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, and Tyr at amino acid
position 279;
any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln,
Arg, Ser, Thr, Trp, and Tyr at
[0040] amino acid position 283; Asn at amino acid position 285; Phe
at amino acid position 286; Asn or Pro at amino acid position 288;
Val at amino acid position 293; any one of Ala, Glu, Gln, and Met
at amino acid position 307; any one of Ile, Pro, and Thr at amino
acid position 308; Pro at amino acid position 309; any one of Ala,
Glu, Ile, Lys, Leu, Met, Ser, Val, and Trp at amino acid position
311; any one of Ala, Asp, and Pro at amino acid position 312; Ala
or Leu at amino acid position 314; Lys at amino acid position 316;
Pro at amino acid position 317; Asn or Thr at amino acid position
318; any one of Phe, His, Lys, Leu, Met, Arg, Ser, and Trp at amino
acid position 332; any one of Asn, Thr, and Trp at amino acid
position 339; Pro at amino acid position 341; any one of Glu, His,
Lys, Gln, Arg, Thr, and Tyr at amino acid position 343; Arg at
amino acid position 375; any one of Gly, Ile, Met, Pro, Thr, and
Val at amino acid position 376; Lys at amino acid position 377; any
one of Asp, Asn, or Val and amino acid position 378; any one of
Ala, Asn, Ser, and Thr at amino acid position 380; any one of Phe,
His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr
at amino acid position 382; any one of Ala, Arg, Asp, Gly, His,
Lys, Ser, and Thr at amino acid position 385; any one of Arg, Asp,
Ile, Lys, Met, Pro, Ser, and Thr at amino acid position 386; any
one of Ala, Arg, His, Pro, Ser, and Thr at amino acid position 387;
any one of Asn, Pro, and Ser at amino acid position 389; Asn at
amino acid position 423; Asn at amino acid position 427; any one of
Leu, Met, Phe, Ser, and Thr at amino acid position 428; any one of
Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,
Val, and Tyr at amino acid position 430; His or Asn at amino acid
position 431; any one of Arg, Gln, His, Ile, Lys, Pro, and Ser at
amino acid position 433; any one of Ala, Gly, His, Phe, Ser, Trp,
and Tyr at amino acid position 434; any one of Arg, Asn, His, Ile,
Leu, Lys, Met, and Thr at amino acid position 436; any one of Lys,
Leu, Thr, and Trp at amino acid position 438; Lys at amino acid
position 440; and Lys at amino acid position 442 as indicated by EU
numbering, in the amino acid sequence of the Fc region contained in
any one of SEQ ID NO: 14, 15, 16, or 17; and [35] the method of any
one of [23] to [34], wherein the antigen-binding molecule is an
antibody.
[0041] In the present invention, the following phrases are used
synonymously: "use of an antigen-binding molecule for eliminating
antigen from plasma, wherein the antigen-binding molecule comprises
an antigen-binding domain whose antigen-binding activity varies
depending on ion concentration conditions, and an Fc region in
which the amino acid at position 238 is Asp and the amino acid at
position 271 is Gly as indicated by EU numbering"; "a method for
treating a disease caused by an antigen, which comprises
administering an antigen-binding molecule comprising an
antigen-binding domain whose antigen-binding activity varies
depending on ion concentration conditions, and an Fc region in
which the amino acid at position 238 is Asp and the amino acid at
position 271 is Gly as indicated by EU numbering"; "a
pharmaceutical composition comprising an antigen-binding molecule
which comprises an antigen-binding domain whose antigen-binding
activity varies depending on ion concentration condition, and an Fc
region in which the amino acid at position 238 is Asp and the amino
acid at position 271 is Gly as indicated by EU numbering"; "use of
an antigen-binding molecule in producing a pharmaceutical
composition, wherein the antigen-binding molecule comprises an
antigen-binding domain whose antigen-binding activity varies
depending on ion concentration condition, and an Fc region in which
the amino acid at position 238 is Asp and the amino acid at
position 271 is Gly as indicated by EU numbering"; and "a process
for producing a pharmaceutical composition, which comprises using
an antigen-binding molecule comprising an antigen-binding domain
whose antigen-binding activity varies depending on ion
concentration condition, and an Fc region in which the amino acid
at position 238 is Asp and the amino acid at position 271 is Gly as
indicated by EU numbering".
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 shows a non-limiting action mechanism for the
elimination of soluble antigen from plasma by administering an
antibody that binds to an antigen in an ion concentration-dependent
manner and whose Fc.gamma. receptor binding is enhanced at a
neutral pH as compared to existing neutralizing antibodies.
[0043] FIG. 2 shows a time course of human IL-6 receptor
concentration in the plasma of human FcRn transgenic mice
administered with Fv4-IgG1, which binds to human IL-6 receptor in a
pH-dependent manner, or H54/L28-IgG1.
[0044] FIG. 3 shows a time course of human IL-6 receptor
concentration in the plasma of human FcRn transgenic mice
administered with Fv4-IgG1 which binds to human IL-6 receptor in a
pH-dependent manner, Fv4-IgG1-F760 which is an Fv4-IgG1 variant
that lacks mouse Fc.gamma.R binding, Fv4-IgG1-F1022 which is an
Fv4-IgG1 variant with enhanced mouse Fc.gamma.R binding, or
Fv4-IgG1-Fuc which is an Fv4-IgG1 antibody with low fucose
content.
[0045] FIG. 4 shows a time course of human IL-6 receptor
concentration in the plasma of human FcRn transgenic mice
administered with Fv4-IgG1 or antigen-binding molecules comprising
as the heavy chain, Fv4-IgG1-F1022 or Fv4-IgG1-F1093 which is a
Fv4-IgG1-F1022 variant with improved FcRn binding in an acidic pH
range.
[0046] FIG. 5 shows a concentration time course of the administered
antigen-binding molecules in the plasma of human FcRn transgenic
mice administered with Fv4-IgG1 or antigen-binding molecules
comprising as the heavy chain, Fv4-IgG1-F1022 or Fv4-IgG1-F1093
which is a Fv4-IgG1-F1022 variant with improved FcRn binding in an
acidic pH range.
[0047] FIG. 6 shows a time course of human IL-6 receptor
concentration in the plasma of human FcRn transgenic mice
administered with Fv4-IgG1, Fv4-IgG1-F1087 which is an Fv4-IgG1
variant with enhanced mouse Fc.gamma.R binding (in particular,
enhanced mouse Fc.gamma.RIIb binding and mouse Fc.gamma.RIII
binding), and Fv4-IgG1-F1182 which is an Fv4-IgG1 variant with
enhanced mouse Fc.gamma.R binding (in particular, enhanced mouse
Fc.gamma.RI binding and mouse Fc.gamma.RIV binding).
[0048] FIG. 7 shows a concentration time course of the administered
antigen-binding molecules in the plasma of human FcRn transgenic
mice administered with Fv4-IgG1, Fv4-IgG1-F1087, and Fv4-IgG1-F1180
and Fv4-IgG1-F1412 which are Fv4-IgG1-F1087 variants with improved
FcRn binding in an acidic pH range.
[0049] FIG. 8 shows a concentration time course of the administered
antigen-binding molecules in the plasma of human FcRn transgenic
mice administered with Fv4-IgG1, Fv4-IgG1-F1182, and Fv4-IgG1-F1181
which is an Fv4-IgG1-F1182 variant with improved FcRn binding in an
acidic pH range.
[0050] FIG. 9 shows a time course of human IL-6 receptor
concentration in the plasma of human FcRn transgenic mice
administered with Fv4-IgG1, Fv4-IgG1-F1087, and Fv4-IgG1-F1180 and
Fv4-IgG1-F1412 which are Fv4-IgG1-F1087 variants with improved FcRn
binding in an acidic pH range.
[0051] FIG. 10 shows a time course of human IL-6 receptor
concentration in the plasma of human FcRn transgenic mice
administered with Fv4-IgG1, Fv4-IgG1-F1182, and Fv4-IgG1-F1181
which is an Fv4-IgG1-F1182 variant with improved FcRn binding in an
acidic pH range.
[0052] FIG. 11 shows a time course of human IL-6 receptor
concentration in the plasma of normal mice administered with
Fv4-mIgG1, Fv4-mIgG1-mF44 which is an Fv4-mIgG1 variant with
enhanced mouse Fc.gamma.RIIb binding and mouse Fc.gamma.RIII
binding, and Fv4-mIgG1-mF46 which is an Fv4-mIgG1 variant with
further enhanced mouse Fc.gamma.RIIb binding and mouse
Fc.gamma.RIII binding.
[0053] FIG. 12 shows a time course of human IL-6 receptor
concentration in the plasma of Fc.gamma.RIII-deficient mice
administered with Fv4-mIgG1, Fv4-mIgG1-mF44 which is an Fv4-mIgG1
variant with enhanced mouse Fc.gamma.RIIb binding and mouse
Fc.gamma.RIII binding, and Fv4-mIgG1-mF46 which is an Fv4-mIgG1
variant with further enhanced mouse Fc.gamma.RIIb binding and mouse
Fc.gamma.RIII binding.
[0054] FIG. 13 shows a time course of human IL-6 receptor
concentration in the plasma of Fc receptor .gamma. chain-deficient
mice administered with Fv4-mIgG1, Fv4-mIgG1-mF44 which is an
Fv4-mIgG1 variant with enhanced mouse Fc.gamma.RIIb binding and
mouse Fc.gamma.RIII binding, and Fv4-mIgG1-mF46 which is an
Fv4-mIgG1 variant with further enhanced mouse Fc.gamma.RIIb binding
and mouse Fc.gamma.RIII binding.
[0055] FIG. 14 shows a time course of human IL-6 receptor
concentration in the plasma of Fc.gamma.RIIb-deficient mice
administered with Fv4-mIgG1, Fv4-mIgG1-mF44 which is an Fv4-mIgG1
variant with enhanced mouse Fc.gamma.RIIb binding and mouse
Fc.gamma.RIII binding, and Fv4-mIgG1-mF46 which is an Fv4-mIgG1
variant with further enhanced mouse Fc.gamma.RIIb binding and mouse
Fc.gamma.RIII binding.
[0056] FIG. 15 shows a result of evaluating the platelet
aggregation ability of the omalizumab-G1d-v3/IgE immune complex by
platelet aggregation assay using platelets derived from donors with
Fc.gamma.RIIa allotype (R/H).
[0057] FIG. 16 shows a result of evaluating the platelet
aggregation ability of the omalizumab-G1d-v3/IgE immune complex by
platelet aggregation assay using platelets derived from donors with
Fc.gamma.RIIa allotype (H/H).
[0058] FIG. 17 shows a result of assessing CD62p expression on the
membrane surface of washed platelets. The black-filled area in the
graph indicates a result of ADP stimulation after reaction with
PBS. The area that is not filled in the graph indicates a result of
ADP stimulation after reaction with the immune complex.
[0059] FIG. 18 shows a result of assessing the expression of active
integrin on the membrane surface of washed platelets. The
black-filled area in the graph indicates a result of ADP
stimulation after reaction with PBS. The area that is not filled in
the graph indicates a result of ADP stimulation after reaction with
the immune complex.
[0060] FIG. 19 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
(IL6R-F652, defined by SEQ ID NO: 61, is an antibody heavy chain
comprising an 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.
[0061] FIG. 20 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 (SEQ ID NO:
63)/GpL16-k0 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 (SEQ ID NO: 61)/IL6R-L 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/GpL16-k0 which does not have
P238D and where an alteration is introduced into IL6R-F652/IL6R-L
which has P238D. Region B contains alterations that exhibit the
effect of enhancing Fc.gamma.RIIb binding when introduced into
GpH7-B3/GpL16-k0 which does not have P238D, but do not exhibit the
effect of enhancing Fc.gamma.RIIb binding when introduced into
IL6R-F652/IL6R-L which has P238D.
[0062] FIG. 21 shows a crystal structure of the
Fc(P238D)/Fc.gamma.RIIb extracellular region complex.
[0063] FIG. 22 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 the least squares
fitting based on the C.alpha. atom pair distances.
[0064] FIG. 23 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 the least squares fitting based on the C.alpha. atom pair
distances.
[0065] FIG. 24 shows that a hydrogen bond can be found between the
main chain of Gly at position 237 (indicated by 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.
[0066] FIG. 25 shows that an electrostatic interaction can be found
between Asp at position 270 (indicated by 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.
[0067] FIG. 26 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 (indicated by 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.
[0068] FIG. 27 shows Glu at position 233 (indicated by 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.
[0069] FIG. 28 shows Ala at position 330 (indicated by 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.
[0070] FIG. 29 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 the least
squares fitting based on the C.alpha. atom pair distances with
respect to Fc Chain B.
[0071] FIG. 30 shows an image of the Fc (P208)/Fc.gamma.RIIb
extracellular region complex determined by X-ray crystal structure
analysis. For each of the CH2 and CH3 domains in the Fc portion,
those on the left side are referred to as domain A and those on the
right side are referred to as domain B.
[0072] FIG. 31 shows comparison after superimposing the structures
of Fc (P208)/Fc.gamma.RIIb extracellular region complex and Fc
(WT)/Fc.gamma.RIIa extracellular region complex (PDB code: 3RY6)
determined by X-ray crystal structure analysis with respect to the
CH2 domain A of the Fc portion by the least squares fitting based
on the C.alpha. atom pair distances. In the diagram, the structure
drawn with heavy line shows the Fc (P208)/Fc.gamma.RIIb
extracellular region complex, while the structure drawn with thin
line indicates the structure of Fc (WT)/Fc.gamma.RIIa extracellular
region complex. Only the CH2 domain A of the Fc portion is drawn
for the Fc (WT)/Fc.gamma.RIIa extracellular region complex.
[0073] FIG. 32 shows in the X-ray crystal structure of the Fc
(P208)/Fc.gamma.RIIb extracellular region complex, a detailed
structure around Asp at position 237 (EU numbering) in the CH2
domain A of the Fc portion, which forms a hydrogen bond with Tyr at
position 160 in Fc.gamma.RIIb at the main chain moiety.
[0074] FIG. 33 shows in the X-ray crystal structure of the Fc
(P208)/Fc.gamma.RIIb extracellular region complex, the structure of
amino acid residues around Asp at position 237 (EU numbering) in
the CH2 domain A of the Fc portion, which forms a hydrogen bond
with Tyr at position 160 in Fc.gamma.RIIb at the main chain
moiety.
[0075] FIG. 34 shows comparison around the loop at positions 266 to
271 (EU numbering) after superimposing the X-ray crystal structures
of the Fc (P238D)/Fc.gamma.RIIb extracellular region complex shown
in Example 10 and the Fc (P208)/Fc.gamma.RIIb extracellular region
complex with respect to the CH2 domain B of the Fc portion by the
least squares fitting based on the C.alpha. atom pair distances.
When compared to Fc (P238D), Fc (P208) has the H268D alteration at
position 268 (EU numbering) and the P271G alteration at position
271 (EU numbering) in the loop.
[0076] FIG. 35 is a diagram showing the structure around Ser239 in
the CH2 domain B of the Fc portion in the X-ray crystal structure
of the Fc (P208)/Fc.gamma.RIIb extracellular region complex, along
with the electron density with 2Fo-Fc coefficient determined by
X-ray crystal structure analysis.
[0077] FIG. 36 shows comparison after superimposing the
three-dimensional structures of the Fc (P208)/Fc.gamma.RIIaR
extracellular region complex and Fc (P208)/Fc.gamma.RIIb
extracellular region complex determined by X-ray crystal structure
analysis by the least squares fitting based on the C.alpha. atom
pair distances.
[0078] FIG. 37 shows comparison around Asp at position 237 (EU
numbering) in the CH2 domain A of the Fc portion between the X-ray
crystal structures of the Fc (P208)/Fc.gamma.RIIaR extracellular
region complex and the Fc (P208)/Fc.gamma.RIIb extracellular region
complex, along with the electron density with 2Fo-Fc coefficient
determined by X-ray crystal structure analysis.
[0079] FIG. 38 shows comparison around Asp at position 237 (EU
numbering) in the CH2 domain B of the Fc portion between the X-ray
crystal structures of the Fc (P208)/Fc.gamma.RIIaR extracellular
region complex and the Fc (P208)/Fc.gamma.RIIb extracellular region
complex, along with the electron density with 2Fo-Fc coefficient
determined by X-ray crystal structure analysis.
[0080] FIG. 39 shows comparison between the constant-region
sequences of G1d and G4d. In the diagram, the amino acids boxed
with thick-frame indicate positions with different amino acid
residues between G1d and G4d.
[0081] FIG. 40 shows the change in plasma antibody concentration of
GA2-IgG1 and GA2-F1087 in normal mice.
[0082] FIG. 41 shows the change in plasma hIgA concentration in
normal mice administered with GA2-IgG1 and GA2-F1087.
[0083] FIG. 42 shows the change in plasma antibody concentration of
278-IgG1 and 278-F1087 in normal mice.
[0084] FIG. 43 shows the change in plasma hIgE (Asp6) concentration
in C57BL/6J mice administered with 278-IgG1 and 278-F1087.
[0085] FIG. 44 shows a time course of anti-human IL-6 receptor
mouse antibody concentration in the plasma of normal mice
administered with Fv4-mIgG1 and Fv4-mIgG1-MB367 which is an
Fv4-mIgG1 variant with enhanced mouse Fc.gamma.RIIb binding.
[0086] FIG. 45 shows a time course of soluble human IL-6 receptor
concentration in the plasma of normal mice administered with
Fv4-mIgG1 and Fv4-mIgG1-MB367 which is an Fv4-mIgG1 variant with
enhanced mouse Fc.gamma.RIIb binding.
MODE FOR CARRYING OUT THE INVENTION
[0087] The definitions and detailed description below are provided
to help the understanding of the present invention illustrated
herein.
Amino Acids
[0088] Herein, amino acids are described in one- or three-letter
codes or both, for example, Ala/A, Leu/L, Arg/R, Lys/K, Asn/N,
Met/M, Asp/D, Phe/F, Cys/C, Pro/P, Gln/Q, Ser/S, Glu/E, Thr/T,
Gly/G, Trp/W, His/H, Tyr/Y, Ile/I, or Val/V.
Alteration of Amino Acids
[0089] For amino acid alterations in the amino acid sequence of an
antigen-binding molecule, known methods such as site-directed
mutagenesis methods (Kunkel et al. (Proc. Natl. Acad. Sci. USA
(1985) 82, 488-492)) and overlap extension PCR may be appropriately
employed. Additions, deletions, and/or substitutions of an amino
acid are added appropriately by these known methods. Substituting
amino acid residues means substituting an amino acid residue with
another amino acid residue for the purpose of altering aspects such
as the following (a) to (c):
(a) backbone structure of a polypeptide in a helical structure
region or a sheet structure region; (b) charge or hydrophobicity at
a target site; or (c) length of a side chain.
[0090] Amino acid residues are classified into the following groups
based on the properties of side chains included in their
structures:
(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
orientation of the chain: Gly and Pro; and (6) aromatic: Trp, Tyr,
and Phe.
[0091] Substitution between amino acid residues within each of
these groups is referred to as conservative substitution. On the
other hand, substitution between amino acid residues from different
amino acid 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 and non-conservative substitutions. Furthermore, a
plurality of known methods may be employed as amino acid alteration
methods for substitution to non-native amino acids (Annu. Rev.
Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad.
Sci. U.S.A. (2003) 100 (11), 6353-6357). For example, a cell-free
translation system (Clover Direct (Protein Express)) containing a
tRNA which has the non-native amino acid bound to a complementary
amber suppressor tRNA of the UAG codon (amber codon), which is one
of the stop codons, is suitably used.
[0092] Furthermore, an expression that uses one-letter amino-acid
codes of the amino acid before alteration and the amino acid after
the alteration before and after a number indicating a specific
position, respectively, may be used appropriately as an expression
for an amino acid alteration. For example, the alteration P238D,
which is used when substituting an amino acid of the Fc region
included in an antibody constant region, expresses substitution of
Pro at position 238 (according to EU numbering) with Asp. That is,
the number shows the position of the amino acid according to EU
numbering, the one-letter amino-acid code written before the number
shows the amino acid before substitution, and the one-letter
amino-acid code written after the number shows the amino acid after
substitution.
And/Or
[0093] As used herein, the term "and/or" means a combination of the
terms before and after the set phrase "and/or", and includes every
combination where "and" and "or" are suitably combined.
Specifically, for example, "the amino acids at positions 326, 328,
and/or 428 are substituted" includes a variation of alterations of
the following amino acids:
amino acid(s) at (a) position 326, (b) position 328, (c) position
428, (d) positions 326 and 328, (e) positions 326 and 428, (f)
positions 328 and 428, and (g) positions 326, 328, and 428.
Antigens
[0094] Herein, "antigens" are not particularly limited in their
structure, as long as they comprise epitopes to which
antigen-binding domains bind. In other words, antigens can be
inorganic or organic substances. 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 RITA, 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, Bcl, 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 O, 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, CNC, 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,
ECAD, 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, Gash, 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, I-309,
TAP, 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
(NF)-alpha, INF-beta, NF-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, PGD2, 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-betaR1 (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 (HVEMATAR, 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-a
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, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A,
WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR,
XIAP, XPD, HMGB1, IgA, A13, CD81, CD97, CD98, DDR1, DKK1, EREG,
Hsp90, IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK9,
prekallikrein, RON, TMEM16F, SOD 1, Chromogranin A, Chromogranin B,
tau, VAP 1, 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, selerostin, 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.
[0095] While receptors are recited as examples of the
above-mentioned antigens, when these receptors exist in soluble
forms in biological fluids such as plasma, they can form complexes
with the antigen-binding molecules of the present invention.
Therefore, as long as the above-mentioned receptors exist in their
soluble forms in biological fluids such as plasma, they may be used
as antigens that may form complexes of the present invention by
binding to an antigen-binding molecule of the present invention. An
example of a non-limiting embodiment of such a soluble receptor is
soluble IL-6R, which is a protein consisting of the amino acids at
positions 1 to 357 in the IL-6R polypeptide sequence of SEQ ID NO:
1 as described in Mullberg et al. (J. Immunol. (1994) 152 (10),
4958-4968).
[0096] Soluble antigens are recited as examples of the
above-mentioned antigens, and the solutions in which the antigens
exist are not limited. Soluble antigens may exist in biological
fluids, or more specifically in all fluids filling the space
between tissues and cells or vessels in organisms. In a
non-limiting embodiment, the antigens to which antigen-binding
molecules of the present invention bind may be present in
extracellular fluids. In vertebrates, extracellular fluid is a
general term for plasma, interstitial fluid, lymph, compact
connective tissue, cerebrospinal fluid, spinal fluid, puncture
fluid, synovial fluid, or such components in the bone and
cartilage, alveolar fluid (bronchoalveolar lavage fluid),
peritoneal fluid, pleural fluid, pericardial fluid, cyst fluid,
aqueous humor (hydatoid), or such transcellular fluids (various
fluids in the glandular cavities and fluids in the digestive tract
cavity and other body cavity fluids produced as a result of active
transport/secretory activities of cells).
Epitope
[0097] "Epitope" means an antigenic determinant in an antigen, and
refers to an antigen site to which the antigen-binding domain of an
antigen-binding molecule disclosed herein binds. Thus, for example,
the epitope can be defined according to its structure.
Alternatively, the epitope may be defined according to the
antigen-binding activity of an antigen-binding molecule that
recognizes the epitope. When the antigen is a peptide or
polypeptide, the epitope can be specified by the amino acid
residues forming the epitope. Alternatively, when the epitope is a
sugar chain, the epitope can be specified by its specific sugar
chain structure.
[0098] A linear epitope is an epitope that contains an epitope
whose primary amino acid sequence is recognized. Such a linear
epitope typically contains at least three and most commonly at
least five, for example, about 8 to 10 or 6 to 20 amino acids in
its specific sequence.
[0099] In contrast to the linear epitope, "conformational epitope"
is an epitope in which the primary amino acid sequence containing
the epitope is not the only determinant of the recognized epitope
(for example, the primary amino acid sequence of a conformational
epitope is not necessarily recognized by an epitope-defining
antibody). Conformational epitopes may contain a greater number of
amino acids compared to linear epitopes. A conformational
epitope-recognizing antibody recognizes the three-dimensional
structure of a peptide or protein. For example, when a protein
molecule folds and forms a three-dimensional structure, amino acids
and/or polypeptide main chains that form a conformational epitope
become aligned, and the epitope is made recognizable by the
antibody. Methods for determining epitope conformations include,
for example, X ray crystallography, two-dimensional nuclear
magnetic resonance, site-specific spin labeling, and electron
paramagnetic resonance, but are not limited thereto. See, for
example, Epitope Mapping Protocols in Methods in Molecular Biology
(1996), Vol. 66, Morris (ed.).
Binding Activity
[0100] Examples of a method for assessing the epitope binding by a
test antigen-binding molecule containing an IL-6R antigen-binding
domain are described below. According to the examples below,
methods for assessing the epitope binding by a test antigen-binding
molecule containing an antigen-binding domain for an antigen other
than IL-6R, can also be appropriately conducted.
[0101] For example, whether a test antigen-binding molecule
containing an IL-6R antigen-binding domain recognizes a linear
epitope in the IL-6R molecule can be confirmed for example as
mentioned below. A linear peptide comprising an amino acid sequence
forming the extracellular domain of IL-6R is synthesized for the
above purpose. The peptide can be synthesized chemically, or
obtained by genetic engineering techniques using a region encoding
the amino acid sequence corresponding to the extracellular domain
in an IL-6R cDNA represented by SEQ ID NO: 2. Then, a test
antigen-binding molecule containing an IL-6R antigen-binding domain
is assessed for its binding activity towards a linear peptide
comprising the amino acid sequence forming the extracellular
domain. For example, an immobilized linear peptide can be used as
an antigen by ELISA to evaluate the binding activity of the
antigen-binding molecule towards the peptide. Alternatively, the
binding activity towards a linear peptide can be assessed based on
the level that the linear peptide inhibits the binding of the
antigen-binding molecule to IL-6R-expressing cells. These tests can
demonstrate the binding activity of the antigen-binding molecule
towards the linear peptide.
[0102] Whether a test antigen-binding molecule containing an IL-6R
antigen-binding domain recognizes a conformational epitope can be
assessed as follows. IL-6R-expressing cells are prepared for the
above purpose. A test antigen-binding molecule containing an IL-6R
antigen-binding domain can be determined to recognize a
conformational epitope when it strongly binds to IL-6R-expressing
cells upon contact, but does not substantially bind to an
immobilized linear peptide comprising an amino acid sequence
forming the extracellular domain of IL-6R. Herein, "not
substantially bind" means that the binding activity is 80% or less,
generally 50% or less, preferably 30% or less, and particularly
preferably 15% or less compared to the binding activity towards
cells expressing human IL-6R.
[0103] Methods for assaying the binding activity of a test
antigen-binding molecule containing an IL-6R antigen-binding domain
towards IL-6R-expressing cells include, for example, the methods
described in Antibodies: A Laboratory Manual (Ed Harlow, David
Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically,
the assessment can be performed based on the principle of ELISA or
fluorescence activated cell sorting (FACS) using IL-6R-expressing
cells as antigen.
[0104] In the ELISA format, the binding activity of a test
antigen-binding molecule containing an IL-6R antigen-binding domain
towards IL-6R-expressing cells can be assessed quantitatively by
comparing the levels of signal generated by enzymatic reaction.
Specifically, a test polypeptide complex is added to an ELISA plate
onto which IL-6R-expressing cells are immobilized. Then, the test
antigen-binding molecule bound to the cells is detected using an
enzyme-labeled antibody that recognizes the test antigen-binding
molecule. Alternatively, when FACS is used, a dilution series of a
test antigen-binding molecule is prepared, and the antibody binding
titer for IL-6R-expressing cells can be determined to compare the
binding activity of the test antigen-binding molecule towards
IL-6R-expressing cells.
[0105] The binding of a test antigen-binding molecule towards an
antigen expressed on the surface of cells suspended in buffer or
the like can be detected using a flow cytometer. Known flow
cytometers include, for example, the following devices:
FACSCanto.TM. II
FACSAria.TM.
FACSArray.TM.
FACSVantage.TM. SE
[0106] FACSCalibur.TM. (all are trade names of BD Biosciences)
EPICS ALTRA HyPerSort
Cytomics FC 500
EPICS XL-MCL ADC EPICS XL ADC
[0107] Cell Lab Quanta/Cell Lab Quanta SC (all are trade names of
Beckman Coulter).
[0108] Preferable methods for assaying the binding activity of a
test antigen-binding molecule containing an IL-6R antigen-binding
domain towards an antigen include, for example, the following
method. First, IL-6R-expressing cells are reacted with a test
antigen-binding molecule, and then this is stained with an
FITC-labeled secondary antibody that recognizes the antigen-binding
molecule. The test antigen-binding molecule is appropriately
diluted with a suitable buffer to prepare the molecule at a desired
concentration. For example, the molecule can be used at a
concentration within the range of 10 .mu.g/ml to 10 ng/ml. Then,
the fluorescence intensity and cell count are determined using
FACSCalibur (BD). The fluorescence intensity obtained by analysis
using the CELL QUEST Software (BD), i.e., the Geometric Mean value,
reflects the quantity of antibody bound to cells. That is, the
binding activity of a test antigen-binding molecule, which is
represented by the quantity of the test antigen-binding molecule
bound, can be determined by measuring the Geometric Mean value.
[0109] Whether a test antigen-binding molecule containing an IL-6R
antigen-binding domain shares a common epitope with another
antigen-binding molecule can be assessed based on the competition
between the two molecules for the same epitope. The competition
between antigen-binding molecules can be detected by cross-blocking
assay or the like. For example, the competitive ELISA assay is a
preferred cross-blocking assay.
[0110] Specifically, in cross-blocking assay, the IL-6R protein
immobilized to the wells of a microtiter plate is pre-incubated in
the presence or absence of a candidate competitor antigen-binding
molecule, and then a test antigen-binding molecule is added
thereto. The quantity of test antigen-binding molecule bound to the
IL-6R protein in the wells is indirectly correlated with the
binding ability of a candidate competitor antigen-binding molecule
that competes for the binding to the same epitope. That is, the
greater the affinity of the competitor antigen-binding molecule for
the same epitope, the lower the binding activity of the test
antigen-binding molecule towards the IL-6R protein-coated
wells.
[0111] The quantity of the test antigen-binding molecule bound to
the wells via the IL-6R protein can be readily determined by
labeling the antigen-binding molecule in advance. For example, a
biotin-labeled antigen-binding molecule is measured using an
avidin/peroxidase conjugate and appropriate substrate. In
particular, cross-blocking assay that uses enzyme labels such as
peroxidase is called competitive ELISA assay. The antigen-binding
molecule can also be labeled with other labeling substances that
enable detection or measurement. Specifically, radiolabels,
fluorescent labels, and such are known.
[0112] When the candidate competitor antigen-binding molecule can
block the binding by a test antigen-binding molecule containing an
IL-6R antigen-binding domain by at least 20%, preferably at least
20 to 50%, and more preferably at least 50% compared to the binding
activity in a control experiment conducted in the absence of the
competitor antigen-binding molecule complex, the test
antigen-binding molecule is determined to substantially bind to the
same epitope bound by the competitor antigen-binding molecule, or
compete for the binding to the same epitope.
[0113] When the structure of an epitope bound by a test
antigen-binding molecule containing an IL-6R antigen-binding domain
has already been identified, whether the test and control
antigen-binding molecules share a common epitope can be assessed by
comparing the binding activities of the two antigen-binding
molecules towards a peptide prepared by introducing amino acid
mutations into the peptide forming the epitope.
[0114] To measure the above binding activities, for example, the
binding activities of test and control antigen-binding molecules
towards a linear peptide into which a mutation is introduced are
compared in the above ELISA format. Besides the ELISA methods, the
binding activity towards the mutant peptide bound to a column can
be determined by flowing test and control antigen-binding molecules
in the column, and then quantifying the antigen-binding molecule
eluted in the elution solution. Methods for adsorbing a mutant
peptide to a column, for example, in the form of a GST fusion
peptide, are known.
[0115] Alternatively, when the identified epitope is a
conformational epitope, whether test and control antigen-binding
molecules share a common epitope can be assessed by the following
method. First, IL-6R-expressing cells and cells expressing IL-6R
with a mutation introduced into the epitope are prepared. The test
and control antigen-binding molecules are added to a cell
suspension prepared by suspending these cells in an appropriate
buffer such as PBS. Then, the cell suspensions are appropriately
washed with a buffer, and an FITC-labeled antibody that recognizes
the test and control antigen-binding molecules is added thereto.
The fluorescence intensity and number of cells stained with the
labeled antibody are determined using FACSCalibur (BD). The test
and control antigen-binding molecules are appropriately diluted
using a suitable buffer, and used at desired concentrations. For
example, they may be used at a concentration within the range of 10
.mu.g/ml to 10 ng/ml. The fluorescence intensity determined by
analysis using the CELL QUEST Software (BD), i.e., the Geometric
Mean value, reflects the quantity of labeled antibody bound to
cells. That is, the binding activities of the test and control
antigen-binding molecules, which are represented by the quantity of
labeled antibody bound, can be determined by measuring the
Geometric Mean value.
[0116] In the above method, whether an antigen-binding molecule
does "not substantially bind to cells expressing mutant IL-6R" can
be assessed, for example, by the following method. First, the test
and control antigen-binding molecules bound to cells expressing
mutant IL-6R are stained with a labeled antibody. Then, the
fluorescence intensity of the cells is determined. When FACSCalibur
is used for fluorescence detection by flow cytometry, the
determined fluorescence intensity can be analyzed using the CELL
QUEST Software. From the Geometric Mean values in the presence and
absence of the polypeptide complex, the comparison value
(.DELTA.Geo-Mean) can be calculated according to the following
formula to determine the ratio of increase in fluorescence
intensity as a result of the binding by the antigen-binding
molecule.
.DELTA.Geo-Mean=Geo-Mean(in the presence of the polypeptide
complex)/Geo-Mean(in the absence of the polypeptide complex)
[0117] The Geometric Mean comparison value (.DELTA.Geo-Mean value
for the mutant IL-6R molecule) determined by the above analysis,
which reflects the quantity of a test antigen-binding molecule
bound to cells expressing mutant IL-6R, is compared to the
.DELTA.Geo-Mean comparison value that reflects the quantity of the
test antigen-binding molecule bound to IL-6R-expressing cells. In
this case, the concentrations of the test antigen-binding molecule
used to determine the .DELTA.Geo-Mean comparison values for
IL-6R-expressing cells and cells expressing mutant IL-6R are
particularly preferably adjusted to be equal or substantially
equal. An antigen-binding molecule that has been confirmed to
recognize an epitope in IL-6R is used as a control antigen-binding
molecule.
[0118] If the .DELTA.Geo-Mean comparison value of a test
antigen-binding molecule for cells expressing mutant IL-6R is
smaller than the .DELTA.Geo-Mean comparison value of the test
antigen-binding molecule for IL-6R-expressing cells by at least
80%, preferably 50%, more preferably 30%, and particularly
preferably 15%, then the test antigen-binding molecule "does not
substantially bind to cells expressing mutant IL-6R". The formula
for determining the Geo-Mean (Geometric Mean) value is described in
the CELL QUEST Software User's Guide (BD biosciences). When the
comparison shows that the comparison values are substantially
equivalent, the epitope for the test and control antigen-binding
molecules can be determined to be the same.
Antigen-Binding Domain
[0119] Herein, an "antigen-binding domain" may be of any structure
as long as it binds to an antigen of interest. Such domains
preferably include, for example:
antibody heavy-chain and light-chain variable regions; a module of
about 35 amino acids called A domain which is contained in the in
vivo cell membrane protein Avimer (WO 2004/044011, WO 2005/040229);
Adnectin containing the 10Fn3 domain which binds to the protein
moiety of fibronectin, a glycoprotein expressed on cell membrane
(WO 2002/032925); Affibody which is composed of a 58-amino acid
three-helix bundle based on the scaffold of the IgG-binding domain
of Protein A (WO 1995/001937); Designed Ankyrin Repeat proteins
(DARPins) which are a region exposed on the molecular surface of
ankyrin repeats (AR) having a structure in which a subunit
consisting of a turn comprising 33 amino acid residues, two
antiparallel helices, and a loop is repeatedly stacked (WO
2002/020565); Anticalins and such, which are domains consisting of
four loops that support one side of a barrel structure composed of
eight circularly arranged antiparallel strands that are highly
conserved among lipocalin molecules such as neutrophil
gelatinase-associated lipocalin (NGAL) (WO 2003/029462); and the
concave region formed by the parallel-sheet structure inside the
horseshoe-shaped structure constituted by stacked repeats of the
leucine-rich-repeat (LRR) module of the variable lymphocyte
receptor (VLR) which does not have the immunoglobulin structure and
is used in the system of acquired immunity in jawless vertebrate
such as lampery and hagfish (WO 2008/016854). Preferred
antigen-binding domains of the present invention include, for
example, those having antibody heavy-chain and light-chain variable
regions. Preferred examples of antigen-binding domains include
"single chain Fv (scFv)", "single chain antibody", "Fv", "single
chain Fv 2 (scFv2)", "Fab", and "F(ab')2".
[0120] The antigen-binding domains of antigen-binding molecules of
the present invention can bind to an identical epitope. Such
epitope can be present, for example, in a protein comprising the
amino acid sequence of SEQ ID NO: 1. Alternatively, each of the
antigen-binding domains of antigen-binding molecules of the present
invention can bind to a different epitope. Herein, the different
epitope can be present in, for example, a protein comprising the
amino acid sequence of SEQ ID NO: 1.
Specific
[0121] With regard to binding of antigen-binding molecules provided
by the present invention to an antigen, the term "specific" means
that one of the molecules that specifically binds to does not
substantially bind to molecules other than its single or plurality
of binding partner molecule(s). Herein, "does not substantially
bind" refers to showing 80% or less, generally 50% or less,
preferably 30% or less and particularly preferably 15% or less
binding activity to molecules other than the binding partner
molecules compared to the binding activity towards the partner
molecule(s), as described in the above-mentioned section on binding
activity. Furthermore, "specific" is also used when an
antigen-binding domain is specific to a particular epitope among
multiple epitopes in an antigen. When an epitope bound by an
antigen-binding domain is contained in multiple different antigens,
antigen-binding molecules containing the antigen-binding domain can
bind to various antigens that have the epitope.
Neutralizing Activity
[0122] In a non-limiting embodiment of the present invention, a
pharmaceutical composition comprising as an active ingredient an
antigen-binding molecule having antigen-neutralizing activity is
provided, wherein the antigen-binding molecule comprises (i) an
antigen-binding domain whose antigen-binding activity varies
depending on ion concentration conditions, (ii) an
Fc.gamma.-binding domain having Fc.gamma.RIIb-selective binding
activity, and (iii) an FcRn-binding domain having FcRn-binding
activity under an acidic pH range condition. Generally,
neutralizing activity refers to activity of inhibiting the
biological activity of a ligand, such as viruses and toxins, having
biological activity on cells. Thus, substances having neutralizing
activity refer to substances that bind to the ligand or the
receptor to which the ligand binds, and inhibits the binding
between the ligand and the receptor. Receptors blocked from binding
with the ligand by the neutralizing activity will not be able to
exhibit biological activity through this receptor. When the
antigen-binding molecule is an antibody, such an antibody having
neutralizing activity is generally called a neutralizing antibody.
Neutralizing activity of a test substance may be measured by
comparing the biological activity in the presence of a ligand
between when the test substance is present and absent.
[0123] For example, major possible ligands for the IL-6 receptor
preferably include IL-6 as shown in SEQ ID NO: 3. The IL-6
receptor, which is an I-type membrane protein with its amino
terminus forming the extracellular domain, forms a hetero-tetramer
with a gp130 receptor which has been induced to dimerize by IL-6
(Heinrich et al. (Biochem. J. (1998) 334, 297-314)). Formation of
the heterotetramer activates Jak which is associated with the gp130
receptor. Jak undergoes autophosphorylation and phosphorylates the
receptor. The phosphorylation site of the receptor and Jak serves
as a binding site for SH2-carrying molecules belonging to the Stat
family such as Stat3; MAP kinase; PI3/Akt; and other SH2-carrying
proteins and adapters. Next, Stat bound to the gp130 receptor is
phosphorylated by Jak. The phosphorylated Stat dimerizes and moves
into the nucleus, and regulates the transcription of target genes.
Jak or Stat can also be involved in signal cascades via receptors
of other classes. Deregulated IL-6 signal cascades are observed in
inflammation and pathological conditions of autoimmune diseases,
and cancers such as prostate cancer and multiple myeloma. Stat3
which may act as an oncogene is constitutively activated in many
cancers. In prostate cancer and multiple myeloma, there is a
crosstalk between the signaling cascade via the IL-6 receptor and
the signaling cascade via the epithelial growth factor receptor
(EGFR) family members (Ishikawa et al. (J. Clin. Exp. Hematopathol.
(2006) 46 (2), 55-66)).
[0124] Such intracellular signaling cascades are different for each
cell type; therefore, appropriate target molecules can be
determined for each target cell of interest, and are not limited to
the above-mentioned factors. Neutralization activity can be
evaluated by measuring the activation of in vivo signaling.
Furthermore, the activation of in vivo signaling can be detected by
using as an index the action of inducing the transcription of a
target gene that exists downstream of the in vivo signaling
cascade. Change in the transcription activity of the target gene
can be detected by the principle of reporter assays. Specifically,
a reporter gene such as green fluorescence protein (GFP) or
luciferase is placed downstream of a promoter region or a
transcription factor of the target gene, its reporter activity is
measured, and thereby change in the transcription activity can be
measured as the reporter activity. Commercially available kits for
measuring the activation of in vivo signaling can be used
appropriately (for example, Mercury Pathway Profiling Luciferase
System (Clontech)).
[0125] Furthermore, for methods of measuring the activity of
neutralizing receptors/ligands of the EGF receptor family and such,
which normally act on signaling cascades that work toward promoting
cell proliferation, the neutralization activity of antigen-binding
molecules can be evaluated by measuring the proliferation activity
of target cells. For example, when cells are promoted to
proliferate by growth factors of the EGF family such as HB-EGF, the
inhibitory effect on the proliferation of such cells based on the
neutralizing activity of an anti-HB-EGF antibody can be suitably
evaluated or measured by the following methods: For evaluating or
measuring the cell proliferation inhibitory activity in vitro, a
method of measuring the incorporation of [.sup.3H]-labeled
thymidine added to the medium by viable cells as an index of DNA
replication ability is used. As more convenient methods, a dye
exclusion method, in which the ability of a cell to exclude a dye
such as trypan blue from the cell is measured under the microscope,
and the MTT method are used. The latter method makes use of the
ability of viable cells to convert MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide),
which is a tetrazolium salt, to a blue formazan product. More
specifically, a test antibody is added as well as a ligand to the
culture solution of a test cell, and after a certain period of
time, the MTT solution is added to the culture solution, and this
is left to stand for a while for incorporation of MTT into the
cell. As a result, MTT, which is a yellow compound, is converted to
a blue compound by the action of succinate dehydrogenase in the
mitochondria of the cell. After dissolving this blue product for
coloration, its absorbance is measured and used as an index for the
number of viable cells. In addition to MTT, reagents such as MTS,
XTT, WST-1, and WST-8 are also commercially available (Nacalai
Tesque, and such) and can be suitably used. For measuring the
activity, a binding antibody which is of the same isotype as the
anti-HB-EGF antibody but does not have the cell proliferation
inhibitory activity can be used as a control antibody in the same
manner as the anti-HB-EGF antibody, and the activity can be
determined when the anti-HB-EGF antibody shows stronger cell
proliferation inhibitory activity than the control antibody.
[0126] Cells that can be preferably used for evaluating the
activity include, for example, cells promoted to proliferate by
HB-EGF such as the ovarian cancer cell line RMG-1, and mouse Ba/F3
cells which have been transformed by a vector for expressing a gene
encoding hEGFR/mG-CSFR, which is a fusion protein in which the
extracellular domain of human EGFR is fused in frame with the
intracellular domain of the mouse G-CSF receptor. In this way,
those skilled in the art can appropriately select cells to be used
for evaluating the activity and use them to measure the cell
proliferation activity as mentioned above.
Antibody
[0127] Herein, "antibody" refers to a natural immunoglobulin or an
immunoglobulin produced by partial or complete synthesis.
Antibodies can be isolated from natural sources such as
naturally-occurring plasma and serum, or culture supernatants of
antibody-producing hybridomas. Alternatively, antibodies can be
partially or completely synthesized using techniques such as
genetic recombination. Preferred antibodies include, for example,
antibodies of an immunoglobulin isotype or subclass belonging
thereto. Known human immunoglobulins include antibodies of the
following nine classes (isotypes): IgG1, IgG2, IgG3, IgG4, IgA1,
IgA2, IgD, IgE, and IgM. Of these isotypes, antibodies of the
present invention include IgG1, IgG2, IgG3, and IgG4. A number of
allotype sequences of human IgG1, human IgG2, human IgG3, and human
IgG4 constant regions due to gene polymorphisms are described in
"Sequences of proteins of immunological interest", NIH Publication
No. 91-3242. Any of such sequences may be used in the present
invention. In particular, for the human IgG1 sequence, the amino
acid sequence at positions 356 to 358 as indicated by EU numbering
may be DEL or EEM. Several allotype sequences due to genetic
polymorphisms have been described in "Sequences of proteins of
immunological interest", NIH Publication No. 91-3242 for the human
IgK (Kappa) constant region and human Ig.lamda., (Lambda) constant
region, and any of the sequences may be used in the present
invention.
[0128] Methods for producing an antibody with desired binding
activity are known to those skilled in the art. Below is an example
that describes a method for producing an antibody that binds to
IL-6R (anti-IL-6R antibody). Antibodies that bind to an antigen
other than IL-6R can also be produced according to the example
described below.
[0129] Anti-IL-6R antibodies can be obtained as polyclonal or
monoclonal antibodies using known methods. The anti-IL-6R
antibodies preferably produced are monoclonal antibodies derived
from mammals. Such mammal-derived monoclonal antibodies include
antibodies produced by hybridomas or host cells transformed with an
expression vector carrying an antibody gene by genetic engineering
techniques. "Humanized antibodies" or "chimeric antibodies" are
included in the monoclonal antibodies of the present invention.
[0130] Monoclonal antibody-producing hybridomas can be produced
using known techniques, for example, as described below.
Specifically, mammals are immunized by conventional immunization
methods using an IL-6R protein as a sensitizing antigen. Resulting
immune cells are fused with known parental cells by conventional
cell fusion methods. Then, hybridomas producing an anti-IL-6R
antibody can be selected by screening for monoclonal
antibody-producing cells using conventional screening methods.
[0131] Specifically, monoclonal antibodies are prepared as
mentioned below. First, the IL-6R gene whose nucleotide sequence is
disclosed in SEQ ID NO: 2 can be expressed to produce an IL-6R
protein shown in SEQ ID NO: 1, which will be used as a sensitizing
antigen for antibody preparation. That is, a gene sequence encoding
IL-6R is inserted into a known expression vector, and appropriate
host cells are transformed with this vector. The desired human
IL-6R protein is purified from the host cells or their culture
supernatants by known methods. In order to obtain soluble IL-6R
from culture supernatants, for example, a protein consisting of the
amino acids at positions 1 to 357 in the IL-6R polypeptide sequence
of SEQ ID NO: 1, such as described in Mullberg et al. (J. Immunol.
(1994) 152 (10), 4958-4968), is expressed as a soluble IL-6R,
instead of the IL-6R protein of SEQ ID NO: 1. Purified native IL-6R
protein can also be used as a sensitizing antigen.
[0132] The purified IL-6R protein can be used as a sensitizing
antigen for immunization of mammals. A partial IL-6R peptide may
also be used as a sensitizing antigen. In this case, a partial
peptide can be prepared by chemical synthesis based on the amino
acid sequence of human IL-6R, or by inserting a partial IL-6R gene
into an expression vector for expression. Alternatively, a partial
peptide can be produced by degrading an IL-6R protein with a
protease. The length and region of the partial IL-6R peptide are
not limited to particular embodiments. A preferred region can be
arbitrarily selected from the amino acid sequence at amino acid
positions 20 to 357 in the amino acid sequence of SEQ ID NO: 1. The
number of amino acids forming a peptide to be used as a sensitizing
antigen is preferably at least five or more, six or more, or seven
or more. More specifically, a peptide of 8 to 50 residues, more
preferably 10 to 30 residues can be used as a sensitizing
antigen.
[0133] For sensitizing antigen, alternatively it is possible to use
a fusion protein prepared by fusing a desired partial polypeptide
or peptide of the IL-6R protein with a different polypeptide. For
example, antibody Fc fragments and peptide tags are preferably used
to produce fusion proteins to be used as sensitizing antigens.
Vectors for expression of such fusion proteins can be constructed
by fusing in frame genes encoding two or more desired polypeptide
fragments and inserting the fusion gene into an expression vector
as described above. Methods for producing fusion proteins are
described in Molecular Cloning 2nd ed. (Sambrook, J et al.,
Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab.
Press). Methods for preparing IL-6R to be used as a sensitizing
antigen, and immunization methods using IL-6R are specifically
described in WO 2003/000883, WO 2004/022754, WO 2006/006693, and
such.
[0134] There is no particular limitation on the mammals to be
immunized with the sensitizing antigen. However, it is preferable
to select the mammals by considering their compatibility with the
parent cells to be used for cell fusion. In general, rodents such
as mice, rats, and hamsters, rabbits, and monkeys are preferably
used.
[0135] The above animals are immunized with a sensitizing antigen
by known methods. Generally performed immunization methods include,
for example, intraperitoneal or subcutaneous injection of a
sensitizing antigen into mammals. Specifically, a sensitizing
antigen is appropriately diluted with PBS (Phosphate-Buffered
Saline), physiological saline, or the like. If desired, a
conventional adjuvant such as Freund's complete adjuvant is mixed
with the antigen, and the mixture is emulsified. Then, the
sensitizing antigen is administered to a mammal several times at 4-
to 21-day intervals. Appropriate carriers may be used in
immunization with the sensitizing antigen. In particular, when a
low-molecular-weight partial peptide is used as the sensitizing
antigen, it is sometimes desirable to couple the sensitizing
antigen peptide to a carrier protein such as albumin or keyhole
limpet hemocyanin for immunization.
[0136] Alternatively, hybridomas producing a desired antibody can
be prepared using DNA immunization as mentioned below. DNA
immunization is an immunization method that confers
immunostimulation by expressing a sensitizing antigen in an animal
immunized as a result of administering a vector DNA constructed to
allow expression of an antigen protein-encoding gene in the animal.
As compared to conventional immunization methods in which a protein
antigen is administered to animals to be immunized, DNA
immunization is expected to be superior in that: [0137]
immunostimulation can be provided while retaining the structure of
a membrane protein such as IL-6R; and [0138] there is no need to
purify the antigen for immunization.
[0139] In order to prepare a monoclonal antibody of the present
invention using DNA immunization, first, a DNA expressing an IL-6R
protein is administered to an animal to be immunized. The
IL-6R-encoding DNA can be synthesized by known methods such as PCR.
The obtained DNA is inserted into an appropriate expression vector,
and then this is administered to an animal to be immunized.
Preferably used expression vectors include, for example,
commercially-available expression vectors such as pcDNA3.1. Vectors
can be administered to an organism using conventional methods. For
example, DNA immunization is performed by using a gene gun to
introduce expression vector-coated gold particles into cells in the
body of an animal to be immunized. Antibodies that recognized IL-6R
can also be produced by the methods described in WO
2003/104453.
[0140] After immunizing a mammal as described above, an increase in
the titer of an IL-6R-binding antibody is confirmed in the serum.
Then, immune cells are collected from the mammal, and then
subjected to cell fusion. In particular, splenocytes are preferably
used as immune cells.
[0141] A mammalian myeloma cell is used as a cell to be fused with
the above-mentioned immune cells. The myeloma cells preferably
comprise a suitable selection marker for screening. A selection
marker confers characteristics to cells for their survival (or
death) under a specific culture condition. Hypoxanthine-guanine
phosphoribosyltransferase deficiency (hereinafter abbreviated as
HGPRT deficiency) and thymidine kinase deficiency (hereinafter
abbreviated as TK deficiency) are known as selection markers. Cells
with HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine
sensitivity (hereinafter abbreviated as HAT sensitivity).
HAT-sensitive cells cannot synthesize DNA in a HAT selection
medium, and are thus killed. However, when the cells are fused with
normal cells, they can continue DNA synthesis using the salvage
pathway of the normal cells, and therefore they can grow even in
the HAT selection medium.
[0142] HGPRT-deficient and TK-deficient cells can be selected in a
medium containing 6-thioguanine, 8-azaguanine (hereinafter
abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal
cells are killed because they incorporate these pyrimidine analogs
into their DNA. Meanwhile, cells that are deficient in these
enzymes can survive in the selection medium, since they cannot
incorporate these pyrimidine analogs. In addition, a selection
marker referred to as G418 resistance provided by the
neomycin-resistant gene confers resistance to 2-deoxystreptamine
antibiotics (gentamycin analogs). Various types of myeloma cells
that are suitable for cell fusion are known.
[0143] For example, myeloma cells including the following cells can
be preferably used: [0144] P3(P3.times.63Ag8.653) (J. Immunol.
(1979) 123 (4), 1548-1550); [0145] P3.times.63Ag8U.1 (Current
Topics in Microbiology and Immunology (1978)81, 1-7); [0146] NS-1
(C. Eur. J. Immunol. (1976)6 (7), 511-519); [0147] MPC-11 (Cell
(1976) 8 (3), 405-415); [0148] SP2/0 (Nature (1978) 276 (5685),
269-270); [0149] FO (J. Immunol. Methods (1980) 35 (1-2), 1-21);
[0150] S194/5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323);
[0151] R210 (Nature (1979) 277 (5692), 131-133), etc.
[0152] Cell fusions between the immunocytes and myeloma cells are
essentially carried out using known methods, for example, a method
by Kohler and Milstein et al. (Methods Enzymol. (1981) 73:
3-46).
[0153] More specifically, cell fusion can be carried out, for
example, in a conventional culture medium in the presence of a cell
fusion-promoting agent. The fusion-promoting agents include, for
example, polyethylene glycol (PEG) and Sendai virus (HVJ). If
required, an auxiliary substance such as dimethyl sulfoxide is also
added to improve fusion efficiency.
[0154] The ratio of immune cells to myeloma cells may be determined
at one's own discretion, preferably, for example, one myeloma cell
for every one to ten immunocytes. Culture media to be used for cell
fusions include, for example, media that are suitable for the
growth of myeloma cell lines, such as RPMI1640 medium and MEM
medium, and other conventional culture medium used for this type of
cell culture. In addition, serum supplements such as fetal calf
serum (FCS) may be preferably added to the culture medium.
[0155] For cell fusion, predetermined amounts of the above immune
cells and myeloma cells are mixed well in the above culture medium.
Then, a PEG solution (for example, the average molecular weight is
about 1,000 to 6,000) prewarmed to about 37.degree. C. is added
thereto at a concentration of generally 30% to 60% (w/v). This is
gently mixed to produce desired fusion cells (hybridomas). Then, an
appropriate culture medium mentioned above is gradually added to
the cells, and this is repeatedly centrifuged to remove the
supernatant. Thus, cell fusion agents and such which are
unfavorable to hybridoma growth can be removed.
[0156] The hybridomas thus obtained can be selected by culture
using a conventional selective medium, for example, HAT medium (a
culture medium containing hypoxanthine, aminopterin, and
thymidine). Cells other than the desired hybridomas (non-fused
cells) can be killed by continuing culture in the above HAT medium
for a sufficient period of time (typically, the period is several
days to several weeks). Then, hybridomas producing the desired
antibody are screened and singly cloned by conventional limiting
dilution methods.
[0157] The hybridomas thus obtained can be selected using a
selection medium based on the selection marker possessed by the
myeloma used for cell fusion. For example, HGPRT- or TK-deficient
cells can be selected by culture using the HAT medium (a culture
medium containing hypoxanthine, aminopterin, and thymidine).
Specifically, when HAT-sensitive myeloma cells are used for cell
fusion, cells successfully fused with normal cells can selectively
proliferate in the HAT medium. Cells other than the desired
hybridomas (non-fused cells) can be killed by continuing culture in
the above HAT medium for a sufficient period of time. Specifically,
desired hybridomas can be selected by culture for generally several
days to several weeks. Then, hybridomas producing the desired
antibody are screened and singly cloned by conventional limiting
dilution methods.
[0158] Desired antibodies can be preferably selected and singly
cloned by screening methods based on known antigen/antibody
reaction. For example, an IL-6R-binding monoclonal antibody can
bind to IL-6R expressed on the cell surface. Such a monoclonal
antibody can be screened by fluorescence activated cell sorting
(FACS). FACS is a system that assesses the binding of an antibody
to cell surface by analyzing cells contacted with a fluorescent
antibody using laser beam, and measuring the fluorescence emitted
from individual cells.
[0159] To screen for hybridomas that produce a monoclonal antibody
of the present invention by FACS, IL-6R-expressing cells are first
prepared. Cells preferably used for screening are mammalian cells
in which IL-6R is forcedly expressed. As control, the activity of
an antibody to bind to cell-surface IL-6R can be selectively
detected using non-transformed mammalian cells as host cells.
Specifically, hybridomas producing an anti-IL-6R monoclonal
antibody can be isolated by selecting hybridomas that produce an
antibody which binds to cells forced to express IL-6R, but not to
host cells.
[0160] Alternatively, the activity of an antibody to bind to
immobilized IL-6R-expressing cells can be assessed based on the
principle of ELISA. For example, IL-6R-expressing cells are
immobilized to the wells of an ELISA plate. Culture supernatants of
hybridomas are contacted with the immobilized cells in the wells,
and antibodies that bind to the immobilized cells are detected.
When the monoclonal antibodies are derived from mouse, antibodies
bound to the cells can be detected using an anti-mouse
immunoglobulin antibody. Hybridomas producing a desired antibody
having the antigen-binding ability are selected by the above
screening, and they can be cloned by a limiting dilution method or
the like.
[0161] Monoclonal antibody-producing hybridomas thus prepared can
be passaged in a conventional culture medium, and stored in liquid
nitrogen for a long period.
[0162] The above hybridomas are cultured by a conventional method,
and desired monoclonal antibodies can be prepared from the culture
supernatants. Alternatively, the hybridomas are administered to and
grown in compatible mammals, and monoclonal antibodies are prepared
from the ascites. The former method is suitable for preparing
antibodies with high purity.
[0163] Antibodies encoded by antibody genes that are cloned from
antibody-producing cells such as the above hybridomas can also be
preferably used. A cloned antibody gene is inserted into an
appropriate vector, and this is introduced into a host to express
the antibody encoded by the gene. Methods for isolating antibody
genes, inserting the genes into vectors, and transforming host
cells have already been established, for example, by Vandamme et
al. (Eur. J. Biochem. (1990) 192(3), 767-775). Methods for
producing recombinant antibodies are also known as described
below.
[0164] For example, a cDNA encoding the variable region (V region)
of an anti-IL-6R antibody is prepared from hybridoma cells
expressing the anti-IL-6R antibody. For this purpose, total RNA is
first extracted from hybridomas. Methods used for extracting mRNAs
from cells include, for example: [0165] the guanidine
ultracentrifugation method (Biochemistry (1979) 18(24), 5294-5299),
and [0166] the AGPC method (Anal. Biochem. (1987) 162(1),
156-159)
[0167] Extracted mRNAs can be purified using the mRNA Purification
Kit (GE Healthcare Bioscience) or such. Alternatively, kits for
extracting total mRNA directly from cells, such as the QuickPrep
mRNA Purification Kit (GE Healthcare Bioscience), are also
commercially available. mRNAs can be prepared from hybridomas using
such kits. cDNAs encoding the antibody V region can be synthesized
from the prepared mRNAs using a reverse transcriptase. cDNAs can be
synthesized using the AMV Reverse Transcriptase First-strand cDNA
Synthesis Kit (Seikagaku Co.) or such. Furthermore, the SMART RACE
cDNA Amplification Kit (Clontech) and the PCR-based 5'-RACE method
(Proc. Natl. Acad. Sci. U.S.A. (1988) 85(23), 8998-9002; Nucleic
Acids Res. (1989) 17(8), 2919-2932) can be appropriately used to
synthesize and amplify cDNAs. In such a cDNA synthesis process,
appropriate restriction enzyme sites described below may be
introduced into both ends of a cDNA.
[0168] The cDNA fragment of interest is purified from the resulting
PCR product, and then this is ligated to a vector DNA. A
recombinant vector is thus constructed, and introduced into E. coli
or such. After colony selection, the desired recombinant vector can
be prepared from the colony-forming E. coli. Then, whether the
recombinant vector has the cDNA nucleotide sequence of interest is
tested by a known method such as the dideoxy nucleotide chain
termination method.
[0169] The 5'-RACE method which uses primers to amplify the
variable region gene is conveniently used for isolating the gene
encoding the variable region. First, a 5'-RACE cDNA library is
constructed by cDNA synthesis using RNAs extracted from hybridoma
cells as a template. A commercially available kit such as the SMART
RACE cDNA Amplification Kit is appropriately used to synthesize the
5'-RACE cDNA library.
[0170] The antibody gene is amplified by PCR using the prepared
5'-RACE cDNA library as a template. Primers for amplifying the
mouse antibody gene can be designed based on known antibody gene
sequences. The nucleotide sequences of the primers vary depending
on the immunoglobulin subclass. Therefore, it is preferable that
the subclass is determined in advance using a commercially
available kit such as the Iso Strip Mouse Monoclonal Antibody
Isotyping Kit (Roche Diagnostics).
[0171] Specifically, for example, primers that allow amplification
of genes encoding .gamma.1, .gamma.2a, .gamma.2b, and .gamma.3
heavy chains and .kappa. and .lamda. light chains are used to
isolate mouse IgG-encoding genes. In general, a primer that anneals
to a constant region site close to the variable region is used as a
3'-side primer to amplify an IgG variable region gene. Meanwhile, a
primer attached to a 5' RACE cDNA library construction kit is used
as a 5'-side primer.
[0172] PCR products thus amplified are used to reshape
immunoglobulins composed of a combination of heavy and light
chains. A desired antibody can be selected using the IL-6R-binding
activity of a reshaped immunoglobulin as an indicator. For example,
when the objective is to isolate an antibody against IL-6R, it is
more preferred that the binding of the antibody to IL-6R is
specific. An IL-6R-binding antibody can be screened, for example,
by the following steps:
(1) contacting an IL-6R-expressing cell with an antibody comprising
the V region encoded by a cDNA isolated from a hybridoma; (2)
detecting the binding of the antibody to the IL-6R-expressing cell;
and (3) selecting an antibody that binds to the IL-6R-expressing
cell.
[0173] Methods for detecting the binding of an antibody to
IL-6R-expressing cells are known. Specifically, the binding of an
antibody to IL-6R-expressing cells can be detected by the
above-described techniques such as FACS Immobilized samples of
IL-6R-expressing cells are appropriately used to assess the binding
activity of an antibody.
[0174] Preferred antibody screening methods that use the binding
activity as an indicator also include panning methods using phage
vectors. Screening methods using phage vectors are advantageous
when the antibody genes are isolated from heavy-chain and
light-chain subclass libraries from a polyclonal
antibody-expressing cell population. Genes encoding the heavy-chain
and light-chain variable regions can be linked by an appropriate
linker sequence to form a single-chain Fv (scFv). Phages presenting
scFv on their surface can be produced by inserting a gene encoding
scFv into a phage vector. The phages are contacted with an antigen
of interest. Then, a DNA encoding scFv having the binding activity
of interest can be isolated by collecting phages bound to the
antigen. This process can be repeated as necessary to enrich scFv
having the binding activity of interest.
[0175] After isolation of the cDNA encoding the V region of the
anti-IL-6R antibody of interest, the cDNA is digested with
restriction enzymes that recognize the restriction sites introduced
into both ends of the cDNA. Preferred restriction enzymes recognize
and cleave a nucleotide sequence that occurs in the nucleotide
sequence of the antibody gene at a low frequency. Furthermore, a
restriction site for an enzyme that produces a sticky end is
preferably introduced into a vector to insert a single-copy
digested fragment in the correct orientation. The cDNA encoding the
V region of the anti-IL-6R antibody is digested as described above,
and this is inserted into an appropriate expression vector to
construct an antibody expression vector. In this case, if a gene
encoding the antibody constant region (C region) and a gene
encoding the above V region are fused in-frame, a chimeric antibody
is obtained. Herein, "chimeric antibody" means that the origin of
the constant region is different from that of the variable region.
Thus, in addition to mouse-human heterochimeric antibodies,
human-human allochimeric antibodies are included in the chimeric
antibodies of the present invention. A chimeric antibody expression
vector can be constructed by inserting the above V region gene into
an expression vector that already has the constant region.
Specifically, for example, a recognition sequence for a restriction
enzyme that excises the above V region gene can be appropriately
placed on the 5' side of an expression vector carrying a DNA
encoding a desired antibody constant region. A chimeric antibody
expression vector is constructed by fusing in frame the two genes
digested with the same combination of restriction enzymes.
[0176] To produce an anti-IL-6R monoclonal antibody, antibody genes
are inserted into an expression vector so that the genes are
expressed under the control of an expression regulatory region. The
expression regulatory region for antibody expression includes, for
example, enhancers and promoters. Furthermore, an appropriate
signal sequence may be attached to the amino terminus so that the
expressed antibody is secreted to the outside of cells. In the
Examples described later, a peptide having the amino acid sequence
MGWSCIILFLVATATGVHS (SEQ ID NO: 4) are used as a signal sequence.
Meanwhile, other appropriate signal sequences may be attached. The
expressed polypeptide is cleaved at the carboxyl terminus of the
above sequence, and the resulting polypeptide is secreted to the
outside of cells as a mature polypeptide. Then, appropriate host
cells are transformed with the expression vector, and recombinant
cells expressing the anti-IL-6R antibody-encoding DNA are
obtained.
[0177] DNAs encoding the antibody heavy chain (H chain) and light
chain (L chain) are separately inserted into different expression
vectors to express the antibody gene. An antibody molecule having
the H and L chains can be expressed by co-transfecting the same
host cell with vectors into which the H-chain and L-chain genes are
respectively inserted. Alternatively, host cells can be transformed
with a single expression vector into which DNAs encoding the H and
L chains are inserted (see WO 1994/011523).
[0178] There are various known host cell/expression vector
combinations for antibody preparation by introducing isolated
antibody genes into appropriate hosts. All of these expression
systems are applicable to isolation of the antigen-binding domains
of the present invention. Appropriate eukaryotic cells used as host
cells include animal cells, plant cells, and fungal cells.
Specifically, the animal cells include, for example, the following
cells.
(1) mammalian cells: CHO (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, 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.
[0179] 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.
[0180] Furthermore, the following cells can be used as fungal
cells: [0181] yeasts: the Saccharomyces genus such as Saccharomyces
serevisiae, and the Pichia genus such as Pichia pastoris; and
[0182] filamentous fungi: the Aspergillus genus such as Aspergillus
niger.
[0183] Furthermore, antibody gene expression systems that utilize
prokaryotic cells are also known. For example, when using bacterial
cells, E. coli cells, Bacillus subtilis cells, and such can
suitably be utilized in the present invention. Expression vectors
carrying the antibody genes of interest are introduced into these
cells by transfection. The transfected cells are cultured in vitro,
and the desired antibody can be prepared from the culture of
transformed cells.
[0184] In addition to the above-described host cells, transgenic
animals can also be used to produce a recombinant antibody. That
is, the antibody can be obtained from an animal into which the gene
encoding the antibody of interest is introduced. For example, the
antibody gene can be constructed as a fusion gene by inserting in
frame into a gene that encodes a protein produced specifically in
milk. Goat .beta.-casein or such can be used, for example, as the
protein secreted in milk. DNA fragments containing the fused gene
inserted with the antibody gene is injected into a goat embryo, and
then this embryo is introduced into a female goat. Desired
antibodies can be obtained as a protein fused with the milk protein
from milk produced by the transgenic goat born from the
embryo-recipient goat (or progeny thereof). In addition, to
increase the volume of milk containing the desired antibody
produced by the transgenic goat, hormones can be administered to
the transgenic goat as necessary (Ebert, K. M. et al.,
Bio/Technology (1994) 12 (7), 699-702).
[0185] When an antigen-binding molecule described herein is
administered to humans, an antigen-binding domain derived from a
genetically recombinant antibody that has been artificially
modified to reduce the heterologous antigenicity against human and
such, can be appropriately used as the antigen-binding domain of
the antigen-binding molecule. Such genetically recombinant
antibodies include, for example, humanized antibodies. These
modified antibodies are appropriately produced by known
methods.
[0186] An antibody variable region used to produce the
antigen-binding domain of an antigen-binding molecule described
herein is generally formed by three complementarity-determining
regions (CDRs) that are separated by four framework regions (FRs).
CDR is a region that substantially determines the binding
specificity of an antibody. The amino acid sequences of CDRs are
highly diverse. On the other hand, the FR-forming amino acid
sequences often have high identity even among antibodies with
different binding specificities. Therefore, generally, the binding
specificity of a certain antibody can be introduced to another
antibody by CDR grafting.
[0187] A humanized antibody is also called a reshaped human
antibody. Specifically, humanized antibodies prepared by grafting
the CDR of a non-human animal antibody such as a mouse antibody to
a human antibody and such are known. Common genetic engineering
techniques for obtaining humanized antibodies are also known.
Specifically, for example, overlap extension PCR is known as a
method for grafting a mouse antibody CDR to a human FR. In overlap
extension PCR, a nucleotide sequence encoding a mouse antibody CDR
to be grafted is added to primers for synthesizing a human antibody
FR. Primers are prepared for each of the four FRs. It is generally
considered that when grafting a mouse CDR to a human FR, selecting
a human FR that has high identity to a mouse FR is advantageous for
maintaining the CDR function. That is, it is generally preferable
to use a human FR comprising an amino acid sequence which has high
identity to the amino acid sequence of the FR adjacent to the mouse
CDR to be grafted.
[0188] Nucleotide sequences to be ligated are designed so that they
will be connected to each other in frame. Human FRs are
individually synthesized using the respective primers. As a result,
products in which the mouse CDR-encoding DNA is attached to the
individual FR-encoding DNAs are obtained. Nucleotide sequences
encoding the mouse CDR of each product are designed so that they
overlap with each other. Then, complementary strand synthesis
reaction is conducted to anneal the overlapping CDR regions of the
products synthesized using a human antibody gene as template. Human
FRs are ligated via the mouse CDR sequences by this reaction.
[0189] The full length V region gene, in which three CDRs and four
FRs are ultimately ligated, is amplified using primers that anneal
to its 5'- or 3'-end, which are added with suitable restriction
enzyme recognition sequences. An expression vector for humanized
antibody can be produced by inserting the DNA obtained as described
above and a DNA that encodes a human antibody C region into an
expression vector so that they will ligate in frame. After the
recombinant vector is transfected into a host to establish
recombinant cells, the recombinant cells are cultured, and the DNA
encoding the humanized antibody is expressed to produce the
humanized antibody in the cell culture (see, European Patent
Publication No. EP 239400 and International Patent Publication No.
WO 1996/002576).
[0190] By qualitatively or quantitatively measuring and evaluating
the antigen-binding activity of the humanized antibody produced as
described above, one can suitably select human antibody FRs that
allow CDRs to form a favorable antigen-binding site when ligated
through the CDRs. Amino acid residues in FRs may be substituted as
necessary, so that the CDRs of a reshaped human antibody form an
appropriate antigen-binding site. For example, amino acid sequence
mutations can be introduced into FRs by applying the PCR method
used for grafting a mouse CDR into a human FR. More specifically,
partial nucleotide sequence mutations can be introduced into
primers that anneal to the FR. Nucleotide sequence mutations are
introduced into the FRs synthesized by using such primers. Mutant
FR sequences having the desired characteristics can be selected by
measuring and evaluating the activity of the amino acid-substituted
mutant antibody to bind to the antigen by the above-mentioned
method (Cancer Res. (1993) 53: 851-856).
[0191] Alternatively, desired human antibodies can be obtained by
immunizing transgenic animals having the entire repertoire of human
antibody genes (see WO 1993/012227; WO 1992/003918; WO 1994/002602;
WO 1994/025585; WO 1996/034096; WO 1996/033735) by DNA
immunization.
[0192] Furthermore, techniques for preparing human antibodies by
panning using human antibody libraries are also known. For example,
the V region of a human antibody is expressed as a single-chain
antibody (scFv) on phage surface by the phage display method.
Phages expressing an scFv that binds to the antigen can be
selected. The DNA sequence encoding the human antibody V region
that binds to the antigen can be determined by analyzing the genes
of selected phages. The DNA sequence of the scFv that binds to the
antigen is determined. An expression vector is prepared by fusing
the V region sequence in frame with the C region sequence of a
desired human antibody, and inserting this into an appropriate
expression vector. The expression vector is introduced into cells
appropriate for expression such as those described above. The human
antibody can be produced by expressing the human antibody-encoding
gene in the cells. These methods are already known (see WO
1992/001047; WO 1992/020791; WO 1993/006213; WO 1993/011236; WO
1993/019172; WO 1995/001438; WO 1995/015388).
[0193] In addition to the techniques described above, techniques of
B cell cloning (identification of each antibody-encoding sequence,
cloning and its isolation; use in constructing expression vector in
order to prepare each antibody (IgG1, IgG2, IgG3, or IgG4 in
particular); and such) such as described in Bernasconi et al.
(Science (2002) 298: 2199-2202) or in WO 2008/081008 can be
appropriately used to isolate antibody genes.
EU Numbering System and Kabat Numbering System
[0194] According to the methods used in the present invention,
amino acid positions assigned to antibody CDR and FR are specified
according to Kabat numbering (Sequences of Proteins of
Immunological Interest (National Institute of Health, Bethesda,
Md., 1987 and 1991)). Herein, when an antigen-binding molecule is
an antibody or antigen-binding fragment, variable region amino
acids are indicated according to Kabat numbering system, while
constant region amino acids are indicated according to EU numbering
system based on Kabat's amino acid positions.
Conditions of Ion Concentration
Conditions of Metal Ion Concentration
[0195] In one embodiment of the present invention, the ion
concentration refers to a metal ion concentration. "Metal ions"
refer to ions of group I elements except hydrogen such as alkaline
metals and copper group elements, group II elements such as
alkaline earth metals and zinc group elements, group III elements
except boron, group IV elements except carbon and silicon, group
VIII elements such as iron group and platinum group elements,
elements belonging to subgroup A of groups V, VI, and VII, and
metal elements such as antimony, bismuth, and polonium. Metal atoms
have the property of releasing valence electrons to become cations.
This is referred to as ionization tendency. Metals with strong
ionization tendency are deemed to be chemically active.
[0196] In the present invention, preferred metal ions include, for
example, calcium ion. Calcium ion is involved in modulation of many
biological phenomena, including contraction of muscles such as
skeletal, smooth, and cardiac muscles; activation of movement,
phagocytosis, and the like of leukocytes; activation of shape
change, secretion, and the like of platelets; activation of
lymphocytes; activation of mast cells including secretion of
histamine; cell responses mediated by catecholamine a receptor or
acetylcholine receptor; exocytosis; release of transmitter
substances from neuron terminals; and axoplasmic flow in neurons.
Known intracellular calcium ion receptors include troponin C,
calmodulin, parvalbumin, and myosin light chain, which have several
calcium ion-binding sites and are believed to be derived from a
common origin in terms of molecular evolution. There are also many
known calcium-binding motifs. Such well-known motifs include, for
example, cadherin domains, EF-hand of calmodulin, C2 domain of
Protein kinase C, Gla domain of blood coagulation protein Factor
IX, C-type lectins of asialoglycoprotein receptor and
mannose-binding receptor, A domains of LDL receptors, annexin,
thrombospondin type 3 domain, and EGF-like domains.
[0197] In the present invention, when the metal ion is calcium ion,
the conditions of calcium ion concentration include low calcium ion
concentration conditions and high calcium ion concentration
conditions. "The antigen-binding activity of an antigen-binding
domain contained in the antigen-binding molecule of the present
invention varies depending on calcium ion concentration conditions"
means that the antigen-binding activity of an antigen-binding
domain contained in the antigen-binding molecule varies due to the
difference in the conditions between low and high calcium ion
concentrations. For example, the antigen-binding activity of an
antigen-binding domain may be higher under a high calcium ion
concentration condition than under a low calcium ion concentration
condition. Alternatively, the antigen-binding activity of an
antigen-binding domain may be, for example, higher under a low
calcium ion concentration condition than under a high calcium ion
concentration condition.
[0198] Herein, the high calcium ion concentration is not
particularly limited to a specific value; however, the
concentration may preferably be selected between 100 .mu.M and 10
mM. In another embodiment, the concentration may be selected
between 200 .mu.M and 5 mM. In an alternative embodiment, the
concentration may be selected between 400 .mu.M and 3 mM. In still
another embodiment, the concentration may be selected between 200
.mu.M and 2 mM. Furthermore, the concentration may be selected
between 400 .mu.M and 1 mM. In particular, a concentration selected
between 500 .mu.M and 2.5 mM, which is close to the plasma (blood)
concentration of calcium ion in vivo, is preferred.
[0199] Herein, the low calcium ion concentration is not
particularly limited to a specific value; however, the
concentration may preferably be selected between 0.1 .mu.M and 30
.mu.M. In another embodiment, the concentration may be selected
between 0.2 .mu.M and 20 .mu.M. In still another embodiment, the
concentration may be selected between 0.5 .mu.M and 10 .mu.M. In an
alternative embodiment, the concentration may be selected between 1
.mu.M and 5 .mu.M. Furthermore, the concentration may be selected
between 2 .mu.M and 4 .mu.M. In particular, a concentration
selected between 1 .mu.M and 5 .mu.M, which is close to the
concentration of ionized calcium in early endosomes in vivo, is
preferred.
[0200] In the present invention, "the antigen-binding activity is
lower under a low calcium ion concentration condition than under a
high calcium ion concentration condition" means that the
antigen-binding activity of an antigen-binding domain or
antigen-binding molecule comprising the domain of the present
invention is weaker at a calcium ion concentration selected between
0.1 .mu.M and 30 .mu.M than at a calcium ion concentration selected
between 100 .mu.M and 10 mM. Preferably, it means that the
antigen-binding activity of an antigen-binding domain or
antigen-binding molecule comprising the domain of the present
invention is weaker at a calcium ion concentration selected between
0.5 .mu.M and 10 .mu.M than at a calcium ion concentration selected
between 200 .mu.M and 5 mM. It particularly preferably means that
the antigen-binding activity at the calcium ion concentration in
the early endosome in vivo is weaker than that at the in vivo
plasma calcium ion concentration; and specifically, it means that
the antigen-binding activity of an antigen-binding molecule is
weaker at a calcium ion concentration selected between 1 .mu.M and
5 .mu.M than at a calcium ion concentration selected between 500
.mu.M and 2.5 mM.
[0201] Whether the antigen-binding activity of an antigen-binding
domain or antigen-binding molecule comprising the domain is changed
depending on metal ion concentrations can be determined, for
example, by the use of known measurement methods such as those
described in the section "Binding Activity" above. For example, in
order to confirm that the antigen-binding activity of an
antigen-binding domain or antigen-binding molecule comprising the
domain becomes higher under a high calcium ion concentration
condition than under a low calcium ion concentration condition, the
antigen-binding activity of the domain or the molecule under low
and high calcium ion concentration conditions is compared.
[0202] In the present invention, the expression "the
antigen-binding activity is lower under a low calcium ion
concentration condition than under a high calcium ion concentration
condition" can also be expressed as "the antigen-binding activity
of an antigen-binding domain or antigen-binding molecule comprising
the domain is higher under a high calcium ion concentration
condition than under a low calcium ion concentration condition". In
the present invention, "the antigen-binding activity is lower under
a low calcium ion concentration condition than under a high calcium
ion concentration condition" is sometimes written as "the
antigen-binding activity is weaker under a low calcium ion
concentration condition than under a high calcium ion concentration
condition". Also, "the antigen-binding activity under a low calcium
ion concentration condition is reduced to be lower than that under
a high calcium ion concentration condition" may be written as "the
antigen-binding activity under a low calcium ion concentration
condition is made weaker than that under a high calcium ion
concentration condition".
[0203] When determining the antigen-binding activity, the
conditions other than calcium ion concentration can be
appropriately selected by those skilled in the art, and are not
particularly limited. For example, the activity can be determined
at 37.degree. C. in HEPES buffer. For example, Biacore (GE
Healthcare) or such can be used for the determination. When the
antigen is a soluble antigen, the antigen-binding activity of an
antigen-binding domain or antigen-binding molecule comprising the
domain can be assessed by flowing the antigen as an analyte over a
chip onto which the antigen-binding domain or antigen-binding
molecule comprising the domain is immobilized. When the antigen is
a membrane antigen, the binding activity of an antigen-binding
domain or antigen-binding molecule comprising the domain to the
membrane antigen can be assessed by flowing the antigen-binding
domain or antigen-binding molecule comprising the domain as an
analyte over a chip onto which the antigen is immobilized.
[0204] As long as the antigen-binding activity of an
antigen-binding molecule of the present invention is weaker under a
low calcium ion concentration condition than under a high calcium
ion concentration condition, the ratio of the antigen-binding
activity between low and high calcium ion concentration conditions
is not particularly limited. However, the ratio of the KD
(dissociation constant) of the antigen-binding molecule for an
antigen at a low calcium ion concentration condition with respect
to the KD at a high calcium ion concentration condition, i.e., the
value of KD (3 .mu.M Ca)/KD (2 mM Ca), is preferably 2 or more,
more preferably 10 or more, and still more preferably 40 or more.
The upper limit of the KD (3 .mu.M Ca)/KD (2 mM Ca) value is not
particularly limited, and may be any value such as 400, 1000, or
10000 as long as the molecule can be produced by techniques known
to those skilled in the art.
[0205] When the antigen is a soluble antigen, KD (dissociation
constant) can be used to represent the antigen-binding activity.
Meanwhile, when the antigen is a membrane antigen, apparent KD
(apparent dissociation constant) can be used to represent the
activity. KD (dissociation constant) and apparent KD (apparent
dissociation constant) can be determined by methods known to those
skilled in the art, for example, using Biacore (GE healthcare),
Scatchard plot, or flow cytometer.
[0206] Alternatively, for example, the dissociation rate constant
(kd) can also be preferably used as an index to represent the ratio
of the antigen-binding activity of an antigen-binding domain or
antigen-binding molecule comprising the domain of the present
invention between low and high calcium concentration conditions.
When the dissociation rate constant (kd) is used instead of the
dissociation constant (KD) as an index to represent the binding
activity ratio, the ratio of the dissociation rate constant (kd)
between low and high calcium concentration conditions, i.e., the
value of kd (low calcium concentration condition)/kd (high calcium
concentration condition), is preferably 2 or more, more preferably
5 or more, still more preferably 10 or more, and yet more
preferably 30 or more. The upper limit of the Kd (low calcium
concentration condition)/kd (high calcium concentration condition)
value is not particularly limited, and can be any value such as 50,
100, or 200 as long as the molecule can be produced by techniques
known to those skilled in the art.
[0207] When the antigen is a soluble antigen, kd (dissociation rate
constant) can be used to represent the antigen-binding activity.
Meanwhile, when the antigen is a membrane antigen, apparent kd
(apparent dissociation rate constant) can be used to represent the
antigen-binding activity. The kd (dissociation rate constant) and
apparent kd (apparent dissociation rate constant) can be determined
by methods known to those skilled in the art, for example, using
Biacore (GE healthcare) or flow cytometer. In the present
invention, when the antigen-binding activity of an antigen-binding
domain or antigen-binding molecule comprising the domain is
determined at different calcium ion concentrations, it is
preferable to use the same conditions except for the calcium
concentrations.
[0208] The methods described in WO 2012/073992 (for example,
paragraph 0200-0213) and such may be presented as examples of a
method of screening for an antigen-binding molecule or an
antigen-binding domain whose antigen-binding activity under low
calcium ion concentration conditions is lower than under high
calcium ion concentration conditions, which is an embodiment
provided by the present invention.
Libraries
[0209] In an embodiment, an antigen-binding domain or
antigen-binding molecule of the present invention can be obtained
from a library that is mainly composed of a plurality of
antigen-binding molecules whose sequences are different from one
another and whose antigen-binding domains have at least one amino
acid residue that alters the antigen-binding activity of the
antigen-binding molecules depending on ion concentrations. The ion
concentrations preferably include, for example, metal ion
concentration and hydrogen ion concentration.
[0210] Herein, a "library" refers to a plurality of antigen-binding
molecules or a plurality of fusion polypeptides containing
antigen-binding molecules, or nucleic acids or polynucleotides
encoding their sequences. The sequences of a plurality of
antigen-binding molecules or a plurality of fusion polypeptides
containing antigen-binding molecules in a library are not
identical, but are different from one another.
[0211] Herein, the phrase "sequences are different from one
another" in the expression "a plurality of antigen-binding
molecules whose sequences are different from one another" means
that the sequences of antigen-binding molecules in a library are
different from one another. Specifically, in a library, the number
of sequences different from one another reflects the number of
independent clones with different sequences, and may also be
referred to as "library size". The library size of a conventional
phage display library ranges from 10.sup.6 to 10.sup.12. The
library size can be increased up to 10.sup.14 by the use of known
techniques such as ribosome display. However, the actual number of
phage particles used in panning selection of a phage library is in
general 10-10000 times greater than the library size. This excess
multiplicity is also referred to as "the number of library
equivalents", and means that there are 10 to 10,000 individual
clones that have the same amino acid sequence. Thus, in the present
invention, the phrase "sequences are different from one another"
means that the sequences of independent antigen-binding molecules
in a library, excluding library equivalents, are different from one
another. More specifically, the above means that there are 10.sup.6
to 10.sup.14 antigen-binding molecules whose sequences are
different from one another, preferably 10.sup.7 to 10.sup.12
molecules, more preferably 10.sup.8 to 10.sup.11, and particularly
preferably 10.sup.8 to 10.sup.10 whose sequences are different from
one another.
[0212] In the present invention, the phrase "a plurality of" in the
expression "a library mainly composed of a plurality of
antigen-binding molecules" generally refers to, in the case of, for
example, antigen-binding molecules, fusion polypeptides,
polynucleotide molecules, vectors, or viruses of the present
invention, a group of two or more types of the substance. For
example, when two or more substances are different from one another
in a particular characteristic, this means that there are two or
more types of the substance. Such examples may include, for
example, mutant amino acids observed at specific amino acid
positions in an amino acid sequence. For example, when there are
two or more antigen-binding molecules of the present invention
whose sequences are substantially the same or preferably the same
except for flexible residues or except for particular mutant amino
acids at hypervariable positions exposed on the surface, there is a
plurality of antigen-binding molecules of the present invention. In
another example, when there are two or more polynucleotide
molecules whose sequences are substantially the same or preferably
the same except for nucleotides encoding flexible residues or
nucleotides encoding mutant amino acids of hypervariable positions
exposed on the surface, there are a plurality of polynucleotide
molecules in the present invention.
[0213] In addition, in the present invention, the phrase "mainly
composed of" in the expression "a library mainly composed of a
plurality of antigen-binding molecules" reflects the number of
antigen-binding molecules whose antigen-binding activity varies
depending on ion concentrations, among independent clones with
different sequences in a library. Specifically, it is preferable
that there are at least 10.sup.4 antigen-binding molecules having
such binding activity in a library. More preferably,
antigen-binding domains of the present invention can be obtained
from a library containing at least 10.sup.5 antigen-binding
molecules having such binding activity. Still more preferably,
antigen-binding domains of the present invention can be obtained
from a library containing at least 10.sup.6 antigen-binding
molecules having such binding activity. Particularly preferably,
antigen-binding domains of the present invention can be obtained
from a library containing at least 10.sup.7 antigen-binding
molecules having such binding activity. Yet more preferably,
antigen-binding domains of the present invention can be obtained
from a library containing at least 10.sup.8 antigen-binding
molecules having such binding activity. Alternatively, this may
also be preferably expressed as the ratio of the number of
antigen-binding molecules whose antigen-binding activity varies
depending on ion concentrations with respect to the number of
independent clones having different sequences in a library.
Specifically, antigen-binding domains of the present invention can
be obtained from a library in which antigen-binding molecules
having such binding activity account for 0.1% to 80%, preferably
0.5% to 60%, more preferably 1% to 40%, still more preferably 2% to
20%, and particularly preferably 4% to 10% of independent clones
with different sequences in the library. In the case of fusion
polypeptides, polynucleotide molecules, or vectors, similar
expressions may be possible using the number of molecules or the
ratio to the total number of molecules. In the case of viruses,
similar expressions may also be possible using the number of
virions or the ratio to total number of virions.
Amino Acids that Alter the Antigen-Binding Activity of
Antigen-Binding Domains Depending on Calcium Ion Concentrations
[0214] Antigen-binding domains or antigen-binding molecules of the
present invention to be screened by the above-described screening
methods may be prepared in any manner. For example, when the metal
ion is calcium ion, it is possible to use preexisting
antigen-binding domains or antigen-binding molecules, preexisting
libraries (phage library, etc.), antibodies or libraries prepared
from hybridomas obtained by immunizing animals or from B cells of
immunized animals, antibodies or libraries obtained by introducing
amino acids capable of chelating calcium (for example, aspartic
acid and glutamic acid) or unnatural amino acid mutations into the
above-described antibodies or libraries (calcium-chelatable amino
acids (such as aspartic acid and glutamic acid), libraries with
increased content of unnatural amino acids, libraries prepared by
introducing calcium-chelatable amino acids (such as aspartic acid
and glutamic acid) or unnatural amino acid mutations at particular
positions, or the like.
[0215] Examples of the amino acids that alter the antigen-binding
activity of antigen-binding molecules depending on ion
concentrations as described above may be any types of amino acids
as long as the amino acids form a calcium-binding motif
Calcium-binding motifs are well known to those skilled in the art
and have been described in details (for example, Springer et al.
(Cell (2000) 102, 275-277); Kawasaki and Kretsinger (Protein Prof
(1995) 2, 305-490); Moncrief et al. (J. Mol. Evol. (1990) 30,
522-562); Chauvaux et al. (Biochem. J. (1990) 265, 261-265);
Bairoch and Cox (FEBS Lett. (1990) 269, 454-456); Davis (New Biol.
(1990) 2, 410-419); Schaefer et al. (Genomics (1995) 25, 638-643);
Economou et al. (EMBO J. (1990) 9, 349-354); Wurzburg et al.
(Structure. (2006) 14, 6, 1049-1058)). Specifically, any known
calcium-binding motifs, including type C lectins such as ASGPR,
CD23, MBR, and DC-SIGN, can be included in antigen-binding
molecules of the present invention. Preferred examples of such
preferred calcium-binding motifs also include, in addition to those
described above, for example, the calcium-binding motif in the
antigen-binding domain of SEQ ID NO: 5.
[0216] Furthermore, as amino acids that alter the antigen-binding
activity of antigen-binding domains included in the antigen-binding
molecules of the present invention depending on calcium ion
concentration conditions, for example, amino acids having
metal-chelating activity may also be preferably used. Examples of
such metal-chelating amino acids include, for example, serine (Ser
(S)), threonine (Thr (T)), asparagine (Asn (N)), glutamine (Gln
(Q)), aspartic acid (Asp (D)), and glutamic acid (Glu (E)).
[0217] Positions in the antigen-binding domains at which the
above-described amino acids are contained are not particularly
limited to particular positions, and may be any positions within
the heavy chain variable region or light chain variable region that
forms an antigen-binding domain, as long as they alter the
antigen-binding activity of antigen-binding molecules depending on
calcium ion concentrations. In a non-limiting embodiment,
antigen-binding domains of the present invention can be obtained
from a library mainly composed of antigen-binding molecules whose
sequences are different from one another and whose heavy chain
antigen-binding domains contain amino acids that alter the
antigen-binding activity of the antigen-binding molecules depending
on calcium ion concentrations. In another non-limiting embodiment,
antigen-binding domains of the present invention can be obtained
from a library mainly composed of antigen-binding molecules whose
sequences are different from one another and whose heavy chain CDR3
domains contain the above-mentioned amino acids. In still another
embodiment, antigen-binding domains of the present invention can be
obtained from a library mainly composed of antigen-binding
molecules whose sequences are different from one another and whose
heavy chain CDR3 domains contain the above-mentioned amino acids at
positions 95, 96, 100a, and/or 101 as indicated according to the
Kabat numbering system.
[0218] Meanwhile, in a non-limiting embodiment of the present
invention, antigen-binding domains of the present invention can be
obtained from a library mainly composed of antigen-binding
molecules whose sequences are different from one another and whose
light chain antigen-binding domains contain amino acids that alter
the antigen-binding activity of antigen-binding molecules depending
on calcium ion concentrations. In another non-limiting embodiment,
antigen-binding domains of the present invention can be obtained
from a library mainly composed of antigen-binding molecules whose
sequences are different from one another and whose light chain CDR1
domains contain the above-mentioned amino acids. In still another
embodiment, antigen-binding domains of the present invention can be
obtained from a library mainly composed of antigen-binding
molecules whose sequences are different from one another and whose
light chain CDR1 domains contain the above-mentioned amino acids at
positions 30, 31, and/or 32 as indicated according to the Kabat
numbering system.
[0219] In another non-limiting embodiment, antigen-binding domains
of the present invention can be obtained from a library mainly
composed of antigen-binding molecules whose sequences are different
from one another and whose light chain CDR2 domains contain the
above-mentioned amino acid residues. In yet another non-limiting
embodiment, the present invention provides libraries mainly
composed of antigen-binding molecules whose sequences are different
from one another and whose light chain CDR2 domains contain the
above-mentioned amino acid residues at position 50 as indicated
according to the Kabat numbering system.
[0220] In still another embodiment of the present invention,
antigen-binding domains of the present invention can be obtained
from a library mainly composed of antigen-binding molecules whose
sequences are different from one another and whose light chain CDR3
domains contain the above-mentioned amino acid residues. In an
alternative embodiment, antigen-binding domains of the present
invention can be obtained from a library mainly composed of
antigen-binding molecules whose sequences are different from one
another and whose light chain CDR3 domains contain the
above-mentioned amino acid residues at position 92 as indicated
according to the Kabat numbering system.
[0221] Furthermore, in a different embodiment of the present
invention, antigen-binding domains of the present invention can be
obtained from a library mainly composed of antigen-binding
molecules whose sequences are different from one another and in
which two or three CDRs selected from the above-described light
chain CDR1, CDR2, and CDR3 contain the aforementioned amino acid
residues. Moreover, antigen-binding domains of the present
invention can be obtained from a library mainly composed of
antigen-binding molecules whose sequences are different from one
another and whose light chains contain the aforementioned amino
acid residues at any one or more of positions 30, 31, 32, 50,
and/or 92 as indicated according to the Kabat numbering system.
[0222] In a particularly preferred embodiment, the framework
sequences of the light chain and/or heavy chain variable region of
an antigen-binding molecule preferably contain human germ line
framework sequences. Thus, in an embodiment of the present
invention, when the framework sequences are completely human
sequences, it is expected that when such an antigen-binding
molecule of the present invention is administered to humans (for
example, to treat diseases), it induces little or no immunogenic
response. In the above sense, the phrase "containing a germ line
sequence" in the present invention means that a part of the
framework sequences in the present invention is identical to a part
of any human germ line framework sequences. For example, when the
heavy chain FR2 sequence of an antigen-binding molecule in the
present invention is a combination of heavy chain FR2 sequences of
different human germ line framework sequences, such a molecule is
also an antigen-binding molecule in the present invention
"containing a germ line sequence".
[0223] Preferred examples of the frameworks include, for example,
fully human framework region sequences currently known, which are
included in the website of V-Base (http://vbase.mrc-cpe.cam.ac.uk/)
or others. Those framework region sequences can be appropriately
used as a germ line sequence contained in an antigen-binding
molecule of the present invention. The germ line sequences may be
categorized according to their similarity (Tomlinson et al. (J.
Mol. Biol. (1992) 227, 776-798); Williams and Winter (Eur. J.
Immunol. (1993) 23, 1456-1461); Cox et al. (Nat. Genetics (1994) 7,
162-168)). Appropriate germ line sequences can be selected from
V.kappa., which is grouped into seven subgroups; V.lamda., which is
grouped into ten subgroups; and VH, which is grouped into seven
subgroups.
[0224] Fully human VH sequences preferably include, but are not
limited to, for example, VH sequences of:
subgroup VH1 (for example, VH1-2, VH1-3, VH1-8, VH1-18, VH1-24,
VH1-45, VH1-46, VH1-58, and VH1-69); subgroup VH2 (for example,
VH2-5, VH2-26, and VH2-70); subgroup VH3 (VH3-7, VH3-9, VH3-11,
VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33,
VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66,
VH3-72, VH3-73, and VH3-74); subgroup VH4 (VH4-4, VH4-28, VH4-31,
VH4-34, VH4-39, VH4-59, and VH4-61); subgroup VH5 (VH5-51);
subgroup VH6 (VH6-1); and subgroup VH7 (VH7-4 and VH7-81). These
are also described in known documents (Matsuda et al. (J. Exp. Med.
(1998) 188, 1973-1975)) and such, and thus persons skilled in the
art can appropriately design antigen-binding molecules of the
present invention based on the information of these sequences. It
is also preferable to use other fully human frameworks or framework
sub-regions.
[0225] Fully human V.kappa. sequences preferably include, but are
not limited to, for example:
A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22,
L23, L24, O2, O4, O8, O12, O14, and O18 grouped into subgroup Vk1;
A1, A2, A3, A5, A7, A17, A18, A19, A23, 01, and 011, grouped into
subgroup Vk2; A11, A27, L2, L6, L10, L16, L20, and L25, grouped
into subgroup Vk3; B3, grouped into subgroup Vk4; B2 (herein also
referred to as Vk5-2), grouped into subgroup Vk5; and A10, A14, and
A26, grouped into subgroup Vk6 (Kawasaki et al. (Eur. J. Immunol.
(2001) 31, 1017-1028); Schable and Zachau (Biol. Chem. Hoppe Seyler
(1993) 374, 1001-1022); Brensing-Kuppers et al. (Gene (1997) 191,
173-181)).
[0226] Fully human V.lamda. sequences preferably include, but are
not limited to, for example:
V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17,
V1-18, V1-19, V1-20, and V1-22, grouped into subgroup VL1; V2-1,
V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19,
grouped into subgroup VL1; V3-2, V3-3, and V3-4, grouped into
subgroup VL3; V4-1, V4-2, V4-3, V4-4, and V4-6, grouped into
subgroup VL4; and V5-1, V5-2, V5-4, and V5-6, grouped into subgroup
VL5 (Kawasaki et al. (Genome Res. (1997) 7, 250-261)).
[0227] Normally, these framework sequences are different from one
another at one or more amino acid residues. These framework
sequences can be used in combination with "at least one amino acid
residue that alters the antigen-binding activity of an
antigen-binding molecule depending on ion concentration conditions"
in the present invention. Other examples of the fully human
frameworks used in combination with "at least one amino acid
residue that alters the antigen-binding activity of an
antigen-binding molecule depending on ion concentration conditions"
in the present invention include, but are not limited to, for
example, KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (for example,
Kabat et al. (1991) supra; Wu et al. (J. Exp. Med. (1970) 132,
211-250)).
[0228] Without being bound by a particular theory, one reason for
the expectation that the use of germ line sequences precludes
adverse immune responses in most individuals is believed to be as
follows. As a result of the process of affinity maturation during
normal immune responses, somatic mutation occurs frequently in the
variable regions of immunoglobulin. Such mutations mostly occur
around CDRs whose sequences are hypervariable, but also affect
residues of framework regions. Such framework mutations do not
exist on the germ line genes, and also they are less likely to be
immunogenic in patients. On the other hand, the normal human
population is exposed to most of the framework sequences expressed
from the germ line genes. As a result of immunotolerance, these
germ line frameworks are expected to have low or no immunogenicity
in patients. To maximize the possibility of immunotolerance,
variable region-encoding genes may be selected from a group of
commonly occurring functional germ line genes.
[0229] Known methods such as site-directed mutagenesis (Kunkel et
al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap
extension PCR can be appropriately employed to produce the
antigen-binding molecules of the present invention in which the
above-described variable region sequences, heavy or light chain
variable region sequences, CDR sequences, or framework sequences
contain amino acids that alter the antigen-binding activity of the
antigen-binding molecules depending on calcium ion concentration
conditions.
[0230] For example, a library which contains a plurality of
antigen-binding molecules of the present invention whose sequences
are different from one another can be constructed by combining
heavy chain variable regions prepared as a randomized variable
region sequence library with a light chain variable region selected
as a framework sequence originally containing at least one amino
acid residue that alters the antigen-binding activity of the
antigen-binding molecule depending on calcium ion concentration
conditions. As a non-limiting example, when the ion concentration
is calcium ion concentration, such preferred libraries include, for
example, those constructed by combining the light chain variable
region sequence of SEQ ID NO: 5 (Vk5-2) and the heavy chain
variable region produced as a randomized variable region sequence
library.
[0231] Alternatively, a light chain variable region sequence
selected as a framework region originally containing at least one
amino acid residue that alters the antigen-binding activity of an
antigen-binding domain or antigen-binding molecule as mentioned
above can be design to contain various amino acid residues other
than the above amino acid residues. In the present invention, such
residues are referred to as flexible residues. The number and
position of flexible residues are not particularly limited as long
as the antigen-binding activity of the antigen-binding domain or
antigen-binding molecule of the present invention varies depending
on ion concentrations. Specifically, the CDR sequences and/or FR
sequences of the heavy chain and/or light chain may contain one or
more flexible residues. For example, when the ion concentration is
calcium ion concentration, non-limiting examples of flexible
residues to be introduced into the light chain variable region
sequence of SEQ ID NO: 5 (Vk5-2) include the amino acid residues
listed in Tables 1 or 2.
TABLE-US-00001 TABLE 1 Kabat NUM- CDR BERING AMINO ACID IN 70% OF
THE TOTAL CDR1 28 S: 100% 29 I: 100% 30 E: 72% N: 14% S: 14% 31 D:
100% 32 D: 100% 33 L: 100% 34 A: 70% N: 30% CDR2 50 E: 100% 51 A:
100% 52 S: 100% 53 H: 5% N: 25% S: 45% T: 25% 54 L: 100% 55 Q: 100%
56 S: 100% CDR3 90 Q: 100% 91 H: 25% S: 15% R: 15% Y: 45% 92 D: 80%
N: 10% S: 10% 93 D: 5% G: 10% N: 25% S: 50% R: 10% 94 S: 50% Y: 50%
95 P: 100% 96 L: 50% Y: 50% (POSITION INDICATES Kabat
NUMBERING)
TABLE-US-00002 TABLE 2 Kabat NUM- CDR BERING AMINO ACID IN 70% OF
THE TOTAL CDR1 28 S: 100% 29 I: 100% 30 E: 83% S: 17% 31 D: 100% 32
D: 100% 33 L: 100% 34 A: 70% N: 30% CDR2 50 H: 100% 51 A: 100% 52
S: 100% 53 H: 5% N: 25% S: 45% T: 25% 54 L: 100% 55 Q: 100% 56 S:
100% CDR3 90 Q: 100% 91 H: 25% S: 15% R: 15% Y: 45% 92 D: 80% N:
10% S: 10% 93 D: 5% G: 10% N: 25% S: 50% R: 10% 94 S: 50% Y: 50% 95
P: 100% 96 L: 50% Y: 50% (POSITION INDICATES Kabat NUMBERING)
[0232] Herein, flexible residues refer to amino acid residue
variations present at hypervariable positions at which several
different amino acids are present on the light chain and heavy
chain variable regions when the amino acid sequences of known
and/or native antibodies or antigen-binding domains are compared.
Hypervariable positions are generally located in the CDR regions.
In an embodiment, the data provided by Kabat, Sequences of Proteins
of Immunological Interest (National Institute of Health Bethesda
Md.) (1987 and 1991) is useful to determine hypervariable positions
in known and/or native antibodies. Furthermore, databases on the
Internet (http://vbase.mrc-cpe.cam.ac.uk/,
http://www.bioinf.org.uk/abs/index.html) provide the collected
sequences of many human light chains and heavy chains and their
locations. The information on the sequences and locations is useful
to determine hypervariable positions in the present invention.
According to the present invention, when a certain amino acid
position has preferably about 2 to about 20 possible amino acid
residue variations, preferably about 3 to about 19, preferably
about 4 to about 18, preferably 5 to 17, preferably 6 to 16,
preferably 7 to 15, preferably 8 to 14, preferably 9 to 13, and
preferably 10 to 12 possible amino acid residue variations, the
position is hypervariable. In some embodiments, a certain amino
acid position may have preferably at least about 2, preferably at
least about 4, preferably at least about 6, preferably at least
about 8, preferably about 10, and preferably about 12 amino acid
residue variations.
[0233] Alternatively, a library containing a plurality of
antigen-binding molecules of the present invention whose sequences
are different from one another can be constructed by combining
heavy chain variable regions produced as a randomized variable
region sequence library with light chain variable regions into
which at least one amino acid residue that alters the
antigen-binding activity of antigen-binding molecules depending on
ion concentrations as mentioned above is introduced. When the ion
concentration is calcium ion concentration, non-limiting examples
of such libraries preferably include, for example, libraries in
which heavy chain variable regions produced as a randomized
variable region sequence library are combined with light chain
variable region sequences in which a particular residue(s) in a
germ line sequence such as SEQ ID NO: 6 (Vk1), SEQ ID NO: 7 (Vk2),
SEQ ID NO: 8 (Vk3), or SEQ ID NO: 9 (Vk4) has been substituted with
at least one amino acid residue that alters the antigen-binding
activity of an antigen-binding molecule depending on calcium ion
concentrations. Non-limiting examples of such amino acid residues
include amino acid residues in light chain CDR1. Furthermore,
non-limiting examples of such amino acid residues include amino
acid residues in light chain CDR2. In addition, non-limiting
examples of such amino acid residues also include amino acid
residues in light chain CDR3.
[0234] Non-limiting examples of such amino acid residues contained
in light chain CDR1 include those at positions 30, 31, and/or 32 in
the CDR1 of light chain variable region as indicated by EU
numbering. Furthermore, non-limiting examples of such amino acid
residues contained in light chain CDR2 include an amino acid
residue at position 50 in the CDR2 of light chain variable region
as indicated by Kabat numbering. Moreover, non-limiting examples of
such amino acid residues contained in light chain CDR3 include an
amino acid residue at position 92 in the CDR3 of light chain
variable region as indicated by Kabat numbering. These amino acid
residues can be contained alone or in combination as long as they
form a calcium-binding motif and/or as long as the antigen-binding
activity of an antigen-binding molecule varies depending on calcium
ion concentrations. Meanwhile, as troponin C, calmodulin,
parvalbumin, and myosin light chain, which have several calcium
ion-binding sites and are believed to be derived from a common
origin in terms of molecular evolution, are known, the light chain
CDR1, CDR2, and/or CDR3 can be designed to have their binding
motifs. For example, it is possible to use cadherin domains, EF
hand of calmodulin, C2 domain of Protein kinase C, Gla domain of
blood coagulation protein Factor IX, C type lectins of
asialoglycoprotein receptor and mannose-binding receptor, A domains
of LDL receptors, annexin, thrombospondin type 3 domain, and
EGF-like domains in an appropriate manner for the above
purposes.
[0235] When heavy chain variable regions produced as a randomized
variable region sequence library and light chain variable regions
into which at least one amino acid residue that alters the
antigen-binding activity of an antigen-binding molecule depending
on ion concentration conditions has been introduced are combined as
described above, the sequences of the light chain variable regions
can be designed to contain flexible residues in the same manner as
described above. The number and position of such flexible residues
are not particularly limited to particular embodiments as long as
the antigen-binding activity of antigen-binding molecules of the
present invention varies depending on ion concentration conditions.
Specifically, the CDR sequences and/or FR sequences of heavy chain
and/or light chain can contain one or more flexible residues. When
the ion concentration is calcium ion concentration, non-limiting
examples of flexible residues to be introduced into the sequence of
light chain variable region include the amino acid residues listed
in Tables 1 and 2.
[0236] The preferred heavy chain variable regions to be combined
include, for example, randomized variable region libraries. Known
methods are combined as appropriate to produce a randomized
variable region library. In a non-limiting embodiment of the
present invention, an immune library constructed based on antibody
genes derived from lymphocytes of animals immunized with a specific
antigen, patients with infections, persons with an elevated
antibody titer in blood as a result of vaccination, cancer
patients, or auto immune disease patients, may be preferably used
as a randomized variable region library.
[0237] In another non-limiting embodiment of the present invention,
a synthetic library produced by replacing the CDR sequences of V
genes in genomic DNA or functional reshaped V genes with a set of
synthetic oligonucleotides containing sequences encoding codon sets
of an appropriate length can also be preferably used as a
randomized variable region library. In this case, since sequence
diversity is observed in the heavy chain CDR3 sequence, it is also
possible to replace the CDR3 sequence only. A criterion of giving
rise to diversity in amino acids in the variable region of an
antigen-binding molecule is that diversity is given to amino acid
residues at surface-exposed positions in the antigen-binding
molecule. The surface-exposed position refers to a position that is
considered to be able to be exposed on the surface and/or contacted
with an antigen, based on structure, ensemble of structures, and/or
modeled structure of an antigen-binding molecule. In general, such
positions are CDRs. Preferably, surface-exposed positions are
determined using coordinates from a three-dimensional model of an
antigen-binding molecule using a computer program such as the
InsightII program (Accelrys). Surface-exposed positions can be
determined using algorithms known in the art (for example, Lee and
Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl.
Cryst. (1983) 16, 548-558)). Determination of surface-exposed
positions can be performed using software suitable for protein
modeling and three-dimensional structural information obtained from
an antibody. Software that can be used for these purposes
preferably includes SYBYL Biopolymer Module software (Tripos
Associates). Generally or preferably, when an algorithm requires a
user input size parameter, the "size" of a probe which is used in
the calculation is set at about 1.4 Angstrom or smaller in radius.
Furthermore, methods for determining surface-exposed regions and
areas using software for personal computers are described by Pacios
(Comput. Chem. (1994) 18 (4), 377-386; J. Mol. Model. (1995) 1,
46-53).
[0238] In another non-limiting embodiment of the present invention,
a naive library, which is constructed from antibody genes derived
from lymphocytes of healthy persons and whose repertoire consists
of naive sequences, which are antibody sequences with no bias, can
also be particularly preferably used as a randomized variable
region library (Gejima et al. (Human Antibodies (2002) 11,
121-129); Cardoso et al. (Scand. J. Immunol. (2000) 51, 337-344)).
Herein, an amino acid sequence comprising a naive sequence refers
to an amino acid sequence obtained from such a naive library.
[0239] In one embodiment of the present invention, an
antigen-binding domain of the present invention can be obtained
from a library containing a plurality of antigen-binding molecules
of the present invention whose sequences are different from one
another, prepared by combining light chain variable regions
constructed as a randomized variable region sequence library with a
heavy chain variable region selected as a framework sequence that
originally contains "at least one amino acid residue that alters
the antigen-binding activity of an antigen-binding molecule
depending on ion concentration conditions". When the ion
concentration is calcium ion concentration, non-limiting examples
of such libraries preferably include those constructed by combining
light chain variable regions constructed as a randomized variable
region sequence library with the sequence of heavy chain variable
region of SEQ ID NO: 10 (6RL#9-IgG1) or SEQ ID NO: 11
(6KC4-1#85-IgG1). Alternatively, such a library can be constructed
by selecting appropriate light chain variable regions from those
having germ line sequences, instead of light chain variable regions
constructed as a randomized variable region sequence library. Such
preferred libraries include, for example, those in which the
sequence of heavy chain variable region of SEQ ID NO: 10
(6RL#9-IgG1) or SEQ ID NO: 11 (6KC4-1#85-IgG1) is combined with
light chain variable regions having germ line sequences.
[0240] Alternatively, the sequence of a heavy chain variable region
selected as a framework sequence that originally contains "at least
one amino acid residue that alters the antigen-binding activity of
an antigen-binding molecule depending on ion concentration
conditions" as mentioned above can be designed to contain flexible
residues. The number and position of the flexible residues are not
particularly limited as long as the antigen-binding activity of an
antigen-binding molecule of the present invention varies depending
on ion concentration conditions. Specifically, the CDR and/or FR
sequences of heavy chain and/or light chain can contain one or more
flexible residues. When the ion concentration is calcium ion
concentration, non-limiting examples of flexible residues to be
introduced into the sequence of heavy chain variable region of SEQ
ID NO: 10 (6RL#9-IgG1) include all amino acid residues of heavy
chain CDR1 and CDR2 and the amino acid residues of the heavy chain
CDR3 except those at position(s) 95, 96, and/or 100a.
Alternatively, non-limiting examples of flexible residues to be
introduced into the sequence of heavy chain variable region of SEQ
ID NO: 11 (6KC4-1#85-IgG1) include all amino acid residues of heavy
chain CDR1 and CDR2 and the amino acid residues of the heavy chain
CDR3 except those at amino acid position(s) 95 and/or 101.
[0241] Alternatively, a library containing a plurality of
antigen-binding molecules whose sequences are different from one
another can be constructed by combining light chain variable
regions constructed as a randomized variable region sequence
library or light chain variable regions having germ line sequences
with heavy chain variable regions into which "at least one amino
acid residue that alters the antigen-binding activity of an
antigen-binding molecule depending on ion concentration conditions"
has been introduced as mentioned above. When the ion concentration
is calcium ion concentration, non-limiting examples of such
libraries preferably include those in which light chain variable
regions constructed as a randomized variable region sequence
library or light chain variable regions having germ line sequences
are combined with the sequence of a heavy chain variable region in
which a particular residue(s) has been substituted with at least
one amino acid residue that alters the antigen-binding activity of
an antigen-binding molecule depending on calcium ion concentration
conditions. Non-limiting examples of such amino acid residues
include amino acid residues of the heavy chain CDR1. Further
non-limiting examples of such amino acid residues include amino
acid residues of the heavy chain CDR2. In addition, non-limiting
examples of such amino acid residues also include amino acid
residues of the heavy chain CDR3. Non-limiting examples of such
amino acid residues of heavy chain CDR3 include the amino acid(s)
at position(s) 95, 96, 100a, and/or 101 in the CDR3 of heavy chain
variable region as indicated by the Kabat numbering. Furthermore,
these amino acid residues can be contained alone or in combination
as long as they form a calcium-binding motif and/or the
antigen-binding activity of an antigen-binding molecule varies
depending on calcium ion concentration conditions.
[0242] When light chain variable regions constructed as a
randomized variable region sequence library or light chain variable
regions having germ line sequence are combined with a heavy chain
variable region into which at least one amino acid residue that
alter the antigen-binding activity of an antigen-binding molecule
depending on ion concentration conditions as mentioned above has
been introduced, the sequence of the heavy chain variable region
can also be designed to contain flexible residues in the same
manner as described above. The number and position of flexible
residues are not particularly limited as long as the
antigen-binding activity of an antigen-binding molecule of the
present invention varies depending on ion concentration conditions.
Specifically, the heavy chain CDR and/or FR sequences may contain
one or more flexible residues. Furthermore, randomized variable
region libraries can be preferably used as amino acid sequences of
CDR1, CDR2, and/or CDR3 of the heavy chain variable region other
than the amino acid residues that alter the antigen-binding
activity of an antigen-binding molecule depending on ion
concentration conditions. When germ line sequences are used as
light chain variable regions, non-limiting examples of such
sequences include those of SEQ ID NO: 6 (Vk1), SEQ ID NO: 7 (Vk2),
SEQ ID NO: 8 (Vk3), and SEQ ID NO: 9 (Vk4).
[0243] Any of the above-described amino acids that alter the
antigen-binding activity of an antigen-binding molecule depending
on calcium ion concentration conditions can be preferably used, as
long as they form a calcium-binding motif. Specifically, such amino
acids include electron-donating amino acids. Preferred examples of
such electron-donating amino acids include serine, threonine,
asparagine, glutamic acid, aspartic acid, and glutamic acid.
Condition of Hydrogen Ion Concentrations
[0244] In an embodiment of the present invention, the condition of
ion concentrations refers to the condition of hydrogen ion
concentrations or pH conditions. In the present invention, the
concentration of proton, i.e., the nucleus of hydrogen atom, is
treated as synonymous with hydrogen index (pH). When the activity
of hydrogen ion in an aqueous solution is represented as aH+, pH is
defined as -log 10aH+. When the ionic strength of the aqueous
solution is low (for example, lower than 10.sup.-3), aH+ is nearly
equal to the hydrogen ion strength. For example, the ionic product
of water at 25.degree. C. and 1 atmosphere is Kw=aH+aOH=10.sup.-14,
and therefore in pure water, aH+=aOH=10.sup.-7. In this case, pH=7
is neutral; an aqueous solution whose pH is lower than 7 is acidic
or whose pH is greater than 7 is alkaline.
[0245] In the present invention, when pH condition is used as the
ion concentration condition, pH conditions include conditions of
high hydrogen ion concentration or low pHs, i.e., an acidic pH
range condition, and conditions of low hydrogen ion concentration
or high pHs, i.e., a neutral pH range condition. "The
antigen-binding activity of an antigen-binding domain contained in
the antigen-binding molecule of the present invention varies
depending on pH condition" means that the antigen-binding activity
of an antigen-binding domain contained in an antigen-binding
molecule varies due to the difference in conditions of a high
hydrogen ion concentration or low pH (an acidic pH range) and a low
hydrogen ion concentration or high pH (a neutral pH range). This
includes, for example, the case where the antigen-binding activity
of an antigen-binding molecule is higher under a neutral pH range
condition than under an acidic pH range condition and the case
where the antigen-binding activity of an antigen-binding molecule
is higher under an acidic pH range condition than under a neutral
pH range condition.
[0246] Herein, neutral pH range is not limited to a specific value
and is preferably selected from between pH 6.7 and pH 10.0. In
another embodiment, the pH can be selected from between pH 6.7 and
pH 9.5. In still another embodiment, the pH can be selected from
between pH 7.0 and pH 9.0. In yet another embodiment, the pH can be
selected from between pH 7.0 and pH 8.0. In particular, the
preferred pH includes pH 7.4, which is close to the pH of plasma
(blood) in vivo.
[0247] Herein, an acidic pH range is not limited to a specific
value and is preferably selected from between pH 4.0 and pH 6.5. In
another embodiment, the pH can be selected from between pH 4.5 and
pH 6.5. In still another embodiment, the pH can be selected from
between pH 5.0 and pH 6.5. In yet another embodiment, the pH can be
selected from between pH 5.5 and pH 6.5. In particular, the
preferred pH includes pH 5.8, which is close to the ionized calcium
concentration in the early endosome in vivo.
[0248] In the present invention, "the antigen-binding activity
under a condition of a high hydrogen ion concentration or low pH
(an acidic pH range) is lower than that under a condition of a low
hydrogen ion concentration or high pH (a neutral pH range)" means
that the antigen-binding activity of antigen-binding domain or
antigen-binding molecule comprising the domain of the present
invention at a pH selected from between pH 4.0 and pH 6.5 is weaker
than that at a pH selected from between pH 6.7 and pH 10.0;
preferably means that the antigen-binding activity of an
antigen-binding domain or antigen-binding molecule comprising the
domain at a pH selected from between pH 4.5 and pH 6.5 is weaker
than that at a pH selected from between pH 6.7 and pH 9.5; more
preferably, means that the antigen-binding activity of an
antigen-binding molecule at a pH selected from between pH 5.0 and
pH 6.5 is weaker than that at a pH selected from between pH 7.0 and
pH 9.0; still more preferably means that the antigen-binding
activity of an antigen-binding molecule at a pH selected from
between pH 5.5 and pH 6.5 is weaker than that at a pH selected from
between pH 7.0 and pH 8.0; particularly preferably means that the
antigen-binding activity at the pH in the early endosome in vivo is
weaker than the antigen-binding activity at the pH of plasma in
vivo; and specifically means that the antigen-binding activity of
an antigen-binding molecule at pH 5.8 is weaker than the
antigen-binding activity at pH 7.4.
[0249] Whether the antigen-binding activity of an antigen-binding
domain or antigen-binding molecule comprising the domain has
changed by the pH condition can be determined, for example, by the
use of known measurement methods such as those described in the
section "Binding Activity" above. For example, the binding activity
is measured under different pH conditions using the measurement
methods described above. For example, the antigen-binding activity
of an antigen-binding domain or antigen-binding molecule comprising
the domain is compared under the conditions of acidic pH range and
neutral pH range to confirm that binding activity of the domain or
the molecule changes to be higher under the condition of neutral pH
range than that under the condition of acidic pH range.
[0250] Furthermore, in the present invention, the expression "the
antigen-binding activity under a condition of high hydrogen ion
concentration or low pH, i.e., under an acidic pH range condition,
is lower than that under a condition of low hydrogen ion
concentration or high pH, i.e., under a neutral pH range condition"
can also be expressed as "the antigen-binding activity of an
antigen-binding domain or antigen-binding molecule comprising the
domain under a condition of low hydrogen ion concentration or high
pH, i.e., under a neutral pH range condition, is higher than that
under a condition of high hydrogen ion concentration or low pH,
i.e., under an acidic pH range condition". In the present
invention, "the antigen-binding activity under a condition of high
hydrogen ion concentration or low pH, i.e., under an acidic pH
range condition, is lower than that under a condition of low
hydrogen ion concentration or high pH, i.e., under a neutral pH
range condition" may be described as "the antigen-binding activity
under a condition of high hydrogen ion concentration or low pH,
i.e., under an acidic pH range condition, is weaker than the
antigen-binding ability under a condition of low hydrogen ion
concentration or high pH, i.e., under a neutral pH range
condition". Alternatively, "the antigen-binding activity under a
condition of high hydrogen ion concentration or low pH, i.e., under
an acidic pH range condition, is reduced to be lower than that
under a condition of low hydrogen ion concentration or high pH,
i.e., under a neutral pH range condition" may be described as "the
antigen-binding activity under a condition of high hydrogen ion
concentration or low pH, i.e., under an acidic pH range condition,
is reduced to be weaker than the antigen-binding ability under a
condition of low hydrogen ion concentration or high pH, i.e., under
a neutral pH range condition".
[0251] The conditions other than hydrogen ion concentration or pH
for measuring the antigen-binding activity may be suitably selected
by those skilled in the art and are not particularly limited.
Measurements can be carried out, for example, at 37.degree. C.
using HEPES buffer. Measurements can be carried out, for example,
using Biacore (GE Healthcare). When the antigen is a soluble
antigen, the antigen-binding activity of antigen-binding domain or
antigen-binding molecule comprising the domain can be determined by
assessing the binding activity to the soluble antigen by flowing
the antigen as an analyte into a chip immobilized with the
antigen-binding domain or the antigen-binding molecule comprising
the domain. When the antigen is a membrane antigen, the binding
activity to the membrane antigen can be assessed by flowing the
antigen-binding domain or the antigen-binding molecule comprising
the domain as an analyte into a chip immobilized with the
antigen.
[0252] As long as the antigen-binding activity of an
antigen-binding molecule of the present invention at a condition of
high hydrogen ion concentration or low pH, i.e., in an acidic pH
range condition is weaker than that at a condition of low hydrogen
ion concentration or high pH, i.e., in a neutral pH range
condition, the ratio of the antigen-binding activity between that
under a condition of high hydrogen ion concentration or low pH,
i.e., under an acidic pH range condition, and under a condition of
low hydrogen ion concentration or high pH, i.e., under a neutral pH
range condition is not particularly limited, and the value of KD
(pH 5.8)/KD (pH 7.4), which is the ratio of the dissociation
constant (KD) for an antigen at a condition of high hydrogen ion
concentration or low pH, i.e., in an acidic pH range condition to
the KD at a condition of low hydrogen ion concentration or high pH,
i.e., in a neutral pH range condition, is preferably 2 or more;
more preferably the value of KD (pH 5.8)/KD (pH 7.4) is 10 or more;
and still more preferably the value of KD (pH 5.8)/KD (pH 7.4) is
40 or more. The upper limit of KD (pH 5.8)/KD (pH 7.4) value is not
particularly limited, and may be any value such as 400, 1000, or
10000, as long as the molecule can be produced by the techniques of
those skilled in the art.
[0253] Alternatively, for example, the dissociation rate constant
(kd) can be suitably used as an index for indicating the ratio of
the antigen-binding activity of an antigen-binding domain or
antigen-binding molecule comprising the domain of the present
invention between that at a condition of high hydrogen ion
concentration or low pH, i.e., in an acidic pH range condition and
at a condition of low hydrogen ion concentration or high pH, i.e.,
in a neutral pH range condition. When kd (dissociation rate
constant) is used as an index for indicating the binding activity
ratio instead of KD (dissociation constant), the value of kd (in an
acidic pH range condition)/kd (in a neutral pH range condition),
which is the ratio of kd (dissociation rate constant) for the
antigen at a condition of high hydrogen ion concentration or low
pH, i.e., in an acidic pH range condition to kd (dissociation rate
constant) at a condition of low hydrogen ion concentration or high
pH, i.e., in a neutral pH range condition, is preferably 2 or more,
more preferably 5 or more, still more preferably 10 or more, and
yet more preferably 30 or more. The upper limit of kd (in an acidic
pH range condition)/kd (in a neutral pH range condition) value is
not particularly limited, and may be any value such as 50, 100, or
200, as long as the molecule can be produced by the techniques of
those skilled in the art.
[0254] When the antigen is a soluble antigen, the dissociation rate
constant (kd) can be used as the value for antigen-binding activity
and when the antigen is a membrane antigen, the apparent
dissociation rate constant (kd) can be used. The dissociation rate
constant (kd) and apparent dissociation rate constant (kd) can be
determined by methods known to those skilled in the art, and
Biacore (GE healthcare), flow cytometer, and such may be used. In
the present invention, when the antigen-binding activity of an
antigen-binding domain or antigen-binding molecule comprising the
domain is measured at different hydrogen ion concentrations, i.e.,
pHs, conditions other than the hydrogen ion concentration, i.e.,
pH, are preferably the same.
[0255] For example, an antigen-binding domain or antigen-binding
molecule whose antigen-binding activity at a condition of high
hydrogen ion concentration or low pH, i.e., in an acidic pH range
condition is lower than that at a condition of low hydrogen ion
concentration or high pH, i.e., in a neutral pH range condition,
which is one embodiment provided by the present invention, can be
obtained via screening of antigen-binding domains or
antigen-binding molecules, comprising the following steps (a) to
(c):
(a) obtaining the antigen-binding activity of an antigen-binding
domain or antigen-binding molecule in an acidic pH range condition;
(b) obtaining the antigen-binding activity of an antigen-binding
domain or antigen-binding molecule in a neutral pH range condition;
and (c) selecting an antigen-binding domain or antigen-binding
molecule whose antigen-binding activity in the acidic pH range
condition is lower than that in the neutral pH range condition.
[0256] Alternatively, an antigen-binding domain or antigen-binding
molecule whose antigen-binding activity at a condition of high
hydrogen ion concentration or low pH, i.e., in an acidic pH range
condition, is lower than that at a condition of low hydrogen ion
concentration or high pH, i.e., in a neutral pH range condition,
which is one embodiment provided by the present invention, can be
obtained via screening of antigen-binding domains or
antigen-binding molecules, or a library thereof, comprising the
following steps (a) to (c):
(a) contacting an antigen-binding domain or antigen-binding
molecule, or a library thereof, in a neutral pH range condition
with an antigen; (b) placing in an acidic pH range condition the
antigen-binding domain or antigen-binding molecule bound to the
antigen in step (a); and (c) isolating the antigen-binding domain
or antigen-binding molecule dissociated in step (b).
[0257] An antigen-binding domain or antigen-binding molecule whose
antigen-binding activity at a condition of high hydrogen ion
concentration or low pH, i.e., in an acidic pH range condition is
lower than that at a condition of low hydrogen ion concentration or
high pH, i.e., in a neutral pH range condition, which is another
embodiment provided by the present invention, can be obtained via
screening of antigen-binding domains or antigen-binding molecules,
or a library thereof, comprising the following steps (a) to
(d):
(a) contacting in an acidic pH range condition an antigen with a
library of antigen-binding domains or antigen-binding molecules;
(b) selecting the antigen-binding domain or antigen-binding
molecule which does not bind to the antigen in step (a); (c)
allowing the antigen-binding domain or antigen-binding molecule
selected in step (b) to bind with the antigen in a neutral pH range
condition; and (d) isolating the antigen-binding domain or
antigen-binding molecule bound to the antigen in step (c).
[0258] An antigen-binding domain or antigen-binding molecule whose
antigen-binding activity at a condition of high hydrogen ion
concentration or low pH, i.e., in an acidic pH range condition, is
lower than that at a condition of low hydrogen ion concentration or
high pH, i.e., in a neutral pH range condition, which is even
another embodiment provided by the present invention, can be
obtained by a screening method comprising the following steps (a)
to (c):
(a) contacting in a neutral pH range condition a library of
antigen-binding domains or antigen-binding molecules with a column
immobilized with an antigen; (b) eluting in an acidic pH range
condition from the column the antigen-binding domain or
antigen-binding molecule bound to the column in step (a); and (c)
isolating the antigen-binding domain or antigen-binding molecule
eluted in step (b).
[0259] An antigen-binding domain or antigen-binding molecule whose
antigen-binding activity at a condition of high hydrogen ion
concentration or low pH, i.e., in an acidic pH range condition, is
lower than that at a condition of low hydrogen ion concentration or
high pH, i.e., in a neutral pH range condition, which is still
another embodiment provided by the present invention, can be
obtained by a screening method comprising the following steps (a)
to (d):
(a) allowing, in an acidic pH range condition, a library of
antigen-binding domains or antigen-binding molecules to pass a
column immobilized with an antigen; (b) collecting the
antigen-binding domain or antigen-binding molecule eluted without
binding to the column in step (a); (c) allowing the antigen-binding
domain or antigen-binding molecule collected in step (b) to bind
with the antigen in a neutral pH range condition; and (d) isolating
the antigen-binding domain or antigen-binding molecule bound to the
antigen in step (c).
[0260] An antigen-binding domain or antigen-binding molecule whose
antigen-binding activity at a high hydrogen ion concentration or
low pH, i.e., in an acidic pH range condition, is lower than that
at a low hydrogen ion concentration or high pH, i.e., in a neutral
pH range condition, which is yet another embodiment provided by the
present invention, can be obtained by a screening method comprising
the following steps (a) to (d):
(a) contacting an antigen with a library of antigen-binding domains
or antigen-binding molecules in a neutral pH range condition; (b)
obtaining the antigen-binding domain or antigen-binding molecule
bound to the antigen in step (a); (c) placing in an acidic pH range
condition the antigen-binding domain or antigen-binding molecule
obtained in step (b); and (d) isolating the antigen-binding domain
or antigen-binding molecule whose antigen-binding activity in step
(c) is weaker than the standard selected in step (b).
[0261] The above-described steps may be repeated twice or more
times. Thus, the present invention provides antigen-binding domains
and antigen-binding molecules whose antigen-binding activity in an
acidic pH range condition is lower than that in a neutral pH range
condition, which are obtained by a screening method that further
comprises the steps of repeating steps (a) to (c) or (a) to (d) in
the above-described screening methods. The number of times that
steps (a) to (c) or (a) to (d) is repeated is not particularly
limited; however, the number is 10 or less in general.
[0262] In the screening methods of the present invention, the
antigen-binding activity of an antigen-binding domain or
antigen-binding molecule at a condition of a high hydrogen ion
concentration or low pH, i.e., in an acidic pH range, is not
particularly limited, as long as it is the antigen-binding activity
at a pH of between 4.0 and 6.5, and includes the antigen-binding
activity at a pH of between 4.5 and 6.6 as the preferred pH. The
antigen-binding activity also includes that at a pH of between 5.0
and 6.5, and that at a pH of between 5.5 and 6.5 as another
preferred pH. The antigen-binding activity also includes that at
the pH in the early endosome in vivo as the more preferred pH, and
specifically, that at pH 5.8. Meanwhile, the antigen-binding
activity of an antigen-binding domain or antigen-binding molecule
at a condition of a low hydrogen ion concentration or high pH,
i.e., in a neutral pH range, is not particularly limited, as long
as it is the antigen-binding activity at a pH of between 6.7 and
10, and includes the antigen-binding activity at a pH of between
6.7 and 9.5 as the preferred pH. The antigen-binding activity also
includes that at a pH of between 7.0 and 9.5 and that at a pH of
between 7.0 and 8.0 as another preferred pH. The antigen-binding
activity also includes that at the pH of plasma in vivo as the more
preferred pH, and specifically, that at pH 7.4.
[0263] The antigen-binding activity of an antigen-binding domain or
antigen-binding molecule can be measured by methods known to those
skilled in the art. Those skilled in the art can suitably determine
conditions other than ionized calcium concentration. The
antigen-binding activity of an antigen-binding domain or
antigen-binding molecule can be assessed based on the dissociation
constant (KD), apparent dissociation constant (KD), dissociation
rate constant (kd), apparent dissociation rate constant (kd), and
such. These can be determined by methods known to those skilled in
the art, for example, using Biacore (GE healthcare), Scatchard
plot, or FACS.
[0264] In the present invention, the step of selecting an
antigen-binding domain or antigen-binding molecule whose
antigen-binding activity at a condition of low hydrogen ion
concentration or high pH, i.e., in a neutral pH range condition, is
higher than that at a condition of high hydrogen ion concentration
or low pH, i.e., in an acidic pH range condition, is synonymous
with the step of selecting an antigen-binding domain or
antigen-binding molecule whose antigen-binding activity at a
condition of high hydrogen ion concentration or low pH, i.e., in an
acidic pH range condition, is lower than that at a condition of low
hydrogen ion concentration or high pH, i.e., in a neutral pH range
condition.
[0265] As long as the antigen-binding activity at a condition of
low hydrogen ion concentration or high pH, i.e., in a neutral pH
range condition, is higher than that at a condition of high
hydrogen ion concentration or low pH, i.e., in an acidic pH range
condition, the difference between the antigen-binding activity at a
condition of low hydrogen ion concentration or high pH, i.e., in a
neutral pH range condition, and that at a condition of high
hydrogen ion concentration or low pH, i.e., in an acidic pH range
condition, is not particularly limited; however, the
antigen-binding activity at a condition of low hydrogen ion
concentration or high pH, i.e., in a neutral pH range condition, is
preferably twice or more, more preferably 10 times or more, and
still more preferably 40 times or more than that at a condition of
high hydrogen ion concentration or low pH, i.e., in an acidic pH
range condition.
Amino Acids that Alter the Antigen-Binding Activity of an
Antigen-Binding Domain Depending on Hydrogen Ion Concentration
Conditions
[0266] The antigen-binding domain or antigen-binding molecule of
the present invention to be screened by the above-described
screening methods may be prepared in any manner. For example,
conventional antigen-binding molecules, conventional libraries
(phage library, etc.), antibodies or libraries prepared from B
cells of immunized animals or from hybridomas obtained by
immunizing animals, antibodies or libraries (libraries with
increased content of amino acids with a side chain pKa of 4.0-8.0
(for example, histidine and glutamic acid) or unnatural amino
acids, libraries introduced with amino acids with a side chain pKa
of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural
amino acid mutations at specific positions, etc.) obtained by
introducing amino acids with a side chain pKa of 4.0-8.0 (for
example, histidine and glutamic acid) or unnatural amino acid
mutations into the above-described antibodies or libraries may be
used.
[0267] Methods for obtaining an antigen-binding domain or
antigen-binding molecule whose antigen-binding activity at a low
hydrogen ion concentration or high pH, i.e., in a neutral pH range
condition, is higher than that at a high hydrogen ion concentration
or low pH, i.e., in an acidic pH range condition, from an
antigen-binding domains or antigen-binding molecules prepared from
hybridomas obtained by immunizing animals or from B cells of
immunized animals preferably include, for example, the
antigen-binding molecule or antigen-binding molecule in which at
least one of the amino acids of the antigen-binding domain or
antigen-binding molecule is substituted with an amino acid with a
side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or an unnatural amino acid mutation, or the antigen-binding
domain or antigen-binding molecule inserted with an amino acid with
a side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or unnatural amino acid, such as those described in WO
2009/125825.
[0268] The sites of introducing mutations of amino acids with a
side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or unnatural amino acids are not particularly limited, and
may be any position as long as the antigen-binding activity in an
acidic pH range becomes weaker than that in a neutral pH range (the
value of KD (in an acidic pH range)/KD (in a neutral pH range) or
kd (in an acidic pH range)/kd (in a neutral pH range) is increased)
as compared to before substitution or insertion. For example, when
the antigen-binding molecule is an antibody, antibody variable
region and CDRs are suitable. Those skilled in the art can
appropriately determine the number of amino acids to be substituted
with or the number of amino acids with a side chain pKa of 4.0-8.0
(for example, histidine and glutamic acid) or unnatural amino acids
to be inserted. It is possible to substitute with a single amino
acid having a side chain pKa of 4.0-8.0 (for example, histidine and
glutamic acid) or a single unnatural amino acid; it is possible to
insert a single amino acid having a side chain pKa of 4.0-8.0 (for
example, histidine and glutamic acid) or a single unnatural amino
acid; it is possible to substitute with two or more amino acids
having a side chain pKa of 4.0-8.0 (for example, histidine and
glutamic acid) or two or more unnatural amino acids; and it is
possible to insert two or more amino acids having a side chain pKa
of 4.0-8.0 (for example, histidine and glutamic acid) or two or
more unnatural amino acids. Alternatively, other amino acids can be
deleted, added, inserted, and/or substituted concomitantly, aside
from the substitution into amino acids having a side chain pKa of
4.0-8.0 (for example, histidine and glutamic acid) or unnatural
amino acids, or the insertion of amino acids having a side chain
pKa of 4.0-8.0 (for example, histidine and glutamic acid) or
unnatural amino acids. Substitution into or insertion of amino
acids with a side chain pKa of 4.0-8.0 (for example, histidine and
glutamic acid) or unnatural amino acids can performed randomly by
methods such as histidine scanning, in which the alanine of alanine
scanning known to those skilled in the art is replaced with
histidine. Antigen-binding molecules exhibiting a greater value of
KD (in an acidic pH range)/KD (in a neutral pH range) or kd (in an
acidic pH range)/kd (in a neutral pH range) as compared to before
the mutation can be selected from antigen-binding domains or
antibodies introduced with random insertions or substitution
mutations of amino acids with a side chain pKa of 4.0-8.0 (for
example, histidine and glutamic acid) or unnatural amino acids.
[0269] Preferred examples of antigen-binding molecules containing
the mutation into amino acids with a side chain pKa of 4.0-8.0 (for
example, histidine and glutamic acid) or unnatural amino acids as
described above and whose antigen-binding activity in an acidic pH
range is lower than that in a neutral pH range include,
antigen-binding molecules whose antigen-binding activity in the
neutral pH range after the mutation into amino acids with a side
chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or
unnatural amino acids is comparable to that before the mutation
into amino acids with a side chain pKa of 4.0-8.0 (for example,
histidine and glutamic acid) or unnatural amino acids. Herein, "an
antigen-binding molecule after the mutation with amino acids having
a side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or unnatural amino acids has an antigen-binding activity
comparable to that before the mutation with amino acids having a
side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or unnatural amino acids" means that, when taking the
antigen-binding activity of an antigen-binding molecule before the
mutation with amino acids having a side chain pKa of 4.0-8.0 (for
example, histidine and glutamic acid) or unnatural amino acids as
100%, the antigen-binding activity of an antigen-binding molecule
after the mutation with amino acids having a side chain pKa of
4.0-8.0 (for example, histidine and glutamic acid) or unnatural
amino acids is at least 10% or more, preferably 50% or more, more
preferably 80% or more, and still more preferably 90% or more. The
antigen-binding activity after the mutation of amino acids with a
side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or unnatural amino acids at pH 7.4 may be higher than that
before the mutation of amino acids with a side chain pKa of 4.0-8.0
(for example, histidine and glutamic acid) or unnatural amino acids
at pH 7.4. If the antigen-binding activity of an antigen-binding
molecule is decreased due to insertion of or substitution into
amino acids with a side chain pKa of 4.0-8.0 (for example,
histidine and glutamic acid) or unnatural amino acids, the
antigen-binding activity can be made to be comparable to that
before the insertion of or substitution into amino acids with a
side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or unnatural amino acids, by introducing a substitution,
deletion, addition, and/or insertion of one or more amino acids of
the antigen-binding molecule. The present invention also includes
antigen-binding molecules whose binding activity has been adjusted
to be comparable by substitution, deletion, addition, and/or
insertion of one or more amino acids after substitution or
insertion of amino acids with a side chain pKa of 4.0-8.0 (for
example, histidine and glutamic acid) or unnatural amino acids.
[0270] In one embodiment of the present invention, a library
containing multiple antigen-binding domains or antigen-binding
molecules of the present invention whose sequences are different
from one another can also be constructed by combining heavy chain
variable regions, produced as a randomized variable region sequence
library, with light chain variable regions introduced with "at
least one amino acid residue that changes the antigen-binding
activity of antigen-binding domain or antigen-binding molecule
depending on the hydrogen ion concentration condition".
[0271] Such amino acid residues include, but are not limited to,
for example, amino acid residues contained in the light chain CDR1.
The amino acid residues also include, but are not limited to, for
example, amino acid residues contained in the light chain CDR2. The
amino acid residues also include, but are not limited to, for
example, amino acid residues contained in the light chain CDR3.
[0272] The above-described amino acid residues contained in the
light chain CDR1 include, but are not limited to, for example,
amino acid residue(s) of position(s) 24, 27, 28, 31, 32, and/or 34
according to Kabat numbering in the CDR1 of light chain variable
region. Meanwhile, the amino acid residues contained in the light
chain CDR2 include, but are not limited to, for example, amino acid
residue(s) of position(s) 50, 51, 52, 53, 54, 55, and/or 56
according to Kabat numbering in the CDR2 of light chain variable
region. Furthermore, the amino acid residues in the light chain
CDR3 include, but are not limited to, for example, amino acid
residues of position(s) 89, 90, 91, 92, 93, 94, and/or 95A
according to Kabat numbering in the CDR3 of light chain variable
region. Moreover, the amino acid residues can be contained alone or
can be contained in combination of two or more amino acids as long
as they allow the change in the antigen-binding activity of an
antigen-binding molecule depending on the hydrogen ion
concentration condition.
[0273] Even when the heavy chain variable region produced as a
randomized variable region sequence library is combined with the
above-described light chain variable region introduced with "at
least one amino acid residue that changes the antigen-binding
activity of an antigen-binding molecule depending on the hydrogen
ion concentration condition", it is possible to design so that the
flexible residues are contained in the sequence of the light chain
variable region in the same manner as described above. The number
and position of the flexible residues are not particularly limited
to a specific embodiment, as long as the antigen-binding activity
of antigen-binding domain or antigen-binding molecule of the
present invention changes depending on the hydrogen ion
concentration condition. Specifically, the CDR and/or FR sequences
of heavy chain and/or light chain can contain one or more flexible
residues. For example, flexible residues to be introduced into the
sequences of the light chain variable regions include, but are not
limited to, for example, the amino acid residues listed in Tables 3
and 4. Meanwhile, amino acid sequences of light chain variable
regions other than the flexible residues and amino acid residues
that change the antigen-binding activity of an antigen-binding
domain or antigen-binding molecule depending on the hydrogen ion
concentration condition suitably include, but are not limited to,
germ line sequences such as Vk1 (SEQ ID NO: 6), Vk2 (SEQ ID NO: 7),
Vk3 (SEQ ID NO: 8), and Vk4 (SEQ ID NO: 9).
TABLE-US-00003 TABLE 3 POSITION AMINO ACID CDR1 28 S: 100% 29 I:
100% 30 N: 25% S: 25% R: 25% H: 25% 31 S: 100% 32 H: 100% 33 L:
100% 34 A: 50% N: 50% CDR2 50 H: 100% OR A: 25% D: 25% G: 25% K:
25% 51 A: 100% A: 100% 52 S: 100% S: 100% 53 K: 33.3% N: 33.3% S:
33.3% H: 100% 54 L: 100% L: 100% 55 Q: 100% Q: 100% 56 S: 100% S:
100% CDR3 90 Q: 100% OR Q: 100% 91 H: 100% S: 33.3% R: 33.3% Y:
33.3% 92 G: 25% N: 25% S: 25% Y: 25% H: 100% 93 H: 33.3% N: 33.3%
S: 33.3% H: 33.3% N: 33.3% S: 33.3% 94 S: 50% Y: 50% S: 50% Y: 50%
95 P: 100% P: 100% 96 L: 50% Y: 50% L: 50% Y: 50% (Position
indicates Kabat numbering)
TABLE-US-00004 TABLE 4 CDR POSITION AMINO ACID CDR1 28 S: 100% 29
I: 100% 30 H: 30% N: 10% S: 50% R: 10% 31 N: 35% S: 65% 32 H: 40%
N: 20% Y: 40% 33 L: 100% 34 A: 70% N: 30% CDR2 50 A: 25% D: 15% G:
25% H: 30% K: 5% 51 A: 100% 52 S: 100% 53 H: 30% K: 10% N: 15% S:
45% 54 L: 100% 55 Q: 100% 56 S: 100% CDR3 90 Q: 100% 91 H: 30% S:
15% R: 10% Y: 45% 92 G: 20% H: 30% N: 20% S: 15% Y: 15% 93 H: 30%
N: 25% S: 45% 94 S: 50% Y: 50% 95 P: 100% 96 L: 50% Y: 50%
(Position indicates Kabat numbering)
[0274] Any amino acid residue may be suitably used as the
above-described amino acid residues that change the antigen-binding
activity of an antigen-binding domain or antigen-binding molecule
depending on the hydrogen ion concentration conditions.
Specifically, such amino acid residues include amino acids with a
side chain pKa of 4.0-8.0. Such electron-releasing amino acids
preferably include, for example, naturally occurring amino acids
such as histidine and glutamic acid, as well as unnatural amino
acids such as histidine analogs (US2009/0035836), m-NO2-Tyr (pKa
7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Bioorg.
Med. Chem. (2003) 11 (17), 3761-3768). Particularly preferred amino
acid residues include, for example, amino acids with a side chain
pKa of 6.0-7.0. Such electron-releasing amino acid residues
preferably include, for example, histidine.
[0275] The preferred heavy chain variable region that is used in
combination includes, for example, randomized variable region
libraries. Known methods are appropriately combined as a method for
producing a randomized variable region library. In a non-limiting
embodiment of the present invention, an immune library constructed
based on antibody genes derived from animals immunized with
specific antigens, patients with infection or persons with an
elevated antibody titer in blood as a result of vaccination, cancer
patients, or lymphocytes of autoimmune diseases may be suitably
used as a randomized variable region library.
[0276] In another non-limiting embodiment of the present invention,
in the same manner as described above, a synthetic library in which
the CDR sequences of V genes from genomic DNA or functional
reconstructed V genes are replaced with a set of synthetic
oligonucleotides containing the sequences encoding codon sets of an
appropriate length can also be suitably used as a randomized
variable region library. In this case, the CDR3 sequence alone may
be replaced because variety in the gene sequence of heavy chain
CDR3 is observed. The basis for giving rise to amino acid
variations in the variable region of an antigen-binding molecule is
to generate variations of amino acid residues of surface-exposed
positions of the antigen-binding molecule. The surface-exposed
position refers to a position where an amino acid is exposed on the
surface and/or contacted with an antigen based on the conformation,
structural ensemble, and/or modeled structure of an antigen-binding
molecule, and in general, such positions are the CDRs. The
surface-exposed positions are preferably determined using the
coordinates derived from a three-dimensional model of the
antigen-binding molecule using computer programs such as InsightII
program (Accelrys). The surface-exposed positions can be determined
using algorithms known in the art (for example, Lee and Richards
(J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst.
(1983) 16, 548-558)). The surface-exposed positions can be
determined based on the information on the three dimensional
structure of antibodies using software suitable for protein
modeling. Software which is suitably used for this purpose includes
the SYBYL biopolymer module software (Tripos Associates). When the
algorithm requires the input size parameter from the user, the
"size" of probe for use in computation is generally or preferably
set at about 1.4 angstrom or less in radius. Furthermore, a method
for determining surface-exposed region and area using personal
computer software is described by Pacios (Comput. Chem. (1994) 18
(4), 377-386; and J. Mol. Model. (1995) 1, 46-53).
[0277] In still another non-limiting embodiment of the present
invention, a naive library constructed from antibody genes derived
from lymphocytes of healthy persons and consisting of naive
sequences, which are unbiased repertoire of antibody sequences, can
also be particularly suitably used as a randomized variable region
library (Gejima et al. (Human Antibodies (2002) 11, 121-129); and
Cardoso et al. (Scand. J. Immunol. (2000) 51, 337-344)).
FcRn
[0278] Unlike Fc.gamma. receptor belonging to the immunoglobulin
superfamily, human FcRn is structurally similar to polypeptides of
major histocompatibility complex (MHC) class I, exhibiting 22% to
29% sequence identity to class I MHC molecules (Ghetie et al.,
Immunol. Today (1997) 18 (12): 592-598). FcRn is expressed as a
heterodimer consisting of soluble .beta. or light chain (.beta.2
microglobulin) complexed with transmembrane .alpha. or heavy chain.
Like MHC, FcRn .alpha. chain comprises three extracellular domains
(.alpha.1, .alpha.2, and .alpha.3) and its short cytoplasmic domain
anchors the protein onto the cell surface. .alpha.1 and .alpha.2
domains interact with the FcRn-binding domain of the antibody Fc
region (Raghavan et al., Immunity (1994) 1: 303-315).
[0279] FcRn is expressed in maternal placenta and york sac of
mammals, and is involved in mother-to-fetus IgG transfer. In
addition, in neonatal small intestine of rodents, where FcRn is
expressed, FcRn is involved in transfer of maternal IgG across
brush border epithelium from ingested colostrum or milk. FcRn is
expressed in a variety of other tissues and endothelial cell
systems of various species. FcRn is also expressed in adult human
endothelia, muscular blood vessels, and hepatic sinusoidal
capillaries. FcRn is believed to play a role in maintaining the
plasma IgG concentration by mediating recycling of IgG to serum
upon binding to IgG Typically, binding of FcRn to IgG molecules is
strictly pH dependent. The optimal binding is observed in an acidic
pH range below 7.0.
[0280] Human FcRn whose precursor is a polypeptide having the
signal sequence of SEQ ID NO: 12 (the polypeptide with the signal
sequence is shown in SEQ ID NO: 13) forms a complex with human
.beta.2-microglobulin in vivo. Soluble human FcRn complexed with
.beta.2-microglobulin is produced by using conventional recombinant
expression techniques. FcRn-binding domain of the present invention
can be assessed for their binding activity to such a soluble human
FcRn complexed with .beta.2-microglobulin. Herein, unless otherwise
specified, human FcRn refers to a form capable of binding to an
FcRn-binding domain of the present invention. Examples include a
complex between human FcRn and human .beta.2-microglobulin.
Binding Activity of an FcRn-Binding Domain or Antigen-Binding
Molecule Comprising the Domain to FcRn, Human FcRn in
Particular
[0281] The binding activity of an FcRn-binding domain contained in
an antigen-binding molecule provided by the present invention to
FcRn, human FcRn in particular, can be measured by methods known to
those skilled in the art, as described in the section "Binding
Activity" above. Those skilled in the art can appropriately
determine the conditions other than pH. The binding activity of
antigen-binding domain or antigen-binding molecule comprising the
domain to human FcRn can be assessed based on the dissociation
constant (KD), apparent dissociation constant (KD), dissociation
rate (kd), apparent dissociation rate (kd), and such. These can be
measured by methods known to those skilled in the art. For example,
Biacore (GE healthcare), Scatchard plot, or flow cytometer may be
used.
[0282] When the human FcRn-binding activity of an FcRn-binding
domain or antigen-binding molecule comprising the domain of the
present invention is measured, conditions other than the pH are not
particularly limited, and can be appropriately selected by those
skilled in the art. Measurements can be carried out, for example,
at a condition of 37.degree. C. using MES buffer, as described in
WO 2009/125825. Alternatively, the human FcRn-binding activity of
an FcRn-binding domain or antigen-binding molecule comprising the
domain of the present invention can be measured by methods known to
those skilled in the art, and may be measured by using, for
example, Biacore (GE Healthcare) or such. The binding activity of
an FcRn-binding domain or antigen-binding molecule comprising the
domain of the present invention to human FcRn can be assessed by
flowing, as an analyte, human FcRn, or an FcRn-binding domain or
antigen-binding molecule comprising the domain into a chip
immobilized with an FcRn-binding domain or antigen-binding molecule
comprising the domain, or human FcRn.
[0283] In the present invention, the acidic pH range presented as
the condition for having binding activity between FcRn and an
antigen-binding molecule of the present invention or FcRn-binding
domain in the molecule generally refers to pH 4.0 to pH 6.5.
Preferably it refers to pH 5.5 to pH 6.5, and particularly
preferably it refers to pH 5.8 to pH 6.0 which is close to the pH
in an early endosome in vivo. The neutral pH range presented as the
condition for having binding activity between FcRn and an
antigen-binding molecule of the present invention or an
FcRn-binding domain included in such a molecule generally refers to
pH 6.7 to pH 10.0. Neutral pH range is preferably a range indicated
by any pH value from pH 7.0 to pH 8.0, and is preferably selected
from pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0,
and is particularly preferably pH 7.4 which is close to the pH in
plasma (in blood) in vivo. When evaluating the binding affinity
between human FcRn and a human FcRn-binding domain or an
antigen-binding molecule containing that domain at pH 7.4 is
difficult due to low binding affinity, pH 7.0 can be used instead
of pH 7.4. As temperature to be used in assay conditions, binding
affinity between a human FcRn and human FcRn-binding domain or
antigen-binding molecule comprising the domain may be assessed at
any temperature from 10.degree. C. to 50.degree. C. Preferably, a
temperature from 15.degree. C. to 40.degree. C. is used to
determine the binding affinity between a human FcRn and human
FcRn-binding domain or antigen-binding molecule comprising the
domain. More preferably, any temperature from 20.degree. C. to
35.degree. C. such as any one of 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, and 35.degree. C. is also equally used
to determine the binding affinity between a human FcRn-binding
domain or antigen-binding molecule comprising the domain and human
FcRn. A temperature of 25.degree. C. is a non-limiting example of
an embodiment of the present invention.
FcRn-Binding Domain
[0284] Antigen-binding molecules of the present invention have an
FcRn-binding domain having an activity to bind to FcRn under an
acidic pH range condition. The FcRn-binding domain is not
particularly limited as long as the antigen-binding molecules have
an FcRn-binding activity in an acidic pH range, and it may be a
domain that has direct or indirect binding activity to FcRn.
Preferred examples of such an FcRn-binding domain include the Fc
region of IgG immunoglobulin, albumin, albumin domain 3, anti-FcRn
antibody described in Christianson et al. (mAbs (2012) 4 (2),
208-216), anti-FcRn peptide described in WO 2007/098420, anti-FcRn
scaffold molecule, and such which have an activity to directly bind
to FcRn, or molecules that bind to IgG or albumin, and such that
have an activity to indirectly bind to FcRn. If the domain already
has FcRn-binding activity in an acidic pH range, it may preferably
be used as it is. If the domain does not have or has weak
FcRn-binding activity in an acidic pH range, amino acids
constituting an FcRn-binding domain in the antigen-binding molecule
may be altered to confer FcRn-binding activity. Alternatively,
amino acids may be altered in a domain already having FcRn-binding
activity in an acidic pH range to increase the FcRn-binding
activity in an acidic pH range. For amino acid alteration of the
FcRn-binding domain, the alteration of interest can be identified
by comparing the FcRn-binding activities in an acidic pH range
before and after the amino acid alteration.
Fc Region
[0285] An Fc region contains the amino acid sequence derived from
the heavy chain constant region of an antibody. An Fc region is a
portion of the heavy chain constant region of an antibody, starting
from the N terminal end of the hinge region, which corresponds to
the papain cleavage site at an amino acid around position 216
according to the EU numbering system, and contains the hinge, CH2,
and CH3 domains. While the Fc region may be obtained from human
IgG1, it is not limited to a particular subclass of IgG. Examples
of such a non-limiting embodiment of the Fc region include the Fc
regions of human IgG1 (SEQ ID NO: 14), IgG2 (SEQ ID NO: 15), IgG3
(SEQ ID NO: 16), or IgG4 (SEQ ID NO: 17).
FcRn-Binding Domain Having Binding Activity to FcRn Under an Acidic
pH Range Condition
[0286] As described above, in a non-limiting embodiment of the
present invention, an Fc region of a human IgG immunoglobulin is
used as the FcRn-binding domain having binding activity to FcRn
under an acidic pH range condition. An Fc region originally having
FcRn-binding activity under an acidic pH range condition may be
used as it is for this domain, and examples of such Fc regions
include Fc regions of human IgGs (IgG1, IgG2, IgG3, or IgG4, and
variants thereof). When an Fc region has weak or no FcRn-binding
activity under an acidic pH range condition, Fc regions having
desired FcRn-binding activity may be obtained by altering amino
acids of the Fc region. Alternatively, Fc regions having desired or
enhanced FcRn-binding activity under an acidic pH range condition
may be suitably obtained by altering amino acids in the Fc region.
Amino acid alterations of an Fc region that results in such desired
binding activity may be determined by comparing the FcRn-binding
activity under an acidic pH range condition before and after the
amino acid alteration. For example, such amino acid alterations may
be determined by methods described in the above-mentioned section
"Binding activity of an FcRn-binding domain or antigen-binding
molecule comprising the domain to FcRn, human FcRn in
particular".
[0287] Alterations of the Fc region that enhance FcRn-binding
activity under an acidic pH range condition are presented below as
examples of a non-limiting embodiment of such alterations.
Preferred Fc regions (starting Fc regions) of an IgG-type
immunoglobulin for alteration include, for example, those of human
IgGs (IgG1, IgG2, IgG3, and IgG4, and variants thereof). The origin
of starting Fc regions is not limited, and they may be obtained
from human or any nonhuman organisms. Such organisms preferably
include mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits,
dogs, goats, sheep, bovines, horses, camels and organisms selected
from nonhuman primates. In another embodiment, starting Fc regions
can also be obtained from cynomolgus monkeys, marmosets, rhesus
monkeys, chimpanzees, or humans. Starting Fc regions can be
obtained preferably from human IgG1; however, they are not limited
to any particular IgG subclass. This means that an Fc region
represented by human IgG1 (SEQ ID NO: 14), IgG2 (SEQ ID NO: 15),
IgG3 (SEQ ID NO: 16), or IgG4 (SEQ ID NO: 17) can be used
appropriately as a starting Fc region, and herein also means that
an Fc region of an arbitrary IgG class or subclass derived from any
organisms described above can be preferably used as a starting Fc
region. Examples of naturally-occurring IgG variants or modified
forms are described in published documents (Curr. Opin. Biotechnol.
(2009) 20 (6): 685-91; Curr. Opin. Immunol. (2008) 20 (4), 460-470;
Protein Eng. Des. Sel. (2010) 23 (4): 195-202; WO 2009/086320; WO
2008/092117; WO 2007/041635; and WO 2006/105338); however, they are
not limited to the examples.
[0288] Examples of alterations include those with one or more
mutations, for example, mutations by substitution of different
amino acid residues for amino acids of starting Fc regions, by
insertion of one or more amino acid residues into starting Fc
regions, or by deletion of one or more amino acids from starting Fc
region. Preferably, the amino acid sequences of altered Fc regions
comprise at least a part of the amino acid sequence of a non-native
Fc region. Such variants necessarily have sequence identity or
similarity less than 100% to their starting Fc region. In a
preferred embodiment, the variants have amino acid sequence
identity or similarity about 75% to less than 100%, more preferably
about 80% to less than 100%, even more preferably about 85% to less
than 100%, still more preferably about 90% to less than 100%, and
most preferably about 95% to less than 100% to the amino acid
sequence of their starting Fc region. In a non-limiting embodiment
of the present invention, at least one amino acid is different
between a modified Fc region of the present invention and its
starting Fc region. Amino acid difference between a modified Fc
region of the present invention and its starting Fc region can also
be preferably specified based on amino acid differences at
above-described particular amino acid positions according to EU
numbering.
[0289] As long as the Fc region has an FcRn-binding activity under
an acidic pH range condition or can increase the human FcRn-binding
activity under an acidic pH range condition, amino acids at any
position may be modified into other amino acids. When the
antigen-binding molecule of the present invention contains the Fc
region of human IgG1 as the FcRn-binding domain, it is preferable
that the resulting Fc region contains a modification that results
in the effect of enhancing FcRn binding under an acidic pH range
condition as compared to the binding activity of the starting human
IgG1 Fc region. Amino acids that allow such modification include,
for example, amino acid(s) at position(s) 252, 254, 256, 309, 311,
315, 433, and/or 434 according to EU numbering, and their
combination amino acid(s) at position(s) 253, 310, 435, and/or 426
as described in WO 1997/034631. Favorable examples include amino
acid(s) at position(s) 238, 252, 253, 254, 255, 256, 265, 272, 286,
288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376,
378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436,
439, and/or 447 as indicated by EU numbering as described in WO
2000/042072. Similarly, favorable examples of amino acids that
allow such modification include, amino acid(s) at position(s) 251,
252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428,
433, 434, and/or 436 according to EU numbering as described in WO
2002/060919. Furthermore, amino acids that allow such modification
include, for example, amino acid(s) at position(s) 250, 314, and
428 according to EU numbering as described in WO2004/092219. In
addition, favorable examples of amino acids that allow such
modification include amino acid(s) at position(s) 238, 244, 245,
249, 252, 256, 257, 258, 260, 262, 270, 272, 279, 283, 285, 286,
288, 293, 307, 311, 312, 316, 317, 318, 332, 339, 341, 343, 375,
376, 377, 378, 380, 382, 423, 427, 430, 431, 434, 436, 438, 440,
and/or 442 as described in WO 2006/020114. Furthermore, favorable
examples of amino acids that allow such modification include amino
acid(s) at position(s) 251, 252, 307, 308, 378, 428, 430, 434,
and/or 436 according to EU numbering as described in WO
2010/045193. Modification of these amino acids enhances FcRn
binding of the Fc region of an IgG-type immunoglobulin under an
acidic pH range condition.
[0290] When the Fc region of human IgG1 is comprised as the
FcRn-binding domain, a non-limiting embodiment of the modification
that results in the effect of enhancing FcRn binding under an
acidic pH range condition as compared to the binding activity of
the starting Fc region of human IgG1 includes at least one or more
amino acid modifications selected from the group consisting of:
Arg or Leu for the amino acid at position 251; Phe, Ser, Thr, or
Tyr for the amino acid at position 252; Ser or Thr for the amino
acid at position 254; Arg, Gly, Ile, or Leu for the amino acid at
position 255; Ala, Arg, Asn, Asp, Gln, Glu, or Thr for the amino
acid at position 256; Ile or Thr for the amino acid at position
308; Pro for the amino acid at position 309; Glu, Leu, or Ser for
the amino acid at position 311; Ala or Asp for the amino acid at
position 312; Ala or Leu for the amino acid at position 314; Ala,
Arg, Asp, Gly, His, Lys, Ser, or Thr for the amino acid at position
385; Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr for the amino acid
at position 386; Ala, Arg, His, Pro, Ser, or Thr for the amino acid
at position 387; Asn, Pro, or Ser for the amino acid at position
389; Leu, Met, Phe, Ser, or Thr for the amino acid at position 428;
Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid at position
433; His, Phe, or Tyr for the amino acid at position 434; and Arg,
Asn, His, Lys, Met, or Thr for the amino acid at position 436, as
indicated by EU numbering. Meanwhile, the number of amino acids to
be modified is not particularly limited; and amino acid may be
modified at only one site or amino acids may be modified at two or
more sites.
[0291] When the Fc region of human IgG1 is comprised as the
FcRn-binding domain, a non-limiting embodiment of the modification
that results in the effect of enhancing FcRn binding in an acidic
pH range condition as compared to the binding activity of the
starting Fc region of human IgG1 may be modifications including Ile
for the amino acid at position 308, Pro for the amino acid at
position 309, and/or Glu for the amino acid at position 311
according to EU numbering. Another non-limiting embodiment of this
modification may include Thr for the amino acid at position 308,
Pro for the amino acid at position 309, Leu for the amino acid at
position 311, Ala for the amino acid at position 312, and/or Ala
for the amino acid at position 314. Furthermore, another
non-limiting embodiment of this modification may include Ile or Thr
for the amino acid at position 308, Pro for the amino acid at
position 309, Glu, Leu, or Ser for the amino acid at position 311,
Ala for the amino acid at position 312, and/or Ala or Leu for the
amino acid at position 314. Another non-limiting embodiment of this
modification may include Thr for the amino acid at position 308,
Pro for the amino acid at position 309, Ser for the amino acid at
position 311, Asp for the amino acid at position 312, and/or Leu
for the amino acid at position 314.
[0292] When the Fc region of human IgG1 is comprised as the
FcRn-binding domain, a non-limiting embodiment of the modification
that results in the effect of enhancing FcRn binding under an
acidic pH range condition as compared to the binding activity of
the starting Fc region of human IgG1 may be modifications including
Leu for the amino acid at position 251, Tyr for the amino acid at
position 252, Ser or Thr for the amino acid at position 254, Arg
for the amino acid at position 255, and/or Glu for the amino acid
at position 256 according to EU numbering.
[0293] When the Fc region of human IgG1 is comprised as the
FcRn-binding domain, a non-limiting embodiment of the modification
that results in the effect of enhancing FcRn binding under an
acidic pH range condition as compared to the binding activity of
the starting Fc region of human IgG1 may be modifications including
Leu, Met, Phe, Ser, or Thr for the amino acid at position 428, Arg,
Gln, His, Ile, Lys, Pro, or Ser for the amino acid at position 433,
His, Phe, or Tyr for the amino acid at position 434, and/or Arg,
Asn, His, Lys, Met, or Thr for the amino acid at position 436
according to EU numbering. Another non-limiting embodiment of this
modification may include His or Met for the amino acid at position
428, and/or His or Met for the amino acid at position 434.
[0294] When the Fc region of human IgG1 is comprised as the
FcRn-binding domain, a non-limiting embodiment of the modification
that results in the effect of enhancing FcRn binding under an
acidic pH range condition as compared to the binding activity of
the starting Fc region of human IgG1 may be modifications including
Arg for the amino acid at position 385, Thr for the amino acid at
position 386, Arg for the amino acid at position 387, and/or Pro
for the amino acid at position 389 according to EU numbering.
Another non-limiting embodiment of this modification may include
Asp for the amino acid at position 385, Pro for the amino acid at
position 386, and/or Ser for the amino acid at position 389.
[0295] Furthermore, when the Fc region of human IgG1 is comprised
as the FcRn-binding domain, a non-limiting embodiment of the
modification that results in the effect of enhancing FcRn binding
under an acidic pH range condition as compared to the binding
activity of the starting Fc region of human IgG1 include at least
one or more amino acid modifications selected from the group
consisting of:
Gln or Glu for the amino acid at position 250; and Leu or Phe for
the amino acid at position 428 according to EU numbering. The
number of amino acids to be modified is not particularly limited;
and amino acid may be modified at only one site or amino acids may
be modified at two sites.
[0296] When the Fc region of human IgG1 is comprised as the
FcRn-binding domain, a non-limiting embodiment of the modification
that results in the effect of enhancing FcRn binding under an
acidic pH range condition as compared to the binding activity of
the starting Fc region of human IgG1 may be modifications including
Gln for the amino acid at position 250, and/or Leu or Phe for the
amino acid at position 428 according to EU numbering. Another
non-limiting embodiment of this modification may include Glu for
the amino acid at position 250, and/or Leu or Phe for the amino
acid at position 428.
[0297] When the Fc region of human IgG1 is comprised as the
FcRn-binding domain, a non-limiting embodiment of the modification
that results in the effect of enhancing FcRn binding under an
acidic pH range condition as compared to the binding activity of
the starting Fc region of human IgG1 include at least two or more
amino acid modifications selected from the group consisting of:
Asp or Glu for the amino acid at position 251; Tyr for the amino
acid at position 252; Gln for the amino acid at position 307; Pro
for the amino acid at position 308; Val for the amino acid at
position 378; Ala for the amino acid at position 380; Leu for the
amino acid at position 428; Ala or Lys for the amino acid at
position 430; Ala, His, Ser, or Tyr for the amino acid at position
434; and Ile for the amino acid at position 436, as indicated by EU
numbering. The number of amino acids to be modified is not
particularly limited; and amino acid may be modified at only two
sites or amino acids may be modified at three or more sites.
[0298] When the Fc region of human IgG1 is comprised as the
FcRn-binding domain, a non-limiting embodiment of the modification
that results in the effect of enhancing FcRn binding under an
acidic pH range condition as compared to the binding activity of
the starting Fc region of human IgG1 may be modifications including
Gln for the amino acid at position 307, and Ala or Ser for the
amino acid at position 434 according to EU numbering. Another
non-limiting embodiment of this modification may include Pro for
the amino acid at position 308, and Ala for the amino acid at
position 434. Furthermore, another non-limiting embodiment of this
modification may include Tyr for the amino acid at position 252,
and Ala for the amino acid at position 434. A different
non-limiting embodiment of this modification may include Val for
the amino acid at position 378, and Ala for the amino acid at
position 434. Another different non-limiting embodiment of this
modification may include Leu for the amino acid at position 428,
and Ala for the amino acid at position 434. Another different
non-limiting embodiment of this modification may include Ala for
the amino acid at position 434, and Ile for the amino acid at
position 436. Furthermore, another non-limiting embodiment of this
modification may include Pro for the amino acid at position 308,
and Tyr for the amino acid at position 434. In addition, another
non-limiting embodiment of this modification may include Gln for
the amino acid at position 307, and Ile for the amino acid at
position 436.
[0299] When the Fc region of human IgG1 is comprised as the
FcRn-binding domain, a non-limiting embodiment of the modification
that results in the effect of enhancing FcRn binding under an
acidic pH range condition as compared to the binding activity of
the starting Fc region of human IgG1 may be modifications including
any one of Gln for the amino acid at position 307, Ala for the
amino acid at position 380, and Ser for the amino acid at position
434 according to EU numbering. Another non-limiting embodiment of
this modification may include Gln for the amino acid at position
307, Ala for the amino acid at position 380, and Ala for the amino
acid at position 434. Furthermore, another non-limiting embodiment
of this modification may include Tyr for the amino acid at position
252, Pro for the amino acid at position 308, and Tyr for the amino
acid at position 434. A different non-limiting embodiment of this
modification may include Asp for the amino acid at position 251,
Gln for the amino acid at position 307, and His for the amino acid
at position 434.
[0300] When the Fc region of human IgG1 is comprised as the
FcRn-binding domain, a non-limiting embodiment of the modification
that results in the effect of enhancing FcRn binding under an
acidic pH range condition as compared to the binding activity of
the starting Fc region of human IgG1 include modification of at
least two or more amino acids selected from the group consisting
of:
Leu for the amino acid at position 238; Leu for the amino acid at
position 244; Arg for the amino acid at position 245; Pro for the
amino acid at position 249; Tyr for the amino acid at position 252;
Pro for the amino acid at position 256; Ala, Ile, Met, Asn, Ser, or
Val for the amino acid at position 257; Asp for the amino acid at
position 258; Ser for the amino acid at position 260; Leu for the
amino acid at position 262; Lys for the amino acid at position 270;
Leu or Arg for the amino acid at position 272; Ala, Asp, Gly, His,
Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid at
position 279; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro,
Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid at position 283;
Asn for the amino acid at position 285; Phe for the amino acid at
position 286; Asn or Pro for the amino acid at position 288; Val
for the amino acid at position 293; Ala, Glu, or Met for the amino
acid at position 307; Ala, Ile, Lys, Leu, Met, Val, or Trp for the
amino acid at position 311; Pro for the amino acid at position 312;
Lys for the amino acid at position 316; Pro for the amino acid at
position 317; Asn or Thr for the amino acid at position 318; Phe,
His, Lys, Leu, Met, Arg, Ser, or Trp for the amino acid at position
332; Asn, Thr, or Trp for the amino acid at position 339; Pro for
the amino acid at position 341; Glu, His, Lys, Gln, Arg, Thr, or
Tyr for the amino acid at position 343; Arg for the amino acid at
position 375; Gly, Ile, Met, Pro, Thr, or Val for the amino acid at
position 376; Lys for the amino acid at position 377; Asp or Asn
for the amino acid at position 378; Asn, Ser, or Thr for the amino
acid at position 380; Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid at position 382; Asn
for the amino acid at position 423; Asn for the amino acid at
position 427; Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln,
Arg, Ser, Thr, Val, or Tyr for the amino acid at position 430; His
or Asn for the amino acid at position 431; Phe, Gly, His, Trp, or
Tyr for the amino acid at position 434; Ile, Leu, or Thr for the
amino acid at position 436; Lys, Leu, Thr, or Trp for the amino
acid at position 438; Lys for the amino acid at position 440; and
Lys for the amino acid at position 442 according to EU numbering.
The number of amino acids to be modified is not particularly
limited and amino acid at only two sites may be modified and amino
acids at three or more sites may be modified.
[0301] When the Fc region of human IgG1 is comprised as the
FcRn-binding domain, a non-limiting embodiment of the modification
that results in the effect of enhancing FcRn binding under an
acidic pH range condition as compared to the binding activity of
the starting Fc region of human IgG1 may be modifications including
Ile for the amino acid at position 257, and Ile for the amino acid
at position 311 according to EU numbering. Another non-limiting
embodiment of this modification may include Ile for the amino acid
at position 257, and His for the amino acid at position 434.
Another non-limiting embodiment of this modification may include
Val for the amino acid at position 376, and His for the amino acid
at position 434.
Fc.gamma. Receptor
[0302] Fc.gamma. receptor (Fc.gamma.R) refers to a receptor capable
of binding to the Fc region of monoclonal IgG1, IgG2, IgG3, or IgG4
antibodies, and includes all members belonging to the family of
proteins substantially encoded by an Fc.gamma. receptor gene. In
humans, the family includes Fc.gamma.RI (CD64) including isoforms
Fc.gamma.RIa, Fc.gamma.RIb and Fc.gamma.RIc; Fc.gamma.RII (CD32)
including isoforms Fc.gamma.RIIa (including allotype H131 and
R131), Fc.gamma.RIIb (including Fc.gamma.RIIb-1 and
Fc.gamma.RIIb-2), and Fc.gamma.RIIc; and Fc.gamma.RIII (CD16)
including isoform Fc.gamma.RIIIa (including allotype V158 and F158)
and Fc.gamma.RIIIb (including allotype Fc.gamma.RIIIb-NA1 and
Fc.gamma.RIIIb-NA2); as well as all unidentified human Fc.gamma.Rs,
Fc.gamma.R isoforms, and allotypes thereof. However, Fc.gamma.
receptor is not limited to these examples. Without being limited
thereto, Fc.gamma.R includes those derived from humans, mice, rats,
rabbits, and monkeys. Fc.gamma.R may be derived from any organism.
Mouse Fc.gamma.R includes, without being limited to, Fc.gamma.RI
(CD64), Fc.gamma.RII (CD32), Fc.gamma.RIII (CD16), and
Fc.gamma.RIII-2 (Fc.gamma.RIV, CD16-2), as well as all unidentified
mouse Fc.gamma.Rs, Fc.gamma.R isoforms, and allotypes thereof. Such
preferred Fc.gamma. receptors include, for example, human
Fc.gamma.RI (CD64), Fc.gamma.RIIa (CD32), Fc.gamma.RIIb (CD32),
Fc.gamma.RIIIa (CD16), and/or Fc.gamma.RIIIb (CD16). The
polynucleotide sequence and amino acid sequence of Fc.gamma.RI are
shown in SEQ ID NOs: 18 (NM.sub.--000566.3) and 19
(NP.sub.--000557.1), respectively; the polynucleotide sequence and
amino acid sequence of Fc.gamma.RIIa are shown in SEQ ID NOs: 20
(BC020823.1) and 21 (AAH20823.1), respectively; the polynucleotide
sequence and amino acid sequence of Fc.gamma.RIIb are shown in SEQ
ID NOs: 22 (BC146678.1) and 23 (AAI46679.1), respectively; the
polynucleotide sequence and amino acid sequence of Fc.gamma.RIIIa
are shown in SEQ ID NOs: 24 (BC033678.1) and 25 (AAH33678.1),
respectively; and the polynucleotide sequence and amino acid
sequence of Fc.gamma.RIIIb are shown in SEQ ID NOs: 26 (BC128562.1)
and 27 (AAI28563.1), respectively (RefSeq accession number is shown
in each parentheses). Whether an Fc.gamma. receptor has binding
activity to the Fc region of a monoclonal IgG1, IgG2, IgG3, or IgG4
antibody can be assessed by ALPHA screen (Amplified Luminescent
Proximity Homogeneous Assay), surface plasmon resonance (SPR)-based
BIACORE method, and others (Proc. Natl. Acad. Sci. USA (2006)
103(11), 4005-4010), in addition to the above-described FACS and
ELISA formats.
[0303] In Fc.gamma.RI (CD64) including Fc.gamma.RIa, Fc.gamma.RIb,
and Fc.gamma.RIc, 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), .alpha. chain that binds to the Fc portion of
IgG is associated with common .gamma. chain having ITAM responsible
for transduction of intracellular activation signal. Meanwhile, the
cytoplasmic domain of Fc.gamma.RII (CD32) including isoforms
Fc.gamma.RIIa (including allotypes H131 and R131) and Fc.gamma.RIIc
contains ITAM. These receptors are expressed on many immune cells
such as macrophages, mast cells, and antigen-presenting cells. The
activation signal transduced upon binding of these receptors to the
Fc portion of IgG results in enhancement of the phagocytic activity
and inflammatory cytokine production of macrophages, mast cell
degranulation, and the enhanced function of antigen-presenting
cells. Fc.gamma. receptors having the ability to transduce the
activation signal as described above are also referred to as
activating Fc.gamma. receptors.
[0304] Meanwhile, the intracytoplasmic domain of Fc.gamma.RIIb
(including Fc.gamma.RIIb-1 and Fc.gamma.RIIb-2) contains ITIM
responsible for transduction of inhibitory signals. The
crosslinking between Fc.gamma.RIIb and B cell receptor (BCR) on B
cells suppresses the activation signal from BCR, which results in
suppression of antibody production via BCR. The crosslinking of
Fc.gamma.RIII and Fc.gamma.RIIb on macrophages suppresses the
phagocytic activity and inflammatory cytokine production.
Fc.gamma.receptors having the ability to transduce the inhibitory
signal as described above are also referred to as inhibitory
Fc.gamma. receptors.
Binding Activity to the Fc.gamma. Receptor
[0305] The binding activity of an Fc.gamma.R-binding domain, which
is included in an antigen-binding molecule of the present
invention, to any of the human Fc.gamma. receptors, Fc.gamma.RI,
Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIIa, and/or
Fc.gamma.RIIIb, can be confirmed by the above-described FACS and
ELISA format, as well as ALPHA Screen (Amplified Luminescent
Proximity Homogeneous Assay), a BIACORE method using the surface
plasmon resonance (SPR) phenomena, and such (Proc. Natl. Acad. Sci.
USA (2006) 103 (11), 4005-4010).
[0306] ALPHA screen is performed by the ALPHA technology based on
the principle described below using two types of beads: donor and
acceptor beads. A luminescent signal is detected only when
molecules linked to the donor beads interact biologically with
molecules linked to the acceptor beads and when the two beads are
located in close proximity. Excited by laser beam, the
photosensitizer in a donor bead converts oxygen around the bead
into excited singlet oxygen. When the singlet oxygen diffuses
around the donor beads and reaches the acceptor beads located in
close proximity, a chemiluminescent reaction within the acceptor
beads is induced. This reaction ultimately results in light
emission. If molecules linked to the donor beads do not interact
with molecules linked to the acceptor beads, the singlet oxygen
produced by donor beads do not reach the acceptor beads and
chemiluminescent reaction does not occur.
[0307] For example, a biotin-labeled antigen-binding molecule
comprising Fc.gamma.R-binding domain is immobilized to the donor
beads and glutathione S-transferase (GST)-tagged Fc.gamma.receptor
is immobilized to the acceptor beads. In the absence of an
antigen-binding molecule comprising a competitive altered
Fc.gamma.R-binding domain, Fc.gamma. receptor interacts with an
antigen-binding molecule comprising a wild-type Fc.gamma.R-binding
domain, inducing a signal of 520 to 620 nm as a result. The
antigen-binding molecule having a non-tagged altered
Fc.gamma.R-binding domain competes with the antigen-binding
molecule comprising a native Fc.gamma.R-binding domain for the
interaction with Fc.gamma. receptor. The relative binding affinity
can be determined by quantifying the reduction of fluorescence as a
result of competition. Methods for biotinylating the
antigen-binding molecules such as antibodies using Sulfo-NHS-biotin
or the like are known. Appropriate methods for adding the GST tag
to an Fc.gamma. receptor include methods that involve fusing
polypeptides encoding Fc.gamma. and GST in-frame, expressing the
fused gene using cells introduced with a vector to which the gene
is operably linked, and then purifying using a glutathione column.
The induced signal can be preferably analyzed, for example, by
fitting to a one-site competition model based on nonlinear
regression analysis using software such as GRAPHPAD PRISM
(GraphPad; San Diego).
[0308] One of the substances for observing their interaction is
immobilized as a ligand onto the gold thin layer of a sensor chip.
When light is shed on the rear surface of the sensor chip so that
total reflection occurs at the interface between the gold thin
layer and glass, the intensity of reflected light is partially
reduced at a certain site (SPR signal). The other substance for
observing their interaction is injected as an analyte onto the
surface of the sensor chip. The mass of immobilized ligand molecule
increases when the analyte binds to the ligand. This alters the
refraction index of solvent on the surface of the sensor chip. The
change in refraction index causes a positional shift of SPR signal
(conversely, the dissociation shifts the signal back to the
original position). In the Biacore system, the amount of shift
described above (i.e., the change of mass on the sensor chip
surface) is plotted on the vertical axis, and thus the change of
mass over time is shown as measured data (sensorgram). Kinetic
parameters (association rate constant (ka) and dissociation rate
constant (kd)) are determined from the curve of sensorgram, and
affinity (KD) is determined from the ratio between these two
constants Inhibition assay is also preferably used in the BIACORE
methods. Examples of such inhibition assay are described in Proc.
Natl. Acad. Sci. USA (2006) 103(11), 4005-4010. Binding activities
of the Fc.gamma.R-binding domain included in the antigen-binding
molecule of the present invention towards any of the human
Fc.gamma. receptors, Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIIb,
Fc.gamma.RIIIa, and/or Fc.gamma.RIIIb, can be determined from the
amount of binding and KD value for each of the human Fc.gamma.
receptors calculated using the Biacore system according to the
examples described above. Here, the amount of binding of the
various Fc.gamma.Rs to the polypeptides is also represented by
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.
[0309] An acidic pH range condition or neutral pH range condition
may be suitably used for the pH conditions to measure the Fc.gamma.
receptor-binding activity of the Fc.gamma.R-binding domain included
in the antigen-binding molecule of the present invention or an
antigen-binding molecule containing the domain. A neutral pH range
as a condition to measure the Fc.gamma. receptor-binding activity
of the Fc.gamma.R-binding domain of the present invention or an
antigen-binding molecule containing the domain generally refers to
pH 6.7 to pH 10.0. Preferably, it is a range indicated with
arbitrary pH values between pH 7.0 and pH 8.0; and preferably, it
is selected from pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH
7.6, pH 7.7, pH 7.8, pH 7.9, and pH 8.0; and particularly
preferably, it is pH 7.4, which is close to the pH of plasma
(blood) in vivo. In the present invention, an acidic pH range as a
condition for the Fc.gamma.R-binding domain of the present
invention or an antigen-binding molecule containing the domain to
have Fc.gamma. receptor-binding activity generally refers to pH 4.0
to pH 6.5. Preferably, it refers to pH 5.5 to pH 6.5, and
particularly preferably, it refers to pH 5.8 to pH 6.0, which are
close to the pH in the early endosome in vivo. With regard to the
temperature used as a measurement condition, the binding affinity
between the Fc.gamma.R-binding domain or an antigen-binding
molecule containing the domain and a human Fc.gamma. receptor can
be evaluated at any temperature between 10.degree. C. and
50.degree. C. Preferably, a temperature between 15.degree. C. and
40.degree. C. is used to determine the binding affinity between the
Fc.gamma.R-binding domain or an antigen-binding molecule containing
the domain and an Fc.gamma. receptor. More preferably, any
temperature between 20.degree. C. and 35.degree. C. such as any one
of 20.degree. C., 21.degree. C., 22.degree. C., 23.degree. C.,
24.degree. C., 25.degree. C., 26.degree. C., 27.degree. C.,
28.degree. C., 29.degree. C., 30.degree. C., 31.degree. C.,
32.degree. C., 33.degree. C., 34.degree. C., and 35.degree. C. can
be used in a similar manner to determine the binding affinity
between the Fc.gamma.R-binding domain or an antigen-binding
molecule containing the domain and an Fc.gamma. receptor. A
temperature of 25.degree. C. is a non-limiting example in an
embodiment of the present invention.
Fc.gamma.R-Binding Domain
[0310] An antigen-binding molecule of the present invention
comprises an antigen-binding domain whose antigen-binding activity
varies depending on ion concentration conditions, an FcRn-binding
domain having FcRn-binding activity under an acidic pH range
condition, and an Fc.gamma. receptor-binding domain having
selective binding activity to an Fc.gamma. receptor (hereinafter
also referred to as a selective Fc.gamma.R-binding domain).
Preferred examples of the Fc.gamma.R-binding domain include Fc
regions of IgG-type immunoglobulins, Fc.gamma.R-binding domains of
IgG-type immunoglobulins, anti-Fc.gamma.R antibodies, and
anti-Fc.gamma.R scaffold molecules. A domain originally having
Fc.gamma.R-binding activity may be suitably used as it is for the
domain. When the domain has weak or no Fc.gamma.R-binding activity,
Fc.gamma.R-binding activity can be conferred by altering amino
acids forming the Fc.gamma.R-binding domain in the antigen-binding
molecule. Alternatively, Fc.gamma.R-binding activity can be
increased by altering amino acids in the domain originally having
Fc.gamma.R-binding activity. The amino acid alterations of the
Fc.gamma.R-binding domain that results in such desired binding
activity may be discovered by comparing the Fc.gamma.R-binding
activity before and after the amino acid alteration. A non-limiting
embodiment of such Fc.gamma.R-binding domains is, for example, the
Fc.gamma.R-binding domain included in the Fc region of human IgG1
(SEQ ID NO: 14), IgG2 (SEQ ID NO: 15), IgG3 (SEQ ID NO: 16), or
IgG4 (SEQ ID NO: 17). For example, when the Fc region of an IgG
antibody is used as the FcRn-binding domain having FcRn-binding
activity under an acidic pH range condition, the Fc.gamma.R-binding
domain included in the Fc region may be used as the
Fc.gamma.R-binding domain.
Fc.gamma.R-Binding Domain Having Selective Binding Activity to an
Fc.gamma. Receptor
[0311] Whether or not an Fc.gamma.R-binding domain of the present
invention has selective binding activity can be confirmed by
comparing binding activities to the respective Fc.gamma. receptors,
determined by the method described in the above-mentioned section
on binding activity to Fc.gamma.receptors. An Fc.gamma.R-binding
domain with higher binding activity to inhibitory Fc.gamma.
receptors than to activating Fc.gamma. receptors may be used as the
selective Fc.gamma.R-binding domain included in the antigen-binding
molecule provided by the present invention. In a non-limiting
embodiment, an Fc.gamma.R-binding domain with higher binding
activity to Fc.gamma.RIIb (including Fc.gamma.RIIb-1 and
Fc.gamma.RIIb-2) than to an activating Fc.gamma. receptor selected
from the group consisting of Fc.gamma.RI (CD64) including
Fc.gamma.RIa, Fc.gamma.RIb, Fc.gamma.RIc, 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 Fc.gamma.RII (CD32) including isoforms
Fc.gamma.RIIa and Fc.gamma.RIIc (including allotypes H131 and R131)
may be used as a selective Fc.gamma.R-binding domain included in an
antigen-binding molecule provided by the present invention.
Furthermore, in a non-limiting embodiment of the present invention,
an Fc.gamma.R-binding domain with higher binding activity to
Fc.gamma.RIIb-1 and/or Fc.gamma.RIIb-2 than to Fc.gamma.RIa,
Fc.gamma.RIb, and Fc.gamma.RIc, Fc.gamma.RIIIa including allotype
V158, Fc.gamma.RIIIa including allotype F158, Fc.gamma.RIIIb
including allotype Fc.gamma.RIIIb-NA1, Fc.gamma.RIIIb including
allotype Fc.gamma.RIIIb-NA2, Fc.gamma.RIIa including allotype H131,
Fc.gamma.RIIa including allotype R131, and/or Fc.gamma.RIIc may be
used as a selective Fc.gamma.R-binding domain included in an
antigen-binding molecule provided by the present invention. Whether
an Fc.gamma.R-binding domain to be tested has selective binding
activity to Fc.gamma. receptors can be determined by comparing the
value (ratio) obtained by dividing the KD values of the
Fc.gamma.R-binding domain for Fc.gamma.RIa, Fc.gamma.RIb,
Fc.gamma.RIc, Fc.gamma.RIIIa including allotype V158,
Fc.gamma.RIIIa including allotype F158, Fc.gamma.RIIIb including
allotype Fc.gamma.RIIIb-NA1, Fc.gamma.RIIIb including allotype
Fc.gamma.RIIIb-NA2, Fc.gamma.RIIa including allotype H131,
Fc.gamma.RIIa including allotype R131, and/or Fc.gamma.RIIc by the
KD values for Fc.gamma.RIIb-1 and/or Fc.gamma.RIIb-2, wherein the
KD values are determined by the method described in the
above-mentioned section on binding activity to Fc.gamma. receptors,
or more specifically, by comparing the Fc.gamma.R selectivity
indices shown in Equation 1.
Fc.gamma.R selectivity index=KD value for activating Fc.gamma.R/KD
value for inhibitory Fc.gamma.R [Equation 1]
[0312] In Equation 1 mentioned above, "activating Fc.gamma.R"
refers to Fc.gamma.RIa, Fc.gamma.RIb, Fc.gamma.RIc, Fc.gamma.RIIIa
including allotype V158, Fc.gamma.RIIIa including allotype F158,
Fc.gamma.RIIIb including allotype Fc.gamma.RIIIb-NA1,
Fc.gamma.RIIIb including allotype Fc.gamma.RIIIb-NA2, Fc.gamma.RIIa
including allotype H131, Fc.gamma.RIIa including allotype R131,
and/or Fc.gamma.RIIc, and inhibitory Fc.gamma.R refers to
Fc.gamma.RIIb-1 and/or Fc.gamma.RIIb-2. Although the activating
Fc.gamma.R and inhibitory Fc.gamma.R used for the KD value
measurements may be selected from any combination, in a
non-limiting embodiment, a value (ratio) obtained by dividing the
KD value for Fc.gamma.RIIa including allotype H131 by the KD value
for Fc.gamma.RIIb-1 and/or Fc.gamma.RIIb-2 may be used.
[0313] For example, the Fc.gamma.R selectivity indices have values
of, 1.2 or greater, 1.3 or greater, 1.4 or greater, 1.5 or greater,
1.6 or greater, 1.7 or greater, 1.8 or greater, 1.9 or greater, 2
or greater, 3 or greater, 5 or greater, 6 or greater, 7 or greater,
8 or greater, 9 or greater, 10 or greater, 15 or greater, 20 or
greater, 25 or greater, 30 or greater, 35 or greater, 40 or
greater, 45 or greater, 50 or greater, 55 or greater, 60 or
greater, 65 or greater, 70 or greater, 75 or greater, 80 or
greater, 85 or greater, 90 or greater, 95 or greater, 100 or
greater, 110 or greater, 120 or greater, 130 or greater, 140 or
greater, 150 or greater, 160 or greater, 170 or greater, 180 or
greater, 190 or greater, 200 or greater, 210 or greater, 220 or
greater, 230 or greater, 240 or greater, 250 or greater, 260 or
greater, 270 or greater, 280 or greater, 290 or greater, 300 or
greater, 310 or greater, 320 or greater, 330 or greater, 340 or
greater, 350 or greater, 360 or greater, 370 or greater, 380 or
greater, 390 or greater, 400 or greater, 410 or greater, 420 or
greater, 430 or greater, 440 or greater, 450 or greater, 460 or
greater, 470 or greater, 480 or greater, 490 or greater, 500 or
greater, 520 or greater, 540 or greater, 560 or greater, 580 or
greater, 600 or greater, 620 or greater, 640 or greater, 660 or
greater, 680 or greater, 700 or greater, 720 or greater, 740 or
greater, 760 or greater, 780 or greater, 800 or greater, 820 or
greater, 840 or greater, 860 or greater, 880 or greater, 900 or
greater, 920 or greater, 940 or greater, 960 or greater, 980 or
greater, 1000 or greater, 1500 or greater, 2000 or greater, 2500 or
greater, 3000 or greater, 3500 or greater, 4000 or greater, 4500 or
greater, 5000 or greater, 5500 or greater, 6000 or greater, 6500 or
greater, 7000 or greater, 7500 or greater, 8000 or greater, 8500 or
greater, 9000 or greater, 9500 or greater, 10000 or greater, or
100000 or greater.
[0314] A non-limiting embodiment of the selective
Fc.gamma.R-binding domain in an antigen-binding molecule of the
present invention includes, for example, Fc regions produced by
modifying the Fc.gamma.R-binding domain included in an Fc region
presented as human IgG1 (SEQ ID NO: 14), IgG2 (SEQ ID NO: 15), IgG3
(SEQ ID NO: 16), or IgG4 (SEQ ID NO: 17). An example of a method
for producing the modified Fc regions includes the method described
in the above-mentioned section on amino acid alterations. Examples
of such altered Fc regions include an Fc region in which amino acid
at position 238 (EU numbering) is Asp or an Fc region in which
amino acid at position 328 (EU numbering) is Glu in a human IgG
(IgG1, IgG2, IgG3, or IgG4). An Fc region in which amino acid at
position 238 (EU numbering) is Asp or an Fc region in which amino
acid at position 328 (EU numbering) is Glu in a human IgG (IgG1,
IgG2, IgG3, or IgG4), and antigen-binding molecules containing such
an Fc region show higher binding activity to Fc.gamma.RIIb-1 and/or
Fc.gamma.RIIb-2 than to Fc.gamma.RIa, Fc.gamma.RIb, Fc.gamma.RIc,
Fc.gamma.RIIIa including allotype V158, Fc.gamma.RIIIa including
allotype F158, Fc.gamma.RIIIb including allotype
Fc.gamma.RIIIb-NA1, Fc.gamma.RIIIb including allotype
Fc.gamma.RIIIb-NA2, Fc.gamma.RIIa including allotype H131,
Fc.gamma.RIIa including allotype R131, and/or Fc.gamma.RIIc.
[0315] Fc regions containing a selective Fc.gamma.R-binding domain
which are included in the antigen-binding molecules of the present
invention and antigen-binding molecules containing such an Fc
region may also be Fc regions and antigen-binding molecules
containing such an Fc region which maintains or shows reduced
binding activity to activating Fc.gamma.R (Fc.gamma.RIa,
Fc.gamma.RIb, Fc.gamma.RIc, Fc.gamma.RIIIa including allotype V158,
Fc.gamma.RIIIa including allotype F158, Fc.gamma.RIIIb including
allotype Fc.gamma.RIIIb-NA1, Fc.gamma.RIIIb including allotype
Fc.gamma.RIIIb-NA2, Fc.gamma.RIIa including allotype H131,
Fc.gamma.RIIa including allotype R131, and/or Fc.gamma.RIIc) when
compared to an Fc region presented as human IgG1 (SEQ ID NO: 14),
IgG2 (SEQ ID NO: 15), IgG3 (SEQ ID NO: 16), or IgG4 (SEQ ID NO: 17)
(hereinafter referred to as a wild-type Fc region) and an
antigen-binding molecule containing such a wild-type Fc region.
[0316] Compared to a wild-type Fc region and an antigen-binding
molecule containing a wild-type Fc region, the degree of the
aforementioned reduction in binding activity to activating
Fc.gamma.R of an Fc region containing a selective
Fc.gamma.R-binding domain included in an antigen-binding molecule
of the present invention, and an antigen-binding molecule
containing such an Fc region is, for example, 99% or less, 98% or
less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or
less, 92% or less, 91% or less, 90% or less, 88% or less, 86% or
less, 84% or less, 82% or less, 80% or less, 78% or less, 76% or
less, 74% or less, 72% or less, 70% or less, 68% or less, 66% or
less, 64% or less, 62% or less, 60% or less, 58% or less, 56% or
less, 54% or less, 52% or less, 50% or less, 45% or less, 40% or
less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or
less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less,
1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less,
0.1% or less, 0.05% or less, 0.01% or less, or 0.005% or less.
[0317] The Fc regions containing a selective Fc.gamma.R-binding
domain and antigen-binding molecules containing such an Fc region,
which are included in the antigen-binding molecules of the present
invention, may also be Fc regions and antigen-binding molecules
containing such an Fc region which shows enhanced binding activity
to inhibitory Fc.gamma.R (Fc.gamma.RIIb-1 and/or Fc.gamma.RIIb-2)
when compared to an Fc region presented as human IgG1 (SEQ ID NO:
14), IgG2 (SEQ ID NO: 15), IgG3 (SEQ ID NO: 16), or IgG4 (SEQ ID
NO: 17) (hereinafter referred to as a wild-type Fc region) and an
antigen-binding molecule containing such a wild-type Fc region.
[0318] Compared to a wild-type Fc region and an antigen-binding
molecule containing a wild-type Fc region, the degree of the
aforementioned enhancement in binding activity to inhibitory
Fc.gamma.R of an Fc region containing a selective
Fc.gamma.R-binding domain included in an antigen-binding molecule
of the present invention and an antigen-binding molecule containing
such an Fc region is, for example, 101% or greater, 102% or
greater, 103% or greater, 104% or greater, 105% or greater, 106% or
greater, 107% or greater, 108% or greater, 109% or greater, 110% or
greater, 112% or greater, 114% or greater, 116% or greater, 118% or
greater, 120% or greater, 122% or greater, 124% or greater, 126% or
greater, 128% or greater, 130% or greater, 132% or greater, 134% or
greater, 136% or greater, 138% or greater, 140% or greater, 142% or
greater, 144% or greater, 146% or greater, 148% or greater, 150% or
greater, 155% or greater, 160% or greater, 165% or greater, 170% or
greater, 175% or greater, 180% or greater, 185% or greater, 190% or
greater, 195% or greater, 2-fold or greater, 3-fold or greater,
4-fold or greater, 5-fold or greater, 6-fold or greater, 7-fold or
greater, 8-fold or greater, 9-fold or greater, 10-fold or greater,
20-fold or greater, 30-fold or greater, 40-fold or greater, 50-fold
or greater, 60-fold or greater, 70-fold or greater, 80-fold or
greater, 90-fold or greater, 100-fold or greater, 200-fold or
greater, 300-fold or greater, 400-fold or greater, 500-fold or
greater, 600-fold or greater, 700-fold or greater, 800-fold or
greater, 900-fold or greater, 1000-fold or greater, 10000-fold or
greater, or 100000-fold or greater.
[0319] Furthermore, the Fc region containing a selective
Fc.gamma.R-binding domain included in an antigen-binding molecule
of the present invention and the antigen-binding molecule
containing such an Fc region may be an Fc region and an
antigen-binding molecule containing such an Fc region which
maintains or shows reduced binding activity to activating
Fc.gamma.R (Fc.gamma.RIa, Fc.gamma.RIb, Fc.gamma.RIc,
Fc.gamma.RIIIa including allotype V158, Fc.gamma.RIIIa including
allotype F158, Fc.gamma.RIIIb including allotype
Fc.gamma.RIIIb-NA1, Fc.gamma.RIIIb including allotype
Fc.gamma.RIIIb-NA2, Fc.gamma.RIIa including allotype H131,
Fc.gamma.RIIa including allotype R131, and/or Fc.gamma.RIIc) when
compared to an Fc region presented as human IgG1 (SEQ ID NO: 14),
IgG2 (SEQ ID NO: 15), IgG3 (SEQ ID NO: 16), or IgG4 (SEQ ID NO: 17)
(hereinafter referred to as a wild-type Fc region) and an
antigen-binding molecule containing such a wild-type Fc region; and
shows enhanced binding activity to inhibitory Fc.gamma.R
(Fc.gamma.RIIb-1 and/or Fc.gamma.RIIb-2) when compared to an Fc
region presented as human IgG1 (SEQ ID NO: 14), IgG2 (SEQ ID NO:
15), IgG3 (SEQ ID NO: 16), or IgG4 (SEQ ID NO: 17) (hereinafter
referred to as a wild-type Fc region) and an antigen-binding
molecule containing such a wild-type Fc region.
[0320] Furthermore, the Fc region containing a selective
Fc.gamma.R-binding domain included in an antigen-binding molecule
of the present invention and the antigen-binding molecule
containing such an Fc region may be an Fc region and an
antigen-binding molecule containing such an Fc region with higher
degree of enhancement of binding activity to an inhibitory
Fc.gamma. receptor (Fc.gamma.RIIb-1 and/or Fc.gamma.RIIb-2) than to
an activating Fc.gamma. receptor (Fc.gamma.RIa, Fc.gamma.RIb,
Fc.gamma.RIc, Fc.gamma.RIIIa including allotype V158,
Fc.gamma.RIIIa including allotype F158, Fc.gamma.RIIIb including
allotype Fc.gamma.RIIIb-NA1, Fc.gamma.RIIIb including allotype
Fc.gamma.RIIIb-NA2, Fc.gamma.RIIa including allotype H131,
Fc.gamma.RIIa including allotype R131), when compared to an Fc
region presented as human IgG1 (SEQ ID NO: 14), IgG2 (SEQ ID NO:
15), IgG3 (SEQ ID NO: 16), or IgG4 (SEQ ID NO: 17) (hereinafter
referred to as a wild-type Fc region) and an antigen-binding
molecule containing such a wild-type Fc region.
[0321] In the present invention, at least another alteration to the
Fc region may be added to the Fc region in which amino acid at
position 238 (EU numbering) is Asp and the Fc region in which amino
acid at position 328 (EU numbering) is Glu, by the embodiments and
such described in the aforementioned section on amino acid
alterations. In addition to these alterations, additional
alterations may also be added. The additional alterations can be
selected from any of substitutions, deletions, and modifications of
an amino acid, and combinations thereof. For example, alterations
that enhance binding activity to Fc.gamma.RIIb while maintaining or
reducing binding activity to Fc.gamma.RIIa (H type) and
Fc.gamma.RIIa (R type) may be added. Addition of such alterations
improves binding selectivity to Fc.gamma.RIIb over
Fc.gamma.RIIa.
[0322] Among these, alterations that improve binding selectivity to
Fc.gamma.RIIb over Fc.gamma.RIIa (R type) is favorable, and
alterations that improve binding selectivity to Fc.gamma.RIIb over
Fc.gamma.RIIa (H type) is more favorable. Examples of preferred
amino acid substitutions for such alterations include: an
alteration by substituting Gly at position 237 (EU numbering) with
Trp; an alteration by substituting Gly at position 237 (EU
numbering) with Phe; an alteration by substituting Pro at position
238 (EU numbering) with Phe; an alteration by substituting Asn at
position 325 (EU numbering) with Met; an alteration by substituting
Ser at position 267 (EU numbering) with Ile; an alteration by
substituting Leu at position 328 (EU numbering) with Asp; an
alteration by substituting Ser at position 267 (EU numbering) with
Val; an alteration by substituting Leu at position 328 (EU
numbering) with Trp; an alteration by substituting Ser at position
267 (EU numbering) with Gln; an alteration by substituting Ser at
position 267 (EU numbering) with Met; an alteration by substituting
Gly at position 236 (EU numbering) with Asp; an alteration by
substituting Ala at position 327 (EU numbering) with Asn; an
alteration by substituting Asn at position 325 (EU numbering) with
Ser; an alteration by substituting Leu at position 235 (EU
numbering) with Tyr; an alteration by substituting Val at position
266 (EU numbering) with Met; an alteration by substituting Leu at
position 328 (EU numbering) with Tyr; an alteration by substituting
Leu at position 235 (EU numbering) with Trp; an alteration by
substituting Leu at position 235 (EU numbering) with Phe; an
alteration by substituting Ser at position 239 (EU numbering) with
Gly; an alteration by substituting Ala at position 327 (EU
numbering) with Glu; an alteration by substituting Ala at position
327 (EU numbering) with Gly; an alteration by substituting Pro at
position 238 (EU numbering) with Leu; an alteration by substituting
Ser at position 239 (EU numbering) with Leu; an alteration by
substituting Leu at position 328 (EU numbering) with Thr; an
alteration by substituting Leu at position 328 (EU numbering) with
Ser; an alteration by substituting Leu at position 328 (EU
numbering) with Met; an alteration by substituting Pro at position
331 (EU numbering) with Trp; an alteration by substituting Pro at
position 331 (EU numbering) with Tyr; an alteration by substituting
Pro at position 331 (EU numbering) with Phe; an alteration by
substituting Ala at position 327 (EU numbering) with Asp; an
alteration by substituting Leu at position 328 (EU numbering) with
Phe; an alteration by substituting Pro at position 271 (EU
numbering) with Leu; an alteration by substituting Ser at position
267 (EU numbering) with Glu; an alteration by substituting Leu at
position 328 (EU numbering) with Ala; an alteration by substituting
Leu at position 328 (EU numbering) with Ile; an alteration by
substituting Leu at position 328 (EU numbering) with Gln; an
alteration by substituting Leu at position 328 (EU numbering) with
Val; an alteration by substituting Lys at position 326 (EU
numbering) with Trp; an alteration by substituting Lys at position
334 (EU numbering) with Arg; an alteration by substituting His at
position 268 (EU numbering) with Gly; an alteration by substituting
His at position 268 (EU numbering) with Asn; an alteration by
substituting Ser at position 324 (EU numbering) with Val; an
alteration by substituting Val at position 266 (EU numbering) with
Leu; an alteration by substituting Pro at position 271 (EU
numbering) with Gly; an alteration by substituting Ile at position
332 (EU numbering) with Phe; an alteration by substituting Ser at
position 324 (EU numbering) with Ile; an alteration by substituting
Glu at position 333 (EU numbering) with Pro; an alteration by
substituting Tyr at position 300 (EU numbering) with Asp; an
alteration by substituting Ser at position 337 (EU numbering) with
Asp; an alteration by substituting Tyr at position 300 (EU
numbering) with Gln; an alteration by substituting Thr at position
335 (EU numbering) with Asp; an alteration by substituting Ser at
position 239 (EU numbering) with Asn; an alteration by substituting
Lys at position 326 (EU numbering) with Leu; an alteration by
substituting Lys at position 326 (EU numbering) with Ile; an
alteration by substituting Ser at position 239 (EU numbering) with
Glu; an alteration by substituting Lys at position 326 (EU
numbering) with Phe; an alteration by substituting Lys at position
326 (EU numbering) with Val; an alteration by substituting Lys at
position 326 (EU numbering) with Tyr; an alteration by substituting
Ser at position 267 (EU numbering) with Asp; an alteration by
substituting Lys at position 326 (EU numbering) with Pro; an
alteration by substituting Lys at position 326 (EU numbering) with
His; an alteration by substituting Lys at position 334 (EU
numbering) with Ala; an alteration by substituting Lys at position
334 (EU numbering) with Trp; an alteration by substituting His at
position 268 (EU numbering) with Gln; an alteration by substituting
Lys at position 326 (EU numbering) with Gln; an alteration by
substituting Lys at position 326 (EU numbering) with Glu; an
alteration by substituting Lys at position 326 (EU numbering) with
Met; an alteration by substituting Val at position 266 (EU
numbering) with Ile; an alteration by substituting Lys at position
334 (EU numbering) with Glu; an alteration by substituting Tyr at
position 300 (EU numbering) with Glu; an alteration by substituting
Lys at position 334 (EU numbering) with Met; an alteration by
substituting Lys at position 334 (EU numbering) with Val; an
alteration by substituting Lys at position 334 (EU numbering) with
Thr; an alteration by substituting Lys at position 334 (EU
numbering) with Ser; an alteration by substituting Lys at position
334 (EU numbering) with His; an alteration by substituting Lys at
position 334 (EU numbering) with Phe; an alteration by substituting
Lys at position 334 (EU numbering) with Gln; an alteration by
substituting Lys at position 334 (EU numbering) with Pro; an
alteration by substituting Lys at position 334 (EU numbering) with
Tyr; an alteration by substituting Lys at position 334 (EU
numbering) with Ile; an alteration by substituting Gln at position
295 (EU numbering) with Leu; an alteration by substituting Lys at
position 334 (EU numbering) with Leu; an alteration by substituting
Lys at position 334 (EU numbering) with Asn; an alteration by
substituting His at position 268 (EU numbering) with Ala; an
alteration by substituting Ser at position 239 (EU numbering) with
Asp; an alteration by substituting Ser at position 267 (EU
numbering) with Ala; an alteration by substituting Leu at position
234 (EU numbering) with Trp; an alteration by substituting Leu at
position 234 (EU numbering) with Tyr; an alteration by substituting
Gly at position 237 (EU numbering) with Ala; an alteration by
substituting Gly at position 237 (EU numbering) with Asp; an
alteration by substituting Gly at position 237 (EU numbering) with
Glu; an alteration by substituting Gly at position 237 (EU
numbering) with Leu; an alteration by substituting Gly at position
237 (EU numbering) with Met; an alteration by substituting Gly at
position 237 (EU numbering) with Tyr; an alteration by substituting
Ala at position 330 (EU numbering) with Lys; an alteration by
substituting Ala at position 330 (EU numbering) with Arg; an
alteration by substituting Glu at position 233 (EU numbering) with
Asp; an alteration by substituting His at position 268 (EU
numbering) with Asp; an alteration by substituting His at position
268 (EU numbering) with Glu; an alteration by substituting Lys at
position 326 (EU numbering) with Asp; an alteration by substituting
Lys at position 326 (EU numbering) with Ser; an alteration by
substituting Lys at position 326 (EU numbering) with Thr; an
alteration by substituting Val at position 323 (EU numbering) with
Ile; an alteration by substituting Val at position 323 (EU
numbering) with Leu; an alteration by substituting Val at position
323 (EU numbering) with Met; an alteration by substituting Tyr at
position 296 (EU numbering) with Asp; an alteration by substituting
Lys at position 326 (EU numbering) with Ala; an alteration by
substituting Lys at position 326 (EU numbering) with Asn; and an
alteration by substituting Ala at position 330 (EU numbering) with
Met.
[0323] Favorable amino acid substitutions among these alterations
are, for example, an alteration by substituting Gly at position 237
(EU numbering) with Trp; an alteration by substituting Gly at
position 237 (EU numbering) with Phe; an alteration by substituting
Ser at position 267 (EU numbering) with Val; an alteration by
substituting Ser at position 267 (EU numbering) with Gln; an
alteration by substituting His at position 268 (EU numbering) with
Asn; an alteration by substituting Pro at position 271 (EU
numbering) with Gly; an alteration by substituting Lys at position
326 (EU numbering) with Leu; an alteration by substituting Lys at
position 326 (EU numbering) with Gln; an alteration by substituting
Lys at position 326 (EU numbering) with Glu; an alteration by
substituting Lys at position 326 (EU numbering) with Met; an
alteration by substituting Ser at position 239 (EU numbering) with
Asp; an alteration by substituting Ser at position 267 (EU
numbering) with Ala; an alteration by substituting Leu at position
234 (EU numbering) with Trp; an alteration by substituting Leu at
position 234 (EU numbering) with Tyr; an alteration by substituting
Gly at position 237 (EU numbering) with Ala; an alteration by
substituting Gly at position 237 (EU numbering) with Asp; an
alteration by substituting Gly at position 237 (EU numbering) with
Glu; an alteration by substituting Gly at position 237 (EU
numbering) with Leu; an alteration by substituting Gly at position
237 (EU numbering) with Met; an alteration by substituting Gly at
position 237 (EU numbering) with Tyr; an alteration by substituting
Ala at position 330 (EU numbering) with Lys; an alteration by
substituting Ala at position 330 (EU numbering) with Arg; an
alteration by substituting Glu at position 233 (EU numbering) with
Asp; an alteration by substituting His at position 268 (EU
numbering) with Asp; an alteration by substituting His at position
268 (EU numbering) with Glu; an alteration by substituting Lys at
position 326 (EU numbering) with Asp; an alteration by substituting
Lys at position 326 (EU numbering) with Ser; an alteration by
substituting Lys at position 326 (EU numbering) with Thr; an
alteration by substituting Val at position 323 (EU numbering) with
Ile; an alteration by substituting Val at position 323 (EU
numbering) with Leu; an alteration by substituting Val at position
323 (EU numbering) with Met; an alteration by substituting Tyr at
position 296 (EU numbering) with Asp; an alteration by substituting
Lys at position 326 (EU numbering) with Ala; an alteration by
substituting Lys at position 326 (EU numbering) with Asn; and an
alteration by substituting Ala at position 330 (EU numbering) with
Met.
[0324] The above-mentioned alteration may be at one position, or
alterations at two or more positions may be combined. Favorable
examples of such alterations are those described in Tables 13 to
14, Tables 16 to 23, and Tables 25 to 27.
[0325] Fc region produced by altering the Fc.gamma.R-binding domain
included in the Fc region presented as human IgG1 (SEQ ID NO: 14),
IgG2 (SEQ ID NO: 15), IgG3 (SEQ ID NO: 16), or IgG4 (SEQ ID NO: 17)
can be given as an example of another non-limiting embodiment of
the selective Fc.gamma.R-binding domain included in the
antigen-binding molecules of the present invention. A method for
producing the modified Fc regions is, for example, the method
described in the above-mentioned section on amino acid alterations.
Examples of such altered Fc regions include an Fc region in which
amino acid at position 238 (EU numbering) is Asp and amino acid at
position at 271 (EU numbering) is Gly in a human IgG (IgG1, IgG2,
IgG3, or IgG4). An Fc region in which amino acid at position 238
(EU numbering) is Asp and amino acid at position at 271 (EU
numbering) is Gly in a human IgG (IgG1, IgG2, IgG3, or IgG4), and
antigen-binding molecules containing such an Fc region show higher
binding activity to Fc.gamma.RIIb-1 and/or Fc.gamma.RIIb-2 than to
Fc.gamma.RIa, Fc.gamma.RIb, Fc.gamma.RIc, Fc.gamma.RIIIa including
allotype V158, Fc.gamma.RIIIa including allotype F158,
Fc.gamma.RIIIb including allotype Fc.gamma.RIIIb-NA1,
Fc.gamma.RIIIb including allotype Fc.gamma.RIIIb-NA2, Fc.gamma.RIIa
including allotype H131, Fc.gamma.RIIa including allotype R131,
and/or Fc.gamma.RIIc.
[0326] In the present invention, at least another alteration to the
Fc region may be added to the Fc region in which amino acid at
position 238 (EU numbering) is Asp and the amino acid at position
271 (EU numbering) is Gly, by the embodiments and such described in
the aforementioned section on amino acid alterations. In addition
to these alterations, additional alterations may also be added. The
additional alterations can be selected from any of substitutions,
deletions, and modifications of an amino acid, and combinations
thereof. For example, alterations that maintain or reduce binding
activity to activating Fc.gamma. receptors (Fc.gamma.RIa,
Fc.gamma.RIb, Fc.gamma.RIc, Fc.gamma.RIIIa including allotype V158,
Fc.gamma.RIIIa including allotype F158, Fc.gamma.RIIIb including
allotype Fc.gamma.RIIIb-NA1, Fc.gamma.RIIIb including allotype
Fc.gamma.RIIIb-NA2, Fc.gamma.RIIa including allotype H131,
Fc.gamma.RIIa including allotype R131) can be added. Alterations
that enhance binding activity to inhibitory Fc.gamma. receptors
(Fc.gamma.RIIb-1 and/or Fc.gamma.RIIb-2) while maintaining or
reducing binding activity to Fc.gamma.RIIa (H type) and
Fc.gamma.RIIa (R type) may be added. Furthermore, alterations where
the degree of enhancement of binding activity to inhibitory
Fc.gamma. receptors (Fc.gamma.RIIb-1 and/or Fc.gamma.RIIb-2) is
higher than the degree of enhancement of binding activity to
activating Fc.gamma. receptors (Fc.gamma.RIa, Fc.gamma.RIb,
Fc.gamma.RIc, Fc.gamma.RIIIa including allotype V158,
Fc.gamma.RIIIa including allotype F158, Fc.gamma.RIIIb including
allotype Fc.gamma.RIIIb-NA1, Fc.gamma.RIIIb including allotype
Fc.gamma.RIIIb-NA2, Fc.gamma.RIIa including allotype H131,
Fc.gamma.RIIa including allotype R131) may also be added. Addition
of such alterations improves binding selectivity to Fc.gamma.RIIb
over Fc.gamma.RIIa.
[0327] An example of a non-limiting embodiment of the altered Fc
region comprising a selective Fc.gamma.R-binding domain includes an
altered Fc region in which at least one or more amino acid selected
from the group consisting of those at positions 233, 234, 237, 264,
265, 266, 267, 268, 269, 272, 274, 296, 326, 327, 330, 331, 332,
333, 355, 356, 358, 396, 409, and 419 (EU numbering) are
substituted in the Fc region in which amino acid at position 238
(EU numbering) is Asp and amino acid at position 271 (EU numbering)
is Gly in a human IgG (IgG1, IgG2, IgG3, or IgG4).
[0328] In addition, an example of a non-limiting embodiment of the
altered Fc region comprising a selective Fc.gamma.R-binding domain
is an altered Fc region comprising any one or more of
Asp at amino acid position 233, Tyr at amino acid position 234, Asp
at amino acid position 237, Ile at amino acid position 264, Glu at
amino acid position 265, any one of Phe, Met, and Leu at amino acid
position 266, any one of Ala, Glu, Gly, and Gln at amino acid
position 267, any one of Asp, Glu, and Gln at amino acid position
268, Asp at amino acid position 269, any one of Asp, Phe, Ile, Met,
Asn, Pro, and Gln at amino acid position 272, Gln at 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 or Arg at amino
acid position 330, Ser at amino acid position 331, Thr at amino
acid position 332, any one of Thr, Lys, and Arg at amino acid
position 333, Gln at amino acid position 355, Glu at amino acid
position 356, Met at amino acid position 358, any one of Asp, Glu,
Phe, Ile, Lys, Leu, Met, Gln, Arg, and Tyr at amino acid position
396, Arg at amino acid position 409, Glu at amino acid position
419, shown by EU numbering, in the Fc region in which amino acid at
position 238 is Asp and amino acid at position 271 (EU numbering)
is Gly in a human IgG (IgG1, IgG2, IgG3, or IgG4).
[0329] Examples of a non-limiting embodiment of Fc region which
further comprises at least another alteration to the Fc region and
further comprises additional alterations mentioned above include Fc
regions shown in Tables 5-1 to 5-7.
TABLE-US-00005 TABLE 5-1 ALTERED Fc REGION ALTERED AMINO ACID(EU
NUMBERING) BP208 E233D/G237D/P238D/H268D/P271G/A330R BP209
G237D/P238D/H268D/P271G/K326A/A330R BP210
G237D/P238D/H268D/P271G/A330R BP211
E233D/P238D/H268D/P271G/K326A/A330R BP212
E233D/P238D/H268D/P271G/Y296D/A330R BP213
E233D/P238D/H268D/P271G/A330R BP214
E233D/L234Y/G237D/P238D/Y296D/K326D/A330K BP215
G237D/P238D/H268D/P271G/Y296D/A330K BP216
G237D/P238D/S267Q/H268D/P271G/A330K BP217
G237D/P238D/S267Q/H268D/P271G/Y296D/A330K BP218
G237D/P238D/H268D/P271G/K326D/A330K BP219
L234Y/G237D/P238D/H268D/P271G/A330K BP220
E233D/G237D/P238D/H268D/P271G/Y296D/A330K BP221
L234Y/G237D/P238D/Y296D/K326A/A330R BP222
L234Y/G237D/P238D/P271G/K326A/A330R BP223
L234Y/G237D/P238D/H268D/P271G/K326A/A330R BP224
L234Y/G237D/P238D/S267Q/H268D/P271G/K326A/A330R BP225
L234Y/G237D/P238D/K326D/A330R BP226
L234Y/G237D/P238D/P271G/K326D/A330R BP227
L234Y/G237D/P238D/H268D/P271G/K326D/A330R BP228
L234Y/G237D/P238D/S267Q/H268D/P271G/K326D/A330R BP229
E233D/L234Y/G237D/P238D/P271G/K326A/A330R BP230
E233D/G237D/P238D/H268D/P271G/Y296D/A330R BP231
G237D/P238D/H268D/P271G/Y296D/A330R BP232
L234Y/G237D/P238D/P271G/K326A/A330K BP233
L234Y/G237D/P238D/P271G/A330K BP234
E233D/L234Y/G237D/P238D/S267Q/H268D/P271G/Y296D/K326D/A330K BP235
E233D/L234Y/G237D/P238D/H268D/P271G/Y296D/K326D/A330R BP236
E233D/L234Y/G237D/P238D/S267Q/H268D/P271G/Y296D/K326D/A330R BP237
E233D/L234Y/G237D/P238D/S267Q/H268D/P271G/Y296D/K326A/A330K
(Table 5-2 is a continuation table of Table 5-1.)
TABLE-US-00006 TABLE 5-2 ALTERED Fc REGION ALTERED AMINO ACID (EU
NUMBERING) BP238
E233D/L234Y/G237D/P238D/H268D/P271G/Y296D/K326A/A330R BP239
E233D/L234Y/G237D/P238D/S267Q/H268D/P271G/Y296D/K326A/A330R BP240
E233D/G237D/P238D/S267Q/H268D/P271G/A330R BP241
E233D/G237D/P238D/H268D/P271G/K326D/A330R BP242
E233D/G237D/P238D/H268D/P271G/K326A/A330R BP243
E233D/L234Y/G237D/P238D/H268D/P271G/A330R BP244
E233D/G237D/P238D/S267Q/H268D/P271G/Y296D/A330R BP245
E233D/G237D/P238D/S267Q/H268D/P271G/Y296D/K326D/A330R BP246
E233D/G237D/P238D/S267Q/H268D/P271G/Y296D/K326A/A330R BP247
E233D/G237D/P238D/H268D/P271G/Y296D/K326D/A330R BP248
E233D/G237D/P238D/H268D/P271G/Y296D/K326A/A330R BP249
E233D/L234Y/G237D/P238D/H268D/P271G/Y296D/A330R BP262
G237D/P238D/H268E/P271G BP264
E233D/G237D/P238D/H268E/P271G/Y296D/A330R BP265
G237D/P238D/H268E/P271G/Y296D/A330R BP266
E233D/G237D/P238D/H268E/P271G/A330R BP267
E233D/G237D/P238D/H268E/P271G BP268
E233D/G237D/P238D/H268E/P271G/Y296D BP269
G237D/P238D/H268E/P271G/Y296D BP300
E233D/G237D/P238D/V264I/H268E/P271G BP313
E233D/G237D/P238D/D265E/H268E/P271G BP333
E233D/G237D/P238D/V266F/H268E/P271G BP338
E233D/G237D/P238D/V266L/H268E/P271G BP339
E233D/G237D/P238D/V266M/H268E/P271G BP348
E233D/G237D/P238D/S267A/H268E/P271G BP350
E233D/G237D/P238D/S267E/H268E/P271G BP352
E233D/G237D/P238D/S267G/H268E/P271G BP367
E233D/G237D/P238D/H268E/E269D/P271G BP384
E233D/G237D/P238D/H268D/P271G/Y296D/A330R/K334R BP390
E233D/G237D/P238D/H268D/P271G/Y296D/A330R/I332S BP391
E233D/G237D/P238D/H268D/P271G/Y296D/A330R/I332T
(Table 5-3 is a continuation table of Table 5-2.)
TABLE-US-00007 TABLE 5-3 ALTERED Fc REGION ALTERED AMINO ACID (EU
NUMBERING) BP392 E233D/G237D/P238D/H268D/P271G/Y296D/A330R/I332K
BP393 E233D/G237D/P238D/H268D/P271G/Y296D/A330R/I332R BP423
E233D/G237D/P238D/S267A/H268E/P271G/A330R BP425
E233D/G237D/P238D/V266L/S267A/H268E/P271G/A330R BP426
E233D/G237D/P238D/S267A/H268E/E269D/P271G/A330R BP427
E233D/G237D/P238D/S267A/H268E/E269Y/P271G/A330R BP428
E233D/G237D/P238D/S267G/H268E/P271G/A330R BP429
E233D/G237D/P238D/V264I/S267G/H268E/P271G/A330R BP430
E233D/G237D/P238D/V266L/S267G/H268E/P271G/A330R BP431
E233D/G237D/P238D/S267G/H268E/E269D/P271G/A330R BP432
E233D/G237D/P238D/S267G/H268E/E269Y/P271G/A330R BP433
E233D/G237D/P238D/H268D/P271G/Y296D/A330K/I332T BP434
E233D/G237D/P238D/H268D/P271G/Y296D/K326D/A330R/I332T BP435
E233D/G237D/P238D/H268D/P271G/Y296D/K326A/A330R/I332T BP436
E233D/G237D/P238D/S267A/H268E/P271G/Y296D/A330R/I332T BP437
G237D/P238D/S267A/H268E/P271G/Y296D/A330R/I332T BP438
E233D/G237D/P238D/S267A/H268E/P271G/A330R/I332T BP439
E233D/G237D/P238D/V264I/V266L/S267A/H268E/P271G/A330R BP440
E233D/G237D/P238D/V264I/H268E/P271G/A330R BP441
E233D/G237D/P238D/V266L/H268E/P271G/A330R BP442
E233D/G237D/P238D/H268E/E269D/P271G/A330R BP443
E233D/G237D/P238D/V266L/H268E/E269D/P271G/A330R BP444
E233D/G237D/P238D/H268E/E269N/P271G/A330R BP445
E233D/G237D/P238D/V264I/S267A/H268E/P271G/A330R BP446
E233D/G237D/P238D/S267A/H268E/E269N/P271G/A330R BP447
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396A BP448
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396D BP449
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396E BP450
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396F BP451
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396G BP452
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396H
(Table 5-4 is a continuation table of Table 5-3.)
TABLE-US-00008 TABLE 5-4 ALTERED Fc REGION ALTERED AMINO ACID (EU
NUMBERING) BP453 E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396I
BP454 E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396K BP455
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396L BP456
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396M BP457
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396N BP458
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396Q BP459
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396R BP460
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396S BP461
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396T BP462
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396V BP463
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396W BP464
E233D/G237D/P238D/S267A/H268E/P271G/A330R/P396Y BP465
E233D/G237D/P238D/H268D/P271G/Y296D/A330R/E333K BP466
E233D/G237D/P238D/H268D/P271G/Y296D/A330R/E333R BP467
E233D/G237D/P238D/H268D/P271G/Y296D/A330R/E334S BP468
E233D/G237D/P238D/H268D/P271G/Y296D/A330R/E334T BP469
E233D/G237D/P238D/H268D/P271G/Y296D/A330R/E333S BP470
E233D/G237D/P238D/H268D/P271G/Y296D/A330R/E333T BP471
E233D/G237D/P238D/H268D/P271G/Y296D/A330R/P331S BP472
E233D/G237D/P238D/H268D/P271G/Y296D/A330S BP473
E233D/G237D/P238D/H268D/P271G/Y296D/A327G/A330R BP474
E233D/G237D/P238D/H268D/P271G/Y296D/A330R/P331S BP475
E233D/G237D/P238D/H268D/P271G/Y296D/A327G/A330S BP476
E233D/G237D/P238D/H268D/P271G/Y296D/A327G/A330S/P331S BP477
E233D/G237D/P238D/H268D/P271G/Y296D/A327G/A330R/P331S BP478
E233D/G237D/P238D/H268D/P271G/Y296D/A330R +
S131C/K133R/G137E/G138S/Q196K/I199T/N203D/K214R/P217S + 219-221
DELETION + K222Y/T223G/H224P/T225P BP479
E233D/G237D/P238D/V264I/V266L/S267A/H268E/P271G BP480
E233D/G237D/P238D/V266L/H268E/E269D/P271G BP481
E233D/G237D/P238D/V264I/S267A/II268E/P271G
(Table 5-5 is a continuation table of Table 5-4.)
TABLE-US-00009 TABLE 5-5 ALTERED Fc REGION ALTERED AMINO ACID (EU
NUMBERING) BP482 E233D/G237D/P238D/S267A/H268E/E269N/P271G BP483
E233D/G237D/P238D/V266L/S267A/H268E/P271G BP484
E233D/G237D/P238D/S267A/H268E/E269D/P271G BP485
E233D/G237D/P238D/S267A/H268E/E269Y/P271G BP487
E233D/G237D/P238D/V264I/S267A/H268E/P271G/A330R/P396M BP488
E233D/G237D/P238D/V264I/S267A/H268E/P271G/Y296D/A330R BP489
E233D/G237D/P238D/V264I/S267A/H268E/P271G/Y296D/A330R/P396M BP490
G237D/P238D/V264I/S267A/H268E/P271G/A330R BP491
G237D/P238D/V264I/S267A/H268E/P271G/Y296D/A330R BP492
P238D/V264I/S267A/H268E/P271G BP493
P238D/V264I/S267A/H268E/P271G/Y296D BP494
G237D/P238D/S267A/H268E/P271G/Y296D/A330R BP495
G237D/P238D/S267G/H268E/P271G/Y296D/A330R BP496
E233D/G237D/P238D/V264I/S267A/H268E/P271G/Y296D BP497
E233D/G237D/P238D/V264I/S267A/H268E/P271G/A327G/A330R BP498
E233D/G237D/P238D/V264I/S267A/H268E/P271G/A330R/P396L BP499
E233D/G237D/P238D/V264I/S267A/H268E/P271G/Y296D/A330R/P396L BP500
G237D/P238D/V264I/S267A/H268E/P271G/Y296D BP501
G237D/P238D/V264I/S267A/H268E/P271G BP502
E233D/G237D/P238D/V264I/S267A/H268E/P271G/Y296D/A327G/A330R BP503
E233D/G237D/P238D/V264I/S267A/H268E/P271G/Y296D/A327G/A330R/P396M
BP504 E233D/G237D/P238D/V264I/S267A/H268E/P271G/E272P BP505
E233D/G237D/P238D/V264I/S267A/H268E/P271G/E272D BP506
E233D/G237D/P238D/V264I/S267A/H268E/P271G/E272P/Y296D/A330R BP507
E233D/G237D/P238D/V264I/S267A/H268E/P271G/E272P/A330R BP508
E233D/G237D/P238D/V264I/S267A/H268E/P271G/E272P/Y296D BP509
E233D/G237D/P238D/V264I/S267A/H268E/P271G/E272D/Y296D BP510
G237D/P238D/V264I/S267A/H268E/P271G/E272P/A330R BP511
G237D/P238D/V264I/S267A/H268E/P271G/E272P/Y296D/A330R BP513
E233D/G237D/P238D/H268E/E272D/P271G
(Table 5-6 is a continuation table of Table 5-5.)
TABLE-US-00010 TABLE 5-6 ALTERED Fc REGION ALTERED AMINO ACIDS (EU
NUMBERING) BP514 E233D/G237D/P238D/H268E/E272F/P271G BP517
E233D/G237D/P238D/H268E/E272I/P271G BP520
E233D/G237D/P238D/H268E/E272M/P271G BP521
E233D/G237D/P238D/H268E/E272N/P271G BP523
E233D/G237D/P238D/H268E/E272Q/P271G BP531
E233D/G237D/P238D/V264I/S267G/H268E/P271G/Y296D/A330R/P396M BP532
E233D/G237D/P238D/V264I/H268E/P271G/Y296D/A330R/P396M BP533
E233D/G237D/P238D/V264I/S267G/H268E/P271G/Y296D/A330R/P396L BP534
E233D/G237D/P238D/V264I/H268E/P271G/Y296D/A330R/P396L BP535
E233D/G237D/P238D/V264I/S267G/H268E/P271G/Y296D/A327G/A330R/P396M
BP536 E233D/G237D/P238D/V264I/H268E/P271G/Y296D/A327G/A330R/P396M
BP537 G237D/P238D/V264I/S267G/H268E/P271G/A330R BP538
G237D/P238D/V264I/H268E/P271G/A330R BP539
G237D/P238D/V264I/S267G/H268E/P271G/E272P/Y296D/A330R BP540
G237D/P238D/V264I/H268E/P271G/E272P/Y296D/A330R BP549
G237D/P238D/S267G/H268E/P271G/A330R BP550
G237D/P238D/V264I/S267G/H268E/P271G/E272D/Y296D/A330R BP551
G237D/P238D/V264I/H268E/P271G/E272D/Y296D/A330R BP552
E233D/G237D/P238D/V264I/S267A/H268E/P271G/E272D/Y296D/A330R BP553
E233D/G237D/P238D/V264I/S267A/H268E/P271G/E272D/A330R BP554
G237D/P238D/V264I/S267A/H268E/P271G/E272D/A330R BP555
G237D/P238D/V264I/S267A/H268E/P271G/E272D/Y296D/A330R BP556
G237D/P238D/V264I/S267G/H268E/P271G/Y296D/A330R BP557
G237D/P238D/S267G/H268D/P271G/Y296D/A330R BP558
G237D/P238D/V264I/S267G/H268E/P271G/E272D/A330R BP559
P238D/V264I/S267A/H268E/P271G/E272D/Y296D BP560
P238D/S267G/H268E/P271G/Y296D/A330R BP561
E233D/G237D/P238D/H268D/P271G/E272D/Y296D/A330R BP562
G237D/P238D/H268D/P271G/E272D/Y296D/A330R BP563
E233D/G237D/P238D/H268E/P271G/E272D/Y296D/A330R
(Table 5-7 is a continuation table of Table 5-6.)
TABLE-US-00011 TABLE 5-7 ALTERED Fc REGION ALTERED AMINO ACIDS (EU
NUMBERING) BP564 G237D/P238D/H268E/P271G/E272D/Y296D/A330R BP565
E233D/G237D/P238D/S267A/H268E/P271G/Y296D/A330R BP567
E233D/P238D/V264I/S267A/H268E/P271G/Y296D BP568
E233D/P238D/V264I/S267A/H268E/P271G
Antigen-Binding Molecule
[0330] In the present invention, "an antigen-binding molecule" is
used in the broadest sense to refer to a molecule comprising an
antigen-binding domain whose antigen-binding activity varies
depending on ion concentration conditions, an FcRn-binding domain
having FcRn-binding activity under an acidic pH range condition,
and an Fc.gamma. receptor-binding domain having selective binding
activity to an Fc.gamma. receptor (a selective Fc.gamma.R-binding
domain). Specifically, the antigen-binding molecules include
various types of molecules as long as they exhibit antigen-binding
activity. Antibodies are examples of molecules in which an
antigen-binding domain whose antigen-binding activity varies
depending on ion concentration conditions, an FcRn-binding domain
having FcRn-binding activity under an acidic pH range condition,
and an Fc.gamma. receptor-binding domain having selective binding
activity to an Fc.gamma. receptor (a selective Fc.gamma.R-binding
domain) are linked together. Antibodies may include single
monoclonal antibodies (including agonistic antibodies and
antagonistic antibodies), human antibodies, humanized antibodies,
chimeric antibodies, and such. Alternatively, when used as antibody
fragments, they preferably include antigen-binding domains and
antigen-binding fragments (for example, Fab, F(ab')2, scFv, and
Fv). Scaffold molecules where three dimensional structures, such as
already-known stable .alpha./.beta. barrel protein structure, are
used as a scaffold (base) and only some portions of the structures
are made into libraries to construct antigen-binding domains are
also included in antigen-binding molecules of the present
invention.
[0331] An antigen-binding molecule of the present invention may
contain at least some portions of an Fc region that mediates the
binding to FcRn and binding to Fc.gamma. receptor and/or complement
receptor. In a non-limiting embodiment, the antigen-binding
molecule includes, for example, antibodies and Fc fusion proteins.
A fusion protein refers to a chimeric polypeptide comprising a
polypeptide having a first amino acid sequence that is linked to a
polypeptide having a second amino acid sequence that would not
naturally link in nature. For example, a fusion protein may
comprise the amino acid sequence of at least a portion of an Fc
region (for example, a portion of an Fc region responsible for the
binding to FcRn, a portion of an Fc region responsible for the
binding to Fc.gamma. receptor, or a portion of an Fc region
responsible for the binding to complement) and a non-immunoglobulin
polypeptide containing, for example, the amino acid sequence of the
ligand-binding domain of a receptor or a receptor-binding domain of
a ligand. The amino acid sequences may be present in separate
proteins that are transported together to a fusion protein, or
generally may be present in a single protein; however, they are
included in a new rearrangement in a fusion polypeptide. Fusion
proteins can be produced, for example, by chemical synthesis, or by
genetic recombination techniques to express a polynucleotide
encoding peptide regions in a desired arrangement.
[0332] Respective domains of the present invention such as the
antigen-binding domain, the FcRn-binding domain having FcRn-binding
activity under an acidic pH range condition, and the Fc.gamma.
receptor-binding domain having selective binding activity to an
Fc.gamma. receptor (selective Fc.gamma.R-binding domain), can be
linked together via linkers or directly via polypeptide bonds. The
linkers comprise arbitrary peptide linkers that can be introduced
by genetic engineering, synthetic linkers, and linkers disclosed
in, for example, Protein Engineering (1996) 9(3), 299-305. However,
peptide linkers are preferred in the present invention. The length
of the peptide linkers is not particularly limited, and can be
suitably selected by those skilled in the art according to the
purpose. The length is preferably five amino acids or more (without
particular limitation, the upper limit is generally 30 amino acids
or less, preferably 20 amino acids or less), and particularly
preferably 15 amino acids.
[0333] For example, such peptide linkers preferably include:
TABLE-US-00012 Ser Gly.cndot.Ser Gly.cndot.Gly.cndot.Ser
Ser.cndot.Gly.cndot.Gly (SEQ ID NO: 28)
Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID NO: 29)
Ser.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID NO: 30)
Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID NO: 31)
Ser.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID NO: 32)
Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID NO:
33) Ser.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID
NO: 34)
Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser
(SEQ ID NO: 35)
Ser.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly
(Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID NO: 30))n
(Ser.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID NO: 31))n
where n is an integer of 1 or larger. The length or sequences of
peptide linkers can be selected accordingly by those skilled in the
art depending on the purpose.
[0334] Synthetic linkers (chemical crosslinking agents) is
routinely used to crosslink peptides, and for example: N-hydroxy
succinimide (NHS), disuccinimidyl suberate (DSS),
bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidyl
propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP),
ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol
bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl
tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),
bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), and
bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).
These crosslinking agents are commercially available.
[0335] When multiple linkers for linking the respective domains are
used, they may all be of the same type, or may be of different
types.
[0336] In addition to the linkers exemplified above, linkers with
peptide tags such as His tag, HA tag, myc tag, and FLAG tag may
also be suitably used. Furthermore, hydrogen bonding, disulfide
bonding, covalent bonding, ionic interaction, and properties of
binding with each other as a result of combination thereof may be
suitably used. For example, the affinity between CH1 and CL of
antibody may be used, and Fc regions originating from the
above-described bispecific antibodies may also be used for hetero
Fc region association. Moreover, disulfide bonds formed between
domains may also be suitably used.
[0337] In order to link the respective domains via peptide linkage,
polynucleotides encoding the domains are linked in frame. Known
methods for linking polynucleotides in frame include techniques
such as ligation of restriction fragments, fusion PCR, and
overlapping PCR. Such methods can be appropriately used alone or in
combination to produce the antigen-binding molecules of the present
invention. In the present invention, the terms "linked" and
"fused", or "linkage" and "fusion" are used interchangeably. These
terms mean that two or more elements or components such as
polypeptides are linked together to form a single structure by any
means including the above-described chemical linking means and
recombination techniques. When two or more domains, elements, or
components are polypeptides, linking in frame means linking two or
more units of reading frames to form a longer continuous reading
frame while maintaining the correct reading frames of the
polypeptides. When two molecules of Fab are used as an
antigen-binding domain, an antibody, which is an antigen-binding
molecule of the present invention where the antigen-binding domain
is linked in frame via peptide bonds without a linker to an
FcRn-binding domain having FcRn-binding activity under an acidic pH
range condition and the Fc.gamma. receptor-binding domain having
selective binding activity to an Fc.gamma. receptor (selective
Fc.gamma.R-binding domain), may be used as a preferred
antigen-binding molecule of the present application. Examples of
non-limiting multiple embodiments of the antigen-binding molecule
of the present invention including the antibody structure are shown
below:
(1) an antibody which comprises F(ab')2 comprising two variable
regions and having antigen-binding activity that varies depending
on ion concentration conditions and an Fc region having
FcRn-binding activity under an acidic pH range condition and
selective binding activity to an Fc.gamma. receptor; (2) an
antibody which comprises F(ab')2 wherein one of the variable
regions forming F(ab')2 has antigen-binding activity that varies
depending on ion concentration conditions and the other variable
region has selective binding activity to an Fc.gamma. receptor, and
an Fc region having FcRn-binding activity under an acidic pH range
condition; and (3) an antibody which comprises F(ab')2 wherein one
of the variable regions forming F(ab')2 has antigen-binding
activity that varies depending on ion concentration conditions and
the other variable region has FcRn-binding activity under an acidic
pH range condition, and an Fc region having selective binding
activity to an Fc.gamma. receptor.
[0338] When an antibody comprises the above-mentioned structure of
(3), a variable region having FcRn-binding activity that varies
depending on pH conditions can be used preferably as the variable
region having FcRn-binding activity under an acidic pH range
condition. Without being bound by a particular theory, if a
variable region whose FcRn-binding activity varies depending on pH
conditions is used and if the variable region does not have
FcRn-binding activity under a neutral pH range condition, the
antibody is released from FcRn at the cell surface when the
antibody bound to FcRn in the acidic endosome is transported to the
cell surface and can be easily recycled into the plasma.
Bispecific Antibodies and Methods for Producing them
[0339] Methods for producing bispecific antibodies may be applied
as an embodiment of the method for preparing antibodies comprising
the structures of (2) and (3) mentioned above. Bispecific
antibodies are antibodies comprising two types of variable regions
that bind specifically to different epitopes. IgG-type bispecific
antibodies can be secreted from a hybrid hybridoma (quadroma)
produced by fusing two types of hybridomas that produce IgG
antibodies (Milstein et al., Nature (1983) 305, 537-540).
[0340] When a bispecific antibody is produced by using
recombination techniques such as those described in the
above-mentioned section on antibodies, one may adopt a method that
introduces genes encoding heavy chains containing the two types of
variable regions of interest into cells to co-express them.
However, even when only the heavy-chain combination is considered,
such a co-expression method will produce a mixture of (i) a
combination of a pair of heavy chains in which one of the heavy
chains contains a variable region that binds to a first epitope and
the other heavy chain contains a variable region that binds to a
second epitope, (ii) a combination of a pair of heavy chains which
include only heavy chains containing a variable region that binds
to the first epitope, and (iii) a combination of a pair of heavy
chains which include only heavy chains containing a variable region
that binds to the second epitope, which are present at a molecular
ratio of 2:1:1. It is difficult to purify antigen-binding molecules
containing the desired combination of heavy chains from the mixture
of three types of heavy chain combinations.
[0341] When producing bispecific antibodies using recombination
techniques such as described above, bispecific antibodies
comprising the hetero combination of heavy chains can be
preferentially secreted by altering the CH3 domain that constitutes
a heavy chain using appropriate amino acid substitutions.
Specifically, it is a method of enhancing heterogeneous heavy chain
formation and inhibiting homogeneous heavy chain formation by
substituting amino acid side chain in one heavy chain CH3 domain
with a bulker side chain (knob (meaning "projection")) while
substituting amino acid side chain in the other heavy chain CH3
domain with a smaller side chain (hole (meaning "void")) so that
the "knob" is placed in the "hole" (WO 1996/027011, Ridgway et al.
(Protein Engineering (1996) 9, 617-621), Merchant et al. (Nat.
Biotech. (1998) 16, 677-681)).
[0342] Furthermore, known techniques for producing bispecific
antibodies include those in which a means for regulating
polypeptide association or association to form heteromeric
multimers constituted by polypeptides is applied to the association
of heavy chains. Specifically, to produce bispecific antibodies,
one can use methods for regulating heavy chain association by
altering amino acid residues forming interface between heavy chains
so as to form two heavy chains with different sequences, while
inhibiting the association of heavy chains having an identical
sequence (WO 2006/106905). Such methods can be used to produce
bispecific antibodies.
[0343] In a non-limiting embodiment of the present invention, two
polypeptides constituting an Fc region derived from a bispecific
antibody described above can be suitably used as the Fc region
contained in an antigen-binding molecule. More specifically, two
polypeptides constituting an Fc region may be suitably used, in
which, of the amino acid sequence of one of the polypeptides, the
amino acid at position 349 as indicated by EU numbering is Cys and
the amino acid at position 366 is Trp, and of the amino acid
sequence of the other of the polypeptides, the amino acid at
position 356 as indicated by EU numbering is Cys, the amino acid at
position 366 is Ser, the amino acid at position 368 is Ala, and the
amino acid at position 407 is Val.
[0344] In another non-limiting embodiment of the present invention,
two polypeptides constituting an Fc region, in which, of the amino
acid sequence of one of the polypeptides, the amino acid at
position 409 according to EU numbering is Asp, and of the amino
acid sequence of the other of the polypeptides, the amino acid at
position 399 according to EU numbering is Lys, may be suitably used
as the Fc region. In the above embodiment, the amino acid at
position 409 may be Glu instead of Asp, and the amino acid at
position 399 may be Arg instead of Lys. Moreover, in addition to
the amino acid Lys at position 399, Asp may suitably be added as
amino acid at position 360 or Asp may suitably be added as amino
acid at position 392.
[0345] In still another non-limiting embodiment of the present
invention, two polypeptides constituting an Fc region, in which, of
the amino acid sequence of one of the polypeptides, the amino acid
at position 370 according to EU numbering is Glu, and of the amino
acid sequence of the other of the polypeptides, the amino acid at
position 357 according to EU numbering is Lys, may be suitably used
as the Fc region.
[0346] In yet another non-limiting embodiment of the present
invention, two polypeptides constituting an Fc region, in which, of
the amino acid sequence of one of the polypeptides, the amino acid
at position 439 according to EU numbering is Glu, and of the amino
acid sequence of the other of the polypeptides, the amino acid at
position 356 according to EU numbering is Lys, may be suitably used
as the Fc region.
[0347] In still yet another non-limiting embodiment of the present
invention, any of the embodiments indicated below, in which the
above have been combined, may be suitably used as the Fc
region:
(i) two polypeptides constituting an Fc region, in which, of the
amino acid sequence of one of the polypeptides, the amino acid at
position 409 according to EU numbering is Asp and the amino acid at
position 370 is Glu, and of the amino acid sequence of the other of
the polypeptides, the amino acid at position 399 according to EU
numbering is Lys and the amino acid at position 357 is Lys (in this
embodiment, the amino acid at position 370 according to EU
numbering may be Asp instead of Glu, and the amino acid Asp at
position 392 according to EU numbering may be used instead of the
amino acid Glu at position 370 according to EU numbering); (ii) two
polypeptides constituting an Fc region, in which, of the amino acid
sequence of one of the polypeptides, the amino acid at position 409
according to EU numbering is Asp and the amino acid at position 439
is Glu, and of the amino acid sequence of the other of the
polypeptides, the amino acid at position 399 according to EU
numbering is Lys and the amino acid at position 356 is Lys (in this
embodiment, the amino acid Asp at position 360 according to EU
numbering, the amino acid Asp at position 392 according to EU
numbering, or the amino acid Asp at position 439 according to EU
numbering may be used instead of the amino acid Glu at position 439
according to EU numbering); (iii) two polypeptides constituting an
Fc region, in which, of the amino acid sequence of one of the
polypeptides, the amino acid at position 370 according to EU
numbering is Glu and the amino acid at position 439 is Glu, and of
the amino acid sequence of the other of the polypeptides, the amino
acid at position 357 according to EU numbering is Lys and the amino
acid at position 356 is Lys; and two polypeptides constituting an
Fc region, in which, of the amino acid sequence of one of the
polypeptides, the amino acid at position 409 according to EU
numbering is Asp, the amino acid at position 370 is Glu, and the
amino acid at position 439 is Glu, and of the amino acid sequence
of the other of the polypeptides, the amino acid at position 399
according to EU numbering is Lys, the amino acid at position 357 is
Lys, and the amino acid at position 356 is Lys (in this embodiment,
the amino acid at position 370 according to EU numbering may not be
substituted to Glu, and furthermore, when the amino acid at
position 370 is not substituted to Glu, the amino acid at position
439 may be Asp instead of Glu, or the amino acid Asp at position
392 may be used instead of the amino acid Glu at position 439).
[0348] Further, in another non-limiting embodiment of the present
invention, two polypeptides constituting an Fc region, in which, of
the amino acid sequence of one of the polypeptides, the amino acid
at position 356 according to EU numbering is Lys, and of the amino
acid sequence of the other of the polypeptides, the amino acid at
position 435 according to EU numbering is Arg and the amino acid at
position 439 is Glu, may also be suitably used.
[0349] In still another non-limiting embodiment of the present
invention, two polypeptides constituting an Fc region, in which, of
the amino acid sequence of one of the polypeptides, the amino acid
at position 356 according to EU numbering is Lys and the amino acid
at position 357 is Lys, and of the amino acid sequence of the other
of the polypeptides, the amino acid at position 370 according to EU
numbering is Glu, the amino acid at position 435 is Arg, and the
amino acid at position 439 is Glu, may also be suitably used.
[0350] Furthermore, in addition to the above-mentioned technique of
associating heterologous heavy chains, the CrossMab technology
which is known as a technology for associating heterologous light
chains, in which a light chain forming a variable region that binds
to a first epitope and a light chain forming a variable region that
binds to a second epitope are respectively associated with a heavy
chain forming a variable region that binds to the first epitope and
a heavy chain forming a variable region that binds to the second
epitope (Scaefer et al. (Proc. Natl. Acad. Sci. U.S.A. (2011) 108,
11187-11192)), may also be used to produce the antigen-binding
molecules provided by the present invention.
Improvement of Pharmacokinetics
[0351] In the present invention, the "ability to eliminate antigens
in plasma" refers to the ability to eliminate from the plasma
antigens that are present in the plasma when the antigen-binding
molecules are administered in vivo or when the antigen-binding
molecules are secreted in vivo. Accordingly, in the present
invention, "ability of antigen-binding molecules to eliminate
antigens in plasma is increased" means that when the
antigen-binding molecules are administered, the rate of antigen
elimination from plasma is accelerated as compared to when an
antigen-binding molecule comprising an antigen-binding domain whose
antigen-binding activity does not vary depending on ion
concentrations, an antigen-binding molecule comprising an
FcRn-binding domain without FcRn-binding activity under an acidic
pH range condition, or an antigen-binding molecule comprising an
Fc.gamma. receptor-binding domain without selective binding
activity to an Fc.gamma. receptor is administered. Whether or not
the ability of an antigen-binding molecule to eliminate antigens in
the plasma increased can be determined, for example, by
administering soluble antigens and the antigen-binding molecule in
vivo, and then measuring the plasma concentration of the soluble
antigen after administration. When the concentration of the soluble
antigens in the plasma is decreased after administration of the
soluble antigens and the antigen-binding molecules comprising an
antigen-binding domain whose antigen-binding activity varies
depending on ion concentration conditions, the FcRn-binding domain
having FcRn-binding activity under an acidic pH range condition,
and an Fc.gamma. receptor-binding domain having selective binding
activity to an Fc.gamma. receptor (a selective Fc.gamma.R-binding
domain), the ability of the antigen-binding molecules to eliminate
antigens in the plasma is judged to be increased. The soluble
antigen may be an antigen that is bound to an antigen-binding
molecule or an antigen that is not bound to an antigen-binding
molecule in the plasma, and its concentration can be determined as
a "plasma concentration of the antigen bound to the antigen-binding
molecule" or as a "plasma concentration of the antigen not bound to
the antigen-binding molecule", respectively (the latter is
synonymous with "free antigen concentration in plasma"). The "total
antigen concentration in plasma" means the sum of concentrations of
the antigen-binding molecule-bound antigen and the antigen not
bound by an antigen-binding molecule, or the "free antigen
concentration in plasma" which is the concentration of the antigen
not bound by an antigen-binding molecule. Thus, the soluble antigen
concentration can be determined as the "total antigen concentration
in plasma". Various methods for measuring the "total antigen
concentration in plasma" or the "free antigen concentration in
plasma" are well known in the art as described hereinafter.
[0352] In the present invention, "enhancement of pharmacokinetics",
"improvement of pharmacokinetics", and "superior pharmacokinetics"
can be restated as "enhancement of plasma (blood) retention",
"improvement of plasma (blood) retention", "superior plasma (blood)
retention", and "prolonged plasma (blood) retention". These terms
are synonymous.
[0353] In the present invention, "improvement of pharmacokinetics"
means not only prolongation of the period until elimination from
the plasma (for example, until the antigen-binding molecule is
degraded intracellularly or the like and cannot return to the
plasma) after administration of the antigen-binding molecule to
humans, or non-human animals such as mice, rats, monkeys, rabbits,
and dogs, but also prolongation of the plasma retention of the
antigen-binding molecule in a form that allows antigen binding (for
example, in an antigen-free form of the antigen-binding molecule)
during the period of administration to elimination due to
degradation. Human IgG having native Fc region can bind to FcRn
from non-human animals. For example, mouse can be preferably used
to be administered in order to confirm the property of the
antigen-binding molecule of the invention since human IgG having
native Fc region can bind to mouse FcRn stronger than to human FcRn
(Int Immunol. (2001) 13(12): 1551-1559). As another example, mouse
in which its native FcRn genes are disrupted and a transgene for
human FcRn gene is harbored to be expressed (Methods Mol Biol.
2010; 602: 93-104) can also be preferably used to be administered
in order to confirm the property of the antigen-binding molecule of
the invention described hereinafter. Specifically, "improvement of
pharmacokinetics" also includes prolongation of the period until
elimination due to degradation of the antigen-binding molecule not
bound to antigens (the antigen-free form of antigen-binding
molecule). The antigen-binding molecule in plasma cannot bind to a
new antigen if the antigen-binding molecule has already bound to an
antigen. Thus, the longer the period that the antigen-binding
molecule is not bound to an antigen, the longer the period that it
can bind to a new antigen (the higher the chance of binding to
another antigen). This enables reduction of the time period that an
antigen is free of the antigen-binding molecule in vivo and
prolongation of the period that an antigen is bound to the
antigen-binding molecule. The plasma concentration of the
antigen-free form of antigen-binding molecule can be increased and
the period that the antigen is bound to the antigen-binding
molecule can be prolonged by accelerating the antigen elimination
from the plasma by administration of the antigen-binding molecule.
Specifically, "improvement of the pharmacokinetics of
antigen-binding molecule" in the present invention includes the
improvement of a pharmacokinetic parameter of the antigen-free form
of the antigen-binding molecule (any of prolongation of the
half-life in plasma, prolongation of mean retention time in plasma,
and impairment of plasma clearance), prolongation of the period
that the antigen is bound to the antigen-binding molecule after
administration of the antigen-binding molecule, and acceleration of
antigen-binding molecule-mediated antigen elimination from the
plasma. The improvement of pharmacokinetics of antigen-binding
molecule can be assessed by determining any one of the parameters,
half-life in plasma, mean plasma retention time, and plasma
clearance for the antigen-binding molecule or the antigen-free form
thereof ("Pharmacokinetics: Enshu-niyoru Rikai (Understanding
through practice)" Nanzando). For example, the plasma concentration
of the antigen-binding molecule or antigen-free form thereof is
determined after administration of the antigen-binding molecule to
mice, rats, monkeys, rabbits, dogs, or humans. Then, each parameter
is determined. When the plasma half-life or mean plasma retention
time is prolonged, the pharmacokinetics of the antigen-binding
molecule can be judged to be improved. The parameters can be
determined by methods known to those skilled in the art. The
parameters can be appropriately assessed, for example, by
noncompartmental analysis using the pharmacokinetics analysis
software WinNonlin (Pharsight) according to the appended
instruction manual. The plasma concentration of antigen-free
antigen-binding molecule can be determined by methods known to
those skilled in the art, for example, using the assay method
described in Clin Pharmacol. 2008 April; 48 (4): 406-417.
[0354] In the present invention, "improvement of pharmacokinetics"
also includes prolongation of the period that an antigen is bound
to an antigen-binding molecule after administration of the
antigen-binding molecule. Whether the period that an antigen is
bound to the antigen-binding molecule after administration of the
antigen-binding molecule is prolonged can be assessed by
determining the plasma concentration of free antigen. The
prolongation can be judged based on the determined plasma
concentration of free antigen or the time period required for an
increase in the ratio of free antigen concentration to the total
antigen concentration.
[0355] The plasma concentration of free antigen not bound to the
antigen-binding molecule or the ratio of free antigen concentration
to the total concentration can be determined by methods known to
those skilled in the art, for example, by the method used in Pharm
Res. 2006 January; 23 (1): 95-103. Alternatively, when an antigen
exhibits a particular function in vivo, whether the antigen is
bound to an antigen-binding molecule that neutralizes the antigen
function (antagonistic molecule) can be assessed by testing whether
the antigen function is neutralized. Whether the antigen function
is neutralized can be assessed by assaying an in vivo marker that
reflects the antigen function. Whether the antigen is bound to an
antigen-binding molecule that activates the antigen function
(agonistic molecule) can be assessed by assaying an in vivo marker
that reflects the antigen function.
[0356] Determination of the plasma concentration of free antigen
and ratio of the amount of free antigen in plasma to the amount of
total antigen in plasma, in vivo marker assay, and such
measurements are not particularly limited; however, the assays are
preferably carried out after a certain period of time has passed
after administration of the antigen-binding molecule. In the
present invention, the period after administration of the
antigen-binding molecule is not particularly limited; those skilled
in the art can determine the appropriate period depending on the
properties and the like of the administered antigen-binding
molecule. Such periods include, for example, one day after
administration of the antigen-binding molecule, three days after
administration of the antigen-binding molecule, seven days after
administration of the antigen-binding molecule, 14 days after
administration of the antigen-binding molecule, and 28 days after
administration of the antigen-binding molecule. In the present
invention, the concept "plasma antigen concentration" comprises
both "total antigen concentration in plasma" which is the sum of
antigen-binding molecule bound antigen and non-bound antigen
concentration or "free antigen concentration in plasma" which is
antigen-binding molecule non-bound antigen concentration.
[0357] The total antigen concentration in the plasma can be lowered
by administration, as antigen-binding molecule, of the
antigen-binding molecule of the present invention by 2-fold,
5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold,
1,000-fold, or even higher as compared to administration of an
antigen-binding molecule containing an antigen-binding domain whose
antigen-binding activity is ion concentration-independent or an
antigen-binding molecule containing an Fc region with an impaired
Fc.gamma.R-binding activity, or compared to when the
antigen-binding domain molecule of the present invention is not
administered.
[0358] Molar antigen/antigen-binding molecule ratio can be
calculated as shown below:
value A: Molar antigen concentration at each time point value B:
Molar antigen-binding molecule concentration at each time point
value C: Molar antigen concentration per molar antigen-binding
molecule concentration (molar antigen/antigen-binding molecule
ratio) at each time point C=A/B.
[0359] Smaller value C indicates higher efficiency of antigen
elimination per antigen-binding molecule whereas higher value C
indicates lower efficiency of antigen elimination per
antigen-binding molecule.
[0360] Molar antigen/antigen-binding molecule ratio can be
calculated as described above.
[0361] A 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold,
200-fold, 500-fold, 1,000-fold or even greater reduction of molar
antigen/antigen-binding molecule ratio can be achieved by
administration of an antigen-binding molecule of the present
invention as compared to when an antigen-binding molecule
comprising an antigen-binding domain whose antigen-binding activity
does not vary depending on ion concentrations, an antigen-binding
molecule comprising an FcRn-binding domain without FcRn-binding
activity under an acidic pH range condition, or an antigen-binding
molecule comprising an Fc.gamma. receptor-binding domain without
selective binding activity to an Fc.gamma. receptor is administered
as the antigen-binding molecule.
[0362] In the present invention, an antigen-binding molecule
comprising an antigen-binding domain whose antigen-binding activity
does not vary depending on ion concentrations, an antigen-binding
molecule comprising an FcRn-binding domain without FcRn-binding
activity under an acidic pH range condition, or an antigen-binding
molecule comprising an Fc.gamma. receptor-binding domain without
selective binding activity to an Fc.gamma. receptor is used as a
reference antigen-binding molecule to be compared with the
antigen-binding molecules of the present invention.
[0363] When evaluating the effect of an FcRn-binding domain having
FcRn-binding activity under an acidic pH range condition, reduction
of total antigen concentration in plasma or molar antigen/antibody
ratio can be assessed by either antigen and antibody co-injection
model or steady-state antigen infusion model using human FcRn
transgenic mouse line 32 or line 276 (Jackson Laboratories, Methods
Mol. Biol. (2010) 602, 93-104), when the antigen-binding molecule
does not cross-react with the mouse counterpart antigen. When an
antigen-binding molecule cross-react with mouse counterpart, they
can be assessed by simply injecting antigen-binding molecule to
human FcRn transgenic mouse line 32 or line 276 (Jackson
Laboratories). In co-injection model, mixture of antigen-binding
molecule and antigen is administered to the mouse. In steady-state
antigen infusion model, infusion pump containing antigen solution
is implanted to the mouse to achieve constant plasma antigen
concentration, and then antigen-binding molecule is injected to the
mouse. Test antigen-binding molecule is administered at same
dosage. Total antigen concentration in plasma, free antigen
concentration in plasma and plasma antigen-binding molecule
concentration is measured at appropriate time point using method
known to those skilled in the art.
[0364] For assessing the effects of an Fc.gamma. receptor-binding
domain having selective binding activity to Fc.gamma. receptors,
when an antigen-binding molecule does not cross-react with a mouse
counterpart antigen, total antigen concentration in plasma or
decrease in antigen/antibody mole ratio can be assessed by either
the antigen-antibody simultaneous injection model or the
steady-state antigen injection model using the conventionally used
C57BL/6J mice (Charles River Japan). When an antigen-binding
molecule cross-reacts with the mouse counterpart, the
antigen-binding molecule can simply be injected to conventionally
used C57BL/6J mice (Charles River Japan) to carry out the
assessment.
[0365] In the co-injection model, a mixture of the antigen-binding
molecule and antigen is administered to mice. In the steady-state
antigen infusion model, an infusion pump filled with an antigen
solution is embedded into mice to achieve a constant plasma antigen
concentration, and then the antigen-binding molecule is injected
into the mice. Test antigen-binding molecules are administered at
the same dose. The total antigen concentration in plasma, free
antigen concentration in plasma, and antigen-binding molecule
concentration in plasma are measured at appropriate time points
using methods known to those skilled in the art.
[0366] Total or free antigen concentration in plasma and molar
antigen/antigen-binding molecule ratio can be measured at 2, 4, 7,
14, 28, 56, or 84 days after administration to evaluate the
long-term effect of the present invention. In other words, a long
term plasma antigen concentration is determined by measuring total
or free antigen concentration in plasma and molar
antigen/antigen-binding molecule ratio at 2, 4, 7, 14, 28, 56, or
84 days after administration of an antigen-binding molecule in
order to evaluate the property of the antigen-binding molecule of
the present invention. Whether the reduction of plasma antigen
concentration or molar antigen/antigen-binding molecule ratio is
achieved by antigen-binding molecule described in the present
invention can be determined by the evaluation of the reduction at
any one or more of the time points described above.
[0367] Total or free antigen concentration in plasma and molar
antigen/antigen-binding molecule ratio can be measured at 15
minutes, 1, 2, 4, 8, 12, or 24 hours after administration to
evaluate the short-term effect of the present invention. In other
words, a short term plasma antigen concentration is determined by
measuring total or free antigen concentration in plasma and molar
antigen/antigen-binding molecule ratio at 15 minutes, 1, 2, 4, 8,
12, or 24 hours after administration of an antigen-binding molecule
in order to evaluate the property of the antigen-binding molecule
of the present invention.
[0368] Route of administration of an antigen-binding molecule of
the present invention can be selected from intradermal,
intravenous, intravitreal, subcutaneous, intraperitoneal,
parenteral and intramuscular injection.
[0369] In the present invention, improvement of pharmacokinetics of
antigen-binding molecule in human is preferred. When the plasma
retention in human is difficult to determine, it may be predicted
based on the plasma retention in mice (for example, normal mice,
human antigen-expressing transgenic mice, human FcRn-expressing
transgenic mice) or monkeys (for example, cynomolgus monkeys).
[0370] "The improvement of the pharmacokinetics and prolonged
plasma retention of an antigen-binding molecule" in the present
invention means improvement of any pharmacokinetic parameter (any
of prolongation of the half-life in plasma, prolongation of mean
retention time in plasma, reduction of plasma clearance, and
bioavailability) after in vivo administration of the
antigen-binding molecule, or an increase in the concentration of
the antigen-binding molecule in the plasma in an appropriate time
after administration. It may be determined by measuring any
parameter such as half-life in plasma, mean retention time in
plasma, plasma clearance, and bioavailability of the
antigen-binding molecule (Pharmacokinetics: Enshu-niyoru Rikai
(Understanding through practice), (Nanzando)). For example, when an
antigen-binding molecule is administered to mice (normal mice and
human FcRn transgenic mice), rats, monkeys, rabbits, dogs, humans,
and so on, and the concentration of the antigen-binding molecule in
the plasma is determined and each of the parameters is calculated,
the pharmacokinetics of the antigen-binding molecule can be judged
to be improved when the plasma half-life or mean retention time in
the plasma is prolonged. These parameters can be determined by
methods known to those skilled in the art. For example, the
parameters can be appropriately assessed by non-compartmental
analysis using pharmacokinetics analysis software WinNonlin
(Pharsight) according to the attached instruction manual.
[0371] Four types of Fc.gamma.Rs, Fc.gamma.RI, Fc.gamma.RIIb,
Fc.gamma.RIII, and Fc.gamma.RIV, have been identified in mice. In
humans as well, as corresponding Fc.gamma.Rs, Fc.gamma.RI,
Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIIa, Fc.gamma.RIIIa, and
Fc.gamma.RIIIb have been identified. Fc.gamma.RIIb which is
considered to be the only inhibitory type among these Fc.gamma.Rs
is conserved in both humans and mice. The other Fc.gamma.Rs, except
for Fc.gamma.RIIIb, transmit activation signals via the
immunoreceptor tyrosine-based activating motif (ITAM), whereas
Fc.gamma.RIIb transmits inhibitory signals via the immunoreceptor
tyrosine-based inhibitory motif (ITIM) present inside the cells
(Nat. Rev. Immunol. (2008) 8, 34-47).
[0372] Fc.gamma.RIIb1 and Fc.gamma.RIIb2 have been reported as
splicing variants of Fc.gamma.RIIb. In both humans and mice,
Fc.gamma.RIIb1 has a longer intracellular domain than
Fc.gamma.RIIb2. Fc.gamma.RIIb1 has been confirmed to be expressed
in B cells, and Fc.gamma.RIIb2 has been confirmed to be expressed
in macrophages, mast cells, dendritic cells, basophils,
neutrophils, and eosinophils (J. Clin. Immunol. (2005) 25 (1),
1-18).
[0373] So far, in humans, dysfunction and decreased expression of
Fc.gamma.RIIb have been reported to be correlated with onset of
autoimmune diseases. For example, it has been reported that in some
SLE patients, binding of transcriptional activators is attenuated
due to polymorphism in an expression promoter region of
Fc.gamma.RIIb, which results in the decreased Fc.gamma.RIIb
expression (Hum. Genet. (2005) 117, 220-227; J. Immunol. (2004)
172, 7192-7199; and J. Immunol. (2004) 172, 7186-7191).
Furthermore, among SLE patients, two types of allotypes have been
reported, where the amino acid at position 233 is Ile or Thr in
Fc.gamma.RIIb. This position exists in the transmembrane region of
Fc.gamma.RIIb, and it is reported that Fc.gamma.RIIb is less likely
to exist in the lipid raft when the amino acid at position 233 is
Thr compared to when this amino acid is Ile, and as a result,
signal transduction function of Fc.gamma.RIIb decreases (Nat. Med.
(2005) 11, 1056-1058; Hum. Mol. Genet., (2005) 14, 2881-2892). In
mice as well, knockout mice produced by disrupting the
Fc.gamma.RIIb gene in C57BL/6 mice has been reported to present
SLE-like symptoms such as autoantibody production and
glomerulonephritis (Immunity 13 (2000) 277-285; J. Exp. Med. (2002)
195, 1167-1174). Furthermore, so far, reduced expression level of
Fc.gamma.RIIb and such have been reported in mice considered to be
models with natural onset of SLE (Immunogenetics (2000) 51,
429-435; Int. Immunol. (1999) 11, 1685-1691; Curr. Biol. (2000) 10,
227-230; J. Immunol. (2002) 169, 4340-4346). From these reports,
Fc.gamma.RIIb is considered to regulate humoral immunity in mice as
in humans.
[0374] When an antibody carrying an Fc of the present invention
eliminates antigens via Fc.gamma.RIIb, the endocytosis function of
Fc.gamma.RIIb is considered to be making the most important
contribution among the functions of Fc.gamma.RIIb. As described
above, Fc.gamma.RIIb1 and Fc.gamma.RIIb2 exist as splicing variants
of Fc.gamma.RIIb, but it is reported that the latter is mainly
involved in the endocytosis of an immune complex of an antibody and
antigen (J. Immunol. (1994), 152 574-585; Science (1992) 256,
1808-1812; Cell (1989) 58, 317-327). So far, mouse Fc.gamma.RIIb2
has been reported to be incorporated into a clathrin-coated pit and
endocytosed (Cell (1989) 58, 317-327). Furthermore, it has been
reported that a dileucine motif is necessary for
Fc.gamma.RIIb2-mediated endocytosis, and the dileucine motif is
conserved in both humans and mice (EMBO J. (1994) 13 (13),
2963-2969). From these, Fc.gamma.RIIb2 may have an endocytotic
ability in humans as in mice.
[0375] On the other hand, unlike Fc.gamma.RIIb2, it has been
reported that Fc.gamma.RIIb1 does not cause endocytosis.
Fc.gamma.RIIb1 has an inserted sequence in its intracellular domain
that is not found in Fc.gamma.RIIb2. It is considered that this
sequence inhibits the uptake of Fc.gamma.RIIb1 into a
clathrin-coated pit, and as a result endocytosis is inhibited (J.
Cell. Biol. (1992) 116, 875-888; J. Cell. Biol. (1989) 109,
3291-3302). In humans as well, Fc.gamma.RIIb1 has an insertion
sequence at a site similar to that of Fc.gamma.RIIb2 as in mice;
therefore, difference in the endocytotic ability between
Fc.gamma.RIIb1 and Fc.gamma.RIIb2 is presumed to be caused by a
similar mechanism. Furthermore, in both humans and mice,
approximately 40% of immune complexes on the cell surface is
reported to be taken up into the cell in 20 minutes (Mol. Immunol.
(2011) 49, 329-337; Science (1992) 256, 1808-1812). Therefore, in
humans as well, Fc.gamma.RIIb2 is presumed to uptake immune
complexes into cells at rates similar to those in mice.
[0376] Since Fc.gamma.RIIb is the only one that has ITIM inside the
cell in both humans and mice among the Fc.gamma.R family and the
distribution of expressing cells are the same, it is presumed that
its function in immune control is similar. Furthermore, considering
the fact that immune complexes are taken up into cells at similar
rates in humans and mice, antigen elimination effects of antibodies
mediated by Fc.gamma.RIIb in humans may be predictable using mice.
Antigen-binding molecules mF44 and mF46 have properties of binding
to soluble antigens in a pH-dependent manner, and have enhanced
affinity to mouse Fc.gamma.RIIb and Fc.gamma.RIII compared to mIgG1
which is an antigen-binding molecule having the property of binding
to a soluble antigen in a pH-dependent manner. Indeed, it is shown
in Example 5 that antigen clearance increased when mF44 or mF46 was
administered to normal mice compared to when mIgG1 was
administered.
[0377] Furthermore, in the later-described Example 6, a similar
experiment was carried out using Fc receptor .gamma.
chain-deficient mice. It has been reported that Fc.gamma.Rs other
than Fc.gamma.RIIb are expressed only in the co-presence of a gamma
chain in mice. Thus, only Fc.gamma.RIIb is expressed in the Fc
receptor .gamma. chain-deficient mice. Administration of mF44 or
mF46, which are antigen-binding molecules having the property of
binding to soluble antigens in a pH-dependent manner, to Fc
receptor .gamma. chain-deficient mice enables assessment of antigen
elimination-acceleration effects when Fc.gamma.RIIb-binding is
selectively enhanced. From the results of Example 6, when mF44 or
mF46 (which are antigen-binding molecules having the property of
binding to soluble antigens in a pH-dependent manner) was
administered to Fc receptor .gamma. chain-deficient mice, antigen
clearance was shown to increase compared to when mIgG1 (which is an
antigen-binding molecule having the property of binding to soluble
antigens in a pH-dependent manner) was administered to the mice.
Furthermore, the results of Example 6 shows that when administered
to Fc receptor .gamma. chain-deficient mice, mF44 or mF46 cause
similar degrees of antigen elimination as when administered to
normal mice.
[0378] In Example 6, a similar experiment was performed using
Fc.gamma.RIII-deficient mice. Since mIgG1, mF44, and mF46 bind only
to Fc.gamma.RIIb and Fc.gamma.RIII among the mFc.gamma.Rs,
administration of the antibodies to Fc.gamma.RIII-deficient mice
enables assessment of antigen elimination-accelerating effects when
Fc.gamma.RIIb-binding is selectively enhanced. The results of
Example 6 indicate that when mF44 or mF46 was administered to
Fc.gamma.RIII-deficient mice, antigen clearance was increased
compared to when mIgG1 was administered to the mice antigen
clearance. Furthermore, the results of Example 6 showed that when
administered to Fc.gamma.RIII-deficient mice, mF44 and mF46 cause
similar degrees of antigen elimination as when administered to Fc
receptor .gamma. chain-deficient mice and when administered to
normal mice.
[0379] These results revealed that antigen elimination can be
accelerated by enhancing selective binding to Fc.gamma.RIIb alone
without enhancing binding to active Fc.gamma.Rs.
[0380] In addition to the reported documents discussed so far,
based on the above-mentioned assessment results using mice, it is
considered that uptake of immune complexes into cells via
Fc.gamma.RIIb takes place in vivo in humans as in mice, and as a
result, antibodies that have Fc with selectively enhanced binding
to human Fc.gamma.RIIb can accelerate elimination of its antigens.
Furthermore, as discussed above, since uptake of immune complexes
into cells via Fc.gamma.RIIb is considered to take place at similar
rates in mice and humans, effects of accelerating antigen
elimination comparable to those of antibodies having Fc with
enhanced affinity to mouse Fc.gamma.RIIb may be achieved in vivo in
humans by using Fc in which affinity to human Fc.gamma.RIIb is
enhanced to a similar extent.
[0381] As described in WO 2009/125825, Fv4-IgG1 is an antibody that
results from conferring to a humanized anti-IL-6 receptor antibody
H54/L28-IgG1 the activity to bind to the antigen in a pH-dependent
manner, i.e., altering the variable region to confer the property
to bind to an antigen at pH 7.4 and dissociate from the antigen at
pH 5.8. WO 2009/125825 showed that the elimination of soluble human
IL-6 receptor is greatly accelerated in mice co-administered with
Fv4 IgG1 and soluble human IL-6 receptor as the antigen as compared
to mice co-administered with H54/L28-IgG1 and the antigen. Herein,
heavy-chain H54-IgG1 and light-chain L28-CK included in
H54/L28-IgG1 are shown in SEQ ID NO: 36 and SEQ ID NO: 37,
respectively; and heavy chain VH3-IgG1 and light-chain VL3-CK
included in Fv4-IgG1 are shown in SEQ ID NO: 38 and SEQ ID NO: 39,
respectively.
[0382] Soluble human IL-6 receptor bound to an antibody
H54/L28-IgG1, which binds to soluble human IL-6 receptor, is
recycled to the plasma along with the antibody via FcRn. Meanwhile,
antibody Fv4-IgG1 which binds to soluble human IL-6 receptor in a
pH-dependent manner dissociates from the soluble human IL-6
receptor that has been bound to the antibody under an acidic
condition in the endosome. Since the dissociated soluble human IL-6
receptor is degraded in the lysosome, elimination of the soluble
human IL-6 receptor can be greatly accelerated, and the antibody
Fv4-IgG1 which binds to the soluble human IL-6 receptor in a
pH-dependent manner is recycled to the plasma after binding to FcRn
in the endosome. Since the recycled antibody can bind to a soluble
human IL-6 receptor again, binding to the antigen (soluble human
IL-6 receptor) and recycling to the plasma via FcRn are repeated.
As a result, a single antibody molecule can repeatedly bind to the
soluble human IL-6 receptor multiple times (FIG. 1).
[0383] On the other hand, as disclosed in the present invention, it
was found that plasma concentration of the soluble antigen can be
reduced greatly by administration of an antigen-binding molecule
with enhanced Fc.gamma.R-binding activity of the Fc.gamma.
receptor-binding domain included in the antigen-binding molecule
which comprises an antigen-binding domain in which antigen-binding
activity changes depending on the ion concentration condition such
as pH, an FcRn-binding domain having FcRn-binding activity under an
acidic pH range condition, and an Fc.gamma. receptor-binding
domain.
[0384] While not being restricted to a particular theory, the
unexpected decrease in soluble antigen concentration in plasma
observed by administration of an antigen-binding molecule with
enhanced binding to Fc.gamma.Rs, which comprises an antigen-binding
domain in which antigen-binding activity changes depending on the
ion-concentration condition such as pH and an FcRn-binding domain
having FcRn-binding activity under an acidic pH range condition can
be explained as follows.
[0385] As described above, an antigen-binding molecule such as
Fv4-IgG1 comprising an antigen-binding domain in which
antigen-binding activity changes depending on the ion-concentration
condition may be able to bind repeatedly to the antigen multiple
times, but the effect of dissociating the soluble antigen in the
endosome to accelerate the antigen elimination from plasma may be
dependent on the rate of uptake of the complex of the antigen and
antigen-binding molecule into the endosome. The antigen-binding
molecules with enhanced binding activities to various Fc.gamma.Rs,
which comprise an antigen-binding domain in which antigen-binding
activity changes depending on the ion-concentration condition, are
actively taken up into cells by binding to various Fc.gamma.Rs
expressed on the cell membrane, and can circulate in the plasma
again by recycling via binding between FcRn and the FcRn-binding
domain in the molecule having binding activity to FcRn under an
acidic pH range condition. More specifically, since the
aforementioned antigen-binding molecules that formed complexes with
soluble antigens in plasma are taken up actively into cells via
Fc.gamma.Rs expressed on the cell membrane, the effect of
accelerating elimination of soluble antigens in plasma may be more
pronounced than that of antigen-binding molecules whose binding
activities to various Fc.gamma.Rs are not enhanced.
[0386] Fc.gamma.R-binding activities of antibodies that bind to
membrane antigens have an important role in cytotoxic activity of
the antibodies. Therefore, when cytotoxic activity is necessary for
an antibody to be used as a pharmaceutical, a human IgG1 isotype
which has high Fc.gamma.R-binding activity is used, and the
technique of enhancing the Fc.gamma.R-binding activities of the
antibody to enhance the cytotoxic activity of the antibody is
widely utilized. On the other hand, the role of Fc.gamma.R-binding
activities of antibodies that bind to soluble antigens and are used
as pharmaceuticals had not been known, and differences in
physiological effects on organisms administered with human IgG1
with high Fc.gamma.R-binding activities and human IgG2 and human
IgG4 with low Fc.gamma.R-binding activities, due to their
differences in Fc.gamma.R-binding activities, had not been fully
examined so far. As described later in the Examples, it was
actually confirmed that in the plasma of individuals administered
with antibodies whose Fc.gamma.R-binding activities have been lost,
changes in soluble-antigen concentration were not affected. On the
other hand, in the present invention, the concentration of soluble
antigens in the plasma was found to be greatly reduced in
individuals administered with antigen-binding molecules with
enhanced Fc.gamma.R-binding activities and comprising an
antigen-binding domain whose binding activity to soluble antigens
changes depending on the ion concentration condition. More
specifically, by combining an FcRn-binding domain having an
FcRn-binding activity under an acidic pH range condition and an
antigen-binding domain whose binding to soluble antigens changes
depending on the ion concentration condition, which are domains
included in antigen-binding molecules targeting soluble antigens,
an advantage of enhancing binding to Fc.gamma.R was found for the
first time.
Ex Vivo Method of Eliminating the Antigens from Plasma
[0387] An example of a non-limiting embodiment of the use of an
antigen-binding molecule for the method of eliminating the antigens
from plasma, which is provided by the present invention, includes
use of the antigen-binding molecule for a so-called ex vivo method
of eliminating the antigens from plasma, which comprises contacting
the antigen-binding molecule of the present invention with plasma
isolated from subjects to allow forming immune complexes, and
allowing the immune complexes to contact cells expressing Fc.gamma.
receptors and FcRn. The speed of antigen elimination from the
plasma can also be promoted by substituting/combining a method for
administering antigen-binding molecules in vivo with a so-called ex
vivo method, in which the plasma containing antigen-binding
molecules and antigens that bind to the antigen-binding molecules
is temporarily taken out of the body and then contacted with cells
expressing FcRn and Fc.gamma. receptors for a certain period of
time, and the plasma containing extracellularly recycled (or
re-secreted or re-circulated) antigen-binding molecules that are
not bound to antigen is returned to the body.
[0388] Furthermore, an example of a non-limiting embodiment of the
use of an antigen-binding molecule in the method provided by the
present invention for eliminating antigens from plasma includes use
of the antigen-binding molecule in a so-called ex vivo method for
eliminating antigens from the plasma, which includes contacting an
immune complex present in the plasma isolated from a subject to
whom the antigen-binding molecules of the present invention are
administered with cells expressing FcRn and Fc.gamma.
receptors.
[0389] Whether or not the antigen is eliminated from plasma can be
confirmed, for example, by assessing whether or not the rate of
antigen elimination in plasma is accelerated as compared to when an
antigen-binding molecule comprising an antigen-binding domain whose
antigen-binding activity does not vary depending on ion
concentrations, an antigen-binding molecule comprising an
FcRn-binding domain without FcRn-binding activity under an acidic
pH range condition, or an antigen-binding molecule comprising an
Fc.gamma. receptor-binding domain without selective binding
activity to an Fc.gamma. receptor is used as a control instead of
an antigen-binding molecule of the present invention.
Methods for Producing Antigen-Binding Molecules
[0390] The present invention provides a method for producing an
antigen-binding molecule having the function of eliminating
antigens in plasma, wherein the method comprises the steps of (a)
to (e) below:
(a) obtaining an antigen-binding domain whose antigen-binding
activity varies depending on ion concentration conditions; (b)
obtaining a gene encoding the antigen-binding domain selected in
step (a); (c) operably linking the gene obtained in step (b) with a
gene encoding an FcRn-binding domain having FcRn-binding activity
under an acidic pH range condition and an Fc.gamma.
receptor-binding domain having selective binding activity to an
Fc.gamma. receptor; (d) culturing host cells containing the gene
operably linked in step (c); and (e) isolating an antigen-binding
molecule from the culture solution obtained in step (d).
[0391] In a non-limiting embodiment of the present invention, after
isolating a polynucleotide encoding an antigen-binding domain whose
binding activity changes depending on the condition selected as
described above, the polynucleotide is inserted into an appropriate
expression vector. For example, when the antigen-binding domain is
an antibody variable region, once a cDNA encoding the variable
region is obtained, the cDNA is digested with restriction enzymes
that recognize the restriction sites inserted at the two ends of
the cDNA. Preferably, the restriction enzymes recognize and digest
a nucleotide sequence that appears at a low frequency in the
nucleotide sequence composing the gene of the antigen-binding
molecule. Furthermore, restriction enzymes that provide cohesive
ends are preferably inserted to insert a single copy of a digested
fragment into the vector in the correct orientation. The cDNA
encoding a variable region of an antigen-binding molecule digested
as described above is inserted into an appropriate expression
vector to obtain an expression vector for the antigen-binding
molecule of the present invention.
[0392] The polynucleotide encoding an antigen-binding domain
obtained as described above is operably linked to the gene encoding
an FcRn-binding domain having FcRn-binding activity under an acidic
pH range condition and an Fc.gamma. receptor-binding domain having
selective binding activity to an Fc.gamma. receptor, which are
described in the sections "FcRn-binding domain having FcRn-binding
activity under an acidic pH range condition" and "Fc.gamma.
receptor-binding domain having selective binding activity to an
Fc.gamma. receptor", respectively. When linking, the genes can be
directly linked in-frame, or the polynucleotides encoding each
domain may be linked in-frame via linkers. In addition to each of
the above-mentioned domains, it may be operably linked with a gene
encoding the Fc.gamma.R-binding domain described in the
above-mentioned section "Fc.gamma.R-binding domain".
[0393] When an antibody is used as the antigen-binding molecule of
the present invention, a polynucleotide encoding an antibody Fc
region may be used appropriately as the above-mentioned
"FcRn-binding domain having FcRn-binding activity under an acidic
pH range condition and Fc.gamma. receptor-binding domain having
selective binding activity to an Fc.gamma. receptor". Fc regions
whose "FcRn-binding activity under an acidic pH range condition"
and "selective binding activity to an Fc.gamma. receptor" are
appropriately modified through modification of the polynucleotides
may also be used. Examples of non-limiting embodiments of such
modifications are shown in the above-mentioned sections
"FcRn-binding domain having FcRn-binding activity under an acidic
pH range condition" and "Fc.gamma. receptor-binding domain having
selective binding activity to an Fc.gamma. receptor",
respectively.
[0394] To produce an antigen-binding molecule of interest, a
polynucleotide encoding the antigen-binding molecule is inserted in
a manner operably linked to a regulatory sequence into an
expression vector. Regulatory sequences include, for example,
enhancers and promoters. Furthermore, an appropriate signal
sequence may be linked to the amino terminus so that the expressed
antigen-binding molecule is secreted to the outside of the cells.
As signal sequence, for example, a peptide having the amino acid
sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 4) is used; however, it is
also possible to link other appropriate signal sequences. The
expressed polypeptide is cleaved at the carboxyl terminus of the
above-described sequence, and the cleaved polypeptide is secreted
as a mature polypeptide to the outside of cells. Then, appropriate
host cells are transformed with this expression vector so that
recombinant cells expressing the polynucleotide encoding the
antigen-binding molecule of interest can be obtained. The
antigen-binding molecules of the present invention can be produced
from the recombinant cells by following the methods described above
in the section on antibodies.
[0395] For a nucleic acid, "operably linked" means that the nucleic
acid has a functional relationship with another nucleic acid
sequence. For example, a DNA encoding a presequence or a secretory
leader is operably linked to a DNA encoding a certain polypeptide
if it is to be expressed as a precursor protein involved in the
secretion of the polypeptide. A promoter or enhancer is operably
linked to a coding sequence if it affects the transcription of the
coding sequence. A ribosome binding site is operably linked to a
coding sequence if it is in a position that facilitates
translation. Generally, "operably linked" means that the linked DNA
sequences are contiguous, and in the case of a secretory leader, it
means that the linked DNA sequences are contiguous and in a reading
frame. However, enhancers do not have to be contiguous. Linking is
accomplished by ligation at suitable restriction sites. If such
sites do not exist, synthetic oligonucleotide adaptors or linkers
are used in accordance with conventional practice. Furthermore,
linked nucleic acids may be produced by the above-mentioned overlap
extension PCR technique.
[0396] In a non-limiting embodiment of the present invention, after
isolating a polynucleotide encoding the above-described
antigen-binding molecule whose antigen-binding activity varies
depending on a selected condition, a variant of the polynucleotide
is inserted into an appropriate expression vector. Such variants
preferably include those prepared via humanization based on the
polynucleotide sequence encoding an antigen-binding molecule of the
present invention obtained by screening as a randomized variable
region library a synthetic library or an immune library constructed
originating from nonhuman animals. The same methods as described
above for producing above-described humanized antibodies can be
used as a method for producing humanized antigen-binding molecule
variants.
[0397] In another embodiment, such variants preferably include
those obtained by introducing an alteration that increases the
antigen affinity (affinity maturation) of an antigen-binding
molecule of the present invention into an isolated polynucleotide
sequence for the molecule obtained by screening using a synthetic
library or a naive library as a randomized variable region library.
Such variants can be obtained by various known procedures for
affinity maturation, including CDR mutagenesis (Yang et al. (J.
Mol. Biol. (1995) 254, 392-403)), chain shuffling (Marks et al.
(Bio/Technology (1992) 10, 779-783)), use of E. coli mutant strains
(Low et al. (J. Mol. Biol. (1996) 250, 359-368)), DNA shuffling
(Patten et al. (Curr. Opin. Biotechnol. (1997) 8, 724-733)), phage
display (Thompson et al. (J. Mol. Biol. (1996) 256, 77-88)), and
sexual PCR (Clameri et al. (Nature (1998) 391, 288-291)).
[0398] In an embodiment of variants of the present invention,
polynucleotides encoding antigen-binding molecules which have a
heavy chain where a polynucleotide encoding an Fc region modified
to have an amino acid mutation as described above is linked in
frame to a polynucleotide encoding the above-described
antigen-binding domain whose binding activity varies depending on a
selected condition.
[0399] The present invention provides methods for producing
antigen-binding molecules, comprising collecting the
antigen-binding molecules from culture media of cells introduced
with vectors in which a polynucleotide encoding an Fc region is
operably linked in frame to a polynucleotide encoding an
antigen-binding domain whose binding activity varies depending on
ion concentration condition. Furthermore, the present invention
also provides methods for producing antigen-binding molecules,
comprising collecting the antigen-binding molecules from culture
media of cells introduced with vectors constructed by operably
linking a polynucleotide encoding an antigen-binding domain whose
binding activity varies depending on ion concentration condition to
a polynucleotide encoding an Fc region which is in advance operably
linked to a vector.
[0400] In the "Methods for producing antigen-binding molecules" of
the present invention, known methods may be employed as methods for
assessing antigen elimination from plasma by the antigen-binding
molecules. An antigen-binding molecule of the present invention is
administered to each group of non-human animals such as mice at an
appropriate age in month. As described later in the section
"Pharmaceutical composition", antigen-binding molecule may be
systemically or locally administered by intravenous injection,
intramuscular injection, intraperitoneal injection, subcutaneous
injection, intracranial injection, or such, as compositions in the
dosage form for injections, transnasal administration,
transpulmonary administration, or transdermal administration.
[0401] Spectroscopic methods such as nuclear magnetic resonance
(NMR) or mass spectrometry (MS) analyses including SELDI(-TOF),
MALDI(-TOF), 1D gel-based analysis, 2D gel-based analysis, liquid
chromatography (for example, high-pressure liquid chromatography
(HPLC) or low-pressure liquid chromatography (LPLC)), thin layer
chromatography, and LC-MS-based techniques may be used to measure
the concentrations. Examples of appropriate LCMS techniques include
ICAT (registered trademark) (Applied Biosystems) and iTRAQ
(registered trademark) (Applied Biosystems). A method for detecting
antigen fragments that have been produced by further digestion of a
targeted antigen by an appropriate enzyme may also be employed when
appropriate. Furthermore, the antigen concentration may be measured
by a direct or indirect detection method. More specifically, the
antigen may be detected directly or indirectly via interaction with
a ligand or ligands such as enzymes, binding, receptors or
transport proteins, antibodies, peptides, aptamers or
oligonucleotides, or any synthetic chemical receptors or compounds
that can bind specifically to the antigen. The ligand can be
modified with a detectable label such as a luminescent label,
fluorescent label, or radioactive label, and/or an affinity tag. An
immunological method may be given as such an example.
[0402] A preferred measurement method may be, for example, an
immunological method that uses an antibody that binds to an epitope
present in the antigen. Examples of such an immunological method
include enzyme immunoassay (ELISA, EIA), fluoroimmunoassay (FIA),
radioimmunoassay (RIA), luminescence immunoassay (LIA), enzyme
antibody technique, fluorescent antibody technique,
immunochromatography method, immunoturbidimetry, latex
turbidimetry, and latex agglutination measurement method.
Furthermore, measurements in these immunological methods may be
carried out manually by hand or using a device such as an analyzer.
Immunological method in the present invention may be carried out
according to a known method such as the sandwich method. For
example, a first antibody immobilized onto a carrier is allowed to
react simultaneously or sequentially with a biological sample and a
second antibody modified by a labeling substance. The
above-mentioned reaction leads to formation of a complex comprising
the first antibody immobilized onto a carrier, the antigen, and a
second antibody modified by a labeling substance, and
quantification of the labeling substance linked to the second
antibody included in this complex enables measurement of the amount
(concentration) of the antigen included in the biological
sample.
[0403] For example, in the case of enzyme immunoassay, a microplate
onto which a first antibody is immobilized, serially diluted
biological samples, a secondary antibody modified by an enzyme such
as HRP, washing buffer, and a solution containing a substrate to
which an enzyme such as HRP reacts are preferably used. In a
non-limiting embodiment of the measurement, a substrate is allowed
to react under an optimal condition with the enzyme which modifies
the secondary antibody, and the amount of the enzyme reaction
product can be determined by an optical method. In the case of
fluoroimmunoassay, an optical waveguide onto which a first antibody
is immobilized, serially diluted biological samples, a secondary
antibody modified by a fluorescent substance, and washing buffer
are preferably used. In a non-limiting embodiment of the
measurement, the intensity of the fluorescence emitted by the
fluorescent substance through irradiation of excitation light onto
the fluorescent substance modifying the secondary antibody may be
measured.
[0404] Furthermore, in the case of radioimmunoassay, the amount of
radiation emitted by the radioactive substance is measured. In the
case of luminescence immunoassay, the amount of luminescence
emitted by the luminescent reaction system is measured.
Furthermore, in the case of immunoturbidimetry, latex turbidimetry,
latex agglutination measurement method, and such, transmitted light
or scattered light is measured by the end-point method or the rate
method. When immunochromatography measurements are made by visual
observation, the color of the labeled substance that appears on the
test line is determined by visual observation. Instead of such
measurement by visual observation, an instrument such as an
analyzer may be used when appropriate.
Pharmaceutical Composition
[0405] The present invention relates to pharmaceutical compositions
comprising antigen-binding molecules of the present invention,
antigen-binding molecules produced by alteration methods of the
present invention, or antigen-binding molecules produced by
production methods of the present invention. Antigen-binding
molecules of the present invention or antigen-binding molecules
produced by production methods of the present invention are useful
as pharmaceutical compositions since they, when administered, have
the strong effect to reduce the plasma antigen concentration as
compared to typical antigen-binding molecules, and exhibit the
improved in vivo immune response, pharmacokinetics, and others in
animals administered with the molecules. The pharmaceutical
compositions of the present invention may comprise pharmaceutically
acceptable carriers.
[0406] In the present invention, pharmaceutical compositions
generally refer to agents for treating or preventing, or
testing/diagnosing diseases.
[0407] The pharmaceutical compositions of the present invention can
be formulated by methods known to those skilled in the art. For
example, they can be used parenterally, in the form of injections
of sterile solutions or suspensions including water or other
pharmaceutically acceptable liquid. For example, such compositions
can be formulated by mixing in the form of unit dose required in
the generally approved medicine manufacturing practice, by
appropriately combining with pharmacologically acceptable carriers
or media, specifically with sterile water, physiological saline,
vegetable oil, emulsifier, suspension, surfactant, stabilizer,
flavoring agent, excipient, vehicle, preservative, binder, or such.
In such formulations, the amount of active ingredient is adjusted
to obtain an appropriate amount in a pre-determined range.
[0408] Sterile compositions for injection can be formulated using
vehicles such as distilled water for injection, according to
standard formulation practice. Aqueous solutions for injection
include, for example, physiological saline and isotonic solutions
containing dextrose or other adjuvants (for example, D-sorbitol,
D-mannose, D-mannitol, and sodium chloride). It is also possible to
use in combination appropriate solubilizers, for example, alcohols
(ethanol and such), polyalcohols (propylene glycol, polyethylene
glycol, and such), non-ionic surfactants (polysorbate 80.TM.,
HCO-50, and such).
[0409] Oils include sesame oil and soybean oils. Benzyl benzoate
and/or benzyl alcohol can be used in combination as solubilizers.
It is also possible to combine buffers (for example, phosphate
buffer and sodium acetate buffer), soothing agents (for example,
procaine hydrochloride), stabilizers (for example, benzyl alcohol
and phenol), and/or antioxidants. Appropriate ampules are filled
with the prepared injections.
[0410] The pharmaceutical compositions of the present invention are
preferably administered parenterally. For example, the compositions
in the dosage form for injections, transnasal administration,
transpulmonary administration, or transdermal administration are
administered. For example, they can be administered systemically or
locally by intravenous injection, intramuscular injection,
intraperitoneal injection, subcutaneous injection, or such.
[0411] Administration methods can be appropriately selected in
consideration of the patient's age and symptoms. The dose of a
pharmaceutical composition containing an antigen-binding molecule
can be, for example, from 0.0001 mg to 1000 mg/kg for each
administration. Alternatively, the dose can be, for example, from
0.001 to 100000 mg per patient. However, the present invention is
not limited by the numeric values described above. The doses and
administration methods vary depending on the patient's weight, age,
symptoms, and such. Those skilled in the art can set appropriate
doses and administration methods in consideration of the factors
described above.
[0412] Furthermore, the present invention provides kits for use in
the methods of the present invention, which comprise at least an
antigen-binding molecule of the present invention. In addition to
the above, pharmaceutically acceptable carriers, media, instruction
manuals describing the using method, and such may be packaged into
the kits.
[0413] Furthermore, the present invention relates to pharmaceutical
agents for eliminating, from the plasma, complexes containing two
or more antigenic binding units and two or more antigen-binding
molecules present in the plasma, which contain as an active
ingredient the antigen-binding molecules of the present invention
or the antigen-binding molecules produced by the production methods
of the present invention.
[0414] The present invention relates to methods for treating a
disease, which includes administering to subjects (patients, human
subjects, etc.) the antigen-binding molecules of the present
invention or the antigen-binding molecules produced by the
production methods of the present invention. A non-limiting example
of the disease includes cancer and inflammatory diseases.
[0415] The present invention also relates to use of the
antigen-binding molecules of the present invention or the
antigen-binding molecules produced by the production methods of the
present invention in the manufacture of a pharmaceutical agent for
eliminating from the plasma complexes containing two or more
antigenic binding units and two or more antigen-binding molecules
present in the plasma.
[0416] The present invention further relates to use of the
antigen-binding molecules of the present invention or the
antigen-binding molecules produced by the production methods of the
present invention for eliminating, from the plasma, complexes
containing two or more antigenic binding units and two or more
antigen-binding molecules present in the plasma.
[0417] In addition, the present invention relates to
antigen-binding molecules of the present invention and
antigen-binding molecules produced by the production methods of
present invention for use in the methods of the present
invention.
[0418] Amino acids contained in the amino acid sequences of the
present invention may be post-translationally modified (for
example, the modification of an N-terminal glutamine into a
pyroglutamic acid by pyroglutamylation is well-known to those
skilled in the art). Naturally, such post-translationally modified
amino acids are included in the amino acid sequences in the present
invention.
[0419] All prior art documents cited in the specification are
incorporated herein by reference.
[0420] Herein below, the present invention will be specifically
described with reference to the Examples, but it is not to be
construed as being limited thereto.
EXAMPLES
Example 1
Preparation of Antigen-Binding Molecules Whose Mouse
Fc.gamma.R-Binding Activity Under a Neutral pH Range Condition is
Higher than the Binding Activity of Native Human IgG Fc Region
[0421] (1-1) pH-Dependent Human IL-6 Receptor-Binding
Antibodies
[0422] H54/L28-IgG1 which comprises H54-IgG1 (SEQ ID NO: 36) and
L28-CK (SEQ ID NO: 37) described in WO2009/125825 is a humanized
anti-IL-6 receptor antibody. Meanwhile, Fv4-IgG1 which comprises
VH3-IgG1 (SEQ ID NO: 38) and VL3-CK (SEQ ID NO: 39) is a humanized
anti-IL-6 receptor antibody resulting from conferring, to
H54/L28-IgG1, the property of binding to soluble human IL-6
receptor in a pH-dependent manner (which binds at pH 7.4 and
dissociates at pH 5.8). The in vivo mouse test described in
WO2009/125825 demonstrated that, in the group administered with a
mixture of Fv4-IgG1 and soluble human IL-6 receptor as the antigen,
the elimination of soluble human IL-6 receptor from plasma was
significantly accelerated as compared to the group administered
with a mixture of H54/L28-IgG1 and soluble human IL-6 receptor as
the antigen.
[0423] The soluble human IL-6 receptor bound to H54/L28-IgG1, which
is an antibody that binds to a soluble human IL-6 receptor, is,
together with the antibody, recycled to plasma by FcRn. Meanwhile,
Fv4-IgG1, which is an antibody that binds to a soluble human IL-6
receptor in a pH dependent manner, dissociates soluble human IL-6
receptor under the acidic condition in the endosome. The
dissociated soluble human IL-6 receptor is degraded in the
lysosomes, thus this enables considerable acceleration of the
elimination of soluble human IL-6 receptor. Furthermore, after
binding to FcRn in the endosome, Fv4-IgG1, which is an antibody
that binds to a soluble human IL-6 receptor in a pH dependent
manner, is recycled to the plasma. Since the recycled antibody can
bind to soluble human IL-6 receptor again, the antibody repeatedly
binds to the antigen (soluble human IL-6 receptor) and is recycled
by FcRn to the plasma. It is thought that, as a result, a single
antibody molecule can bind repeatedly several times to soluble
human IL-6 receptor (FIG. 1).
(1-2) Preparation of an Anti-Human IL-6 Receptor Antibody with
Enhanced Mouse Fc.gamma.R Binding and Anti-Human IL-6 Receptor
Antibody without Mouse Fc.gamma.R Binding
[0424] VH3-IgG1-F1022 (SEQ ID NO: 40), an antigen-binding molecule
with enhanced mouse Fc.gamma.R binding, was prepared by
substituting Asp for Lys at position 326 (EU numbering) and Tyr for
Leu at position 328 (EU numbering) in VH3-IgG1. Fv4-IgG1-F1022
containing VH3-IgG1-F1022 as the heavy chain and VL3-CK as the
light chain was produced using the method described in Reference
Example 1.
[0425] Meanwhile, VH3-IgG1-F760 (SEQ ID NO: 41), an antigen-binding
molecule without mouse Fc.gamma.R binding, was prepared by
substituting Arg for Leu at position 235 and Lys for Ser at
position 239 (EU numbering) in VH3-IgG1. Fv4-IgG1-F760 containing
VH3-IgG1-F760 as the heavy chain and VL3-CK as the light chain was
produced using the method described in Reference Example 1.
(1-3) Assessment of Mouse Fc.gamma.R-Binding Activity
[0426] VH3/L(WT)-IgG1, VH3/L(WT)-IgG1-F1022, and
VH3/L(WT)-IgG1-F760, which contain VH3-IgG1, VH3-IgG1-F1022, and
VH3-IgG1-F760 as the heavy chain, and L(WT)-CK (SEQ ID NO: 42) as
the light chain, were produced using the method described in
Reference Example 1. These antibodies were kinetically analyzed for
their mouse Fc.gamma.R binding as described below.
(1-4) Kinetic Analysis of Mouse Fc.gamma.R Binding
[0427] The binding of antibodies to mouse Fc.gamma.RI,
Fc.gamma.RIIb, Fc.gamma.RIII, and Fc.gamma.RIV (hereinafter,
referred to as mouse Fc.gamma.Rs) (R & D systems,
SinoBiological, or prepared by the method described in Reference
Example 2) was kinetically analyzed using Biacore T100 and T200 (GE
Healthcare). An appropriate amount of protein L (ACTIGEN or
BioVision) was immobilized onto a Sensor chip CM4 (GE Healthcare)
by an amino coupling method, and antibodies of interest were
captured thereto. Then, diluted solutions of mouse Fc.gamma.Rs and
a running buffer as a blank were injected, and the mouse
Fc.gamma.Rs were allowed to interact with antibodies captured onto
the sensor chip. The running buffer used was 20 mmol/l ACES, 150
mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4. This buffer was also used
to dilute the mouse Fc.gamma.Rs. The sensor chip was regenerated
using 10 mmol/1 glycine-HCl, pH 1.5. All measurements were carried
out at 25.degree. C. The binding rate constant ka (l/Ms) and
dissociation rate constant kd (l/s), which are kinetic parameters,
were calculated from the sensorgrams obtained by the measurement.
KD (M) of each antibody for human Fc.gamma.R was calculated based
on the values. Each parameter was calculated using Biacore T100 or
T200 Evaluation Software (GE Healthcare).
[0428] The result shown in Table 6 was obtained by the measurement.
VH3/L (WT)-IgG1-F1022 was demonstrated to have increased binding
activity to mFc.gamma.RI, mFc.gamma.RII, and mFc.gamma.RIII as
compared to VH3/L (WT)-IgG1. Regarding VH3/L (WT)-IgG1-F760, the
binding to the various mouse Fc.gamma.Rs was undetectable,
demonstrating that VH3/L (WT)-IgG1-F760 lacks the binding activity
to the various mouse Fc.gamma.Rs. In the table, VH3/L (WT)-IgG1,
VH3/L (WT)-IgG1-F1022, and VH3/L (WT)-IgG1-F760 are shown as IgG1,
F1022, and F760, respectively.
TABLE-US-00013 TABLE 6 VARIANT KD (M) NAME mFc .gamma. RI mFc
.gamma. RII mFc .gamma. RIIII mFc .gamma. RIV IgG1 5.3E-08 9.8E-07
2.4E-06 8.6E-08 F1022 7.6E-09 1.0E-08 5.5E-09 1.4E-07 F760 NOT NOT
NOT NOT DETECTED DETECTED DETECTED DETECTED
(1-5) Preparation of Antibodies with Low Fucose Content
[0429] Known methods for increasing the Fc.gamma.R-binding activity
of antibodies include methods for making sugar chains linked to an
antibody be sugar chains with low fucose content (J. Biol. Chem.
(2003) 278, 3466-3473) in addition to methods for introducing an
amino acid alteration into the Fc region of an antibody. An
Fv4-IgG1 with low fucose content (hereinafter, abbreviated as
Fv4-IgG1-Fuc) was produced by expressing Fv4-IgG1 using fucose
transporter gene-deficient CHO cells (WO 2006/067913) as host cells
according to the method described in Reference Example 1. It has
been reported that, of the mFc.gamma.Rs (mouse Fc.gamma.
receptors), antibodies with low fucose content have selectively
increased Fc.gamma.RIV-binding activity (Science, 2005, 310 (5753)
1510-1512).
Example 2
Effect of Eliminating Antigens from Plasma by Antigen-Binding
Molecules Whose Fc.gamma.R-Binding Activity is Higher than the
Binding Activity of Native Human IgG Fc Region
[0430] (2-1) Effect of H54/L28-IgG1 and Fv4-IgG1 to Eliminate
Antigens from Plasma
[0431] H54/L28-IgG1, which is an anti-human IL-6 receptor antibody,
and Fv4-IgG1 having the property of binding to human IL-6 receptor
in a pH-dependent manner were produced by the method described in
Reference Example 1. In vivo infusion tests were carried out using
the produced H54/L28-IgG1 and Fv4-IgG1 by the method described
below.
(2-1-1) In Vivo Infusion Tests Using Human FcRn Transgenic Mice
[0432] An animal model in which the soluble human IL-6 receptor
concentration is maintained constant in plasma was created by
implanting an infusion pump (MINI-OSMOTIC PUMP MODEL2004, alzet)
containing soluble human IL-6 receptor under the skin on the back
of human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32+/+
mouse, Jackson Laboratories, Methods Mol Biol. (2010) 602, 93-104).
The in vivo dynamics after administration of an anti-human IL-6
receptor antibody was assessed in the animal model. To suppress the
production of neutralizing antibodies against soluble human IL-6
receptor, an anti-mouse CD4 monoclonal antibody (prepared by a
known method) was administered once at 20 mg/kg into the caudal
vein. Then, an infusion pump containing 92.8 .mu.g/ml soluble human
IL-6 receptor was subcutaneously implanted on the back of the mice.
Three days after implantation of the infusion pump, an anti-human
IL-6 receptor antibody was administered once at 1 mg/kg into the
caudal vein. The blood was collected from the mice 15 minutes,
seven hours, one day, two days, four days, and seven days after
administration of the anti-human IL-6 receptor antibody.
Immediately, the collected blood was centrifuged at 15,000 rpm and
4.degree. C. for 15 minutes to prepare plasma. The isolated plasma
was stored in a freezer set at -20.degree. C. or below until
use.
(2-1-2) Determination of the hsIL-6R Soluble Human IL-6 Receptor
Concentration in Plasma by an Electrochemiluminescent Method
[0433] The hsIL-6R soluble human IL-6 receptor concentrations in
mouse plasma were determined by an electrochemiluminescent method.
hsIL-6R soluble human IL-6 receptor standard curve samples prepared
at 2000, 1000, 500, 250, 125, 62.5, and 31.25 pg/ml and assay
samples of mouse plasma diluted 50 times or more were mixed with
Monoclonal Anti-human IL-6R Antibody (R&D), Biotinylated
Anti-human IL-6 R Antibody (R&D), Tocilizumab, which had been
ruthenated with SULFO-TAG NHS Ester (Meso Scale Discovery). The
mixtures were incubated at 37.degree. C. overnight. Tocilizumab was
prepared at a final concentration of 333 .mu.g/ml. Then, the
reaction mixtures were aliquoted in an MA400 PR Streptavidin Plate
(Meso Scale Discovery). The solution reacted at room temperature
for one hour was washed out, and then Read Buffer T (.times.4)
(Meso Scale Discovery) was aliquoted Immediately thereafter, the
measurement was carried out using SECTOR PR 400 Reader (Meso Scale
Discovery). The concentration of hsIL-6R soluble human IL-6
receptor was determined based on the response of the standard curve
using analysis software SOFTmax PRO (Molecular Devices).
[0434] A time course of the monitored human IL-6 receptor
concentration is shown in FIG. 2. As compared to H54/L28-IgG1,
Fv4-IgG1 that binds to human IL-6 receptor in a pH-dependent manner
could reduce the human IL-6 receptor concentration, but could not
reduce it below the baseline without antibody administration. That
is, the administered antibody which binds to an antigen in a
pH-dependent manner could not reduce the antigen concentration in
plasma below the level prior to antibody administration.
(2-2) The Effect of Eliminating an Antigen from Plasma by an
Antibody with Increased or Reduced Fc.gamma.R-Binding Activity
[0435] Whether the time course of human IL-6 receptor concentration
is influenced by increasing or reducing the Fc.gamma.R-binding
activity of Fv4-IgG1, which is a pH-dependent human IL-6
receptor-binding antibody, was assessed by the method described
below. Using Fv4-IgG1, Fv4-IgG1-F760, Fv4-IgG1-F1022, and
Fv4-IgG1-Fuc prepared as described in Example 1, in vivo infusion
tests were performed by the method described below.
(2-2-1) In Vivo Infusion Tests Using Human FcRn Transgenic Mice
[0436] A animal model in which the soluble human IL-6 receptor
concentration is maintained constant in plasma was created by
implanting an infusion pump (MINI-OSMOTIC PUMP MODEL2004, alzet)
containing soluble human IL-6 receptor under the skin on the back
of human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32+/+
mouse, Jackson Laboratories, Methods Mol Biol. (2010) 602, 93-104).
In the animal model, an anti-human IL-6 receptor antibody was
administered simultaneously with Sanglopor (CSL Behring) which is a
human immunoglobulin preparation, to assess the in vivo dynamics of
the soluble human IL-6 receptor after antibody administration. To
suppress the production of neutralizing antibodies against soluble
human IL-6 receptor, an anti-mouse CD4 monoclonal antibody
(prepared by a known method) was administered once at 20 mg/kg into
the caudal vein. Then, an infusion pump containing 92.8 .mu.g/ml
soluble human IL-6 receptor was subcutaneously implanted on the
back of the mice. Three days after implantation of the infusion
pump, an anti-human IL-6 receptor antibody and Sanglopor were
administered once at 1 mg/kg and 1000 mg/kg, respectively, into the
caudal vein. The blood was collected from the mice 15 minutes,
seven hours, one day, two days, four days, seven days, 14 days, and
21 days after administration of the anti-human IL-6 receptor
antibody. The blood was collected from the mice 15 minutes, seven
hours, one day, two days, three days, seven days, 14 days, and 21
days after administration of the anti-human IL-6 receptor antibody.
Immediately, the collected blood was centrifuged at 15,000 rpm and
4.degree. C. for 15 minutes to prepare the plasma. The isolated
plasma was stored in a freezer set at -20.degree. C. or below until
use.
(2-2-2) Determination of the Soluble Human IL-6 Receptor (hsIL-6R)
Concentration in Plasma by an Electrochemiluminescent Method
[0437] The hsIL-6R soluble human IL-6 receptor concentrations in
mouse plasma were determined by the same electrochemiluminescent
method as described in (2-1-2).
[0438] The result is shown in FIG. 3. The time course of human IL-6
receptor concentration in plasma of mice administered with
Fv4-IgG1-F760, from which the mouse Fc.gamma.R binding of Fv4-IgG1
is deleted, was demonstrated to be comparable to that in mice
administered with Fv4-IgG1. The cytotoxic activity to a membrane
antigen depends on the Fc.gamma.R binding, and thus the cytotoxic
activity is lost when eliminating the Fc.gamma.R binding. On the
other hand, even when administering an antibody, from which mouse
Fc.gamma.R binding is deleted, against human IL-6 receptor which is
a soluble antigen, there was no effect on the time course of human
IL-6 receptor concentration in the plasma of the administered mice.
Thus, it would be thought that the Fc.gamma.R binding of an
antibody against the soluble antigen has no contribution to the
time course of antigen concentration in the plasma of mice
administered with the antibody.
[0439] Surprisingly, however, the human IL-6 receptor concentration
in the plasma of mice administered with Fv4-IgG1-F1022 with
enhanced mouse Fc.gamma.R binding was considerably reduced as
compared to the human IL-6 receptor concentration in the plasma of
mice administered with Fv4-IgG1. As to the degree of reduction, the
concentration was confirmed to be decreased below the baseline
human IL-6 receptor concentration without antibody administration.
In particular, the human IL-6 receptor concentration in the plasma
of mice administered with Fv4-IgG1-F1022 was reduced down to about
1/100 three days after administration as compared to the case of
Fv4-IgG1 administration. This finding demonstrates that, by
administering to mice an antibody that binds to human IL-6 receptor
in a pH-dependent manner and whose Fc.gamma.R binding has been
enhanced, the human IL-6 receptor concentration in the plasma of
the mice can be significantly reduced, and as to the degree of
reduction, the antigen concentration in plasma can be reduced below
the level before antibody administration.
[0440] Furthermore, it was also demonstrated that, as compared to
mice administered with Fv4-IgG1, the human IL-6 receptor
concentration in plasma was reduced in mice administered with
Fv4-IgG1-Fuc which has sugar chains with low fucose content and
with increased mouse Fc.gamma.R IV-binding activity. In particular,
the human IL-6 receptor concentration in the plasma of mice
administered with Fv4-IgG1-Fuc was reduced down to about 1/2 seven
days after administration as compared to the case of Fv4-IgG1
administration. The above finding demonstrates that, by
administering to mice a pH-dependent antigen-binding molecule that
binds to human IL-6 receptor in a pH-dependent manner and whose
Fc.gamma.R binding has been enhanced, the soluble antigen
concentration in the plasma of the mice can be reduced. In this
case, methods for enhancing the Fc.gamma.R binding are not
particularly limited to introduction of amino acid alterations. It
was demonstrated that such enhancement can be achieved, for
example, by using a human IgG Fc region to which a sugar chain with
low fucose content is linked at position 297 (EU numbering);
however, the effect of Fv4-IgG1-Fuc to reduce antigen concentration
was smaller than Fv4-F1022. Based on this result, it would be
thought that, of several Fc.gamma.Rs (Fc.gamma.RI, II, III, and IV
for mouse), mFc.gamma.IV, to which the binding of Fv4-IgG1-Fuc is
enhanced, does not have a large contribution to the reduction of
antigen concentration as an Fc.gamma.R.
[0441] Thus, it was revealed that, by administering to an
individual an antibody that binds to a soluble antigen in a
pH-dependent manner and whose Fc.gamma.R binding has been enhanced,
the soluble antigen concentration in the plasma of the individual
can be markedly reduced.
[0442] Without being bound by a particular theory, the unexpected
reduction of soluble antigen concentration in plasma, which was
observed when administering an antigen-binding molecule that
comprises an antigen-binding domain whose Fc.gamma.R binding has
been enhanced and whose antigen-binding activity is altered
depending on the ion concentration condition such as pH and an
FcRn-binding domain that has FcRn-binding activity under an acidic
pH range condition, can be explained as follows.
[0443] IgG antibodies that are non-specifically incorporated into
cells return to the cell surface by binding to FcRn under the
acidic condition in the endosome, and then dissociate from FcRn
under the neutral condition in plasma. In such a case, when an
antibody that neutralizes the function of a soluble antigen by
binding to the antigen is administered to mice in which the
concentration of the soluble antigen is maintained constant in
plasma, the soluble antigen in plasma forms a complex with the
antibody administered. The soluble antigen incorporated into cells
while remaining as the complex is thought to be recycled, in a
state bound to the antibody, to the plasma together with the
antibody, because the Fc region of the antibody binds to FcRn under
the acidic condition in the endosome.
[0444] Meanwhile, when the antibody against the soluble antigen is
an antibody that binds to the antigen in a pH-dependent manner
(i.e., an antibody that dissociates the soluble antigen under the
acidic condition in the endosome), the soluble antigen that is
non-specifically incorporated into cells while remaining as a
complex with the antibody, is dissociated from the antibody in the
endosome and degraded in the lysosome in the cell; thus, the
soluble antigen is not recycled to the plasma. That is, it is
thought that Fv4-IgG1 incorporated as a complex with the soluble
antigen into cells can dissociate the soluble antigen in the
endosome and thus accelerate the elimination of the soluble
antigen.
[0445] As described above, antigen-binding molecules such as
Fv4-IgG1, which contain an antigen-binding domain whose
antigen-binding activity is altered depending on the ion
concentration, are thought to be capable of binding to antigens
repeatedly several times. The effect to accelerate the elimination
of soluble antigens from the plasma by dissociating them in the
endosome is thought to depend on the rate of incorporation of the
antigen/antigen-binding molecule complex into the endosome. An
antigen-binding molecule that contains an antigen-binding domain
whose binding activity to various Fc.gamma.Rs has been increased
and whose antigen-binding activity is altered depending on the
condition of ion concentration, is actively incorporated into cells
by binding to various Fc.gamma.Rs expressed on the cell membrane,
and can be shuttled back to plasma by recycling via the binding
between FcRn and the FcRn-binding domain comprised in the molecule,
which has FcRn-binding activity under an acidic pH range condition.
That is, it is thought that, since the above antigen-binding
molecule which forms a complex with a soluble antigen in plasma is
actively incorporated into cells via Fc.gamma.R expressed on the
cell membrane, its effect to accelerate the elimination of the
soluble antigen from plasma is more markedly shown than
antigen-binding molecules whose binding activity to various
Fc.gamma.Rs has not been increased.
[0446] The Fc.gamma.R-binding activity of an antibody that binds to
a membrane antigen plays an important role in the cytotoxic
activity of the antibody. Thus, when it is necessary for an
antibody used as a pharmaceutical agent to have cytotoxic activity,
a human IgG1 isotype with strong Fc.gamma.R-binding activity is
used. In addition, techniques to enhance the cytotoxic activity of
such antibodies by increasing the Fc.gamma.R-binding activity of
the antibodies are used commonly in the art.
[0447] Meanwhile, the role of the Fc.gamma.R-binding activity of
antibodies that bind to soluble antigens and which are used as
pharmaceutical agents has not been known in the art. There has been
no sufficient assessment on what difference in the effect on the
living organism administered with the antibodies is caused by the
difference in the Fc.gamma.R-binding activity between human IgG1
with high Fc.gamma.R-binding activity and human IgG2 and human IgG4
with low Fc.gamma.R-binding activity. Actually, it was demonstrated
in the present Example that there was no influence on the time
course of soluble antigen concentration in the plasma of the
individuals administered with an antibody that lacks
Fc.gamma.R-binding activity. Meanwhile, in the present invention,
it was revealed that the soluble antigen concentration was
significantly reduced in the plasma of the individuals administered
with an antigen-binding molecule whose Fc.gamma.R-binding activity
has been increased and which contains an antigen-binding domain
whose soluble antigen-binding activity is altered depending on the
ion concentration condition. Specifically, it can be said that the
present inventors revealed for the first time the benefit of the
enhancement of Fc.gamma.R binding by combining an FcRn-binding
domain that has FcRn-binding activity under an acidic pH range
condition with an antigen-binding domain whose soluble antigen
binding is altered depending on the ion concentration condition,
comprised in an antigen-binding molecule targeted to a soluble
antigen.
Example 3
Effect of Eliminating Antigens from Plasma by Antigen-Binding
Molecules Whose Fc.gamma.R-Binding Activity is Greater than that of
Native Human IgG Fc Region and Whose Human FcRn-Binding Activity
has been Increased Under an Acidic pH Range Condition
[0448] (3-1) Preparation of Antigen-Binding Molecules Whose
Fc.gamma.R-Binding Activity is Greater than the Binding Activity of
Native Human IgG Fc Region and Whose Human FcRn-Binding Activity
has been Increased Under an Acidic pH Range Condition
[0449] A reported method for improving the retention of IgG
antibody in plasma is to improve the FcRn binding under an acidic
pH range condition. It is thought that, when the FcRn binding under
an acidic pH range condition is improved by introducing an amino
acid substitution into the Fc region of an IgG antibody, this
increases the recycling efficiency from the endosome to plasma,
resulting in an improvement of the plasma retention of the IgG
antibody.
[0450] There are many reports on amino acid alterations to improve
the plasma retention by improving the human FcRn-binding activity
under an acidic pH range condition. Such alterations include, for
example:
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 WO2010/106180; WO2010/045193; WO2009/058492;
WO2008/022152; WO2006/050166, WO2006/053301, WO2006/031370;
WO2005/123780; WO2005/047327; WO2005/037867; WO2004/035752; and
WO2002/060919.
[0451] VH3-IgG1-F1093 (SEQ ID NO: 43) with a substitution of Leu
for Met at position 428 and Ser for Asn at position 434 (EU
numbering) in VH3-IgG1-F1022 was prepared to improve the
pharmacodynamics of Fv4-IgG1-F1022 that was demonstrated to
produce, when administered, the effect of significantly reducing
the soluble antigen concentration in plasma, as described in
Example 2. Fv4-IgG1-F1093 comprising VH3-IgG1-F1093 as the heavy
chain and VL3-CK as the light chain was constructed using the
method described in Reference Example 1.
(3-2) Effect of Eliminating Antigens from Plasma by Antigen-Binding
Molecules Whose Fc.gamma.R-Binding Activity is Greater than that of
Native Human IgG Fc Region and Whose Human FcRn-Binding Activity
has been Increased Under an Acidic pH Range Condition
[0452] An in vivo infusion test was carried out for Fv4-IgG1-F1093
by the same method as described in Example (2-1-1) using human FcRn
transgenic mice in which the soluble human IL-6 receptor
concentration is maintained constant in plasma. Soluble human IL-6
receptor concentrations in the plasma of the mice were determined
by the method described in Example (2-1-2). The result is shown in
FIG. 4.
(3-2-1) Determination of the Anti-Human IL-6 Receptor Antibody
Concentration in Plasma by the ELISA Method
[0453] Anti-human IL-6 receptor antibody concentrations in mouse
plasma were determined by the ELISA method. First, an anti-Human
IgG (.gamma.-chain specific) F(ab')2 Fragment of Antibody (SIGMA)
was aliquoted in a Nunc-Immuno Plate, MaxiSoup (Nalge nunc
International). The plate was allowed to stand at 4.degree. C.
overnight to prepare a plate immobilized with the anti-human IgG.
Standard curve samples containing an anti-human IL-6 receptor
antibody (concentration in plasma: 0.8, 0.4, 0.2, 0.1, 0.05, 0.025,
and 0.0125 .mu.g/ml) and assay samples of mouse plasma diluted 100
times or more were prepared. 100 .mu.l each of the standard curve
and assay samples were combined with 200 .mu.l of 20 ng/ml soluble
human IL-6 receptor. The resulting mixtures were allowed to stand
at room temperature for one hour, and aliquoted to each well of the
plate immobilized with the anti-human IgG The plate was allowed to
stand at room temperature for another one hour. Then, Biotinylated
Anti-human IL-6 R Antibody (R&D) was reacted thereto at room
temperature for one hour. Next, Streptavidin-PolyHRP80
(Stereospecific Detection Technologies) was reacted thereto at room
temperature for one hour. The chromogenic reaction of the reaction
solution was performed using as a substrate TMB One Component HRP
Microwell Substrate (BioFX Laboratories). After terminating the
reaction with 1N sulfuric acid (Showa Chemical), the absorbance at
450 nm of the reaction solution of each well was measured with a
microplate reader. Antibody concentrations in mouse plasma were
determined based on the absorbance of the standard curve using the
analysis software SOFTmax PRO (Molecular Devices).
[0454] The result is shown in FIG. 5.
(3-3) Improvement of Pharmacodynamics by Increasing the Human
FcRn-Binding Activity Under an Acidic pH Range Condition
[0455] As shown in FIG. 5, in the group administered with
Fv4-IgG1-F1022 resulting from the enhancement of the
Fc.gamma.R-binding activity of Fv4-IgG1 under a neutral pH range
condition, the plasma retention of the administered antibody was
demonstrated to be reduced as compared to the group administered
with Fv4-IgG1. Meanwhile, in the group administered with
Fv4-IgG1-F1093 resulting from the enhancement of the human
FcRn-binding activity of Fv4-IgG1-F1022 under an acidic pH range
condition, the plasma retention of the administered antibody was
demonstrated to be significantly improved as compared to the group
administered with Fv4-IgG1-F1022.
[0456] Furthermore, as shown in FIG. 4, the time course of the
soluble human IL-6 receptor concentration in the plasma of the
Fv4-IgG1-F1022-administered group was equivalent to that of the
Fv4-IgG1-F1093-administered group, up to three days after antibody
administration. On day three after administration, as compared to
the Fv4-IgG1-administered group, the soluble human IL-6 receptor
concentration in plasma was reduced as much as about 100 times in
both of the Fv4-IgG1-F1022 and Fv4-IgG1-F1093-administered groups.
However, on day seven after antibody administration, the soluble
human IL-6 receptor concentration in plasma was observed to be
elevated in the Fv4-IgG1-F1022-administered group as compared to on
day three after administration. On the other hand, in the
Fv4-IgG1-F1093-administered group, an increase in the plasma
concentration of soluble human IL-6 receptor was not observed,
showing that the effect to reduce the soluble human IL-6 receptor
concentration was sustained in this administration group.
[0457] Specifically, Fv4-IgG1-F1093, when administered, reduced the
soluble human IL-6 receptor concentration in the plasma of the
administered individual down to about 1/100 as compared to
Fv4-IgG1, and in addition, it sustained this condition for a long
period. Thus, Fv4-IgG1-F1093 was demonstrated to be a highly
excellent antigen-binding molecule. Without being bound by a
particular theory, the phenomenon observed herein can be explained
as follows. Fv4-IgG1-F1022 in which the Fc.gamma.R-binding activity
of Fv4-IgG1 has been increased under a neutral pH range condition
is thought to be incorporated in a large amount mainly into cells
expressing Fc.gamma.R on the cell membrane. The incorporated
antibody is transferred into the endosome, and by binding to FcRn
in the endosome, the antibody is recycled to the plasma. When the
FcRn-binding activity of the antibody is not high enough under the
condition at acidic pH in the endosome, the antibody incorporated
into the endosome is thought to be incapable of sufficient
recycling. Specifically, a possible reason for the reduced plasma
retention of Fv4-IgG1-F1022 relative to Fv4-IgG1 would be that the
FcRn-binding activity under an acidic pH range condition is
insufficient for sufficient recycling of the endosome-incorporated
antibody to the plasma by FcRn binding, and the antibody that was
not recycled was degraded in the lysosome.
[0458] On the other hand, as with Fv4-IgG1-F1022, Fv4-IgG1-F1093
resulting from the enhancement of the human FcRn-binding activity
of Fv4-IgG1-F1022 under an acidic pH range condition is thought to
be incorporated in a large amount mainly into cells expressing
Fc.gamma.R on the cell membrane. An antibody incorporated and
transferred into the endosome is recycled to the plasma by binding
to FcRn in the endosome. Since its human FcRn-binding activity
under an acidic pH range condition is enhanced, Fv4-IgG1-F1093 is
thought to have sufficient FcRn-binding activity in the endosome.
Thus, after incorporation into cells, most of Fv4-IgG1-F1093 is
recycled to the plasma. Thus, it would be thought that the plasma
retention of Fv4-IgG1-F1093 was improved in administered
individuals as compared to Fv4-IgG1-F1022.
[0459] On the other hand, it has been known that the plasma
retention of ordinary antibodies is improved when their
FcRn-binding activity is improved under an acidic pH range
condition. However, it is thought that, when the antibody retention
in plasma is improved, the plasma retention of antibody-bound
antigens is also improved, and this results in an increase of the
antigen concentration in plasma. In actual, as described in
WO2010/088444, Antibody 18E introduced with the alteration YTE into
Antibody 18, which is a human IgG1 antibody against IL-6, to
increase the FcRn-binding activity under an acidic pH range
condition, showed improved antibody retention in the plasma of
cynomolgus monkeys, and at the same time, the concentration of the
IL-6 antigen was also elevated in the plasma.
[0460] Surprisingly, however, when administering Fv4-IgG1-F1093
introduced with an alteration similar to YTE for increasing the
FcRn-binding activity under an acidic pH range condition into
Fv4-F1022 that binds to the antigen in a pH-dependent manner and
has increased Fc.gamma.R-binding activity, the plasma retention of
the antibody was significantly improved in the administered
individuals without increasing the concentration of soluble human
IL-6 receptor which is the antigen. Rather, on day seven after
antibody administration, the soluble human IL-6 receptor
concentration remained low in the individuals administered with
Fv4-IgG1-F1093 as compared to those administered with
Fv4-F1022.
[0461] Without being bound by a particular theory, the phenomenon
observed herein can be explained as follows. When administered to a
living organism, an antibody without pH-dependent antigen binding
is non-specifically incorporated into cells. Antigens that remain
to be bound to the antibody are recycled to the plasma in the same
extent as the antibody. Meanwhile, for an antibody with increased
FcRn-binding activity under an acidic pH range condition, the
extent of recycling to the plasma in a living organism administered
with the antibody is higher than that of an antibody without
increased FcRn-binding activity, and this results in an increased
extent of recycling of antigens bound to the antigen to the plasma
in the living organism. Thus, due to the improved plasma retention
of the antibody administered in the living organism, the plasma
concentration of the antigen to which the antibody binds is thought
to be also increased in the living organism.
[0462] Meanwhile, when administered to a living organism, an
antibody that binds to an antigen in a pH-dependent manner and
which has increased Fc.gamma.R-binding activity is mainly
incorporated into cells expressing Fc.gamma.R on the cell membrane,
and this reduces the plasma retention. Furthermore, after being
incorporated into the cells while bound to the antibody, the
antigen is dissociated from the antibody in the endosome and then
degraded in the lysosome, resulting in a decrease of the antigen
concentration in plasma in the living organism. When the
FcRn-binding activity is increased under an acidic pH range
condition, the antibody retention in plasma, even if worsened due
to increased Fc.gamma.R-binding activity, is improved by an
increase in the rate of recycling by FcRn. In this case, since the
antigen bound to the antibody that binds to the antigen in a
pH-dependent manner is dissociated from the antibody in the
endosome and directly degraded in the lysosome, it is not thought
that the antigen concentration is increased in the plasma.
Furthermore, the improved plasma retention of the antibody
administered to the living organism is thought to allow the antigen
elimination effect of the antibody to be sustained, and the antigen
concentration to be maintained low for a longer period.
[0463] The above findings demonstrate that the plasma retention of
an administered antibody is improved in a living organism
administered with the antibody in which the human FcRn-binding
activity under an acidic pH range condition is enhanced in an
antigen-binding molecule whose Fc.gamma.R-binding activity is
higher than that of native human IgG Fc region. Furthermore, it was
revealed that, in this case, the antibody retention in plasma is
improved without deteriorating the antigen-elimination effect.
Example 4
Further Assessment of the Effect of Eliminating Antigens from
Plasma Antigen-Binding Molecules Whose Fc.gamma.R-Binding Activity
is Greater than that of Native Human IgG Fc Region and Whose Human
FcRn-Binding Activity has been Increased Under an Acidic pH Range
Condition
(4-1) The Antigen Elimination Effect of an Antibody Whose
Fc.gamma.R-Binding Activity is Enhanced
[0464] As described in Example 2, the antigen concentration in
plasma was significantly reduced in the group administered with
Fv4-IgG1-F1022 with enhanced mouse Fc.gamma.R binding. Meanwhile,
as shown in Example 3, the reduced plasma retention observed in the
Fv4-IgG1-F1022-administered group was markedly improved by
increasing the human FcRn-binding activity of Fv4-IgG1-F1022 under
an acidic pH range condition. Next, the effect of eliminating
soluble antigens from plasma by enhancing mouse Fc.gamma.R binding
and the effect of improving the plasma retention of an antibody by
enhancing the human FcRn binding activity under an acidic pH range
condition were further assessed as described below.
(4-2) Preparation of an Anti-Human IL-6 Receptor Antibody with
Enhanced Mouse Fc.gamma.R Binding
[0465] VH3-IgG1-F1087 (SEQ ID NO: 44) resulting from substituting
Asp for Lys at position 326 (EU numbering) in VH3-IgG1, and
VH3-IgG1-F1182 (SEQ ID NO: 45) resulting from substituting Asp for
Ser at position 239 and Glu for Ile at position 332 (EU numbering)
in VH3-IgG1, were prepared as antigen-binding molecules with
enhanced mouse Fc.gamma.R binding. Fv4-IgG1-F1087 that contains
VH3-IgG1-F1087 as the heavy chain and VL3-CK as the light chain,
and Fv4-IgG1-F1182 that contains VH3-IgG1-F1182 as the heavy chain
and VL3-CK as the light chain, were produced using the method
described in Reference Example 1.
(4-3) Assessment of Mouse Fc.gamma.R-Binding Activity
[0466] VH3/L (WT)-IgG1-F1087 and VH3/L (WT)-IgG1-F1182 which
contain VH3-IgG1-F1087 and VH3-IgG1-F1182 as the heavy chain,
respectively, and L (WT)-CK (SEQ ID NO: 42) as the light chain,
were prepared by the method described in Reference Example 1. These
antibodies, and VH3/L (WT)-IgG1-F1022 and VH3/L (WT)-IgG1 were
assessed for their mouse Fc.gamma.R-binding activity by the method
described in Reference Example 2. The result is shown in Table 7.
In addition, the ratio of the increase in the mouse
Fc.gamma.R-binding activity of each variant relative to the IgG1
before alteration is shown in Table 8. In the table, VH3/L
(WT)-IgG1, VH3/L (WT)-IgG1-F1022, VH3/L (WT)-IgG1-F1087, and VH3/L
(WT)-IgG1-F1182 are shown as IgG1, F1022, F1087, and F1182,
respectively.
TABLE-US-00014 TABLE 7 VARIANT KD (M) NAME mFc .gamma. RI mFc
.gamma. RIIb mFc .gamma. RIII mFc .gamma. RIV IgG1 5.3E-08 9.8E-07
2.4E-06 8.6E-08 F1022 7.6E-09 1.0E-08 5.5E-09 1.4E-07 F1087 2.9E-08
5.6E-08 5.2E-08 3.3E-07 F1182 2.4E-09 1.1E-07 4.8E-07 5.3E-10
TABLE-US-00015 TABLE 8 VARIANT RATIO OF BINDING TO IgG1 NAME mFc
.gamma. RI mFc .gamma. RIIb mFc .gamma. RIII mFc .gamma. RIV IgG1
1.0 1.0 1.0 1.0 F1022 7.0 93.6 440.5 0.6 F1087 1.8 17.5 46.2 0.3
F1182 22.1 9.1 5.0 162.3
[0467] As shown in Table 8, it was demonstrated that F1087 and
F1022 had increased binding activity to mouse Fc.gamma.RI, mouse
Fc.gamma.RIIb, and mouse Fc.gamma.RIII as compared to IgG1, whereas
their mouse Fc.gamma.RIV-binding activity was not increased.
Regarding the binding activity of F1087 to mouse Fc.gamma.RI, mouse
Fc.gamma.RIIb, mouse Fc.gamma.RIII, and mouse Fc.gamma.RIV, the
extent of its increase was revealed to be smaller than that of
F1022. Meanwhile, it was shown that the binding activity of F1182
to mouse Fc.gamma.RI and mouse Fc.gamma.RIV was considerably
increased, whereas the extent of increase in its binding activity
to Fc.gamma.RIIb and Fc.gamma.RIII was smaller than those of F1022
and F1087. As mentioned above, these three types of variants showed
enhanced binding to some mouse Fc.gamma.Rs; however, it was shown
that the Fc.gamma.R to which the binding activity is selectively
increased and the extent of the increase vary depending on the
variant.
(4-4) The Effect of Eliminating Antigens from the Plasma of
Fv4-IgG1-F1087 and Fv4-IgG1-F1182
[0468] By the same method as described in Example 2, in vivo
infusion tests using human FcRn transgenic mice were carried out to
determine the soluble IL-6 receptor concentrations in the plasma of
the mice. The result is shown in FIG. 6.
[0469] In both of the groups administered with Fv4-IgG1-F1087 and
Fv4-IgG1-F1182 in vivo, which have increased mouse
Fc.gamma.R-binding activity as compared to Fv4-IgG1, the in vivo
plasma concentration of soluble human IL-6 receptor could be
reduced as compared to the group administered with Fv4-IgG1. The
effect to reduce the plasma concentration of soluble human IL-6
receptor was high especially in the group administered with
Fv4-IgG1-F1087 which has enhanced binding to mouse Fc.gamma.RII and
mouse Fc.gamma.RIII. Meanwhile, the effect of F1182 administration
to reduce the plasma concentration of soluble human IL-6 receptor
was small in the group administered with F1182 in vivo which has
considerably increased binding activity to mouse Fc.gamma.RI and
mouse Fc.gamma.RIV (as well as several-fold enhanced binding to
mouse Fc.gamma.RII and mouse Fc.gamma.RIII). It was thought from
these results that the mouse Fc.gamma.Rs that more significantly
contribute by an effect that efficiently decreases the antigen
concentration in the plasma of mice administered with a
pH-dependent antigen-binding antibody, are mouse Fc.gamma.RII
and/or mouse Fc.gamma.RIII. Specifically, it is thought that the
plasma antigen concentration can be more efficiently reduced in
vivo by administering into a living organism a pH-dependent
antigen-binding antibody with enhanced binding to mouse
Fc.gamma.RII and/or mouse Fc.gamma.RIII.
(4-5) Preparation of Antigen-Binding Molecules Whose
Fc.gamma.R-Binding Activity is Greater than the Binding Activity of
Native Human IgG Fc Region and which have Increased Human
FcRn-Binding Activity Under an Acidic pH Range Condition
[0470] As described in Example 3, when compared to human FcRn
transgenic mice administered with Fv4-IgG1-F1022, the plasma
retention of an antibody is markedly improved in human FcRn
transgenic mice administered with Fv4-IgG1-F1093 resulting from
increasing the human FcRn-binding activity under an acidic pH range
condition of Fv4-IgG1-F1022 in which the mouse Fc.gamma.R-binding
activity has been increased. Whether this effect is also observed
in human FcRn transgenic mice administered with Fv4-IgG1-F1087 and
Fv4-IgG1-F1182, and whether the same effect is observed in mice
administered with variants whose human FcRn-binding activity has
been increased under an acidic pH range condition by addition of an
alteration distinct from the alteration assessed in Example 3 were
assessed as follows.
[0471] VH3-IgG1-F1180 (SEQ ID NO: 46) and VH3-IgG1-F1181 (SEQ ID
NO: 47) were prepared by substituting Leu for Met at position 428
and Ser for Asn at position 434 (EU numbering) in the heavy chains
VH3-IgG1-F1087 and VH3-IgG1-F1182, in order to increase their human
FcRn-binding activity of Fv4-IgG1-F1087 and Fv4-IgG1-F1182 under an
acidic pH range condition. Furthermore, VH3-IgG1-F1412 (SEQ ID NO:
48) was prepared by substituting Ala for Asn at position 434 (EU
numbering) in the heavy chain VH3-IgG1-F1087, in order to increase
the human FcRn-binding activity of Fv4-IgG1-F1087 under an acidic
pH range condition. Fv4-IgG1-F1180, Fv4-IgG1-F1181, and
Fv4-IgG1-F1412, which contain the above heavy chains and VL3-CK as
the light chain, were prepared using the method described in
Reference Example 1.
(4-6) Improvement of Pharmacodynamics of Antibodies by Increasing
Human FcRn-Binding Activity Under an Acidic pH Range Condition
[0472] In vivo infusion tests were carried out by administering
Fv4-IgG1-F1180, Fv4-IgG1-F1181, and Fv4-IgG1-F1412 to human FcRn
transgenic mice according to the same method as described in
Example 2 to determine the soluble IL-6 receptor concentrations in
the plasma of the mice. The results on the soluble IL-6 receptor
concentrations in the plasma of the mouse groups administered with
Fv4-IgG1-F1087, Fv4-IgG1-F1180, Fv4-IgG1-F1412, and Fv4-IgG1 are
shown in FIG. 9. The results on the soluble IL-6 receptor
concentrations in the plasma of the mouse groups administered with
Fv4-IgG1-F1182, Fv4-IgG1-F1181, and Fv4-IgG1 are shown in FIG. 10.
Meanwhile, the plasma antibody concentrations in the mouse groups
were measured by the method described in Example 3. The results on
the plasma antibody concentrations of Fv4-IgG1-F1087,
Fv4-IgG1-F1180, Fv4-IgG1-F1412, and Fv4-IgG1 in the mouse groups
are shown in FIG. 7; and the results on the plasma antibody
concentrations of Fv4-IgG1-F1182, Fv4-IgG1-F1181, and Fv4-IgG1 are
shown in FIG. 8.
[0473] It was confirmed that, as compared to the group of mice
administered with Fv4-IgG1-F1182, the plasma retention of
antibodies was improved in the group of mice administered with
Fv4-IgG1-F1181 resulting from increasing the human FcRn-binding
activity of Fv4-IgG1-F1182 in an acidic pH range. Meanwhile, the
soluble IL-6 receptor concentration in the plasma of the mouse
groups administered with Fv4-IgG1-F1181 was comparable to that in
the group of mice administered with Fv4-IgG1-F1182. When compared
to the mouse groups administered with Fv4-IgG1, the soluble IL-6
receptor concentration in the plasma was decreased in both
groups.
[0474] On the other hand, as compared to the group of mice
administered with Fv4-IgG1-F1087, the plasma retention of
antibodies was improved in both groups of mice administered with
Fv4-IgG1-F1180 and Fv4-IgG1-F1412 resulting from increasing the
human FcRn-binding activity of Fv4-IgG1-F1087 in an acidic pH
range, and surprisingly, the plasma retention was improved up to a
level comparable to that of the mouse groups administered with
Fv4-IgG1. Furthermore, the sustainability of the effect of reducing
the soluble IL-6 receptor concentration in plasma was improved by
the improvement of the plasma antibody retention in the groups of
administered mice. Specifically, in the groups of administered
mice, the soluble IL-6 receptor concentrations in plasma 14 days
and 21 days after administration of Fv4-IgG1-F1180 and
Fv4-IgG1-F1412 were significantly reduced as compared to the
concentrations 14 days and 21 days after administration of
Fv4-IgG1-F1087.
[0475] In view of the above, as for the groups of mice administered
with the four examples of antibodies, Fv4-IgG1-F1093,
Fv4-IgG1-F1181, Fv4-IgG1-F1180, and Fv4-IgG1-F1412, it was
demonstrated that the plasma retention can be improved in a living
organism administered with an antibody in which the human
FcRn-binding activity under an acidic pH range condition has been
enhanced in an antigen-binding molecule whose Fc.gamma.R-binding
activity is higher than the binding activity of native human IgG Fc
region. It was also demonstrated that, in the living organism
administered with the antigen-binding molecule, the plasma
retention is improved without deteriorating the effect of
eliminating antigens from the living organism, and rather, the
antigen elimination effect can be sustained.
[0476] It is demonstrated that alteration for enhancing human
FcRn-binding activity under an acidic pH range condition could be
accomplished by the method that substitutes Ala for Asn at position
434 (EU numbering), in addition to the method that substitutes Leu
for Met at position 428 (EU numbering) and Ser for Asn at position
434 (EU numbering). Therefore, alterations used for enhancing human
FcRn-binding activity under an acidic pH range condition are not
particularly limited, and the method that substitutes Leu for Met
at position 428 (EU numbering) and Ser for Asn at position 434 (EU
numbering) in an IgG antibody (Nat. Biotechnol. (2010) 28,
157-159), the method that substitutes Ala for Asn at position 434
(EU numbering) in an IgG antibody (Drug Metab. Dispos. (2010) 38
(4), 600-605), the method that substitutes Tyr for Met at position
252 (EU numbering), Thr for Ser at position 254 (EU numbering), and
Glu for Thr at position 256 (EU numbering) in an IgG antibody (J.
Biol. Chem. (2006) 281, 23514-23524), the method that substitutes
Gln for Thr at position 250 (EU numbering) and Leu for Met at
position 428 (EU numbering) in an IgG antibody (J. Immunol. (2006)
176 (1), 346-356), the method that substitutes His for Asn at
position 434 (EU numbering) in an IgG antibody (Clin. Pharmcol.
Ther. (2011) 89 (2), 283-290), as well as alterations described in
WO 2010/106180, WO 2010/045193, WO 2009/058492, WO 2008/022152, WO
2006/050166, WO 2006/053301, WO 2006/031370, WO 2005/123780, WO
2005/047327, WO 2005/037867, WO 2004/035752, WO 2002/060919 and
such can be used.
(4-7) Preparation of Antigen-Binding Molecules with Increased Human
FcRn-Binding Activity Under an Acidic pH Range Condition and
Suppressed Binding to a Rheumatoid Factor
[0477] In recent years, an antibody molecule resulting from
substituting His for Asn at position 434 (EU numbering) in a
humanized anti-CD4 antibody to improve the plasma retention by
increasing its human FcRn-binding activity under an acidic pH range
condition, has been reported to bind to the rheumatoid factor (RF)
(Clin. Pharmacol. Ther. (2011) 89 (2), 283-290). This antibody has
a human IgG1 Fc region and a substitution of His for Asn at
position 434 (EU numbering) in the FcRn-binding site. The
rheumatoid factor has been demonstrated to recognize and bind to
the substituted portion.
[0478] As shown in (4-6), various alterations have been reported as
alterations for enhancing human FcRn-binding activity under an
acidic pH range condition, and introducing these alterations to the
FcRn-binding site in an Fc region may enhance its affinity to a
rheumatoid factor that recognizes this site.
[0479] However, antigen-binding molecules that have increased human
FcRn-binding activity under an acidic pH range condition but do not
have the binding to the rheumatoid factor can be produced by
introducing into the site of the Fc region an alteration that
reduces the rheumatoid factor-binding activity alone without
reducing the FcRn-binding activity under an acidic pH range
condition.
[0480] Such alterations used for reducing the rheumatoid
factor-binding activity include alterations at positions 248-257,
305-314, 342-352, 380-386, 388, 414-421, 423, 425-437, 439, and
441-444 (EU numbering), preferably those at positions 387, 422,
424, 426, 433, 436, 438, and 440 (EU numbering), and particularly
preferably, an alteration that substitutes Glu or Ser for Val at
position 422, an alteration that substitutes Arg for Ser at
position 424, an alteration that substitutes Asp for His at
position 433, an alteration that substitutes Thr for Tyr at
position 436, an alteration that substitutes Arg or Lys for Gln at
position 438, and an alteration that substitutes Glu or Asp for Ser
at position 440 (EU numbering). These alterations may be used alone
or in combination.
[0481] Alternatively, it is possible to introduce N-type
glycosylation sequences to reduce the rheumatoid factor-binding
activity. Specifically, known N-type glycosylation sequences
include Asn-Xxx-Ser/Thr (Xxx represents an arbitrary amino acid
other than Pro). This sequence can be introduced into the Fc region
to add an N-type sugar chain, and the binding to RF can be
inhibited by the steric hindrance of the N-type sugar chain.
Alterations used for adding an N-type sugar chain preferably
include an alteration that substitutes Asn for Lys at position 248,
an alteration that substitutes Asn for Ser at position 424, an
alteration that substitutes Asn for Tyr at position 436 and Thr for
Gln at position 438, and an alteration that substitutes of Asn for
Qln at position 438, according to EU numbering, particularly
preferably an alteration that substitutes Asn for Ser at position
424 (EU numbering).
Example 5
Effects of Eliminating Antigens from Plasma for Antigen-Binding
Molecules Whose Fc.gamma.R-Binding Activity is Higher than that of
an Fc Region of Native Mouse IgG
[0482] (5-1) Antigen Elimination Effect of Mouse Antibodies with
Enhanced Fc.gamma.R-Binding Activity
[0483] As described in Examples 1 to 4, it was demonstrated that
the elimination of soluble human IL-6 receptor from mouse plasma is
accelerated in the groups of human FcRn transgenic mice
administered with antigen-binding molecules resulting from
increasing the mouse Fc.gamma.R-binding activity of antigen-binding
molecules that have a human antibody Fc region and the property of
binding to human IL-6 receptor in a pH-dependent manner. Whether
this effect is also achieved in normal mice having mouse FcRn that
was administered with antigen-binding molecules that have a mouse
antibody Fc region and the property of binding to human IL-6
receptor in a pH-dependent manner, was assessed in normal mice
having mouse FcRn as follows.
(5-2) Preparation of Mouse Antibodies with Increased
Fc.gamma.R-Binding Activity
[0484] For a mouse IgG1 antibody having the property of binding to
human IL-6 receptor in a pH-dependent manner, the heavy chain
VH3-mIgG1 (SEQ ID NO: 49) and the light chain VL3-mk1 (SEQ ID NO:
50) were constructed using the method described in Reference
Example 1. Meanwhile, to increase the mouse Fc.gamma.R-binding
activity of VH3-mIgG1, VH3-mIgG1-mF44 (SEQ ID NO: 51) was produced
by substituting Asp for Ala at position 327 (EU numbering).
Likewise, VH3-mIgG1-mF46 (SEQ ID NO: 52) was produced by
substituting Asp for Ser at position 239 and Asp for Ala at
position 327, according to EU numbering, in VH3-mIgG1. Fv4-mIgG1,
Fv4-mIgG1-mF44, and Fv4-mIgG1-mF46, which contain VH3-mIgG1,
VH3-mIgG1-mF44, and VH3-mIgG1-mF46, respectively, as the heavy
chain, and VL3-mk1 as the light chain, were prepared using the
method described in Reference Example 1.
(5-3) Assessment of Mouse Fc.gamma.R-Binding Activity
[0485] VH3/L (WT)-mIgG1, VH3/L (WT)-mIgG1-mF44, and VH3/L
(WT)-mIgG1-mF46, which contain VH3-mIgG1, VH3-mIgG1-mF44, and
VH3-mIgG1-mF46, respectively, as the heavy chain, and L (WT)-CK
(SEQ ID NO: 42) as the light chain, were prepared by the method
described in Reference Example 1. These antibodies were assessed
for their mouse Fc.gamma.R-binding activity by the method described
in Reference Example 2. The result is shown in Table 9. In
addition, the ratio of the increase in the mouse Fc.gamma.R-binding
activity of each variant relative to the mIgG1 before alteration is
shown in Table 10. In the table, VH3/L (WT)-mIgG1, VH3/L
(WT)-mIgG1-mF44, and VH3/L (WT)-mIgG1-mF46 are shown as mIgG1,
mF44, and mF46, respectively.
TABLE-US-00016 TABLE 9 VARIANT KD (M) NAME mFc .gamma. RI mFc
.gamma. RIIb mFc .gamma. RIII mFc .gamma. RIV mIgG1 NOT 1.1E-07
2.1E-07 NOT DETECTED DETECTED mF44 NOT 8.9E-09 6.7E-09 NOT DETECTED
DETECTED mF46 NOT 1.2E-09 3.6E-09 NOT DETECTED DETECTED
TABLE-US-00017 TABLE 10 VARIANT BINDING RATIO TO mIgG1 NAME mFc
.gamma. RI mFc .gamma. RIIb mFc .gamma. RIII mFc .gamma. RIV mIgG1
NOT 1.0 1.0 NOT DETECTED DETECTED mF44 NOT 11.9 31.0 NOT DETECTED
DETECTED mF46 NOT 91.4 57.5 NOT DETECTED DETECTED
[0486] The assessment result of Example 4 showing that VH3/L
(WT)-mIgG1 having the Fc region of native mouse IgG1 antibody only
binds to mouse Fc.gamma.RIIb and mouse Fc.gamma.RIII but not to
mouse Fc.gamma.RI and mouse Fc.gamma.RIV, suggests that mouse
Fc.gamma.Rs important for the reduction of antigen concentration
are mouse Fc.gamma.RII and/or mouse Fc.gamma.RIII. VH3/L
(WT)-mIgG-mF44 and VH3/L (WT)-mIgG1-mF46 introduced with an
alteration that is thought to increase the Fc.gamma.R-binding
activity of VH3/L (WT)-mIgG1 was demonstrated to have increased
binding activity to both of mouse Fc.gamma.RIIb and mouse
Fc.gamma.RIII.
(5-4) Assessment of the Effect to Reduce the Soluble IL-6 Receptor
Concentration in the Plasma of Normal Mice
[0487] The effect to eliminate soluble IL-6 receptor from the
plasma of normal mice administered with the anti-human IL-6
receptor antibody Fv4-mIgG1, Fv4-mIgG1-mF44, or Fv4-mIgG1mF46 was
assessed as follows.
[0488] An animal model where the soluble human IL-6 receptor
concentration is maintained in a steady state in plasma was created
by implanting an infusion pump (MINI-OSMOTIC PUMP MODEL2004, alzet)
containing soluble human IL-6 receptor under the skin on the back
of normal mice (C57BL/6J mouse, Charles River Japan). The in vivo
dynamics of soluble human IL-6 receptor after administration of the
anti-human IL-6 receptor antibody was assessed in the animal model.
To suppress the production of antibodies against soluble human IL-6
receptor, an anti-mouse CD4 monoclonal antibody was administered
once at 20 mg/kg into the caudal vein. Then, an infusion pump
containing 92.8 .mu.g/ml soluble human IL-6 receptor was
subcutaneously implanted on the back of the mice. Three days after
implantation of the infusion pump, the anti-human IL-6 receptor
antibody was administered once at 1 mg/kg into the caudal vein. The
blood was collected from the mice 15 minutes, seven hours, one day,
two days, four days, seven days, 14 days (or 15 days), and 21 days
(or 22 days) after administration of the anti-human IL-6 receptor
antibody. Immediately thereafter, the collected blood was
centrifuged at 15,000 rpm and 4.degree. C. for 15 minutes to
prepare the plasma. The isolated plasma was stored in a freezer set
at -20.degree. C. or below until use.
[0489] The soluble human IL-6 receptor concentrations in plasma
were determined by the method described in (2-1-2). The result is
shown in FIG. 11.
[0490] Surprisingly, it was demonstrated that, in mice administered
with mF44 and mF46 introduced with an alteration to increase the
binding activity of mIgG1 (native mouse IgG1) to mouse
Fc.gamma.RIIb and mouse Fc.gamma.RIII, the plasma IL-6 receptor
concentration was markedly reduced as compared to mice administered
with mIgG1. In particular, even on day 21 after administration of
mF44, the plasma IL-6 receptor concentration in the
mF44-administered group was reduced by about 6 times as compared to
the plasma IL-6 receptor concentration in the group without
antibody administration, and about 10 times as compared to the
mIgG1-administered group. On the other hand, on day seven after
administration of mF46, the plasma IL-6 receptor concentration in
the mF46-administered group was markedly reduced by about 30 times
as compared to the plasma IL-6 receptor concentration in the group
without antibody administration, and about 50 times as compared to
the mIgG1-administered group.
[0491] The above findings demonstrate that the elimination of
soluble IL-6 receptor from plasma was also accelerated in mice
administered with antibodies in which the mouse Fc.gamma.R-binding
activity of an antigen-binding molecule having the Fc regions of
mouse IgG1 antibody is increased, as with antibodies in which the
mouse Fc.gamma.R-binding activity of an antigen-binding molecule
having the Fc region of human IgG1 antibody is increased. Without
being bound by a particular theory, the phenomenon observed as
described above can be explained as follows.
[0492] When administered to mice, antibodies that bind to a soluble
antigen in a pH-dependent manner and have increased
Fc.gamma.R-binding activity are actively incorporated mainly into
cells expressing Fc.gamma.R on the cell membrane. The incorporated
antibodies dissociate the soluble antigen under an acidic pH
condition in the endosome, and then recycled to plasma via FcRn.
Thus, a factor that achieves the effect of eliminating the plasma
soluble antigen of such an antibody is the Fc.gamma.R-binding
activity level of the antibody. Specifically, as the
Fc.gamma.R-binding activity is greater, the incorporation into
Fc.gamma.R-expressing cells occurs more actively, and this makes
the elimination of soluble antigens from plasma more rapid.
Furthermore, as long as the Fc.gamma.R-binding activity has been
increased, the effect can be assessed in the same manner regardless
of whether the Fc region contained in an antibody originates from
human or mouse IgG1. Specifically, the assessment can be achieved
for an Fc region of any animal species, such as any of human IgG1,
human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse
IgG2b, mouse IgG3, rat IgG, monkey IgG, and rabbit IgG, as long as
the binding activity to the Fc.gamma.R of the animal species to be
administered has been increased.
Example 6
The Antigen Elimination Effect by Antibodies with the Binding
Activity Increased in an Fc.gamma.RIIb-Selective Manner
[0493] (6-1) The Antigen Elimination Effect of Antibodies in which
the Fc.gamma.RIIb-Binding Activity has been Selectively
Increased
[0494] Fc.gamma.RIII-deficient mice (B6.129P2-FcgrR3tm1Sjv/J mouse,
Jackson Laboratories) express mouse Fc.gamma.RI, mouse
Fc.gamma.RIIb, and mouse Fc.gamma.RIV, but not mouse Fc.gamma.RIII.
Meanwhile, Fc receptor .gamma. chain-deficient mice (Fcer1g mouse,
Taconic, Cell (1994) 76, 519-529) express mouse Fc.gamma.RIIb
alone, but not mouse Fc.gamma.RI, mouse Fc.gamma.RIII, and mouse
Fc.gamma.RIV.
[0495] As described in Example 5, it was demonstrated that mF44 and
mF46 with increased Fc.gamma.R-binding activity of native mouse
IgG1 show selectively enhanced binding to mouse Fc.gamma.RIIb and
mouse Fc.gamma.RIII. It was conceived that, using the selectively
increased binding activity of the antibodies, the condition under
which an antibody with selectively enhanced mouse Fc.gamma.RIIb
binding is administered can be mimicked by administering mF44 and
mF46 to mouse Fc.gamma.RIII-deficient mice or Fc receptor .gamma.
chain-deficient mice which do not express mouse Fc.gamma.RIII.
(6-2) Assessment of the Antigen Elimination Effect by Selective
Enhancement of Binding to Mouse Fc.gamma.RIIb Using
Fc.gamma.RIII-Deficient Mice
[0496] The effect to eliminate soluble IL-6 receptor from plasma in
Fc.gamma.RIII-deficient mice administered with the anti-human IL-6
receptor antibody Fv4-mIgG1, Fv4-mIgG1-mF44, or Fv4-mIgG1-mF46 was
assessed by the same method described in Example 5. The soluble
human IL-6 receptor concentrations in the plasma of the mice were
determined by the method described in Example (2-1-2). The result
is shown in FIG. 12.
[0497] Surprisingly, it was demonstrated that, the plasma IL-6
receptor concentrations in Fc.gamma.RIII-deficient mice
administered with mF44 and mF46, which mimic the condition under
which the mouse Fc.gamma.RIIb-binding activity of mIgG1 (native
mouse IgG1) is selectively increased, were markedly reduced as
compared to the plasma IL-6 receptor concentration in mice
administered with mIgG1. In particular, the plasma IL-6 receptor
concentration of the mF44-administered group was reduced by about
three times as compared to that of the mIgG1-administered group and
the accumulation of antibody concentration due to antibody
administration was suppressed. Meanwhile, on day three after
administration, the plasma IL-6 receptor concentration of the
mF46-administered group was markedly reduced by about six times as
compared to the plasma IL-6 receptor concentration of the group
without antibody administration, and about 25 times as compared to
the plasma IL-6 receptor concentration of the mIgG1-administered
group. This result shows that, as the mouse Fc.gamma.RIIb-binding
activity of an anti-human IL-6 receptor antibody that binds to the
antigen in a pH-dependent manner is greater, the IL-6 receptor
concentration can be reduced more in the plasma of mice
administered with the antibody.
(6-3) Assessment of the Antigen Elimination Effect by Selective
Enhancement of Mouse Fc.gamma.RIIb Binding Using Fc Receptor
.gamma. Chain-Deficient Mice
[0498] The effect to eliminate soluble IL-6 receptor from the
plasma of Fc receptor .gamma. chain-deficient mice administered
with the anti-human IL-6 receptor antibody Fv4-mIgG1,
Fv4-mIgG1-mF44, or Fv4-mIgG1mF46, was assessed by the same method
as described in Example 5. The soluble human IL-6 receptor
concentrations in the plasma of the mice were determined by the
method described in Example (2-1-2). The result is shown in FIG.
13.
[0499] As with the case where mF44 and mF46 were administered to
Fc.gamma.RIII-deficient mice, the plasma IL-6 receptor
concentration in Fc receptor .gamma. chain-deficient mice
administered with mF44 and mF46, which mimic the condition
resulting from the selective increase in the mouse
Fc.gamma.RIIb-binding activity of mIgG1 (native mouse IgG1), was
demonstrated to be markedly reduced as compared to the plasma IL-6
receptor concentration in Fc receptor .gamma. chain-deficient mice
administered with mIgG1. In particular, the plasma IL-6 receptor
concentration in the mF44-administered group was reduced to about
three times that in the mIgG1-administered group, and the
accumulation of antigen concentration due to antibody
administration was suppressed. Meanwhile, on day three after
administration, the plasma IL-6 receptor concentration in the
mF46-administered group was markedly reduced by about five times as
compared to that in the group without antibody administration, and
about 15 times as compared to that in the mIgG1-administered
group.
[0500] The results described in Examples (6-2) and (6-3) show that
the soluble antigen concentration in the plasma is markedly reduced
in the group administered with an antibody that binds to a soluble
antigen in a pH-dependent manner and has selectively increased
mouse Fc.gamma.RIIb-binding activity.
Example 7
The Antigen Elimination Effect of Antibodies with Selective
Enhancement of the Binding to Fc.gamma.RIII
[0501] (7-1) The Antigen Elimination Effect of Antibodies with
Selectively Enhanced Fc.gamma.RIII Binding
[0502] Fc.gamma.RIIb-deficient mice (Fcgr2b (Fc.gamma.RII) mouse,
Taconic) (Nature (1996) 379 (6563), 346-349) express mouse
Fc.gamma.RI, mouse Fc.gamma.RIII, and mouse Fc.gamma.RIV, but not
mouse Fc.gamma.RIIb. As described in Example 5, it was demonstrated
that mF44 and mF46 resulting from increasing the Fc.gamma.R-binding
activity of native mouse IgG1 show selectively enhanced binding to
mouse Fc.gamma.RIIb and mouse Fc.gamma.RIII. It was conceived that,
based on the use of the selectively increased binding activity of
the antibodies, the condition of administration of an antibody with
selectively enhanced binding to mouse Fc.gamma.RIII can be mimicked
by administering mF44 or mF46 to mouse Fc.gamma.RIIb-deficient mice
which do not express mouse Fc.gamma.RIIb.
[0503] As described in Example 6, the soluble antigen concentration
was reduced in the plasma of Fc.gamma.RIII-deficient mice, which
mimic the condition of administration of an antibody with
selectively increased mouse Fc.gamma.RIIb-binding activity.
Meanwhile, whether the soluble antigen concentration is reduced in
the plasma of Fc.gamma.RIIb-deficient mice, which mimic the
condition of administration of an antibody with selectively
increased mouse Fc.gamma.RIII-binding activity, was assessed by the
test described below.
(7-2) Assessment of the Antigen Elimination Effect by Selective
Enhancement of Mouse Fc.gamma.RIII Binding Using
Fc.gamma.RIIb-Deficient Mice
[0504] The effect to eliminate soluble IL-6 receptor from the
plasma of Fc.gamma.RIIb-deficient mice administered with the
anti-human IL-6 receptor antibody Fv4-mIgG1, Fv4-mIgG1-mF44, or
Fv4-mIgG1mF46, was assessed by the same method as described in
Example 5. The soluble human IL-6 receptor concentrations in plasma
were determined by the method described in Example (2-1-2). The
result is shown in FIG. 14.
[0505] Surprisingly, in the groups administered with mF44 and mF46,
which mimic selective increase of the mouse Fc.gamma.RIII-binding
activity of mIgG1 (native mouse IgG1), the plasma IL-6 receptor
concentration was reduced, but the remarkable reduction was not
confirmed compared to that shown in Example 6.
[0506] Without being bound by a particular theory, based on the
results described in Examples 5, 6, and 7, the following discussion
is possible. The elimination of soluble IL-6 receptor from plasma
was found to be markedly accelerated in normal mice expressing both
mouse Fc.gamma.RIIb and mouse Fc.gamma.RIII that were administered
with mF44 and mF46 with selectively increased binding activity of
mIgG1 (native mouse IgG1) to mouse Fc.gamma.RIIb and mouse
Fc.gamma.RIII. Furthermore, it was revealed that, when mF44 and
mF46 were administered to mice that express mouse Fc.gamma.RIIb but
not mouse Fc.gamma.RIII (i.e., Fc.gamma.RIII-deficient mice and Fc
receptor .gamma. chain-deficient mice), the elimination of soluble
IL-6 receptor from plasma was also accelerated markedly in the
mice. Meanwhile, when mF44 and mF46 were administered to mice that
express mouse Fc.gamma.RIII but not mouse Fc.gamma.RIIb (i.e.,
Fc.gamma.RII-deficient mice), the elimination of soluble IL-6
receptor from plasma was not remarkably accelerated in the
mice.
[0507] From the above findings, it is thought that, the antibodies
mF44 and mF46 in which the binding activity of mIgG1 (native mouse
IgG1) to mouse Fc.gamma.RIIb and mouse Fc.gamma.RIII is increased,
are incorporated into Fc.gamma.R-expressing cells mainly by mouse
Fc.gamma.RIIb, and thus the soluble antigen in the plasma that
binds to the antibodies is eliminated. Meanwhile, the
Fc.gamma.RIII-mediated incorporation of antibody/antigen complexes
into Fc.gamma.R-expressing cells is thought not to significantly
contribute to the elimination of the soluble antigen from plasma.
Furthermore, as shown in Example 4, the plasma concentration of
soluble IL-6 receptor was markedly reduced in mice administered
with Fv4-IgG1-F1087 having increased binding activity to mouse
Fc.gamma.RIIb and mouse Fc.gamma.RIII, in particular. Meanwhile,
the effect to eliminate soluble IL-6 receptor from the plasma of
mice administered with Fv4-IgG1-F1182 with increased binding
activity to mouse Fc.gamma.RI and mouse Fc.gamma.RIV, in
particular, was smaller than that of Fv4-IgG1-F1087.
[0508] Furthermore, as shown in Example 2, in mice administered
with Fv4-IgG1-Fuc whose mouse Fc.gamma.RIV-binding activity has
been considerably increased by having sugar chains with low fucose
content (Science (2005) 310 (5753), 1510-1512), the plasma
concentration of soluble IL-6 receptor was reduced as compared to
that in mice administered with Fv4-IgG1; however, the reduction
effect was as small as about twice. Thus, mouse
Fc.gamma.RIV-mediated incorporation of antibodies into
Fc.gamma.R-expressing cells is thought not to significantly
contribute to the elimination of soluble antigens from plasma.
[0509] In view of the above, it was demonstrate that, of several
mouse Fc.gamma.Rs, mouse Fc.gamma.RIIb plays a major role in
antibody incorporation into Fc.gamma.R-expressing cells in mice.
Thus, it would be thought that mutations to be introduced into the
mouse Fc.gamma.R-binding domain particularly preferably include,
but are not limited to, mutations that enhance the binding to mouse
Fc.gamma.RIIb.
[0510] The above findings demonstrate that, in mice, the
Fc.gamma.RIIb-binding activity of the antibodies to be administered
is more preferably increased to accelerate the elimination of
soluble antigens from the plasma of a living organism by
administering to it antigen-binding molecules that bind to soluble
antigens in a pH-dependent manner and have increased Fc.gamma.R
binding activity. Specifically, when administered to a living
organism, antigen-binding molecules that bind to soluble antigens
in a pH-dependent manner and have increased Fc.gamma.RIIb-binding
activity can accelerate the elimination of the soluble antigens
from plasma and effectively reduce the plasma concentration of
soluble antigens, and thus, the antigen-binding molecules were
revealed to show a very effective action.
Example 8
Assessment of the Platelet Aggregatory Ability of Antibodies
Containing an Fc Region introduced with an Existing Alteration that
Enhances the Fc.gamma.RIIb Binding
[0511] (8-1) Preparation of Antibodies Containing an Fc Region
Introduced with an Existing Alteration that Enhances the
Fc.gamma.RIIb Binding
[0512] As described in Example 7, antigens can be efficiently
eliminated from the plasma of the living organism by administering
antibodies with selectively increased Fc.gamma.RIIb-binding
activity to the living organism. Furthermore, the administration of
antibodies containing an Fc region with selectively increased
Fc.gamma.RIIb-binding activity is thought to be preferred from the
viewpoint of safety and side effects in the living organism
administered with such antibodies.
[0513] However, the mouse Fc.gamma.RIIb binding and mouse
Fc.gamma.RIII binding are both enhanced in mF44 and mF46, and thus
the binding enhancement is not selective for mouse Fc.gamma.RIIb.
Since the homology between mouse Fc.gamma.RIIb and mouse
Fc.gamma.RIII is high, it would be difficult to find an alteration
that enhances the mouse Fc.gamma.RIIb-selective binding while
distinguishing the two. Moreover, there is no previous report on Fc
regions with selectively enhanced mouse Fc.gamma.RIIb binding.
Also, the homology between human Fc.gamma.RIIb and human
Fc.gamma.RIIa (the two allotypes, 131Arg and 131His) is also known
to be high. Moreover, there is no report on Fc regions that contain
an alteration that enhances the human Fc.gamma.RIIb-selective
binding while distinguishing the two (Seung et al., (Mol. Immunol.
(2008) 45, 3926-3933); Greenwood et al., (Eur. J. Immunol. (1993)
23 (5), 1098-1104)). Furthermore, it has been reported that
antibodies with enhanced Fc.gamma.RIIa-binding has increased
platelet aggregation activity and may increase the risk for
developing thrombosis when administered to organisms (Meyer et al.
(J. Thromb. Haemost. (2009), 7 (1), 171-181) and Robles-Carrillo et
al. (J. Immunol. (2010), 185 (3), 1577-1583)). Thus, whether
antibodies with enhanced Fc.gamma.RIIa binding have an increased
platelet aggregatory activity was assessed as follows.
(8-2) Assessment of the Human Fc.gamma.R-Binding Activity of
Antibodies Containing an Fc Region Introduced with an Existing
Alteration that Enhances the Fc.gamma.RIIb Binding
[0514] Antibodies containing an Fc region introduced with an
existing alteration that enhances the human Fc.gamma.RIIb binding
were analyzed for their affinity for human Fc.gamma.RIa, R-type and
H-type Fc.gamma.RIIa, Fc.gamma.RIIb, and Fc.gamma.RIIIa by the
following procedure. An H chain was constructed to have, as the
antibody H chain variable region, the antibody variable region
IL6R-H (SEQ ID NO: 53) against human interleukin 6 receptor which
is disclosed in WO2009/125825, and as the antibody H chain constant
region, IL6R-G1d (SEQ ID NO: 54) that has G1d resulting from
removing the C-terminal Gly and Lys from human IgG1. Then,
IL6R-G1d-v3 (SEQ ID NO: 55) was constructed by altering the Fc
region of IL6R-G1d by the substitution of Glu for Ser at position
267 (EU numbering) and Phe for Leu at position 328 (EU numbering),
as described in Seung et al., (Mol. Immunol. (2008) 45, 3926-3933).
IL6R-L (SEQ ID NO: 56) which is the L chain of anti-human
interleukin 6 receptor antibody was used as a common antibody L
chain, and expressed in combination with respective H chains
according to the method described in Reference Example 1, and the
resulting antibodies were purified. Hereinafter, antibodies
containing IL6R-G1d and IL6R-G1d-v3 as the heavy chain are referred
to as IgG1 and IgG1-v3, respectively.
[0515] Then, the interaction between Fc.gamma.R and the above
antibodies was kinetically analyzed using Biacore T100 (GE
Healthcare). The assay for the interaction was carried out at
25.degree. C. using HBS-EP+(GE Healthcare) as a running buffer. The
chip used was a Series S Sencor Chip CM5 (GE Healthcare)
immobilized with Protein A by an amino coupling method. Each
Fc.gamma.R diluted with the running buffer was allowed to interact
with the antibodies of interest captured onto the chip to measure
the binding of the antibodies to each Fc.gamma.R. After
measurement, 10 mM glycine-HCl (pH 1.5) was reacted to the chip to
wash off the captured antibodies to repeatedly use the regenerated
chip. A sensorgram obtained as a result of the measurement was
analyzed using 1:1 Langmuir binding model with Biacore Evaluation
Software, and binding rate constant ka (L/mol/s) and dissociation
rate constant kd (l/s) were calculated, and the dissociation
constant KD (mol/l) was calculated from these values. The KD values
of IgG1 and IgG1-v3 to each Fc.gamma.R (the KD values of each
antibody to each Fc.gamma.R) are shown in Table 11, while the
relative KD values of IgG1-v3, which are obtained by dividing KD of
IgG1 to each Fc.gamma.R by KD of IgG1-v3 to each Fc.gamma.R, are
shown in Table 12.
TABLE-US-00018 TABLE 11 KD (M) ANTIBODY Fc .gamma. RIa Fc .gamma.
RIIaR Fc .gamma. RIIaH Fc .gamma. RIIb Fc .gamma. RIIIa IgG1
3.4E-10 1.2E-06 7.7E-07 5.3E-06 3.1E-06 IgG1-v3 1.9E-10 2.3E-09
1.5E-06 1.3E-08 8.8E-06
TABLE-US-00019 TABLE 12 Fc .gamma. RIa Fc .gamma. RIIaR Fc .gamma.
RIIaH Fc .gamma. RIIb Fc .gamma. RIIIa KD VALUE 1.8 522 0.51 408
0.35 RATIO
[0516] These results confirmed that compared to the antibody
containing the IgG1 Fc region, the antibody containing an altered
Fc region in which Ser at position 267 has been substituted with
Glu and Leu at position 328 has been substituted with Phe, as
indicated by EU numbering, in the IgG1 Fc region (Mol. Immunol.
(2008) 45, 3926-3933), has 408-times increased affinity to
Fc.gamma.RIIb, and while affinity to Fc.gamma.RIIa H type was
decreased to 0.51 times, affinity to Fc.gamma.RIIa R type was
increased 522 times.
(8-3) Assessment of the Ability to Aggregate Platelets
[0517] Next, whether the increased/reduced Fc.gamma.RIIa affinity
of the antibody containing the Fc region with the substitution of
Glu for Ser at position 267 and Phe for Leu at position 328 (EU
numbering) in the Fc region of IgG1 changes the platelet
aggregatory ability, was assessed using platelets derived from
donors with H-type or R-type Fc.gamma.RIIa. The antibody comprising
as the light chain omalizumab_VL-CK (SEQ ID NO: 58) and
omalizumab_VH-G1d (SEQ ID NO: 57) that contains the heavy chain
variable region of hIgG1 antibody (human IgG1 constant region) that
binds to IgE and the G1d heavy chain constant region, was
constructed using the method described in Reference Example 1.
Furthermore, omalizumab_VH-G1d-v3 (SEQ ID NO: 59) was constructed
by substituting Glu for Ser at position 267 and Phe for Leu at
position 328 (EU numbering) in omalizumab_VH-G1d.
Omalizumab-G1d-v3, which contains omalizumab_VH-G1d-v3 as the heavy
chain and omalizumab_VL-CK as the light chain, was prepared using
the method described in Reference Example 1. This antibody was
assessed for the platelet aggregatory ability.
[0518] 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 A (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 B (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). This yielded washed platelets at a
density of about 300,000/.mu.l. 156 .mu.l of the washed platelets
was aliquoted into assay cuvettes containing a stir bar 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.
[0519] The result of platelet aggregation for each donor with an
Fc.gamma.RIIa polymorphic form (H/H or R/H) obtained from the above
assay is shown in FIGS. 15 and 16. From the result in FIG. 15, it
is shown that platelet aggregation is enhanced when the immune
complex is added to the platelets of a donor with the Fc.gamma.RIIa
polymorphic form (R/H). Meanwhile, as shown in FIG. 16, platelet
aggregation was not enhanced when the immune complex is added to
the platelets of a donor with the Fc.gamma.RIIa polymorphic form
(H/H).
[0520] 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).
Fluorescence intensity for each stain was measured using a flow
cytometer (FACS CantoII, BD bioscience).
[0521] The result on CD62p expression, obtained by the above assay
method, is shown in FIG. 17. The result on the activated integrin
expression is shown in FIG. 18. 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.
[0522] The above results demonstrate that the antibody having the
Fc region introduced with an existing alteration that enhances the
human Fc.gamma.RIIb binding, which is the substitution of Glu for
Ser at position 267 and Phe for Leu at position 328 (EU numbering)
in the Fc region of IgG1, promotes the aggregation of platelets
whose Fc.gamma.RIIa allotype is that in which the amino acid at
position 131 is R, as compared to platelets whose Fc.gamma.RIIa
allotype is that in which the amino acid at position 131 is H. That
is, it was suggested that the risk of developing thrombosis due to
platelet aggregation can be increased when an antibody containing
an Fc region introduced with an existing alteration that enhances
the binding to existing human Fc.gamma.RIIb is administered to
humans having R-type Fc.gamma.RIIa. It was shown that the
antigen-binding molecules containing an Fc region of the present
invention that enhances the Fc.gamma.RIIb binding more selectively
not only improves the antigen retention in plasma, but also
possibly solves the above problems. Thus, the usefulness of the
antigen-binding molecules of the present invention is obvious.
Example 9
Comprehensive Analysis of Fc.gamma.RIIb Binding of Variants
Introduced with an Alteration at the Hinge Portion in Addition to
the P238D Alteration
[0523] 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 from the analysis of naturally-occurring
antibodies. Therefore, in order to find variants that further
enhance Fc.gamma.RIIb binding, alterations were comprehensively
introduced into the altered Fc produced by substituting Pro at
position 238 (EU numbering) with Asp. IL6R-F11 (SEQ ID NO: 60) 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 in
IL6R-G1d (SEQ ID NO: 54) which was used as the antibody H chain.
Furthermore, IL6R-F652 (SEQ ID NO: 61) was prepared by introducing
an alteration of substituting Pro at position 238 (EU numbering)
with Asp into IL6R-F11. 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 L6R-F652 each with 18 amino acids excluding the
original amino acids and Cysteine. IL6R-L (SEQ ID NO: 56) was
utilized as an antibody L chain. These variants were expressed and
purified 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.
[0524] A figure that shows the results of analyzing the interaction
with the respective Fc.gamma.Rs 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 shown 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. 19).
[0525] As a result, it was found that the Fc.gamma.RIIb binding of
eleven types of alterations were enhanced compared with the
antibody before introducing alterations, and they have the effects
of maintaining or enhancing Fc.gamma.RIIa type R-binding. The
activities of these eleven variants to bind Fc.gamma.RIIb and
Fc.gamma.RIIa R are summarized in Table 13. In the table, the
sequence ID numbers refer to those of the H chains of the variants,
and alteration refers to the alteration introduced into IL6R-F11
(SEQ ID NO: 60).
TABLE-US-00020 TABLE 13 RELATIVE RELATIVE BINDING BINDING ACTIVITY
TO ACTIVITY 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
[0526] FIG. 20 shows relative values for the Fc.gamma.RIIb-binding
activity obtained by additionally introducing the above eleven
alterations into a variant carrying the P238D alteration, and
relative values for the Fc.gamma.RIIb-binding activity of a variant
obtained by introducing the alterations into an Fc that does not
contain the P238D. These eleven alterations enhanced the amount of
Fc.gamma.RIIb binding compared with before introduction when they
were further introduced into the P238D variant. 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 (data not
shown).
[0527] These results showed that, based on the effects of
introducing alterations into a naturally-occurring IgG1, it is
difficult to predict the effects of combining and introducing the
same alterations into the variant containing the P238D alteration.
In other words, it would not have been possible to discover these
eight alterations identified this time without this investigation
that introduces the same alterations are combined and introduced
into the variant containing the P238D alteration.
[0528] The results of measuring KD values of the variants indicated
in Table 13 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 14. In the table, alteration
refers to the alteration introduced into IL6R-F11 (SEQ ID NO: 60).
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 (I %) 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 variant 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 14 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: 60) 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 gray-filled
cells in Table 14 show values calculated by using Equation 2 of
Reference Example 2.
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
[0529] Table 14 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 14 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 maintained or decreased binding activities to
Fc.gamma.RIIa type R and type H and enhanced binding activity to
Fc.gamma.RIIb, 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.
TABLE-US-00021 TABLE 14 KD VALUE FOR THE STRONGER OF THE BINDING
ACTIVITIES OF A VARIANT TO Fc.gamma.RIIaR AND Fc.gamma.RIIaH/ KD
VALUE FOR THE KD (IIb) STRONGER OF THE OF PARENT BINDING ACTIVITIES
POLYPEPTIDE/ OF THE PARENT KD (IIb) POLYPEPTIDE TO KD (mol/L) KD
(IIaR)/ KD (IIaH)/ OF ALTERED Fc.gamma.RIIaR AND ALTERATION
Fc.gamma.RIa Fc.gamma.RIIaR Fc.gamma.RIIaH Fc.gamma.RIIb
Fc.gamma.RIIIaV KD (IIb) KD (IIb) POLYPEPTIDE Fc.gamma.RIIaH *
3.2E-10 1.0E-06 6.7E-07 2.6E-06 3.5E-07 0.4 0.3 2.6 0.1 9.0E-10
5.0E-06 5.0E-06 6.8E-06 1.5E-06 0.7 0.7 1.0 1.0 L234W/P238D 6.3E-08
1.6E-05 1.9E-05 2.0E-06 3.7E-05 8.1 9.5 3.4 3.2 L234Y/P238D 7.5E-08
2.6E-05 2.3E-05 1.6E-06 4.5E-05 15.9 14.4 4.2 4.6 G237A/P238D
1.4E-07 3.2E-05 2.1E-05 3.0E-06 3.7E-05 10.5 7.0 2.3 4.2
G237D/P238D 1.4E-07 2.1E-05 2.5E-05 2.0E-06 4.3E-05 10.7 12.8 3.5
4.2 G237E/P238D 3.4E-07 3.8E-05 2.5E-05 3.6E-06 4.1E-05 10.6 7.0
1.9 5.0 G237F/P238D 5.2E-08 1.4E-05 1.6E-05 3.4E-06 4.3E-05 4.1 4.7
2.0 2.8 G237L/P238D 1.2E-07 1.8E-05 1.8E-05 2.6E-06 4.1E-05 6.9 7.1
2.7 3.5 G237M/P238D 5.2E-08 2.2E-05 2.0E-05 2.9E-06 3.7E-05 7.7 7.0
2.4 4.0 G237W/P238D 3.6E-08 7.2E-06 1.2E-05 2.3E-06 3.8E-05 3.1 5.2
2.9 1.4 G237Y/P238D 9.3E-08 7.9E-06 1.5E-05 2.3E-06 4.2E-05 3.4 6.4
2.9 1.6 P238D/S239D 4.9E-09 3.5E-06 1.9E-05 1.4E-06 1.7E-05 2.6
14.0 5.0 0.7
Example 10
X-Ray Crystal Structure Analysis of a Complex Formed Between an Fc
Containing P238D and an Extracellular Region of Fc.gamma.RIIb
[0530] As indicated earlier in Example 9, even though an alteration
that is predicted from the analysis of naturally-occurring IgG1
antibodies to improve 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 at 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,
referred to as Fc (P238D)) and the extracellular region of
Fc.gamma.RIIb was elucidated by X-ray crystal structure analysis,
and this was compared to the three-dimensional structure of the
complex formed between the Fc of a naturally-occurring IgG1
(hereinafter, referred to as Fc (WT)) and the extracellular region
of Fc.gamma.RIIb, and the binding modes were compared. Multiple
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. Immunol. (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, Fc.gamma.RIIa, whose three-dimensional structure in
complex with Fc (WT) has already known, and Fc.gamma.RIIb match 93%
in amino acid sequence of their extracellular region 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.
[0531] 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.
21. The Fc.gamma.RIIb extracellular region is bound between two Fc
CH2 domains, and this was 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. 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 the least squares
fitting based on the C.alpha. atom pair distances with respect to
the Fc.gamma.RIIb extracellular region and the Fc CH2 domain A
(FIG. 22). 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 extracted Fc.gamma.RIIb extracellular region and Fc CH2
domain B were compared for comparison of the interatomic
interaction between Fc.gamma.RIIb and Fc (WT) CH2 domain B with the
interatomic interaction between Fc.gamma.RIIb and Fc (P238D) CH2
domain B. As shown in Table 15, the interatomic interactions
between Fc CH2 domain B and Fc.gamma.RIIb in Fc (P238D) and Fc (WT)
did not match.
TABLE-US-00022 TABLE 15 Fc (P648D) CH2 Fc (WT) CH2 DOMAIN B DOMAIN
B INTERACTION INTERACTION PARTNER PARTNER (DISTANCE (DISTANCE
BETWEEN BETWEEN Fc.gamma.RIIb ATOM ATOMS, .ANG.) ATOMS, .ANG.) 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 O 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) Ala
327 CB (3.63) Arg 131 NH1 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) Arg 131 NH2 Asp 270 CG
(3.20) Asp 265 CA (3.16) Asp 270 OD2 (2.80) Val 266 N (3.37) Asp
270 OD1 (2.87) Ala 327 CB (3.66) Tyr 157 CE1 Leu 234 CG (3.64) Leu
234 CD1 (3.61) Tyr 157 OH Gly 236 O (3.62) Leu 234 CA (3.48) Leu
234 CG (3.45)
[0532] Furthermore, the detailed structures around P238D were
compared by superposing the X-ray crystal structure of Fc
(P238D)/Fc.gamma.RIIb extracellular region complex on the model
structure of the Fc (WT)/Fc.gamma.RIIb extracellular region complex
using the least squares method based on the C.alpha. atomic
distance between Fc CH2 domains A and B alone. As the position of
the amino acid residue at position 238 (EU numbering), i.e., a
mutagenesis position of Fc (P238D), is altered from Fc (WT), the
loop structure around the amino acid residue at position 238
following the hinge region is found to be different between Fc
(P238D) and Fc (WT) (FIG. 23). Pro at position 238 (EU numbering)
is originally located inside Fc (WT), forming a hydrophobic core
with residues around position 238. However, if Pro at position 238
(EU numbering) is altered to highly hydrophilic and charged Asp,
the presence of the altered Asp residue in a hydrophobic core is
energetically disadvantageous 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 region, and this may
have caused this change in the loop structure near the amino acid
residue at position 238. Furthermore, since this loop is not far
from the hinge region crosslinked by S--S bonds, its structural
change will not be limited to a local change, and will affect the
relative positioning between the FcCH2 domain A and domain B. As a
result, the interatomic interactions between Fc.gamma.RIIb and Fc
CH2 domain B were assumed to 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.
[0533] 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 position 237 (EU numbering),
which is adjacent to P238D which is mutated, and Tyr at position
160 in Fc.gamma.RIIb (FIG. 24). 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. Considering that
the amino acid at position 160 is one of the few differences
between Fc.gamma.RIIa and Fc.gamma.RIIb in 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. 25). In
Fc.gamma.RIIa type H, which is one of the allotypes of
Fc.gamma.RIIa, the residue corresponding to Arg at position 131 of
Fc.gamma.RIIb 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 near P238D
due to P238D introduction and the accompanying change in the
relative domain positioning causes formation of new interactions
which is not found in the binding of the naturally-occurring IgG
and Fc.gamma.R, and this could lead to a selective binding profile
of P238D variants for Fc.gamma.RIIb.
[Expression and Purification of Fc (P238D)]
[0534] An Fc containing the P238D alteration was prepared as
follows. First, Cys at position 220 (EU numbering) of hIL6R-IgG1-v1
(SEQ ID NO: 62) was substituted with Ser. Then, genetic sequence of
Fc (P238D) from Glu at position 236 (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 (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, the Cys
residue was substituted with Ser to avoid formation of unnecessary
disulfide bonds.
[Expression and Purification of the Fc.gamma.RIIb Extracellular
Region]
[0535] The Fc.gamma.RIIb extracellular region was prepared
according to the method of Reference Example 2.
[Purification of the Fc (P238D)/Fc.gamma.RIIb Extracellular Region
Complex]
[0536] To 2 mg of the Fc.gamma.RIIb extracellular region sample
obtained for use in 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 sugar
chains were cleaved, except for N-acetylglucosamine directly bound
to Asn of the Fc.gamma.RIIb extracellular region. Next, the
Fc.gamma.RIIb extracellular region sample subjected to sugar chain
cleavage treatment, which was concentrated by ultrafiltration with
5000 MWCO, was purified by gel filtration chromatography
(Superdex200 10/300) 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. The mixture
concentrated by ultrafiltration with 10,000 MWCO was purified by
gel filtration chromatography (Superdex200 10/300) using a column
equilibrated in 20 mM HEPS at pH 7.5 containing 0.05 M NaCl. Thus,
a sample of the Fc (P238D)/Fc.gamma.RIIb extracellular region
complex was obtained.
[Crystallization of the Fc (P238D)/Fc.gamma.RIIb Extracellular
Region Complex]
[0537] Using the sample of the Fc (P238D)/Fc.gamma.RIIb
extracellular region complex which was concentrated to
approximately 10 mg/mL by ultrafiltration with 10,000 MWCO,
crystallization of the complex was carried out by the sitting drop
vapor diffusion method. Hydra II Plus One (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. The crystallization drop after sealing was allowed to remain
at 20.degree. C., and thus thin plate-like crystals were
obtained.
[Measurement of X-Ray Diffraction Data from an Fc
(P238D)/Fc.gamma.RIIb Extracellular Region Complex Crystal]
[0538] 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
single crystal was fished out of the solution using a pin with
attached tiny nylon loop, and frozen in liquid nitrogen. Then, the
X-ray diffraction data of the crystal 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 of the
crystal 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.,
a=90.degree., 13=100.70.degree., .gamma.=90.degree..
[X Ray Crystal Structure Analysis of the Fc (P238D)/Fc.gamma.RIIb
Extracellular Region Complex]
[0539] 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 set
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 set 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 set 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). 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]
[0540] 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 from among them was set as
the model structure for the Fc (WT)/Fc.gamma.RIIb extracellular
region complex.
Example 11
Analysis of Fc.gamma.R Binding of Fc Variants Whose Alteration
Sites were Determined Based on Crystal Structures
[0541] Based on the results of X-ray crystal structure analysis on
the complex formed between Fc (P238D) and the Fc.gamma.RIIb
extracellular region obtained in Example 10, variants were
constructed by comprehensively introducing alterations into sites
on the altered Fc 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 whether combinations of
alterations that further enhance Fc.gamma.RIIb binding in addition
to the P238D alteration can be obtained, was examined.
[0542] IL6R-B3 (SEQ ID NO: 63) was produced by introducing into
IL6R-G1d (SEQ ID NO: 54), 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: 56) was utilized as the common antibody L chain. These
antibody variants expressed were purified according to the method
of Reference Example 1. The binding of these antibody variants 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.
[0543] A figure was produced according to the following method to
show 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, alteration
by substituting Pro at position 238 (EU numbering) with Asp) to
each Fc.gamma.R, and the obtained was then multiplied by 100 and
shown 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. 26).
[0544] As shown in FIG. 26, the results show that of all the
alterations, 24 types of alterations were found to maintain or
enhance 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 16. In the table, alteration refers to the
alteration introduced into IL6R-B3 (SEQ ID NO: 63). The template
used for producing IL6R-B3, IL6R-G1d/IL6R-L, is indicated with an
asterisk (*).
TABLE-US-00023 TABLE 16 RELATIVE BINDING VARIANT NAME ALTERATION
Fc.gamma.RIa Fc.gamma.RIIaR Fc.gamma.RIIaH Fc.gamma.RIIb
Fc.gamma.RIIIa IL6R- * 140 650 1670 62 3348 G1d/IL6R-L
IL6R-B3/IL6R-L 145 625 1601 58 3264 IL6R- P238D 100 100 100 100 100
BF648/IL6R-L IL6R- P238D/E233D 118 103 147 116 147 2B002/IL6R-L
IL6R- P238D/S267A 121 197 128 110 138 BP100/IL6R-L IL6R-
P238D/S267Q 104 165 66 106 86 BP102/IL6R-L IL6R- P238D/S267V 56 163
69 107 77 BP103/IL6R-L IL6R- P238D/H268D 127 150 110 116 127
BP106/IL6R-L IL6R- P238D/H268E 123 147 114 118 129 BP107/IL6R-L
IL6R- P238D/H268N 105 128 127 101 127 BP110/IL6R-L IL6R-
P238D/P271G 119 340 113 157 102 BP112/IL6R-L IL6R- P238D/Y296D 95
87 37 103 96 2B128/IL6R-L IL6R- P238D/V323I 73 92 83 104 94
2B169/IL6R-L IL6R- P238D/V323L 116 117 115 113 122 2B171/IL6R-L
IL6R- P238D/V323M 140 244 179 132 144 2B172/IL6R-L IL6R-
P238D/K326A 117 159 103 119 102 BP136/IL6R-L IL6R- P238D/K326D 124
166 96 118 105 BP117/IL6R-L IL6R- P238D/K326E 125 175 92 114 103
BP120/IL6R-L IL6R- P238D/K326L 113 167 132 103 146 BP126/IL6R-L
IL6R- P238D/K326M 117 181 133 110 145 BP119/IL6R-L IL6R-
P238D/K326N 98 103 97 106 102 BP142/IL6R-L IL6R- P238D/K326Q 118
155 135 113 157 BP121/IL6R-L IL6R- P238D/K326S 101 132 128 104 144
BP118/IL6R-L IL6R- P238D/K326T 110 126 110 108 114 BP116/IL6R-L
IL6R- P238D/A330K 52 101 108 119 120 BP911/IL6R-L IL6R- P238D/A330M
106 101 89 105 91 BP078/IL6R-L IL6R- P238D/A330R 60 81 93 103 97
BP912/IL6R-L
[0545] The results of measuring KD values of the variants shown in
Table 16 for Fc.gamma.RIa, Fc.gamma.RIIaR, Fc.gamma.RIIaH,
Fc.gamma.RIIb, and Fc.gamma.RIIIa V types by the method of
Reference Example 2 are summarized in Table 13. In the table,
alteration refers to the alteration introduced into IL6R-B3 (SEQ ID
NO: 63). 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 17. Here, parent polypeptide refers
to the variant which has IL6R-B3 (SEQ ID NO: 63) 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 gray-filled cells in Table 17 show values calculated by using
Equation 2 of Reference Example 2.
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
TABLE-US-00024 TABLE 17 ##STR00001## ##STR00002##
[0546] Table 17 shows that in comparison with IL6R-B3, 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 17 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.
[0547] With regard to the promising variants among the obtained
combination variants, the factors leading to their effects were
investigated using the crystal structure. FIG. 27 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 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
of Fc.gamma.RIIb may be decreased, and consequently this is
speculated to contribute to improvement of binding free energy.
[0548] Similarly, FIG. 28 shows the surrounding 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 surrounding 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 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 or Glu at position 86 in Fc.gamma.RIIb.
[0549] FIG. 29 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 the
least squares fitting based on the C.alpha. 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, the alteration produced by substituting Pro at position
271 (EU numbering) with Gly gives flexibility to this loop
structure, and is speculated to contribute to enhancement of
binding by reducing the energetic barrier when allowing an optimum
structure to form during interaction with Fc.gamma.RIIb.
Example 12
Examination of the Combinatorial Effect of Alterations that Enhance
Fc.gamma.RIIb Binding when Combined with P238D
[0550] Of the alterations obtained in Examples 9 and 11, 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 its effect was examined.
[0551] Particularly good alterations selected from Tables 13 and 17
were introduced into the antibody H chain IL6R-BF648 in a similar
manner to the method of Example 11. IL6R-L was utilized as the
antibody L chain, the expressed antibodies were purified according
to the method of Reference Example 1. The binding to each of the
Fc.gamma.Rs (Fc.gamma.RIa, Fc.gamma.RIIa H type, Fc.gamma.RIIa R
type, Fc.gamma.RIIb, and Fc.gamma.RIIIa V type) was comprehensively
evaluated by the method of Reference Example 2.
[0552] According to the following method, relative binding
activities were calculated for the results of analyzing
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 substitution of Pro at position
238 (EU numbering) with Asp to each Fc.gamma.R, and multiplied by
100; and then the value was shown as the relative binding activity
value of each variant to each Fc.gamma.R (Table 18). In the table,
alteration refers to the alteration introduced into IL6R-B3 (SEQ ID
NO: 63). The template used for producing IL6R-B3, IL6R-G1d/IL6R-L,
is indicated with an asterisk (*).
TABLE-US-00025 TABLE 18-1 VARIANT RELATIVE BINDING ACTIVITY NAME
ALTERATION FcgRIa FcgRIIaR FcgRIIaH FcgRIIb FcgRIIIaV
IL6R-G1d/IL6R-L * 140 650 1670 62 3348 IL6R-B3/IL5R-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
[0553] Table 18-2 is a continuation table of Table 18-1.
TABLE-US-00026 TABLE 18-2 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/A330K 43 614 368 175 143
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/A330K 69 672 395 178 249
IL6R-BP195/IL6R-L
E233D/L234Y/G237D/P238D/H268D/P271G/Y296D/K326A/A330K 68 661 344
181 221 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/K326A/A330K 71 642 294
179 206 IL6R-BP198/IL6R-L E233D/L234Y/P238D/H268D/P271G/K326A/A330K
104 449 188 164 157 IL6R-BP199/IL6R-L E233D/P238D/K326A/A330R 112
172 116 144 103 IL6R-BP200/IL6R-L
E233D/L234Y/G237D/P238D/P271G/K326A/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
[0554] The results of measuring KD values of the variants shown in
Table 18 for Fc.gamma.RIa, Fc.gamma.RIIaR, Fc.gamma.RIIaH,
Fc.gamma.RIIb, and Fc.gamma.RIIIaV types by the method of Reference
Example 2 are summarized in Tables 19-1 and 19-2. In the table,
alteration refers to the alteration introduced into IL6R-B3 (SEQ ID
NO: 63). 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 the variant for
Fc.gamma.RIIaR by the KD value of the variant for Fc.gamma.RIIb,
and the value obtained by dividing the KD value of the 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 Tables 19-1 and 19-2. Here, parent
polypeptide refers to the variant which has IL6R-B3 (SEQ ID NO: 63)
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 of gray-filled cells in
Tables 19-1 and 19-2 show values calculated by using Equation 2 of
Reference Example 2.
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
[0555] Tables 19-1 and 19-2 show 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
Tables 19-1 and 19-2 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.
TABLE-US-00027 TABLE 19-1 ##STR00003## ##STR00004##
[0556] Table 19-2 is a continuation table of Table 19-1.
TABLE-US-00028 TABLE 19-2 ##STR00005## ##STR00006##
Example 13
Preparation of Variants with Enhanced Fc.gamma.RIIb Binding
[0557] As shown in Example 8, when enhancing the Fc.gamma.RIIb
binding, it is preferable that the Fc.gamma.RIIb binding is
enhanced while maximally suppressing the binding to other
activating Fc.gamma.Rs. Thus, the present inventors additionally
produced variants with enhanced Fc.gamma.RIIb binding or improved
selectivity to Fc.gamma.RIIb by combining alterations that enhance
the Fc.gamma.RIIb binding or improving the selectivity to
Fc.gamma.RIIb. Specifically, the alterations described in Examples
9, 11, and 12 which were found to be effective when combined with
alteration P238D, were combined with one another, on the basis of
the P238D alteration which showed the excellent effect to enhance
the Fc.gamma.RIIb binding and to improve the selectivity to
Fc.gamma.RIIb.
[0558] Variants were produced by combining the Fc regions of
IL6R-G1d (SEQ ID NO: 54) and IL6R-B3 (SEQ ID NO: 63) with
alterations E233D, L234Y, G237D, S267Q, H268D, P271G, Y296D, K326D,
K326A, A330R, and A330K described in Examples 9, 11, and 12 which
were found to be effective when combined with alteration P238D.
Using IL6R-L (SEQ ID NO: 56) as the antibody L chain, antibodies
comprising the above-described variants in the heavy chain were
expressed and purified according to the method described in
Reference Example 1. The resulting variants were respectively
assessed for the 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 described in Reference Example 2.
[0559] The KD of each variant to each Fc.gamma.R is shown in Table
20. "Alteration" refers to an alteration introduced into IL6R-B3
(SEQ ID NO: 63). IL6R-B3/IL6R-L which is used as the template to
produce each variant is indicated by asterisk (*). "KD (IIaR)/KD
(IIb)" in the table shows the value obtained by dividing the KD of
each variant for Fc.gamma.RIIaR by the KD of each variant for
Fc.gamma.RIIb. The greater the value, the higher the selectivity to
Fc.gamma.RIIb. "KD (I %) of parent polypeptide/KD (I %) of altered
polypeptide" shows the 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. 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 Fc.gamma.RIIaR by the KD value
of each variant for Fc.gamma.RIIaR. In Table 20, the numeral in the
gray-filled cells indicates that the binding of Fc.gamma.R 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.
TABLE-US-00029 TABLE 20 ##STR00007## ##STR00008##
[0560] When taking the binding to each Fc.gamma.R by IL6R-B3/IL6R-L
resulting from introducing the K439E alteration into the
IL6R-G1d/IL6R-L containing the sequence of native human IgG1 as 1,
the binding of IL6R-G1d/IL6R-L to Fc.gamma.RIa as 1.3 times; the
binding of IL6R-G1d/IL6R-L to Fc.gamma.RIIaR was 1.1 times; the
binding of IL6R-G1d/IL6R-L to Fc.gamma.RIIaH was 1.1 times, the
binding of IL6R-G1d/IL6R-L to Fc.gamma.RIIb binding was 1.2 times,
and the binding of IL6R-G1d/IL6R-L to Fc.gamma.RIIIaV was 0.9
times. Thus, for any given Fc.gamma.R type, the binding of
IL6R-B3/IL6R-L to Fc.gamma.R was comparable to the binding of
IL6R-G1d/IL6R-L to Fc.gamma.R. Thus, the comparison of the binding
of each variant to each Fc.gamma.R with that of IL6R-B3/IL6R-L
prior to introduction of the alteration is assumed to be equivalent
to the comparison of the binding of each variant to each Fc.gamma.R
with the binding to each Fc.gamma.R by IL6R-G1d/IL6R-L containing
the sequence of native human IgG1. For this reason, in the
subsequent Examples below, the binding activity of each variant to
each Fc.gamma.R will be compared to that to each Fc.gamma.R by
IL6R-B3/IL6R-L prior to introduction of the alteration. Table 20
shows that all the variants have increased Fc.gamma.RIIb binding
activity as compared to IL6R-B3 prior to introduction of the
alteration. The binding activity of IL6R-BF648/IL6R-L, which was
the lowest, was increased by 2.6 times, while the binding activity
of IL6R-BP230/IL6R-L, which is the highest, was increased by 147.6
times. Regarding the value of KD (IIaR)/KD (IIb) that represents
the selectivity, the value for IL6R-BP234/IL6R-L, which was the
lowest, was 10.0, while the value for IL6R-BP231/IL6R-L, which was
the highest, was 32.2. Compared to 0.3 for IL6R-B3/IL6R-L prior to
introduction of the alteration, these values imply that all the
variants have improved selectivity. All the variants showed lower
binding activity to Fc.gamma.RIa, Fc.gamma.RIIaH, and
Fc.gamma.RIIIaV than that of IL6R-B3/IL6R-L prior to introduction
of the alteration.
Example 14
X-Ray Crystal Structure Analysis of the Complexes of Fc.gamma.RIIb
Extracellular Region or Fc.gamma.RIIaR Extracellular Region and Fc
Region with Enhanced Fc.gamma.RIIb Binding
[0561] As shown in Example 13, the Fc.gamma.RIIb binding of variant
IL6R-BP230/IL6R-L, whose Fc.gamma.RIIb binding was enhanced most,
was enhanced to about 150 times as compared to IL6R-B3/IL6R-L prior
to introduction of the alteration, while the enhancement of its
Fc.gamma.RIIaR binding was suppressed to an extent of about 1.9
times. Thus, IL6R-BP230/IL6R-L is a variant excellent in both
Fc.gamma.RIIb binding and selectivity. However, the present
inventors sought a possibility to create more preferable variants
with further enhanced Fc.gamma.RIIb binding while suppressing the
Fc.gamma.RIIaR binding as possible.
[0562] As shown in FIG. 25 described in Example 10, in the Fc
region with alteration P238D, Asp at position 270 (EU numbering) in
its CH2 domain B forms a tight electrostatic interaction with Arg
at position 131 in Fc.gamma.RIIb. This amino acid residue at
position 131 is His in Fc.gamma.RIIIa and Fc.gamma.RIIaH, while it
is Arg in Fc.gamma.RIIaR like in Fc.gamma.RIIb. Thus, there is no
difference between Fc.gamma.RIIaR and Fc.gamma.RIIb in terms of the
interaction of the amino acid residue at position 131 with Asp at
position 270 (EU numbering) in the CH2 domain B. This is assumed to
be a major factor for the poor selectivity between the
Fc.gamma.RIIb binding and Fc.gamma.RIIaR binding of the Fc
region.
[0563] On the other hand, the extracellular regions of
Fc.gamma.RIIa and Fc.gamma.RIIb are 93% identical in amino acid
sequence, and thus they have very high homology. Based on the
crystal structure analysis of the complex of the Fc region of
native IgG1 (hereinafter abbreviated as Fc (WT)) and the
extracellular region of Fc.gamma.RIIaR (J. Imunol. (2011) 187,
3208-3217), a difference found around the interface between the two
interacting with each other was only three amino acids (Gln127,
Leu132, Phe160) between Fc.gamma.RIIaR and Fc.gamma.RIIb. Thus, the
present inventors predicted that it was extremely difficult to
improve the selectivity of the Fc region between the Fc.gamma.RIIb
binding and Fc.gamma.RIIaR binding.
[0564] In this context, the present inventors conceived that, in
order to further enhance the Fc.gamma.RIIb-binding activity of the
Fc region, and to improve the selectivity of the Fc regions between
the binding to Fc.gamma.RIIb and Fc.gamma.RIIaR binding, it was
important to clarify subtle differences between Fc
region-Fc.gamma.RIIb interaction and Fc region-Fc.gamma.RIIaR
interaction by analyzing not only the three-dimensional structure
of the complex of the Fc region with enhanced Fc.gamma.RIIb binding
and the extracellular region of Fc.gamma.RIIb but also the
three-dimensional structure of the complex of the Fc region with
enhanced Fc.gamma.RIIb binding and the extracellular region of
Fc.gamma.RIIaR. First, the present inventors analyzed the X-ray
crystal structure of the complex of the extracellular region of
Fc.gamma.RIIb or Fc.gamma.RIIaR and Fc (P208) resulting from
eliminating the K439E alteration from the Fc region of
IL6R-BP208/IL6R-L created as described in Example 12, which was the
variant used as the base in producing IL6R-BP230/IL6R-L.
(14-1) X-Ray Crystal Structure Analysis of the Complex of Fc (P208)
and the Extracellular Region of Fc.gamma.RIIb
[Expression and Purification of Fc (P208)]
[0565] Fc (P208) was prepared as described below. First, IL6R-P208
was produced by substituting Lys for Glu at position 439 (EU
numbering) in IL6R-BP208, as is in the case of the sequence of
native human IgG1. Then, the gene sequence of Fc (P208), which was
cloned by PCR from Glu at position 216 (EU numbering) to the C
terminus using as a template a DNA encoding a variant with a
substitution of Ser for Cys at position 220 (EU numbering), was
cloned. Expression vector construction, expression, and
purification were achieved according to the method described in
Reference Example 1. Meanwhile, Cys at position 220 (EU numbering)
in ordinary IgG1 forms a disulfide bond to a Cys in the L chain.
When preparing the Fc region alone, the L chain is not
co-expressed. Thus, Cys at position 220 was substituted by Ser to
avoid unnecessary disulfide bond formation.
[Expression and Purification of the Extracellular Region of
Fc.gamma.RIIb]
[0566] The extracellular region of Fc.gamma.RIIb was prepared
according to the method described in Reference Example 2.
[Purification of the Fc (P208)/Fc.gamma.RIIb Extracellular Region
Complex]
[0567] 0.15 mg of the purified product of Endo F1 (Protein Science
(1996) 5, 2617-2622) expressed in E. coli as a fusion protein with
glutathione S-transferase was added 1.5 mg of a crystallization
sample of the extracellular region of Fc.gamma.RIIb. This added
sample in 0.1 M Bis-Tris buffer (pH 6.5) was allowed to stand at
room temperature for three days to cleave off N-type sugar chains
except N-acetylglucosamine directly linked to the Asn in the sample
of the extracellular region of Fc.gamma.RIIb. Then, the sample of
the extracellular region of Fc.gamma.RIIb subjected to the sugar
chain cleavage treatment was concentrated with a 5000MWCO
ultrafiltration filter, and purified by chromatography with a gel
filtration column (Superdex200 10/300) equilibrated with 20 mM
HEPES (pH7.5)/0.1 M NaCl. Furthermore, to the purified
Fc.gamma.RIIb extracellular region fraction with its sugar chains
cleaved, Fc (P208) was added so that the molar ratio of the
Fc.gamma.RIIb extracellular region would be present in slight
excess. The mixture concentrated by ultrafiltration with 10,000
MWCO was purified by chromatography with a gel filtration column
(Superdex200 10/300) equilibrated with 25 mM HEPES (pH 7.5), 0.1 M
NaCl. The purified fraction prepared as described above was used as
a sample of Fc (P208)/Fc.gamma.RIIb extracellular region complex in
the subsequent assessment.
[Crystallization of the Complex of Fc (P208)/Fc.gamma.RIIb
Extracellular Region]
[0568] A sample of Fc (P208)/Fc.gamma.RIIb extracellular region
complex concentrated to about 10 mg/ml with a 10000 MWCO
ultrafiltration filter was 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 of 0.1 M Bis-Tris (pH 6.5), 19%(w/v) PEG3350,
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 similar condition were crushed with Seed Bead (Hampton
Research) to prepare a seed crystal solution. The crystallization
drops were added with 0.15 .mu.l of a diluted solution prepared
from the seed solution and allowed to stand at 20.degree. C. in
sealed reservoir wells. This yielded plate-like crystals.
[X-Ray Diffraction Data Measurements from an Fc
(P208)/Fc.gamma.RIIb Extracellular Region Complex Crystal]
[0569] A single crystal of Fc (P208)/Fc.gamma.RIIb extracellular
region complex prepared as described above 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. Then, the
single 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 collected at Spring-8
BL32XU. During the measurement, the crystal was constantly placed
in a nitrogen stream at -178.degree. C. to maintain in a frozen
state. A total of 300 X-ray diffraction images from the single
crystal were collected using CCD detector MX-225HE (RAYONIX)
attached to a beam line with rotating the single crystal
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,
the diffraction intensity data of the single crystal up to 2.81
.ANG. resolution was obtained. The crystal belongs to the space
group C222.sub.1 with lattice constant a=156.69 .ANG., b=260.17
.ANG., c=56.85 .ANG., .alpha.=90.degree., .beta.=90.degree., and
.gamma.=90.degree..
[X-Ray Crystal Structure Analysis of Fc (P208)/Fc.gamma.RIIb
Extracellular Region Complex]
[0570] The structure of Fc (P208)/Fc.gamma.RIIb extracellular
region complex 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.RIIb extracellular region complex. The
amino acid residue portions of the A chain positions 239-340 and
the B chain positions 239-340, which were taken out as a separate
coordinate from the structural coordinate of PDB code: 3SGJ for the
crystal structure of Fc (WT)/Fc.gamma.RIIIa extracellular region
complex, were used respectively as models for searching the CH2
domain of the Fc region. Likewise, the amino acid residue portions
of the A chain positions 341-444 and the B chain positions 341-443,
which were taken out as a single coordinate from the structural
coordinate of PDB code: 3SGJ, were used as a model for searching
the CH3 domain of the Fc region. Finally, the amino acid residue
portions of the A chain positions 6-178, which were taken out from
the structural coordinate of PDB code: 2FCB for the crystal
structure of the extracellular region of Fc.gamma.RIIb, were used
as a model for searching Fc (P208). The present inventors tried to
determine the orientations and positions of the respective search
models of the CH3 domain of the Fc region, the extracellular region
of Fc.gamma.RIIb, and the CH2 domain of the Fc region in the
crystal lattices based on the 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 A was determined based on 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 complex of Fc (P208)/Fc.gamma.RIIb extracellular region. The
crystallographic reliability factor R value of the structural model
for the data of diffracted intensity at 25 to 3.0 .ANG. was 42.6%
and Free R value was 43.7% after rigid body refinement of the
obtained initial structural model which moves the two CH2 domains
and two CH3 domains of the Fc region, and the extracellular region
of Fc.gamma.RIIb. Then, structural model refinement was achieved by
repeating structural refinement using program REFMAC5 (Acta Cryst.
(2011) D67, 355-367) followed by revision of the structural 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 region calculated
according to the structural model, and the phases calculated
according to the structural 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 4786
non-hydrogen atoms became 24.5% and 28.2% to 27259 diffraction
intensity data from 25 .ANG. to 2.81 .ANG. resolution,
respectively.
[0571] The three-dimensional structure of the complex of Fc
(P208)/Fc.gamma.RIIb extracellular region was determined at a
resolution of 2.81 .ANG. by structure analysis. The structure
obtained by the analysis is shown in FIG. 30. Fc.gamma.RIIb
extracellular region was revealed to be bound between the two CH2
domains of the Fc region, which resembles the three-dimensional
structures of the previously analyzed complexes of Fc (WT), which
is the Fc of native IgG, and each of the extracellular regions 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), and Fc.gamma.RIIa (J. Immunol.
(2011) 187 (6), 3208-3217).
[0572] A close observation of the complex of Fc
(P208)/Fc.gamma.RIIb extracellular region revealed a change in the
loop structure at positions 233 to 239 (EU numbering) following the
hinge region in the CH2 domain A of the Fc region due to an
influence of the introduced the G237D and P238D alterations as
compared to the complex of Fc (WT)/Fc.gamma.RIIaR extracellular
region (FIG. 31). This leads to that the main chain of Asp at
position 237 (EU numbering) in Fc (P208) formed a tight hydrogen
bond to the side chain of Tyr at position 160 in Fc.gamma.RIIb
(FIG. 32). In both Fc.gamma.RIIaH and Fc.gamma.RIIaR, the amino
acid residue at position 160 is Phe, which is incapable of forming
such a hydrogen bond. This suggests that the above described
hydrogen bond has important contribution to the enhancement of the
Fc.gamma.RIIb binding and the acquisition of the selectivity
against Fc.gamma.RIIa binding of Fc (P208), i.e., improvement of
the Fc.gamma.RIIb-binding activity and reduction of
Fc.gamma.RIIa-binding activity of Fc (P208).
[0573] On the other hand, the side chain of Asp at position 237 (EU
numbering) in Fc (P208) forms neither particularly significant
interaction in the Fc.gamma.RIIb binding nor interaction with other
residues within the Fc region. Ile at position 332, Glu at position
333, and Lys at position 334 (EU numbering) in the Fc region are
located close to Asp at position 237 (EU numbering) (FIG. 33). When
the amino acid residues of these positions are substituted by
hydrophilic residues to form an interaction with the side chain of
Asp at position 237 (EU numbering) in Fc (P208) and the loop
structure can be stabilized by the interaction, this can lead to
reduction of the entropic energy loss due to the hydrogen bonding
between the Fc region and Tyr at position 160 in Fc.gamma.RIIb and
thereby to an increase in the binding free energy, i.e., an
increase in the binding activity.
[0574] When the X-ray crystal structure of the complex of Fc
(P238D) with the P238D alteration and Fc.gamma.RIIb extracellular
region described in Example 10 is compared to the X-ray crystal
structure of the complex of Fc (P208) and Fc.gamma.RIIb
extracellular region, alterations are observed at five portions in
Fc (P208) as compared to Fc (P238D) and most of the changes are
seen only at the side chain level. Meanwhile, a positional
deviation at the main chain level due to the Pro-to-Gly alteration
at position 271 (EU numbering) is also observed in the CH2 domain B
of the Fc region, and in addition there is a structural change in
the loop at positions 266 to 270 (EU numbering) (FIG. 34). As
described in Example 11, it is suggested that, when Asp at position
270 (EU numbering) in Fc (P238D) forms a tight electrostatic
interaction with Arg at position 131 in Fc.gamma.RIIb, the
interaction can induce stereochemical stress at Pro at position 271
(EU numbering). The experiment described herein suggests that the
structural change observed with the alteration to Gly for the amino
acid at position 271 (EU numbering) is assumed to be a result of
elimination of the structural distortion accumulated at Pro prior
to the alteration and the elimination results in an increase in the
free energy for the Fc.gamma.RIIb binding, i.e., an increase in the
binding activity.
[0575] Furthermore, it was demonstrated that, due to the change of
the loop structure at positions 266 to 271 (EU numbering), Arg at
position 292 (EU numbering) underwent a structural change with two
states. In this case, it is suggested that the electrostatic
interaction (FIG. 34) formed between Arg at position 292 (EU
numbering) and Asp at position 268 (EU numbering) which is an
altered residue in Fc (P208) can contribute to the stabilization of
the loop structure. Since the electrostatic interaction formed
between Asp at position 270 (EU numbering) in the loop and Arg at
position 131 in Fc.gamma.RIIb largely contribute to the binding
activity of Fc (P208) to Fc.gamma.RIIb, the stabilization of the
loop structure in the binding conformation was likely to reduce the
entropic energy loss upon binding. Thus, the alteration is expected
to result in an increase in the binding free energy, i.e., an
increase in the binding activity.
[0576] Moreover, the possibility of alteration to increase the
activity was scrutinized based on the result of structural
analysis. Ser at position 239 (EU numbering) was found as a
candidate for the site to introduce alteration. As shown in FIG.
35, Ser at position 239 (EU numbering) in the CH2 domain B is
present at the position toward which Lys at position 117 in
Fc.gamma.RIIb extends most naturally in structure. However, since
the electron density was not observed for Lys at position 117 in
Fc.gamma.RIIb by the analysis described above, the Lys has no
definite structure. In this situation, Lys117 is likely to have
only a limited effect on the interaction with Fc (P208). When Ser
at position 239 (EU numbering) in the CH2 domain B is substituted
with negatively charged Asp or Glu, such an alteration is expected
to cause an electrostatic interaction with the positively charged
Lys at position 117 in Fc.gamma.RIIb, thereby resulting in improved
Fc.gamma.RIIb-binding activity.
[0577] On the other hand, an observation of the structure of Ser at
position 239 (EU numbering) in the CH2 domain A revealed that, by
forming a hydrogen bond to the main chain of Gly at position 236
(EU numbering), the side chain of this Ser stabilized the loop
structure at positions 233 to 239, including Asp at position 237
(EU numbering) that forms a hydrogen bond to the side chain of Tyr
at position 160 in Fc.gamma.RIIb, following the hinge region (FIG.
32). The stabilization of the loop structure in the binding
conformation can reduce the entropic energy loss upon binding, and
result in an increase in the binding free energy, i.e., an
improvement of the binding activity. Meanwhile, when Ser at
position 239 (EU numbering) in the CH2 domain A is substituted with
Asp or Glu, the loop structure may become unstable due to loss of
the hydrogen bond to the main chain of Gly at position 236 (EU
numbering). In addition, the alteration may result in electrostatic
repulsion to Asp at position 265 (EU numbering) in close proximity,
leading to further destabilization of the loop structure. The
energy for the destabilization corresponds to loss of free energy
for the Fc.gamma.RIIb binding, which may result in reduction in the
binding activity.
(14-2) X-Ray Crystal Structure Analysis of the Complex of Fc (P208)
and Fc.gamma.RIIaR Extracellular Region
[Expression and Purification of the Extracellular Region of
Fc.gamma.RIIaR]
[0578] The extracellular region of Fc.gamma.RIIaR was prepared
according to the method described in Reference Example 2.
[Purification of the Complex of Fc (P208)/Fc.gamma.RIIaR Type
Extracellular Region]
[0579] 1.5 mg of purified sample of the extracellular region of
Fc.gamma.RIIaR was added with 0.15 mg of the purified product of
Endo F1 (Protein Science (1996) 5, 2617-2622) expressed in E. coli
as a fusion protein with S-transferase, 20 .mu.l of 5 U/ml Endo F2
(QA-bio), and 20 .mu.l of 5 U/ml Endo F3 (QA-bio). After 9 days of
incubation at room temperature in 0.1 M Na acetate buffer (pH 4.5),
the sample was further added with 0.07 mg of the above-described
Endo F1, 7.5 .mu.l of the above-described Endo F2, and 7.5 .mu.l of
the above-described Endo F3, and was incubated for three days to
cleave off N-type sugar chains except N-acetylglucosamine directly
linked to the Asn in the sample of the extracellular region of
Fc.gamma.RIIaR. Then, the sample of the extracellular region of
Fc.gamma.RIIaR concentrated with a 10000 MWCO ultrafiltration
filter and subjected to the above-described sugar chain cleavage
treatment was purified by chromatography with a gel filtration
column (Superdex200 10/300) equilibrated with 25 mM HEPES (pH 7),
0.1 M NaCl. Next, to the purified Fc.gamma.RIIaR extracellular
region fraction with its sugar chains cleaved, Fc (P208) was added
so that the molar ratio of the Fc.gamma.RIIb extracellular region
would be present in slight excess. The mixture concentrated by
ultrafiltration with 10,000 MWCO was purified by chromatography
with a gel filtration column (Superdex200 10/300) equilibrated with
25 mM HEPES (pH 7), 0.1 M NaCl. The purified fraction prepared as
described above was used as a sample of Fc (P208)/Fc.gamma.RIIaR
type extracellular region complex in the subsequent assessment.
[Crystallization of the Complex of Fc (P208)/Fc.gamma.RIIaR Type
Extracellular Region]
[0580] 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 tight sealed and allowed to stand at 20.degree. C. This
yielded plate-like crystals.
[X-Ray Diffraction Data Measurement from Fc (P208)/Fc.gamma.RIIaR
Extracellular Region Complex Crystal]
[0581] A single crystal of Fc (P208)/Fc.gamma.RIIaR extracellular
region complex prepared as described above 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 collected 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.RIIaR Type
Extracellular Region Complex]
[0582] The structure of Fc (P208)/Fc.gamma.RIIaR type extracellular
region complex 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.RIIaR 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 (14-1), the orientation and position of Fc
(P208)/Fc.gamma.RIIaR extracellular region complex in the crystal
lattices were determined based on the rotation function and
translation function. The crystallographic reliability factor R
value of the structural model for the data of diffracted intensity
at 25 to 3.0 .ANG. was 38.4% and Free R value was 30.0% after rigid
body refinement of the obtained initial structural model which
moves the two CH2 domains and two CH3 domains of the Fc region, and
the extracellular region of Fc.gamma.RIIaR. Then, structural model
refinement was achieved by repeating structural refinement using
program REFMAC5 (Acta Cryst. (2011) D67, 355-367) followed by
revision of the structural model performed using program Coot (Acta
Cryst. (2010) D66, 486-501) with reference to the electron density
maps where the coefficients Fo-Fc and 2Fo-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 38.0% to 24838 diffraction intensity data from 25
.ANG. to 2.87 .ANG. resolution, respectively.
[0583] The three-dimensional structure of the complex of Fc
(P208)/Fc.gamma.RIIaR extracellular region was determined at a
resolution of 2.87 .ANG. by structure analysis. A comparison of the
crystal structure between the complex of Fc (P208)/Fc.gamma.RIIaR
type extracellular region and the complex of Fc
(P208)/Fc.gamma.RIIb extracellular region described in Example
(14-1) detected almost no difference at the level of overall
structure (FIG. 36), reflecting the very high amino acid identity
between the two Fc.gamma. receptors.
[0584] However, a precise observation of the structures at the
electron density level detected some differences that can lead to
improvement of the selectivity between the Fc.gamma.RIIb binding
and the Fc.gamma.RIIaR binding of the Fc region. The amino acid
residue at position 160 in Fc.gamma.RIIaR is not Tyr but Phe. As
shown in FIG. 37, the hydrogen bond between the main chain of the
amino acid residue at position 237 (EU numbering) in the CH2 domain
A of the Fc region and Tyr at position 160 in Fc.gamma.RIIb, though
formed upon binding between Fc.gamma.RIIb and the Fc region with
alteration P238D, is expected not to be formed upon binding between
Fc.gamma.RIIb and the Fc region with alteration P238D. The absence
of the hydrogen bond formation can be a major factor for improving
the selectivity between the Fc.gamma.RIIb binding and the
Fc.gamma.RIIaR binding of the Fc region introduced with alteration
P238D. Further comparison at the electron density level showed
that, in the Fc region/Fc.gamma.RIIb complex, electron density was
clearly observable for the side chains of Leu at positions 235 (EU
numbering) and 234 (EU numbering), whereas the electron density of
the side chains was unclear in the Fc region/Fc.gamma.RIIaR
complex. This suggests that the loop near position 237 (EU
numbering) becomes flexible due to the reduced interaction with
FcgRIIaR around this position. Meanwhile, a structural comparison
of the CH2 domain B of the Fc region (FIG. 38) in same region
revealed that, in the complex of the Fc region and Fc.gamma.RIIb,
electron density was observable up to Asp at position 237 (EU
numbering), whereas, in the complex structure of the Fc region and
Fc.gamma.RIIaR, electron density was observable up to three
residues prior to Asp at position 237 (EU numbering), i.e., up to
around Leu at position 234 (EU numbering), suggesting that
Fc.gamma.RIIaR binding forms an interaction over a larger region as
compared to the FcgRIIb binding. The finding described above
suggests the possibility that, in the CH2 domain A of the Fc
region, the region from position 234 to 238 (EU numbering) has a
large contribution to the binding between the Fc region and
Fc.gamma.RIIb, while in the CH2 domain B of the Fc region the
region from position 234 to 238 (EU numbering) has a large
contribution to the binding between the Fc region and
Fc.gamma.RIIaR.
Example 15
Fc Variants for which Alteration Sites were Determined Based on
Crystal Structure
[0585] As described in Example 14, Asp at position 268 (EU
numbering) was suggested to electrostatically interact with Arg at
position 292 (EU numbering) (FIG. 34) as a result of the local
structural change due to introduction of the alteration P271G in
domain B of the variant with enhanced Fc.gamma.RIIb binding (P208).
There is a possibility that the loop structure at positions 266 to
271 (EU numbering) is stabilized by the formation of the
interaction, resulting in enhancement of the Fc.gamma.RIIb binding.
Thus, the present inventors assessed whether the Fc.gamma.RIIb
binding of the variant could be enhanced by additional
stabilization of its loop structure due to enhancement of the
electrostatic interaction by substituting Glu for Asp at position
268 (EU numbering) in the variant. On the other hand, as shown in
FIG. 33, Tyr at position 160 (EU numbering) in Fc.gamma.RIIb
interacts with the main chain of Asp at position 237 (EU numbering)
in domain A of P208. Meanwhile, the side chain of Asp at position
237 (EU numbering) is located close to Ile at position 332, Glu at
position 333, and Lys at position 334 (EU numbering) in the
molecule without forming any particularly significant interaction.
Thus, the present inventors also assessed whether the interaction
with Tyr at position 160 in Fc.gamma.RIIb can be enhanced through
stabilization of the loop structure at positions 266 to 271 (EU
numbering) due to increased interaction with the side chain of Asp
at position 237 (EU numbering) by substituting hydrophilic amino
acid residues at the positions described above.
[0586] Variants of IL6R-BP230/IL6R-L prepared as described in
Example 13 were produced by introducing with each of the
alterations H268E, I332T, I332S, 1332E, I332K, E333K, E333R, E333S,
E333T, K334S, K334T, and K334E. IL6R-L (SEQ ID NO: 56) was used as
the antibody L chain. Antibodies containing the light chain of
IL6R-L and the above-described heavy chain variants were expressed
and purified according to the method 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, FcgRIIb,
or FcgRIIIaV) by the method described in Reference Example 2.
[0587] The KD of each variant to each Fc.gamma.R is shown in Table
21. In the table, "alteration" refers to an alteration introduced
into IL6R-BP3 (SEQ ID NO: 63). IL6R-B3/IL6R-L which is used as the
template to produce IL6R-BP230 is indicated by 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 Fc.gamma.RIIb by the KD value of each variant
for Fc.gamma.RIIb. 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 Fc.gamma.R IIaR by the KD value
of each variant for Fc.gamma.R IIaR. KD (IIaR)/KD (IIb) shows the
value obtained by dividing the KD of each variant for
Fc.gamma.RIIaR by the KD of the variant for Fc.gamma.RIIb. The
greater the value, the higher the selectivity to Fc.gamma.RIIb. In
Table 21, the numeral in the gray-filled cells indicates that the
binding of Fc.gamma.R 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.
TABLE-US-00030 TABLE 21 ##STR00009##
[0588] Both Fc.gamma.RIIb-binding activity and Fc.gamma.RIIb
selectivity of IL6R-BP264/IL6R-L, IL6R-BP465/IL6R-L,
IL6R-BP466/IL6R-L, and IL6R-BP470, resulting from introducing
alterations H268E, E333K, E333R, and E333T, respectively, into
IL6R-BP230/IL6R-L were increased as compared to those of
IL6R-BP230/IL6R-L. The Fc.gamma.RIIb selectivity of
IL6R-BP391/IL6R-L introduced with the I332T alteration was reduced
while its Fc.gamma.RIIb-binding activity was increased as compared
to IL6R-BP230/IL6R-L.
Example 16
Comprehensive Introduction of Alterations at Amino Acid Residues
Around Position 271 (EU Numbering)
[0589] In the structural comparison between Fc (P208) and
Fc.gamma.RIIb and Fc (P238D)/Fc.gamma.RIIb, the most significant
difference is found in the structure around position 271 (EU
numbering) in the CH2 domain B of the Fc region (FIG. 33). As
described in Example 11, it is suggested that, when, in Fc (P238D),
Asp at position 270 (EU numbering) forms a tight electrostatic
interaction with Arg at position 131 in Fc.gamma.RIIb, the
interaction can induce stereochemical stress at Pro at position 271
(EU numbering). In the structure of Fc (P208)/Fc.gamma.RIIb, due to
the substitution of Gly for Pro at position 271 (EU numbering), a
positional deviation occurred at the main chain level so as to
eliminate the structural distortion, resulting in a large
structural change around position 271. There is a possibility that
additional stabilization of the changed structure around position
271 further reduces the entropic energy loss caused by the binding
upon formation of an electrostatic interaction with Arg at position
131 in Fc.gamma.RIIb. Thus, alterations that enhance the
Fc.gamma.RIIb binding or increase the Fc.gamma.RIIb selectivity of
the Fc region were sought by comprehensive introduction of
alterations at amino acid residues around position 271 (EU
numbering).
[0590] IL6R-BP267 was constructed as a template in comprehensive
introduction of alterations by introducing alterations E233D,
G237D, P238D, H268E, and P271G into IL6R-B3 (SEQ ID NO: 63). IL6R-L
(SEQ ID NO: 56) was used as the antibody L chain. Antibodies
containing the light chain of IL6R-L and the above-described heavy
chain variants were expressed and purified according to the method
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, FcgRIIb, or FcgRIIIaV) by the
method described in Reference Example 2. The amino acids at
positions 264, 265, 266, 267, 269, and 272 (EU numbering) in
IL6R-BP267 were substituted with each of 18 types of amino acids,
except Cys and the amino acid prior to substitution. IL6R-L (SEQ ID
NO: 56) was used as the antibody L chain. Antibodies containing the
light chain of IL6R-L and the above-described heavy chain variants
were expressed and purified according to the method 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
described in Reference Example 2. Variants whose Fc.gamma.RIIb
binding has been enhanced or Fc.gamma.RIIb selectivity has been
increased as compared to the Fc.gamma.RIIb binding or Fc.gamma.IIb
selectivity of IL6R-BP267/IL6R-L prior to introduction of the
alterations are shown in Table 22.
TABLE-US-00031 TABLE 22 ##STR00010##
[0591] The KD value of each variant to each Fc.gamma.R is shown in
Table 22. In the table, "alteration" refers to an alteration
introduced into IL6R-B3, which was used as a template.
IL6R-B3/IL6R-L which is used as the template to produce IL6R-BP267
is indicated by asterisk (*). In the table, KD (I %) of parent
polypeptide/KD (I %) of altered polypeptide shows the 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.
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 Fc.gamma.RIIaR by the KD of each variant for
Fc.gamma.R IIaR. KD (IIaR)/KD (I %) shows the value obtained by
dividing the KD value of each variant for Fc.gamma.RIIaR by the KD
value of the variant for Fc.gamma.RIIb. The greater the value, the
higher the selectivity to Fc.gamma.RIIb. In Table 22, the numeral
in the gray-filled cells indicates that the binding of Fc.gamma.R
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.
[0592] All the binding activities of variants shown in Table 22 to
Fc.gamma.RIa, Fc.gamma.RIIaH, and Fc.gamma.RIIIaV were comparable
or reduced as compared to that of IL6R-B3/IL6R-L. Meanwhile, the
Fc.gamma.RIIb-binding activity of variants resulting from adding
alterations S267A, V264I, E269D, S267E, V266F, S267G, and V266M,
respectively, to IL6R-BP267/IL6R-L were 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. This demonstrates that the S267A, S267G,
E272M, E272Q, D265E, E272D, E272N, V266L, E272I, and E272F
alterations produce the effect to improve the Fc.gamma.RIIb
selectivity.
Example 17
Enhancement of the Fc.gamma.RIIb Binding by Introduction of
Alterations into CH3 Region
[0593] A substitution alteration of Leu for Pro at position 396 (EU
numbering) has been reported to enhance the Fc.gamma.RIIb binding
(Cancer Res. (2007) 67, 8882-8890). The amino acid at position 396
(EU numbering) is present at a position which is not directly
involved in the interaction with Fc.gamma.R. However, the amino
acid can be assumed to have an effect on the interaction with
Fc.gamma.R by changing the antibody structure. Thus, the present
inventors assessed whether the Fc.gamma.RIIb binding of the Fc
region is enhanced or its Fc.gamma.RIIb selectivity is increased by
comprehensive introduction of amino acid alterations at position
396 (EU numbering) in the Fc region.
[0594] IL6R-BP423 was constructed as a template in comprehensive
introduction of alterations by introducing alterations E233D,
G237D, P238D, S267A, H268E, P271G, and A330R into IL6R-B3 (SEQ ID
NO: 63). Variants, in which the amino acid at position 396 (EU
numbering) in IL6R-BP423 was substituted with each of 18 types of
amino acids, except cysteine and the amino acid prior to
substitution, were constructed. IL6R-L (SEQ ID NO: 56) was used as
the antibody L chain. Antibodies containing the light chain of
IL6R-L and the above-described heavy chain variants were expressed
and purified according to the method 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 described in
Reference Example 2. The binding of the resulting variants to each
Fc.gamma.R is shown in Table 23.
TABLE-US-00032 TABLE 23 ##STR00011##
[0595] In the table, "alteration introduced into IL6R-BP423" refers
to an alteration introduced into IL6R-BP423, which was used as a
template. IL6R-B3/IL6R-L which is 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
Fc.gamma.RIIb by the KD value of each variant for Fc.gamma.RIIb.
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
Fc.gamma.R IIaR. KD (IIaR)/KD (IIb) shows the value obtained by
dividing the KD of each variant for Fc.gamma.RIIaR by the KD of the
variant for Fc.gamma.RIIb. The greater the value, the higher the
selectivity to Fc.gamma.RIIb. In Table 23, the numeral in the
gray-filled cells indicates that the binding of Fc.gamma.R 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.
[0596] The result shown in Table 23 demonstrates that: the
Fc.gamma.RIIb-binding activity of IL6R-BP456/IL6R-L resulting from
introducing alteration P396M into IL6R-BP423/IL6R-L,
IL6R-BP455/IL6R-L resulting from introducing alteration P396L into
IL6R-BP423/IL6R-L, IL6R-BP464/IL6R-L resulting from introducing
alteration P396Y into IL6R-BP423/IL6R-L, IL6R-BP450/IL6R-L
resulting from introducing alteration P396F into IL6R-BP423/IL6R-L,
IL6R-BP448/IL6R-L resulting from introducing alteration P396D into
IL6R-BP423/IL6R-L, IL6R-BP458/IL6R-L resulting from introducing
alteration P396Q into IL6R-BP423/IL6R-L, IL6R-BP453/IL6R-L
resulting from introducing alteration P396I into IL6R-BP423/IL6R-L,
IL6R-BP449/IL6R-L resulting from introducing alteration P396E into
IL6R-BP423/IL6R-L, IL6R-BP454/IL6R-L resulting from introducing
alteration P396K into IL6R-BP423/IL6R-L, and IL6R-BP459/IL6R-L
resulting from introducing alteration 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 alteration
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 Fc.gamma.RIIb selectivity. As seen in
Table 23, the binding activity of the prepared variants to
Fc.gamma.RIa, Fc.gamma.RIIaH, and Fc.gamma.RIIIaV was all lower
than that of IL6R-B3/IL6R-L, which was the parent polypeptide.
Example 18
Preparation of Variants with Enhanced Fc.gamma.RIIb Binding Using
Subclass Sequences
[0597] The Fc.gamma.R binding profile varies depending on the
subclass of human IgG The present inventors assessed whether the
difference in the binding activity to each Fc.gamma.R between IgG1
and IgG4 could be utilized to increase the Fc.gamma.RIIb-binding
activity and/or improve the selectivity. First, IgG1 and IgG4 were
analyzed for their binding activity to each Fc.gamma.R. IL6R-G4d
(SEQ ID NO: 64) containing G4d was constructed as the antibody H
chain. G4d is an Fc region that 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: 56) was used as the
antibody L chain. Antibodies containing the light chain of IL6R-L
and IL6R-G1d/IL6R-L or IL6R-G4d/IL6R-L were expressed and purified
according to the method 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 described in
Reference Example 2. The binding of the resulting variants to each
FcgR is summarized in Table 24.
TABLE-US-00033 TABLE 24 KD KD KD KD KD AGAINST AGAINST AGAINST
AGAINST AGAINST VARIANT Fc.gamma.RIa Fc.gamma.RIIaR Fc.gamma.RIIaH
Fc.gamma.RIIb Fc.gamma.RIIIaV NAME (mol/L) (mol/L) (mol/L) (mol/L)
(mol/L) IL6R-G1d/ 1.20E-10 9.70E-07 6.50E-07 3.90E-06 4.20E-07
IL6R-L IL6R-G4d/ 6.60E-10 2.10E-06 3.40E-06 2.60E-06 3.40E-06
IL6R-L
[0598] It was demonstrated that the Fc.gamma.RIIb binding of
IL6R-G4d/IL6R-L was 1.5 times stronger than that of IL6R-G1d/IL6R-L
whereas the Fc.gamma.RIIaR binding of IL6R-G4d/IL6R-L was 2.2 times
weaker than that of IL6R-G1d/IL6R-L. Meanwhile, the binding
activity of IL6R-G4d/IL6R-L to Fc.gamma.RIa, Fc.gamma.RIIaH, and
Fc.gamma.RIIIaV was lower than that of IL6R-G1d/IL6R-L. The result
described above revealed that IL6R-G4d had preferable
characteristics as compared to IL6R-G1d in terms of both
FcgRIIb-binding activity and selectivity.
[0599] FIG. 39 is an alignment to compare the CH1 sequences of G1d
and G4d up to the C terminus (positions 118 to 445 (EU numbering)).
In FIG. 39, amino acid residues that are different between G1d and
G4d are filled with black. The present inventors assessed whether
the Fc.gamma.RIIb binding could be further increased and/or the
Fc.gamma.RIIb selectivity could be further improved by selecting,
from the above-described different amino acids, some portions that
are predicted to be involved in the interaction with Fc.gamma.R,
and grafting at least one amino acid residue or more of the G4d
sequence, which confers a property preferable from the viewpoint of
both Fc.gamma.RIIb-binding activity and selectivity, to a variant
with enhanced Fc.gamma.RIIb binding.
[0600] Specifically, the present inventors produced:
IL6R-BP473 resulting from introducing alteration A327G into
IL6R-BP230; IL6R-BP472 resulting from introducing alteration A330S
into IL6R-BP230; IL6R-BP471 resulting from introducing alteration
P331S into IL6R-BP230; IL6R-BP474 resulting from introducing
alterations A330S and P331S into IL6R-BP230; IL6R-BP475 resulting
from introducing alterations A327G and A330S into IL6R-BP230;
IL6R-BP476 resulting from introducing alterations A327G, A330S, and
P331S into IL6R-BP230; and IL6R-BP477 resulting from introducing
alterations A327G and P331S into IL6R-BP230. Furthermore, to
construct IL6R-BP478, the amino acids from Ala at position 118 to
Thr at position 225 (EU numbering) in IL6R-BP230 was substituted
with the amino acids of the G4d sequence from Ala at position 118
to Pro at position 222 (EU numbering). IL6R-L (SEQ ID NO: 56) was
used as the antibody L chain. Antibodies containing the light chain
of IL6R-L and the heavy chain variants described above were
purified according to the method described in Reference Example 1.
The purified antibodies were assessed for their binding activity to
each Fc.gamma.R (Fc.gamma.RIa, Fc.gamma.RIIaH, Fc.gamma.RIIaR,
Fc.gamma.RIIb, or Fc.gamma.RIIIaV) by the method described in
Reference Example 2.
[0601] The KD value of each variant to each Fc.gamma.R is shown in
Table 25. "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 Fc.gamma.RIIb by the KD value of
each variant for Fc.gamma.RIIb. In the table, "alteration
introduced into 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, in which the
G4d sequence from Ala at position 118 up to Pro at position 222 (EU
numbering) has been substituted for the segment from Ala at
position 118 up to Thr at position 225 (EU numbering) in
IL6R-BP230, 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 Fc.gamma.R IIaR by the KD value
of the variant for Fc.gamma.R IIaR. KD (IIaR)/KD (IIb) shows the
value obtained by dividing the KD of each variant for
Fc.gamma.RIIaR by the KD of the variant for Fc.gamma.RIIb. The
greater the value, the higher the selectivity to Fc.gamma.RIIb. In
Table 25, the numeral in the gray-filled cells indicates that the
binding of Fc.gamma.R 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.
TABLE-US-00034 TABLE 25 ##STR00012##
[0602] Of the variants shown in Table 25, IL6R-BP473/IL6R-L
introduced with the alteration A327G showed Fc.gamma.RIIb binding
increased by 1.2 times compared to that of IL6R-BP230/IL6R-L.
IL6R-BP478/IL6R-L produced by substituting the amino acids from Ala
at position 118 to Thr at position 225 (EU numbering) of IL6R-BP230
with the amino acids from Ala at position 118 to Pro at position
222 (EU numbering) of G4d sequence, has 1.1 times enhanced binding
to Fc.gamma.RIIb than that of IL6R-BP230/IL6R-L, and binding of
IL6R-BP478/IL6R-L to Fc.gamma.RIIaR is decreased to 0.9 times that
of IL6R-BP230/IL6R-L. Binding activities of all variants to
Fc.gamma.RIa, Fc.gamma.RIIaH, and Fc.gamma.RIIIaV were lower than
those of the parent polypeptide IL6R-B3/IL6R-L.
[0603] In the examination carried out so far, introducing the A327G
alteration, which substitutes the amino acid in the human IgG4
sequence for the amino acid at position 327 (EU numbering) in
variant IL6R-BP230/IL6R-L, was shown to enhance
Fc.gamma.RIIb-binding activity. A further examination was performed
for amino acids that do not match between the IgG4 and IgG1
sequences and those other than the amino acid at position 327 (EU
numbering). Specifically, variants were produced by introducing the
following alterations into IL6R-BP230, which was used 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, as indicated by EU
numbering respectively. 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.
[0604] The KD of each variant to each Fc.gamma.R is shown in Table
26. In the table, "KD (IIb) of the parent polypeptide/KD (IIb) of
the altered polypeptide" represents the value obtained by dividing
the KD value of IL6R-B3/IL6R-L to Fc.gamma.RIIb by the KD value of
each variant to Fc.gamma.RIIb. In the table, "alteration of
IL6R-BP230" refers to an alteration introduced into IL6R-BP230.
IL6R-B3/IL6R-L used as the template to produce IL6R-BP230 is
indicated with * 1. "KD (IIaR) of the parent polypeptide/KD (IIaR)
of the altered polypeptide" represents the value obtained by
dividing the KD value of IL6R-B3/IL6R-L to Fc.gamma.RIIaR by the KD
value of the same variant to Fc.gamma.RIIaR. KD (IIaR)/KD (IIb)
represents the value obtained by dividing KD of each variant to
Fc.gamma.RIIaR by KD of the same variant to Fc.gamma.RIIb. The
greater the value, the higher the selectivity to Fc.gamma.RIIb. In
Table 26, the numeral in the cell filled with gray indicates that
the binding of Fc.gamma.R to IgG was weak, and it was determined
that the analysis could not be correctly performed by kinetic
analysis, and thus was calculated using:
KD=CR.sub.max/(R.sub.eq-RI)-C [Equation 2]
described in Reference Example 2.
TABLE-US-00035 TABLE 26 ##STR00013##
[0605] As shown in Table 26, IL6R-BP541/IL6R-L resulting from
introducing K274Q (each represented by EU numbering) into
IL6R-BP230/IL6R-L, IL6R-BP544/IL6R-L resulting from introducing
R355Q into IL6R-BP230/IL6R-L, IL6R-BP545/IL6R-L resulting from
introducing D356E into IL6R-BP230/IL6R-L, and IL6R-BP546/IL6R-L
resulting from introducing L358M into IL6R-BP230/IL6R-L, showed
enhanced Fc.gamma.RIIb binding as compared to IL6R-BP230/IL6R-L
prior to the introduction of alteration. Of them, IL6R-BP544/IL6R-L
resulting from introducing R355Q (each represented by EU numbering)
into IL6R-BP230/IL6R-L, IL6R-BP545/IL6R-L resulting from
introducing D356E into IL6R-BP230/IL6R-L, and IL6R-BP546/IL6R-L
resulting from introducing L358M into IL6R-BP230/IL6R-L, were shown
to have an increased KD(IIaR)/KD(IIb) value and improved
selectivity to Fc.gamma.RIIb, as compared to IL6R-BP230/IL6R-L
prior to the introduction of alteration.
Example 19
Assessment of Combinations of Alterations that Enhance the
Fc.gamma.RIIb Binding or Improve the Fc.gamma.RIIb Selectivity
[0606] Additional combinations of the alterations which had been
found by the evaluation described above to improve the
Fc.gamma.RIIb binding or Fc.gamma.RIIb selectivity were assessed.
Specifically, the alterations that had been assessed to be
effective in enhancing the Fc.gamma.RIIb binding and/or improving
the Fc.gamma.RIIb selectivity were introduced in combination into
IL6R-B3 (SEQ ID NO: 63). Furthermore, existing alterations S267E
and L328F that enhance the Fc.gamma.RIIb binding (Seung et al.,
(Mol. Immunol. (2008) 45, 3926-3933)) were introduced into IL6R-B3
to produce IL6R-BP253 as a comparison control. IL6R-L (SEQ ID NO:
56) was used as the antibody L chain. Antibodies containing the
light chain of IL6R-L and the above-described heavy chain variants
were expressed and purified according to the method as 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
described in Reference Example 2.
[0607] The KD of each variant to each Fc.gamma.R is shown in Table
27. In the table, "alteration" refers to an alteration introduced
into IL6R-B3 (SEQ ID NO: 63). IL6R-B3/IL6R-L which is used as the
template to produce each variant is indicated by asterisk (*). 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
Fc.gamma.RIIb by the KD value of each variant for Fc.gamma.RIIb.
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 Fc.gamma.R IIaR by the KD of the variant for
Fc.gamma.RIIaR. KD (IIaR)/KD (IIb) shows the value obtained by
dividing the KD of each variant for Fc.gamma.RIIaR by the KD of the
variant for Fc.gamma.RIIb. The greater the value, the higher the
selectivity to Fc.gamma.RIIb as compared to Fc.gamma.RIIaR.
Meanwhile, KD (IIaH)/KD (IIb) shows the value obtained by dividing
the KD of each variant for Fc.gamma.RIIaH by the KD of the variant
for Fc.gamma.RIIb. The greater the value, the higher the
selectivity to Fc.gamma.RIIb as compared to Fc.gamma.RIIaH. In
Table 27, the numeral in the gray-filled cells indicates that the
binding of Fc.gamma.R 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.
TABLE-US-00036 TABLE 27 ##STR00014## ##STR00015## ##STR00016##
##STR00017##
[0608] Of the variants shown in Table 27, IL6R-BP253/IL6R-L added
with the existing alterations that enhance the Fc.gamma.RIIb
binding exhibited Fc.gamma.RIIb- and Fc.gamma.RIIaR-binding
activities increased to 277 times and 529 times those of
IL6R-B3/IL6R-L prior to introduction of the alterations,
respectively. Furthermore, the Fc.gamma.RIa-binding activity of
IL6R-BP253/IL6R-L was also greater than that of IL6R-B3/IL6R-L.
Meanwhile, the Fc.gamma.RIIaH binding and Fc.gamma.RIIIaV binding
of IL6R-BP253/IL6R-L were reduced as compared to those of
IL6R-B3/IL6R-L. Among other variants, IL6R-BP436/IL6R-L and
IL6R-BP438/IL6R-L showed an Fc.gamma.RIa binding slightly enhanced
as compared to that of IL6R-B3/IL6R-L prior to introduction of the
alterations. All other variants showed a reduced Fc.gamma.RIa
binding. In addition, all the variants exhibited reduced
Fc.gamma.RIIaH binding and Fc.gamma.RIIIaV binding as compared to
those of IL6R-B3/IL6R-L.
[0609] 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-491/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. Of the above, 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. Thus, it can be said that these variants are superior
in both of the level and selectivity of enhancement of the
Fc.gamma.RIIb binding activity compared to the prior art.
[0610] 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 (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.
Furthermore, 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. From
these results, all of the variants shown in Table 27 have been
shown to be variants with improved selectivity to Fc.gamma.RIIb as
compared to the known 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, and IL6R-BP500/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; 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.
Accordingly, these variants can be considered to have better
properties in terms of both binding activities and selectivity to
Fc.gamma.RIIb than antibodies produced by existing techniques.
[0611] Without being bound by a particular theory, variants
IL6R-BP568/IL6R-L and IL6R-BP492/IL6R-L which have conserved
Tregitope sequence having higher Treg-inducing ability (De Groot et
al. (Blood (2008) 112, 3303-3311)) and thus considered to have high
Treg-inducing activity than the variants having Y296D,
IL6R-BP567/IL6R-L and IL6R-BP493/IL6R-L, may be more effective.
Regarding the binding activity and selectivity of these variants
for Fc.gamma.RIIb, comparison with the native type shows that
Fc.gamma.RIIaR binding is 1.6 times and Fc.gamma.RIIb binding is
211 times that of the native type for IL6R-BP568/IL6R-L, and
Fc.gamma.RIIaR binding is 1.2 times and Fc.gamma.RIIb binding is
131 times that of the native type for IL6R-BP492/IL6R-L, and these
variants were found to have high binding activity and selectivity
to Fc.gamma.RIIb.
Example 20
Preparation of Antibodies that Bind to Human IgA in a
Calcium-Dependent Manner
[0612] (20-1) Preparation of Human IgA (hIgA)
[0613] Examples 2 to 4 show that molecules that have enhanced mouse
Fc.gamma.R binding and which bind in a pH-dependent manner to human
IL-6 receptor as an antigen, can significantly reduce the
concentration of the antigen in plasma. Then, an additional test
was carried out using antibodies to human IgA as an antigen, in
order to assess the presence of a similar effect of eliminating
soluble antigens from plasma in a living organism administered with
antibodies that have enhanced mouse Fc.gamma.R binding and which
bind in a pH-dependent manner to antigens other than human IL-6
receptor. The antigen, human IgA (hereinafter also referred to as
hIgA) (its variable region is from an anti-human IL6R antibody) was
prepared using the following recombination technique. hIgA was
expressed by culturing host cells containing a recombinant vectors
carrying H (WT)-IgA1 (SEQ ID NO: 65) and L (WT)-CK (SEQ ID NO: 42),
and purified by a method known to those skilled in the art using
ion-exchange chromatography and gel filtration chromatography.
(20-2) Expression and Purification of an Antibody that Binds to
hIgA
[0614] GA2-IgG1 (heavy chain, SEQ ID NO: 66; light chain, SEQ ID
NO: 67) is an antibody that binds to hIgA. A DNA sequence encoding
GA2-IgG1 (heavy chain, SEQ ID NO: 66; light chain, SEQ ID NO: 67)
was inserted into an animal cell expression plasmid by a method
known to those skilled in the art. The antibody was expressed and
purified by the method described below. Cells of the human fetal
kidney cell-derived FreeStyle 293-F line (Invitrogen) were
suspended in FreeStyle 293 Expression Medium (Invitrogen). The cell
suspension was plated at a cell density of 1.33.times.10.sup.6
cells/ml in 3 ml to each well of a 6-well plate. Then, the prepared
plasmid was introduced into cells by the lipofection method. The
cells were cultured in a CO.sub.2 incubator (37.degree. C., 8%
CO.sub.2, 90 rpm) for 4 days. From the isolated culture
supernatant, the antibody was purified by a method known to those
skilled in the art using rProtein A Sepharose.TM. Fast Flow
(Amersham Biosciences). The absorbance (wavelength: 280 nm) of the
solution of the purified antibody was measured using a
spectrophotometer. The antibody concentration was determined using
the extinction coefficient calculated from the measured value by
the PACE method (Protein Science (1995) 4, 2411-2423).
(20-3) Assessment of the Isolated Antibody for its
Calcium-Dependent hIgA-Binding Ability
[0615] The antibody isolated as described in Example (20-2) was
assessed for its hIgA-binding activity (dissociation constant, KD
(M)) using Biacore T200 (GE Healthcare). The binding activity was
measured using as a running buffer, 0.05% tween20, 20 mmol/l ACES,
150 mmol/1 NaCl (pH 7.4 or pH 5.8) containing 3 .mu.M or 1.2 mM
CaCl.sub.2. An appropriate amount of recombinant Protein A/G
(Thermo Scientific) was immobilized onto a Sensor chip CM5 (GE
Healthcare) by an amino coupling method, and the antibody was
allowed to bind thereto. Then, an appropriate concentration of hIgA
(described in (A1-1)) was injected as an analyte and allowed to
interact with the antibody on the sensor chip. The measurement was
carried out at 37.degree. C. After measurement, 10 mmol/L
glycine-HCl (pH 1.5) was injected to regenerate the sensor chip.
From the measurement result, the dissociation constant KD (M) was
calculated by curve fitting analysis and equilibrium analysis using
Biacore T200 Evaluation Software (GE Healthcare). The result is
shown in Table 28. GA2-IgG1 strongly bound to hIgA at a Ca.sup.2+
concentration of 1.2 mM, and weakly bound to hIgA at a Ca.sup.2+
concentration of 3 .mu.M. Meanwhile, at a Ca.sup.2+ concentration
of 1.2 mM, GA2-IgG1 strongly bound to human IgA at pH 7.4, and
weakly bound to human IgA at pH 5.8. In summary, GA2-IgG1 was
demonstrated to bind to human IgA in a pH- and calcium-dependent
manner.
TABLE-US-00037 TABLE 28 Antibody name Condition Fit ka kd KD [M]
GA2-IgG1 pH 7.4, 1.2 mM Ca 1:1binding model 4.0E+05 1.6E-02 3.9E-08
pH 7.4, 3 .mu.M Ca Steady State Affinity -- -- 6.7E-06 pH 5.8, 1.2
mM Ca Steady State Affinity -- -- 4.0E-06 pH 5.8, 3 .mu.M Ca Steady
State Affinity -- -- 5.0E-06
Example 21
Preparation of Antibody Variants that Bind to hIgA in a
Calcium-Dependent Manner
[0616] Next, to further accelerate antigen (hIgA) elimination from
plasma, GA2-F1087 (heavy chain, SEQ ID NO: 68) was produced by
substituting Tyr for Leu at position 328 (EU numbering) in GA2-IgG1
for enhancing the mouse Fc.gamma.R binding of GA2-IgG1 that binds
to hIgA in a calcium-dependent manner. A DNA sequence encoding
GA2-F1087 (heavy chain, SEQ ID NO: 68; light chain, SEQ ID NO: 67)
was inserted into an animal expression plasmid by a method known to
those skilled in the art. Antibody variants were expressed by the
above-described method using the plasmid. The concentrations of the
variants were measured after purification. Antibodies comprising
the above alteration exhibited significantly increased mouse
Fc.gamma.R binding, as shown in Example (4-3).
Example 22
Assessment of the Effect on the Plasma Antigen Retention in Normal
Mice Administered with Ca-Dependent hIgA-Binding Antibodies
(22-1) In Vivo Tests Using Normal Mice
[0617] hIgA (human IgA, prepared as described in Example (20-1))
was administered alone or in combination with an anti-hIgA antibody
to normal mice (C57BL/6J mouse, Charles River Japan). After
administration, the in vivo dynamics of hIgA and anti-hIgA
antibodies was assessed. An hIgA solution (80 .mu.g/ml) or a mixed
solution of hIgA and an anti-hIgA antibody was administered once at
a dose of 10 ml/kg into the caudal vein. The anti-hIgA antibodies
used were GA2-IgG1 and GA2-F1087 described above.
[0618] In all of the mixed solutions, the concentration of hIgA was
80 .mu.g/ml, and the concentration of anti-hIgA antibody was 2.69
mg/ml. In this experiment, the anti-hIgA antibodies were present
significantly in excess over hIgA, and thus most of hIgA was
thought to bind to the antibodies. In the group administered with
GA-hIgG1, from the mice, the blood was collected five minutes,
seven hours, one day, two days, three days, and seven days after
administration. Meanwhile, in the group administered with GA-F1087,
from the mice, the blood was collected five minutes, 30 minutes,
one hour, two hours, one day, three days, and seven days after
administration. The collected blood was immediately centrifuged at
12,000 rpm and 4.degree. C. for 15 minutes to isolate the plasma.
The isolated plasma was stored in a freezer at -20.degree. C. or
below until use.
(22-2) Determination of the Plasma Anti-hIgA Antibody Concentration
in Normal Mice by the ELISA Method
[0619] Anti-hIgA antibody concentrations in mouse plasma were
measured by the ELISA method. First, to prepare an anti-human
IgG-immobilized plate, Anti-Human IgG (.gamma.-chain specific)
F(ab')2 Fragment of Antibody (SIGMA) was aliquoted to each well of
a Nunc-Immuno Plate, MaxiSorp (Nalge nunc International), and the
plate was allowed to stand at 4.degree. C. overnight. Calibration
curve samples of anti-hIgA antibody prepared as standard solutions
for the plasma concentration (0.5, 0.25, 0.125, 0.0625, 0.03125,
0.01563, and 0.007813 .mu.g/ml) and assay samples of mouse plasma
diluted 100 times or more, were aliquoted to the above-mentioned
anti-human IgG-immobilized plate. After one hour of incubation of
the plate at 25.degree. C., Goat Anti-Human IgG (.gamma. chain
specific) Biotin (BIOT) Conjugate (Southern Biotechnology
Associates Inc.) was aliquoted to each well of the plate. Then, the
plate was incubated at 25.degree. C. for one hour. Next,
Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was
aliquoted to each well of the plate. Then, the plate was incubated
at 25.degree. C. for one hour. Chromogenic reaction was performed
using as a substrate TMB One Component HRP Microwell Substrate
(BioFX Laboratories). After terminating the reaction with 1N
sulfuric acid (Showa Chemical), the absorbance of the reaction
solution in each well was measured at 450 nm with a microplate
reader. Anti-hIgA antibody concentrations in mouse plasma were
determined based on the absorbance of the standard curve using the
analysis software SOFTmax PRO (Molecular Devices). A time course of
the antibody concentrations of GA2-IgG1 and GA2-F1087 in the plasma
of normal mice after intravenous administration, which were
measured by the method described above, is shown in FIG. 40. The
results demonstrate that, with respect to the clone GA2-IgG1 that
has pH-dependent, strong hIgA-binding activity, the plasma
concentration of the antibody is not significantly reduced even if
the Fc.gamma.R binding is enhanced.
(22-3) Determination of the Plasma hIgA Concentration by the ELISA
Method
[0620] hIgA concentrations in mouse plasma were measured by the
ELISA method. First, to prepare an anti-human IgA-immobilized
plate, Goat anti-Human IgA Antibody (BETHYL) was aliquoted to each
well of a Nunc-Immuno Plate, MaxiSoup (Nalge nunc International),
and the plate was allowed to stand at 4.degree. C. overnight.
Calibration curve samples of hIgA were prepared as standard
solutions for the plasma concentration (0.4, 0.2, 0.1, 0.05, 0.025,
0.0125, and 0.00625 .mu.g/ml), and used. 100 .mu.l each of the
calibration curve samples and assay samples of mouse plasma diluted
100 times or more, was combined with 200 .mu.l of 500 ng/ml hsL6R.
This was mixed and incubated at room temperature for one hour.
Then, 100 .mu.l of the mixtures was aliquoted to the anti-human
IgA-immobilized plate. The plate was allowed to stand at room
temperature for one hour. Next, Biotinylated Anti-human IL-6 R
Antibody (R&D) was aliquoted to each well of the plate. After
one hour of incubation at room temperature, Streptavidin-PolyHRP80
(Stereospecific Detection Technologies) was aliquoted to each well
of the plate. The plate was incubated at room temperature for one
hour. Chromogenic reaction was performed using as a substrate TMB
One Component HRP Microwell Substrate (BioFX Laboratories). After
terminating the reaction with 1N sulfuric acid (Showa Chemical),
the absorbance of the reaction solution in each well was measured
at 450 nm with a microplate reader. The concentrations in mouse
plasma were determined based on the absorbance of the standard
curve using the analysis software SOFTmax PRO (Molecular Devices).
A time course of the hIgA concentration in the plasma of normal
mice after intravenous administration, which was measured by the
above method, is shown in FIG. 41.
[0621] The result showed that, in mice administered with hIgA in
combination with GA2-IgG1 having a Ca-dependent hIgA-binding
activity of 100 times or more greater, hIgA elimination was
accelerated compared to the administration of hIgA alone.
Meanwhile, in the plasma of mice administered with GA2-F1087 with
enhanced binding to hIgA and Fc.gamma.R, the concentration of hIgA
was reduced below the measurable range (0.006 .mu.g/ml or more) one
day after administration, and thus the hIgA elimination was
significantly accelerated compared to the plasma of mice
administered with GA-IgG1. The findings described in Examples 2 to
7 above demonstrate the increased antigen elimination effect of
antibodies with enhanced Fc.gamma.R binding in mice administered
with IL6R and anti-IL6R antibody. Likewise, a similar effect was
also demonstrated to be achieved in mice administered with the hIgA
antigen and anti-hIgA antibody. From the results obtained so far in
the Examples, antigen elimination in this case may also be mediated
by Fc.gamma.RIIb.
Example 23
Preparation of a pH-Dependent Anti-IgE Antibody
(23-1) Preparation of an Anti-Human IgE Antibody
[0622] In order to prepare a pH-dependent anti-human IgE antibody,
human IgE (heavy chain, SEQ ID NO: 69; light chain, SEQ ID NO: 70)
(its variable region is from an anti-human glypican 3 antibody) was
expressed as an antigen using FreeStyle293 (Life Technologies). The
expressed human IgE was prepared and purified by a general
chromatographic method known to those skilled in the art. An
antibody that binds to human IgE in a pH-dependent manner was
selected from many antibodies isolated. The heavy chain and light
chain variable regions of the selected anti-human IgE antibody were
fused with a human IgG1 heavy chain constant region and a human
light chain constant region, and the resulting antibody gene was
inserted into a vector. Using the vector, a recombinant anti-human
IgE antibody was expressed and purified. The prepared antibody was
named clone 278 (hereinafter referred to as 278-IgG1; heavy chain,
SEQ ID NO: 71, light chain, SEQ ID NO: 72).
(23-2) Assessment of the Anti-Human IgE Antibody for the Human
IgE-Binding Activity and pH-Dependent Binding Activity
[0623] Antibodies capable of dissociating antigens in the endosome
can be produced in such a way that they bind to antigens not only
in a pH-dependent manner but also in a Ca-dependent manner. Thus,
278-IgG1 and the control human IgG1 antibody Xolair (omalizumab,
Novartis) without pH/Ca-dependent IgE-binding ability were assessed
for the pH-dependent binding ability and pH/Ca-dependent binding
ability to human IgE (hIgE). Specifically, 278-IgG1 and Xolair were
assessed for their hIgE-binding activity (dissociation constant, KD
(M)) using Biacore T200 (GE Healthcare). Measurements were carried
out using the following three types of running buffers: [0624] 1.2
mmol/CaCl.sub.2, 0.05% tween20, 20 mmol/l ACES, 150 mmol/l NaCl, pH
7.4 [0625] 1.2 mmol/CaCl.sub.2, 0.05% tween20, 20 mmol/l ACES, 150
mmol/l NaCl, pH 5.8 [0626] 3 .mu.mol/1 CaCl.sub.2, 0.05% tween20,
20 mmol/l ACES, 150 mmol/l NaCl, pH 5.8
[0627] An appropriate amount of a peptide (hereinafter referred to
as "biotinylated GPC3 peptide") resulting from adding biotin to the
C-terminal Lys of a chemically synthesized sequence derived from
human glypican 3 protein (SEQ ID NO: 73) was loaded on a Sensor
chip SA (GE Healthcare), and immobilized on the Sensor chip SA
based on biotin/streptavidin affinity. An appropriate concentration
of human IgE was injected and captured by the biotinylated GPC3
peptide to immobilize human IgE on the chip. An appropriate
concentration of 278-IgG1 was injected as an analyte, and allowed
to interact with human IgE on the sensor chip. Then, 10 mmol/L
glycine-HCl (pH 1.5) was injected to regenerate the sensor chip.
All of the assay for the interaction was performed at 37.degree. C.
Using Biacore T200 Evaluation Software (GE Healthcare), the assay
results were analyzed by curve fitting to determine the binding
rate constant ka (l/Ms) and dissociation rate constant kd (l/s).
Dissociation constant KD (M) was calculated from the above
constants. Then, the pH-dependent binding was assessed by
calculating the KD ratio of each antibody between the conditions of
pH 5.8/1.2 mM C.alpha. and pH 7.4/1.2 mM Ca. The pH/Ca-dependent
binding was assessed by calculating the KD ratio of each antibody
between the conditions of pH 5.8/3 .mu.M C.alpha. and pH 7.4/1.2 mM
Ca. The results are shown in Table 29.
TABLE-US-00038 TABLE 29 pH dependency pH/Ca dependency Antibody
name ka kd KD KD (pH 5.8, 1.2 mM Ca)/ KD (pH 5.8, 3 .mu.M Ca)/
(abbreviated) Buffer condition (1/Ms) (1/s) (M) KD (pH 7.4, 1.2 mM
Ca) KD (pH 7.4, 1.2 mM Ca) Clone 278 pH 7.4, 1.2 mM Ca 1.5E+06
3.6E-03 2.4E-09 842.5 1636.5 pH 5.8, 1.2 mM Ca 1.2E+05 2.3E-01
2.0E-06 pH 5.8, 3 .mu.M Ca 6.2E+04 2.4E-01 3.9E-06 Xolair pH 7.4,
1.2 mM Ca 2.5E+06 1.1E-02 4.4E-09 2.3 2.9 pH 5.8, 1.2 mM Ca 2.4E+06
2.4E-02 9.9E-09 pH 5.8, 3 .mu.M Ca 1.4E+06 1.7E-02 1.3E-08
Example 24
Preparation of an Antibody Variant that Binds to Human IgE in a
pH-Dependent Manner
[0628] Next, for further accelerating the elimination of antigen
(human IgE) from plasma, a DNA sequence encoding 278-F1087 (heavy
chain, SEQ ID NO: 74; light chain, SEQ ID NO: 72) with a
substitution of Tyr for Leu at position 328 (EU numbering) in
278-IgG1 was inserted into an animal expression plasmid by a method
known to those skilled in the art, in order to enhance the mouse
Fc.gamma.R binding of 278-IgG1 that binds to human IgE in a
pH-dependent manner. The antibody variants were expressed by the
above-mentioned method using animal cells introduced with the
plasmid. The concentrations of the antibody variants were
determined after purification.
Example 25
In Vivo Assessment of 278-IgG1
[0629] (25-1) Preparation of Human IgE (hIgE (Asp6)) for In Vivo
Assessment
[0630] hIgE (Asp6) (its variable region is from an anti-human
glypican 3 antibody), which is a human IgE for in vivo assessment,
consisting of the heavy chain (SEQ ID NO: 75) and light chain (SEQ
ID NO: 70), was prepared by the same method as described in Example
(23-1). hIgE (Asp6) is a molecule in which asparagine has been
replaced with aspartic acid in the six N-glycosylation sites in
human IgE, so that time-dependent changes in the concentration of
human IgE as an antigen in the plasma does not affect the
heterogeneity of N-linked sugar chains of human IgE.
(25-2) Assessment of the Effect of Accelerating Human IgE
Elimination from the Plasma of Normal Mice Administered with Clone
278
[0631] As described in Examples 2 to 4, and 22, the antigen
concentration was demonstrated to be significantly reduced in the
plasma of mice administered with the molecules that bind in a
pH-dependent manner to human IL-6 receptor or human IgA as an
antigen, and whose binding to mouse Fc.gamma.R has been enhanced.
An additional test was carried out using antibodies to human IgE as
an antigen to assess whether a similar effect of eliminating
soluble antigens from the plasma of a living organism administered
with antibodies with enhanced mouse Fc.gamma.R binding that bind in
a pH-dependent manner to antigens other than human IL-6 receptor
and human IgA, when the binding to mouse Fc.gamma.R is
enhanced.
[0632] hIgE (Asp6) and anti-human IgE antibodies were assessed for
their in vivo dynamics after administration of hIgE (Asp6) alone,
or hIgE (Asp6) in combination with the anti-hIgE antibodies
(278-IgG1 and 278-F1087) to C57BL/6J mice (Charles river Japan).
hIgE (Asp6) (20 .mu.g/ml) or a mixture of hIgE (Asp6) and an
anti-human IgE antibody was administered once at 10 mL/kg into the
caudal vein (as described in Table 30, all antibodies were prepared
at the same concentration). In this case, each antibody was present
significantly in excess over hIgE (Asp6), and thus almost all of
hIgE (Asp6) was thought to bind to the antibody. In the group
administered with clone 278 (278-IgG1), from the mice, the blood
was collected five minutes, two hours, seven hours, one day, two
days, four days, five days, seven days, 14 days, and 21 days after
administration. In the group administered with 278-F1087, from the
mice, the blood was collected five minutes, 30 minutes, one hour,
two hours, one day, three days, seven days, 14 days, and 21 days
after administration. The collected blood was immediately
centrifuged at 15,000 rpm and 4.degree. C. for 5 minutes to isolate
the plasma. The isolated plasma was stored in a freezer at
-20.degree. C. or below until use.
TABLE-US-00039 TABLE 30 hIgE (Asp6) Anti-hIgE antibody
concentration concentration in administered in administered
Anti-hIgE solution solution antibody (.mu.g/mL) (.mu.g/mL) 278-IgG1
20 100 278-F1087 20 100
(25-3) Determination of the Plasma Anti-Human IgE Antibody
Concentration in Normal Mice
[0633] Anti-hIgE antibody concentrations in mouse plasma were
measured by the ELISA method. Standard curve samples were prepared
at 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, and 0.00625 .mu.g/ml for
plasma concentrations. To secure the homogeneity of the immune
complex between hIgE (Asp6) and anti-hIgE antibody, hIgE (Asp6) was
added at 1 .mu.g/ml to the standard curve samples and assay samples
of mouse plasma. The samples of the 278-hIgG1 administration group
and the corresponding standard curve samples were allowed to stand
at room temperature for 30 minutes. Meanwhile, the samples of the
278-F1087 administration group and the corresponding standard curve
samples were stirred at 37.degree. C. overnight. After incubation
or stirring, the standard curve samples and assay samples of mouse
plasma were aliquoted to an immunoplate (Nunc-Immuno Plate,
MaxiSorp (Nalge nunc International)) immobilized with Anti-Human
Kappa Light Chain Antibody (Bethyl Laboratories), and this was
allowed to stand/stirred at room temperature for two hours (the
samples of the 278-F1087 administration group and the standard
curve samples of 278-F1087), or allowed to stand at 4.degree. C.
overnight (the samples of the 278-hIgG1 administration group and
the standard curve samples of 278-hIgG1). Then, Rabbit anti-Human
IgG (Fc) Secondary antibody, Biotin conjugate (Pierce
Biotechnology) and Streptavidin-Poly HRP80 (Stereospecific
Detection Technologies) were each reacted in succession for one
hour. Chromogenic reaction was performed using as a substrate TMB
One Component HRP Microwell Substrate (BioFX Laboratories). After
terminating the reaction with 1N sulfuric acid (Showa Chemical),
the concentrations in mouse plasma were determined based on the
color development by a method for measuring the absorbance at 450
nm with a microplate reader. The concentrations in mouse plasma
were determined based on the absorbance of the standard curve using
the analysis software SOFTmax PRO (Molecular Devices). A time
course of antibody concentrations in plasma after intravenous
administration, which were determined by the above method, is shown
in FIG. 42. The result demonstrates that, in mice administered with
the variants resulting from enhancing the Fc.gamma.R binding of
278-IgG1 with pH-dependent, strong human IgE-binding activity, the
antibody concentration in the plasma of the mice was not
significantly reduced as compared to that of 278-IgG1.
(25-4) Determination of the Plasma hIgE (Asp6) Concentration in
Normal Mice
[0634] hIgE (Asp6) concentrations in mouse plasma were measured by
the ELISA method. Calibration curve samples were prepared at 192,
96, 48, 24, 12, 6, and 3 ng/ml for plasma concentrations. To secure
the homogeneity of the immune complex between hIgE (Asp6) and
anti-hIgE antibody, in the group administrated with 278-hIgG1,
Xolair (Novartis) was added at 10 .mu.g/ml to the standard curve
and assay samples of mouse plasma, and the mixtures were allowed to
stand at room temperature for 30 munities. In the group
administrated with 278-F1087, 278-F1022 (heavy chain, SEQ ID NO:
76; light chain, SEQ ID NO: 72; prepared in the same manner as
Example 24) or 278-F760 (heavy chain, SEQ ID NO: 77; light chain,
SEQ ID NO: 72; prepared in the same manner as Example A5) was added
at 20 .mu.g/ml, and the mixtures were stirred at 37.degree. C. for
60 hours. The assay samples of mouse plasma were aliquoted to an
immunoplate (MABTECH) immobilized with anti-human IgE, or an
immunoplate (Nunc F96 MicroWell Plate (Nalge nunc International))
immobilized with anti-human IgE (clone 107, MABTECH), and this was
allowed to stand or stirred at room temperature for two hours, or
allowed to stand at 4.degree. C. overnight. Then, the human GPC3
core protein (SEQ ID NO: 78), anti-GPC3 antibody (in-house
preparation) biotinylated with NHS-PEG4-Biotin (Thermo Fisher
Scientific), and Sterptavidin-PolyHRP80 (Stereospecific Detection
Technologies) were each reacted in succession for one hour.
Chromogenic reaction was performed using as a substrate TMB One
Component HRP Microwell Substrate (BioFX Laboratories). After
terminating the reaction with 1N sulfuric acid (Showa Chemical),
the concentrations in mouse plasma were determined based on the
color development by a method for measuring the absorbance at 450
nm with a microplate reader. Alternatively, chromogenic reaction
was performed using as a substrate SuperSignal.RTM. ELISA Pico
Chemiluminescent Substrate (Thermo Fisher Scientific), and the
concentrations in mouse plasma were determined by a method for
measuring the luminescence intensity with a microplate reader. The
concentrations in mouse plasma were determined based on the
absorbance or luminescence intensity of the standard curve using
the analysis software SOFTmax PRO (Molecular Devices). A time
course of hIgE (Asp6) concentrations in plasma after intravenous
administration, which were determined by the above method, is shown
in FIG. 43.
[0635] Regarding the elimination of human IgE alone, the result
demonstrates that, in mice administered with human IgE in
combination with 278-IgG1 having the strong pH-dependent binding
activity, the elimination of human IgE was accelerated as compared
to the administration of human IgE alone. Furthermore, in mice
administered with human IgE in combination with 278-F1087 resulting
from enhancing Fc.gamma.R binding of 278-IgG1, the elimination of
human IgE was demonstrated to be significantly accelerated as
compared to the mice administered with human IgE alone, or human
IgE in combination with 278-IgG1. That is, it was shown that the
antigen elimination was accelerated not only in mice administered
with the above-mentioned anti-IL6R antibody and anti-IgA antibody
with enhanced Fc.gamma.R binding, but also in mice administered
with the anti-IgE antibody with enhanced Fc.gamma.R binding. From
the results obtained so far in the Examples, antigen elimination in
this case may also be mainly mediated by Fc.gamma.RIIb.
Example 26
Effects of Eliminating Antigens from Plasma for Antigen-Binding
Molecules with Fc.gamma.RIIb-Binding Activity Higher than that of
an Fc Region of Native Mouse IgG
[0636] (26-1) Antigen Elimination Effect of Mouse Antibodies with
Selectively Enhanced Fc.gamma.RIIb-Binding Activity
[0637] In Examples 5 to 7, a group of normal mice administered with
an antigen-binding molecule produced by enhancing the mouse
Fc.gamma.R-binding activity of an antigen-binding molecule having a
mouse antibody Fc region and having pH-dependent human IL-6
receptor-binding properties, and a group of Fc receptor .gamma.
chain-deficient mice and a group of Fc.gamma.RIII-deficient mice
which simulates the condition where an antibody with selectively
enhanced mouse Fc.gamma.RIIb-binding activity is administered were
examined. From these results, antigen-binding molecules exhibiting
pH-dependent binding to soluble antigens and having selectively
enhanced Fc.gamma.RIIb-binding activity were shown to be able to
efficiently eliminate soluble antigens in plasma when administered
in vivo. Whether this effect will be seen in normal mice
administered with an antigen-binding molecule comprising a mouse Fc
region with selectively enhanced mouse Fc.gamma.RIIb-binding
activity and having pH-dependent human IL-6 receptor-binding
properties was examined as indicated below.
(26-2) Production of Mouse Antibodies with Selectively Enhanced
Fc.gamma.RIIb-Binding Activity
[0638] VH3-mIgG1 (SEQ ID NO: 49) and VL3-mk1 (SEQ ID NO: 50) were
produced as the heavy chain and light chain, respectively, of a
mouse IgG1 antibody having pH-dependent human IL-6 receptor-binding
properties using the method of Reference Example 1. Furthermore, to
enhance the mouse Fc.gamma.RIIb-binding activity of VH3-mIgG1,
substitutions of Glu for Thr at position 230, Ala for Val at
position 231, Asn for Pro at position 232, Glu for Ser at position
238, and Asp for Ser at position 239 as indicated by EU numbering
were carried out to produce VH3-mIgG1-MB367 (SEQ ID NO: 79).
Fv4-mIgG1 or Fv4-mIgG1-MB367 comprising VL3-mk1 as the light chain
and VH3-mIgG1 or VH3-mIgG1-MB367, respectively, as the heavy chain
was expressed and purified using the method of Reference Example
1.
(26-3) Confirmation of Mouse Fc.gamma.R-Binding Activity
[0639] VH3/L(WT)-mIgG1 or VH3/L(WT)-mIgG1-MB367 comprising L(WT)-CK
(SEQ ID NO: 42) as the light chain and VH3-mIgG1 or
VH3-mIgG1-MB367, respectively, as the heavy chain was expressed and
purified using the method of Reference Example 1. Mouse
Fc.gamma.R-binding activities of these antibodies were assessed by
the method of Reference Example 2, and the results are shown in
Table 31. Furthermore, how much the mouse Fc.gamma.R-binding
activity of each variant is enhanced as compared to mIgG1 before
the alteration is shown in Table 32.
TABLE-US-00040 TABLE 31 KD (M) VARIANT NAME mFc .gamma. RIIb mFc
.gamma. RIII VH3/L (WT)-mIgG1 2.10E-07 2.82E-07 VH3/L
(WT)-mIgG1-MB367 1.32E-09 4.54E-08
TABLE-US-00041 TABLE 32 BINDING RATIO TO mIgG1 VARIANT NAME mFc
.gamma. RIIb mFc .gamma. RIII VH3/L (WT)-mIgG1 1.0 1.0 VH3/L
(WT)-mIgG1-MB367 158.6 6.2
[0640] According to the results shown in Table 32,
VH3/L(WT)-mIgG1-MB367 produced by introducing the five
above-mentioned alterations into VH3/L(WT)-mIgG1 had mouse
Fc.gamma.RIIb-binding activity that was enhanced approximately 160
times and mouse Fc.gamma.RIII-binding activity that was enhanced
6.2 times as compared to before alteration. That is,
VH3/L(WT)-mIgG1-MB367 showed selective and enhanced mouse
Fc.gamma.RIIb-binding activity.
(26-4) Confirmation of Effects of Reducing the Concentration of
Soluble Human IL-6 Receptor in Plasma Using Normal Mice
[0641] Effects of elimination of soluble human IL-6 receptor in
plasma of normal mice administered with Fv4-mIgG1 or
Fv4-mIgG1-MB367 as the anti-human IL-6 receptor antibody were
examined as described below.
(26-4-1) In Vivo Test Using Normal Mice
[0642] Normal mice (C57BL/6J mouse; Charles River Japan) were
co-administered with soluble human IL-6 receptor and anti-human
IL-6 receptor mouse antibody, and then assessed for their in vivo
dynamics. A mixed solution of soluble human IL-6 receptor and an
anti-human IL-6 receptor mouse antibody was administered once at a
dose of 10 mL/kg into the tail vein. In this case, the soluble
human IL-6 receptor and the anti-human IL-6 receptor mouse antibody
were administered at a dose of 50 .mu.g/kg and 1 mg/kg,
respectively. The above-mentioned Fv4-mIgG1 or Fv4-mIgG1-MB367 was
used as an anti-human IL-6 receptor mouse antibody. Blood was
collected from the mice 5 minutes, 7 hours, 1 day, 2 days, 3 days,
7 days, 14 days, 21 days, and 28 days after administration of the
anti-human IL-6 receptor mouse antibody. The collected blood
samples were 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 at -20.degree. C. or below until
measurement.
(26-4-2) ELISA Determination of the Anti-Human IL-6 Receptor Mouse
Antibody Concentration in Plasma
[0643] The anti-human IL-6 receptor mouse antibody concentration in
mouse plasma was determined by ELISA. First, soluble human IL-6
receptor was aliquoted into a Nunc-Immuno Plate, MaxiSoup (Nalge
nunc International) and allowed to stand overnight at 4.degree. C.
to prepare a soluble human IL-6 receptor-immobilized plate.
Calibration curve samples containing an anti-human IL-6 receptor
mouse antibody were prepared at plasma concentrations of 2.50,
1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 .mu.g/mL, and mouse
plasma assay samples diluted 100 times or higher were prepared. 100
.mu.L of these calibration curve samples and plasma assay samples
were aliquoted into each well of the soluble human IL-6
receptor-immobilized plate, and this was stirred at room
temperature for two hours. Subsequently, color development reaction
of a reaction solution which was allowed to react at room
temperature for two hours with Anti-mouse IgG-peroxidase antibody
(SIGMA) was carried out using TMB One Component HRP Microwell
Substrate (BioFX Laboratories) as a substrate. After stopping the
reaction by adding 1N Sulfuric acid (Showa Chemical), absorbance of
the reaction solution in each well at 450 nm was measured by a
microplate reader. The antibody concentration in mouse plasma was
calculated based on the absorbance from the calibration curve using
the analytical software SOFTmax PRO (Molecular Devices). The
results are shown in FIG. 44.
(26-4-3) Determination of Soluble Human IL-6 Receptor Concentration
in Plasma by an Electrochemiluminescent Method
[0644] An hsIL-6R concentration in mouse plasma was determined by
an electrochemiluminescent method. Calibration curve samples of
hsIL-6R were prepared at plasma concentrations of 12.5, 6.25, 3.13,
1.56, 0.781, 0.391, and 0.195 ng/mL. Mouse plasma assay samples
were prepared by 50 times or higher dilution. 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 (Chugai
Pharmaceutical Co. Ltd.) were mixed in and was allowed to react
overnight at 37.degree. C. Then, a Streptavidin Gold Multi-ARRAY
Plate (Meso Scale Discovery) was blocked with a PBS-Tween solution
containing 0.5% BSA (w/v) at 5.degree. C. overnight, and the mixed
solution was aliquoted into the plate. After further reacting the
plate for two hours at room temperature, the plate was washed.
Then, Read Buffer T (.times.2) (Meso Scale Discovery) was aliquoted
into the plate and measurements were performed immediately using
SECTOR Imager 2400 (Meso Scale Discovery). hSIL-6R concentrations
were calculated based on the response from the calibration curve
using the analytical software SOFTmax PRO (Molecular Devices). The
results are shown in FIG. 45.
[0645] As shown in FIG. 44, in the group administered with
Fv4-mIgG1-MB367 having selectively enhanced mouse
Fc.gamma.RIIb-binding activity, while the time-course change in
plasma antibody concentration was equivalent to that in the group
administered with Fv4-mIgG1, decrease in plasma retention of
antibody due to enhancement of mouse Fc.gamma.RIIb-binding activity
was not observed. Meanwhile, as shown in FIG. 45, in the group
administered with Fv4-mIgG1-MB367 having selectively enhanced mouse
Fc.gamma.RIIb-binding activity, plasma concentration of the soluble
IL-6 receptor was remarkably decreased as compared to that of the
Fv4-mIgG1-administered group.
[0646] The above showed that when administered in vivo, an
antigen-binding molecule which binds to a soluble antigen in a
pH-dependent manner and has selectively enhanced
Fc.gamma.RIIb-binding activity can efficiently eliminate soluble
antigens in plasma in normal mice as well. Without being restricted
to a particular theory, the phenomena observed here may be
explained as follows.
[0647] When an antibody that binds to a soluble antigen in a
pH-dependent manner and has enhanced Fc.gamma.RIIb-binding activity
is administered to a mouse, the antibody is actively taken up
mainly by cells expressing Fc.gamma.RIIb on its cell membrane. The
internalized antibody dissociates from the soluble antigen under
the acidic pH condition in the endosome, and is then recycled to
the plasma via FcRn. Therefore, one of the elements that brings
about an effect of eliminating soluble antigens in plasma due to
such antibody may include the strength of Fc.gamma.RIIb-binding
activity of the antibody. That is, a stronger Fc.gamma.RIIb-binding
activity leads to a more active internalization into
Fc.gamma.RIIb-expressing cells, and soluble antigens in the plasma
can be eliminated quickly. Furthermore, such effects can be
verified in a similar manner regardless of whether the Fc region
included in the antibody is derived from a human IgG1 or a mouse
IgG1, as long as Fc.gamma.RIIb-binding activity is enhanced. More
specifically, verification can be carried out using the Fc region
of any animal species, such as from human IgG1, human IgG2, human
IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b, mouse IgG3,
rat IgG, monkey IgG, or rabbit IgG as long as binding activity is
enhanced for Fc.gamma.RIIb of the animal species to which it is
administered.
Reference Example 1
Construction of Antibody Expression Vectors; and Expression and
Purification of Antibodies
[0648] 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 cells (Invitrogen) for antibody expression.
The obtained culture supernatant was collected, and then passed
through a 0.22 .mu.m MILLEX(R)-GV filter (Millipore), or through a
0.45 .mu.m MILLEX(R)-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
rProtein A Sepharose Fast Flow (GE Healthcare) or Protein G
Sepharose 4 Fast Flow (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
[0649] 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.sub.--000566 (Version No. NM.sub.--000566.3), Fc.gamma.RIIa
was produced based on the sequence of NCBI Accession No.
NM.sub.--001136219 (Version No. NM.sub.--001136219.1),
Fc.gamma.RIIb was produced based on the sequence of NCBI Accession
No. NM.sub.--004001 (Version No. NM.sub.--004001.3), Fc.gamma.RIIIa
was produced based on the sequence of NCBI Accession No.
NM.sub.--001127593 (Version No. NM.sub.--001127593.1), and
Fc.gamma.RIIIb was produced based on the sequence of NCBI Accession
No. NM.sub.--000570 (Version No. NM.sub.--000570.3), and a His tag
was attached to the C terminus. Furthermore, the existence of
polymorphism is known for Fc.gamma.RIIa, Fc.gamma.RIIIa, and
Fc.gamma.RIIIb, and the polymorphic sites were produced by
referring to Warmerdam et al. (J. Exp. Med., 1990, 172: 19-25) for
Fc.gamma.RIIa; Wu et al. (J. Clin. Invest., 1997, 100 (5):
1059-1070) for Fc.gamma.RIIIa; and Ory et al. (J. Clin. Invest.,
1989, 84, 1688-1691) for Fc.gamma.RIIIb.
[0650] 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 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 Kifunesine 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 FF) in step 1,
affinity column chromatography (HisTrap HP) for His tag in step 2,
gel filtration column chromatography (Superdex200) in step 3, and
aseptic chromatography in step 4. However, for Fc.gamma.RI, anion
exchange column chromatography using Q sepharose FF 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).
[0651] Analysis of interaction between each altered antibody and
the Fc.gamma. receptor prepared as mentioned above was carried out
using Biacore T100 (GE Healthcare), Biacore T200 (GE Healthcare),
Biacore A100, and Biacore 4000. HBS-EP+(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.
[0652] 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.
[0653] 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
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 (l/s) were calculated; and from those values the
dissociation constants KD (mol/L) were calculated.
[0654] 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 T100 Software Handbook BR1006-48
Edition AE.
[0655] The behavior of interacting molecules according to the 1:1
binding model on Biacore can be described by Equation 3 shown
below.
R.sub.eq=CR.sub.max/(KD+C)+RI [Equation 3]
Req: a plot of steady-state binding levels against analyte
concentration C: concentration RI: bulk refractive index
contribution in the sample Rmax: analyte binding capacity of the
surface
[0656] 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]
[0657] KD can be calculated by substituting the values of Rmax, 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. Rmax 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 Rmax 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.
Sequence CWU 1
1
811468PRTHomo sapiens 1Met Leu Ala Val Gly Cys Ala Leu Leu Ala Ala
Leu Leu Ala Ala Pro 1 5 10 15 Gly Ala Ala Leu Ala Pro Arg Arg Cys
Pro Ala Gln Glu Val Ala Arg 20 25 30 Gly Val Leu Thr Ser Leu Pro
Gly Asp Ser Val Thr Leu Thr Cys Pro 35 40 45 Gly Val Glu Pro Glu
Asp Asn Ala Thr Val His Trp Val Leu Arg Lys 50 55 60 Pro Ala Ala
Gly Ser His Pro Ser Arg Trp Ala Gly Met Gly Arg Arg 65 70 75 80 Leu
Leu Leu Arg Ser Val Gln Leu His Asp Ser Gly Asn Tyr Ser Cys 85 90
95 Tyr Arg Ala Gly Arg Pro Ala Gly Thr Val His Leu Leu Val Asp Val
100 105 110 Pro Pro Glu Glu Pro Gln Leu Ser Cys Phe Arg Lys Ser Pro
Leu Ser 115 120 125 Asn Val Val Cys Glu Trp Gly Pro Arg Ser Thr Pro
Ser Leu Thr Thr 130 135 140 Lys Ala Val Leu Leu Val Arg Lys Phe Gln
Asn Ser Pro Ala Glu Asp 145 150 155 160 Phe Gln Glu Pro Cys Gln Tyr
Ser Gln Glu Ser Gln Lys Phe Ser Cys 165 170 175 Gln Leu Ala Val Pro
Glu Gly Asp Ser Ser Phe Tyr Ile Val Ser Met 180 185 190 Cys Val Ala
Ser Ser Val Gly Ser Lys Phe Ser Lys Thr Gln Thr Phe 195 200 205 Gln
Gly Cys Gly Ile Leu Gln Pro Asp Pro Pro Ala Asn Ile Thr Val 210 215
220 Thr Ala Val Ala Arg Asn Pro Arg Trp Leu Ser Val Thr Trp Gln Asp
225 230 235 240 Pro His Ser Trp Asn Ser Ser Phe Tyr Arg Leu Arg Phe
Glu Leu Arg 245 250 255 Tyr Arg Ala Glu Arg Ser Lys Thr Phe Thr Thr
Trp Met Val Lys Asp 260 265 270 Leu Gln His His Cys Val Ile His Asp
Ala Trp Ser Gly Leu Arg His 275 280 285 Val Val Gln Leu Arg Ala Gln
Glu Glu Phe Gly Gln Gly Glu Trp Ser 290 295 300 Glu Trp Ser Pro Glu
Ala Met Gly Thr Pro Trp Thr Glu Ser Arg Ser 305 310 315 320 Pro Pro
Ala Glu Asn Glu Val Ser Thr Pro Met Gln Ala Leu Thr Thr 325 330 335
Asn Lys Asp Asp Asp Asn Ile Leu Phe Arg Asp Ser Ala Asn Ala Thr 340
345 350 Ser Leu Pro Val Gln Asp Ser Ser Ser Val Pro Leu Pro Thr Phe
Leu 355 360 365 Val Ala Gly Gly Ser Leu Ala Phe Gly Thr Leu Leu Cys
Ile Ala Ile 370 375 380 Val Leu Arg Phe Lys Lys Thr Trp Lys Leu Arg
Ala Leu Lys Glu Gly 385 390 395 400 Lys Thr Ser Met His Pro Pro Tyr
Ser Leu Gly Gln Leu Val Pro Glu 405 410 415 Arg Pro Arg Pro Thr Pro
Val Leu Val Pro Leu Ile Ser Pro Pro Val 420 425 430 Ser Pro Ser Ser
Leu Gly Ser Asp Asn Thr Ser Ser His Asn Arg Pro 435 440 445 Asp Ala
Arg Asp Pro Arg Ser Pro Tyr Asp Ile Ser Asn Thr Asp Tyr 450 455 460
Phe Phe Pro Arg 465 21407DNAHomo sapiens 2atgctggccg tcggctgcgc
gctgctggct gccctgctgg ccgcgccggg agcggcgctg 60gccccaaggc gctgccctgc
gcaggaggtg gcgagaggcg tgctgaccag tctgccagga 120gacagcgtga
ctctgacctg cccgggggta gagccggaag acaatgccac tgttcactgg
180gtgctcagga agccggctgc aggctcccac cccagcagat gggctggcat
gggaaggagg 240ctgctgctga ggtcggtgca gctccacgac tctggaaact
attcatgcta ccgggccggc 300cgcccagctg ggactgtgca cttgctggtg
gatgttcccc ccgaggagcc ccagctctcc 360tgcttccgga agagccccct
cagcaatgtt gtttgtgagt ggggtcctcg gagcacccca 420tccctgacga
caaaggctgt gctcttggtg aggaagtttc agaacagtcc ggccgaagac
480ttccaggagc cgtgccagta ttcccaggag tcccagaagt tctcctgcca
gttagcagtc 540ccggagggag acagctcttt ctacatagtg tccatgtgcg
tcgccagtag tgtcgggagc 600aagttcagca aaactcaaac ctttcagggt
tgtggaatct tgcagcctga tccgcctgcc 660aacatcacag tcactgccgt
ggccagaaac ccccgctggc tcagtgtcac ctggcaagac 720ccccactcct
ggaactcatc tttctacaga ctacggtttg agctcagata tcgggctgaa
780cggtcaaaga cattcacaac atggatggtc aaggacctcc agcatcactg
tgtcatccac 840gacgcctgga gcggcctgag gcacgtggtg cagcttcgtg
cccaggagga gttcgggcaa 900ggcgagtgga gcgagtggag cccggaggcc
atgggcacgc cttggacaga atccaggagt 960cctccagctg agaacgaggt
gtccaccccc atgcaggcac ttactactaa taaagacgat 1020gataatattc
tcttcagaga ttctgcaaat gcgacaagcc tcccagtgca agattcttct
1080tcagtaccac tgcccacatt cctggttgct ggagggagcc tggccttcgg
aacgctcctc 1140tgcattgcca ttgttctgag gttcaagaag acgtggaagc
tgcgggctct gaaggaaggc 1200aagacaagca tgcatccgcc gtactctttg
gggcagctgg tcccggagag gcctcgaccc 1260accccagtgc ttgttcctct
catctcccca ccggtgtccc ccagcagcct ggggtctgac 1320aatacctcga
gccacaaccg accagatgcc agggacccac ggagccctta tgacatcagc
1380aatacagact acttcttccc cagatag 14073107PRTHomo sapiens 3Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr 20
25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln
Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105 419PRTArtificial SequenceAn artificially
synthesized sequence 4Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser 5107PRTArtificial
SequenceAn artificially synthesized sequence 5Glu Thr Thr Leu Thr
Gln Ser Pro Ala Phe Met Ser Ala Thr Pro Gly 1 5 10 15 Asp Lys Val
Asn Ile Ser Cys Lys Ala Ser Gln Asp Ile Asp Asp Asp 20 25 30 Met
Asn Trp Tyr Gln Gln Lys Pro Gly Glu Ala Ala Ile Phe Ile Ile 35 40
45 Gln Glu Ala Thr Thr Leu Val Pro Gly Ile Ser Pro Arg Phe Ser Gly
50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Ile
Glu Ser 65 70 75 80 Glu Asp Ala Ala Tyr Tyr Phe Cys Leu Gln His Asp
Asn Phe Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 105 6107PRTArtificial SequenceAn artificially synthesized
sequence 6Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe 85 90 95 Thr Phe
Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 7112PRTArtificial
SequenceAn artificially synthesized sequence 7Asp Ile Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn
Gly Asp Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Met Gln Val 85 90 95 Leu Arg Asn Pro Leu Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Gln 100 105 110 8107PRTArtificial SequenceAn
artificially synthesized sequence 8Glu Ile Val Met Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser
Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp
Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65
70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp
Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 9112PRTArtificial SequenceAn artificially synthesized sequence
9Asp Ile Val Met Thr Gln Ser Pro Glu Ser Leu Val Leu Ser Leu Gly 1
5 10 15 Gly Thr Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Val Leu Tyr
Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys
Pro Gly Gln 35 40 45 Pro Pro Thr Leu Leu Phe Ser Trp Ala Ser Ile
Arg Asp Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Ala Ser Gly Ser
Gly Thr Glu Phe Thr Leu Thr 65 70 75 80 Ile Ser Asp Leu Gln Ala Glu
Asp Ala Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Arg Ala Pro
Ser Phe Gly Gln Gly Thr Lys Leu Gln Ile Lys 100 105 110
10121PRTArtificial SequenceAn artificially synthesized sequence
10Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp
Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Asp Pro
Gly Gly Gly Glu Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 11126PRTArtificial SequenceAn
artificially synthesized sequence 11Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Glu Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr
Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Ala Pro Tyr Tyr Tyr Asp Ser Ser Gly
Tyr Thr Asp Ala 100 105 110 Phe Asp Ile Trp Gly Gln Gly Thr Met Val
Thr Val Ser Ser 115 120 125 12365PRTHomo sapiens 12Met Gly Val Pro
Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe 1 5 10 15 Leu Leu
Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr 20 25 30
His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp 35
40 45 Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser
Leu 50 55 60 Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu
Asn Gln Val 65 70 75 80 Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu
Arg Ile Lys Glu Lys 85 90 95 Leu Phe Leu Glu Ala Phe Lys Ala Leu
Gly Gly Lys Gly Pro Tyr Thr 100 105 110 Leu Gln Gly Leu Leu Gly Cys
Glu Leu Gly Pro Asp Asn Thr Ser Val 115 120 125 Pro Thr Ala Lys Phe
Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp 130 135 140 Leu Lys Gln
Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile 145 150 155 160
Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr 165
170 175 Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu
Arg 180 185 190 Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met
Arg Leu Lys 195 200 205 Ala Arg Pro Ser Ser Pro Gly Phe Ser Val Leu
Thr Cys Ser Ala Phe 210 215 220 Ser Phe Tyr Pro Pro Glu Leu Gln Leu
Arg Phe Leu Arg Asn Gly Leu 225 230 235 240 Ala Ala Gly Thr Gly Gln
Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser 245 250 255 Phe His Ala Ser
Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His 260 265 270 Tyr Cys
Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val 275 280 285
Glu Leu Glu Ser Pro Ala Lys Ser Ser Val Leu Val Val Gly Ile Val 290
295 300 Ile Gly Val Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu
Leu 305 310 315 320 Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro Trp
Ile Ser Leu Arg 325 330 335 Gly Asp Asp Thr Gly Val Leu Leu Pro Thr
Pro Gly Glu Ala Gln Asp 340 345 350 Ala Asp Leu Lys Asp Val Asn Val
Ile Pro Ala Thr Ala 355 360 365 13119PRTHomo sapiens 13Met Ser Arg
Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly
Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg 20 25
30 His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser
35 40 45 Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn
Gly Glu 50 55 60 Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe
Ser Lys Asp Trp 65 70 75 80 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe
Thr Pro Thr Glu Lys Asp 85 90 95 Glu Tyr Ala Cys Arg Val Asn His
Val Thr Leu Ser Gln Pro Lys Ile 100 105 110 Val Lys Trp Asp Arg Asp
Met 115 14330PRTArtificial SequenceAn artificially synthesized
sequence 14Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225
230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 325 330 15326PRTArtificial SequenceAn
artificially synthesized sequence 15Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65
70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser
Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185
190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310
315 320 Ser Leu Ser Pro Gly Lys 325 16377PRTHomo sapiens 16Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro
Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro
Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu
Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150
155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val
Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg
Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275
280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro
Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser
Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser
Leu Ser Pro Gly Lys 370 375 17327PRTArtificial SequenceAn
artificially synthesized sequence 17Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65
70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser
Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185
190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310
315 320 Leu Ser Leu Ser Leu Gly Lys 325 181125DNAHomo sapiens
18atgtggttct 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
112519374PRTHomo sapiens 19Met Trp Phe Leu Thr Thr Leu Leu Leu Trp
Val Pro Val Asp Gly Gln 1 5 10 15 Val Asp Thr Thr Lys Ala Val Ile
Thr Leu Gln Pro Pro Trp Val Ser 20 25 30 Val Phe Gln Glu Glu Thr
Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45 Pro Gly Ser Ser
Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60 Thr Ser
Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser 65 70 75 80
Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85
90 95 Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser
Arg 100 105 110 Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His
Ala Trp Lys 115 120 125 Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg
Asn Gly Lys Ala Phe 130 135 140 Lys Phe Phe His Trp Asn Ser Asn Leu
Thr Ile Leu Lys Thr Asn Ile 145 150 155 160 Ser His Asn Gly Thr Tyr
His Cys Ser Gly Met Gly Lys His Arg Tyr 165 170 175 Thr Ser Ala Gly
Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190 Val Leu
Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205
Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210
215 220 Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg
Asn 225 230 235 240 Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg
Glu Asp Ser Gly 245 250 255 Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp
Gly Asn Val Leu Lys Arg 260 265 270 Ser Pro Glu Leu Glu Leu Gln Val
Leu Gly Leu Gln Leu Pro Thr Pro 275 280 285 Val Trp Phe His Val Leu
Phe Tyr Leu Ala Val Gly Ile Met Phe Leu 290 295 300 Val Asn Thr Val
Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys 305 310 315 320 Lys
Lys Trp Asp Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys Lys 325 330
335 Val Ile Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys
340 345 350 Cys Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu Gly Val His
Arg Lys 355 360 365 Glu Pro Gln Gly Ala Thr 370 20951DNAHomo
sapiens 20atgactatgg 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 95121316PRTHomo
sapiens 21Met Thr Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg
Asn Leu 1 5 10 15 Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu
Ala Ser Ala Asp 20 25 30 Ser Gln Ala Ala Pro Pro Lys Ala Val Leu
Lys Leu Glu Pro Pro Trp 35 40 45 Ile Asn Val Leu Gln Glu Asp Ser
Val Thr Leu Thr Cys Gln Gly Ala 50 55 60 Arg Ser Pro Glu Ser Asp
Ser Ile Gln Trp Phe His Asn Gly Asn Leu 65 70 75 80 Ile Pro Thr His
Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn 85 90 95 Asp Ser
Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp 100 105 110
Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro 115
120 125 His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His
Ser 130 135 140 Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln
Asn Gly Lys 145 150 155 160 Ser Gln Lys Phe Ser His Leu Asp Pro Thr
Phe Ser Ile Pro Gln Ala 165 170 175 Asn His Ser His Ser Gly Asp Tyr
His Cys Thr Gly Asn Ile Gly Tyr 180 185 190 Thr Leu Phe Ser Ser Lys
Pro Val Thr Ile Thr Val Gln Val Pro Ser 195 200 205 Met Gly Ser Ser
Ser Pro Met Gly Val Ile Val Ala Val Val Ile Ala 210 215 220 Thr Ala
Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys 225 230 235
240 Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala
245 250 255 Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys
Arg Gln 260 265 270 Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp
Gly Gly Tyr Met 275 280 285 Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp
Asp Lys Asn Ile Tyr Leu 290 295 300 Thr Leu Pro Pro Asn Asp His Val
Asn Ser Asn Asn 305 310 315 22876DNAHomo sapiens 22atgggaatcc
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 87623291PRTHomo sapiens 23Met Gly Ile
Leu Ser Phe Leu Pro Val Leu Ala Thr Glu Ser Asp Trp 1 5 10 15 Ala
Asp Cys Lys Ser Pro Gln Pro Trp Gly His Met Leu Leu Trp Thr 20 25
30 Ala Val Leu Phe Leu Ala Pro Val Ala Gly Thr Pro Ala Ala Pro Pro
35 40 45 Lys Ala Val Leu Lys Leu Glu Pro Gln Trp Ile Asn Val Leu
Gln Glu 50 55 60 Asp Ser Val Thr Leu Thr Cys Arg Gly Thr His Ser
Pro Glu Ser Asp 65 70 75 80 Ser Ile Gln Trp Phe His Asn Gly Asn Leu
Ile Pro Thr His Thr Gln 85 90 95 Pro Ser Tyr Arg Phe Lys Ala Asn
Asn Asn Asp Ser Gly Glu Tyr Thr 100 105 110 Cys Gln Thr Gly Gln Thr
Ser Leu Ser Asp Pro Val His Leu Thr Val 115 120 125 Leu Ser Glu Trp
Leu Val Leu Gln Thr Pro His Leu Glu Phe Gln Glu 130 135 140 Gly Glu
Thr Ile Val Leu Arg Cys His Ser Trp Lys Asp Lys Pro Leu 145 150 155
160 Val Lys Val Thr Phe Phe Gln Asn Gly Lys Ser Lys Lys Phe Ser Arg
165 170 175 Ser Asp Pro Asn Phe Ser Ile Pro Gln Ala Asn His Ser His
Ser Gly 180 185 190 Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Leu
Tyr Ser Ser Lys 195 200 205 Pro Val Thr Ile Thr Val Gln Ala Pro Ser
Ser Ser Pro Met Gly Ile 210 215 220 Ile Val Ala Val Val Thr Gly Ile
Ala Val Ala Ala Ile Val Ala Ala 225 230 235 240 Val Val Ala Leu Ile
Tyr Cys Arg Lys Lys Arg Ile Ser Ala Asn Pro 245 250 255 Thr Asn Pro
Asp Glu Ala Asp Lys Val Gly Ala Glu Asn Thr Ile Thr 260 265 270 Tyr
Ser Leu Leu Met His Pro Asp Ala Leu Glu Glu Pro Asp Asp Gln 275 280
285 Asn Arg Ile 290 24765DNAHomo sapiens 24atgtggcagc 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 76525254PRTHomo sapiens
25Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1
5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu
Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu
Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln
Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser
Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser Gly Glu Tyr
Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln
Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg
Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His
Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135
140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro
145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg
Gly Leu Val 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn
Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ser Val Ser Thr Ile Ser Ser
Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met
Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val
Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 225 230 235 240 Lys Asp
His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 250
26702DNAHomo sapiens 26atgtggcagc 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
70227233PRTHomo sapiens 27Met Trp Gln Leu Leu Leu Pro Thr Ala Leu
Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro
Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Ser Val Leu
Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser
Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu
Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80
Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85
90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu
Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His
Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val
Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Asp Arg Lys Tyr Phe His His
Asn Ser Asp Phe His Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp
Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175 Gly Ser Lys Asn
Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu
Ala Val Ser Thr Ile Ser Ser Phe Ser Pro Pro Gly Tyr Gln 195 200 205
Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210
215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile 225 230
284PRTArtificial SequenceAn artificially synthesized sequence 28Gly
Gly Gly Ser 1 294PRTArtificial SequenceAn artificially synthesized
sequence 29Ser Gly Gly Gly 1 305PRTArtificial SequenceAn
artificially synthesized sequence 30Gly Gly Gly Gly Ser 1 5
315PRTArtificial SequenceAn artificially synthesized sequence 31Ser
Gly Gly Gly Gly 1 5 326PRTArtificial SequenceAn artificially
synthesized sequence 32Gly Gly Gly Gly Gly Ser 1 5 336PRTArtificial
SequenceAn artificially synthesized sequence 33Ser Gly Gly Gly Gly
Gly 1 5 347PRTArtificial SequenceAn artificially synthesized
sequence 34Gly Gly Gly Gly Gly Gly Ser 1 5 357PRTArtificial
SequenceAn artificially synthesized sequence 35Ser Gly Gly Gly Gly
Gly Gly 1 5 36447PRTArtificial SequenceAn artificially synthesized
sequence 36Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser
Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro
Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly
Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile
Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg
Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235
240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360
365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 37214PRTArtificial
SequenceAn artificially synthesized sequence 37Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val
Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40
45 Tyr Tyr Gly Ser Glu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu
Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Asn
Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Glu Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 38447PRTArtificial
SequenceAn artificially synthesized sequence 38Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His
Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40
45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met
Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295
300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser
Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445
39214PRTArtificial SequenceAn artificially synthesized sequence
39Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Thr Asp Ile Ser Ser
His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu
Leu Leu Ile 35 40 45 Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr
Cys Gly Gln Gly Asn Arg Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
40447PRTArtificial SequenceAn artificially synthesized sequence
40Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Asp
Ala Tyr Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 435 440 445 41447PRTArtificial SequenceAn
artificially synthesized sequence 41Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys
Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser
Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly
Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60
Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr
Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Arg Gly Gly Pro 225 230 235 240 Lys Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445 42214PRTArtificial SequenceAn artificially synthesized sequence
42Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr
Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
43447PRTArtificial SequenceAn artificially synthesized sequence
43Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Asp
Ala Tyr Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Leu His Glu 420 425 430 Ala Leu His Ser His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 435 440 445 44447PRTArtificial SequenceAn
artificially synthesized sequence 44Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys
Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser
Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly
Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60
Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr
Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Tyr Pro Ala Pro Ile Glu
Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 435 440 445 45447PRTArtificial SequenceAn
artificially synthesized sequence 45Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys
Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser
Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly
Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60
Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr
Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro 225 230 235 240 Asp Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu
Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445 46447PRTArtificial SequenceAn artificially synthesized sequence
46Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Tyr Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Leu His Glu 420 425 430 Ala Leu His Ser His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 435 440 445 47447PRTArtificial SequenceAn
artificially synthesized sequence 47Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys
Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser
Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly
Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60
Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr
Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro 225 230 235 240 Asp Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu
Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 420 425 430
Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445 48447PRTArtificial SequenceAn artificially synthesized sequence
48Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Tyr Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Ala His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 435 440 445 49443PRTMus musculus 49Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr
Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25
30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp
35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro
Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr
Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro
Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys
Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155
160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro
Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala
His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro
Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu
Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp
Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val
Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp
Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280
285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile
290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg
Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr
Thr Ile Pro Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val
Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile
Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr
Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400
Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405
410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn
His
His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440
50214PRTMus musculus 50Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala
Ser Thr Asp Ile Ser Ser His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Tyr Gly Ser His
Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu
Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Ala Asp Ala Ala
100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr
Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu
Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu
Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu
Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe
Asn Arg Asn Glu Cys 210 51443PRTArtificial SequenceAn artificially
synthesized sequence 51Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser
Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg
Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser
Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg
Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly
100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser
Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser
Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu
Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser
Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr
Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro
Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser
Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215
220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro
225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro
Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro
Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His
Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr
Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp
Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala
Asp Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335
Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340
345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp
Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly
Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp
Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val
Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser
Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser
Leu Ser His Ser Pro Gly Lys 435 440 52443PRTArtificial SequenceAn
artificially synthesized sequence 52Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys
Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser
Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly
Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60
Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr
Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr
Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala
Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly
Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly
Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185
190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser
195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly
Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Asp Val
Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile
Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser
Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp
Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln
Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met
His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310
315 320 Ser Ala Asp Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro
Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys
Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp
Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln
Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser
Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr
Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430
Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440
53119PRTArtificial SequenceAn artificially synthesized sequence
53Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 54447PRTArtificial SequenceAn artificially
synthesized sequence 54Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser
Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg
Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser
Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg
Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly
100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215
220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340
345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445
55447PRTArtificial SequenceAn artificially synthesized sequence
55Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Phe Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 435 440 445 56214PRTHomo sapiens 56Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr 20 25
30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln
Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115
120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly
Glu Cys 210 57449PRTArtificial SequenceAn artificially synthesized
sequence 57Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser
Ile Thr Ser Gly 20 25 30 Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala Ser Ile Thr Tyr Asp Gly
Ser Thr Asn Tyr Asn Pro Ser Val 50 55 60 Lys Gly Arg Ile Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val Trp Gly 100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115
120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235
240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro
58218PRTArtificial SequenceAn artificially synthesized sequence
58Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr
Asp 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu
Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135
140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys 210 215 59449PRTArtificial SequenceAn artificially
synthesized sequence 59Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser
Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp Asn Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala Ser Ile Thr
Tyr Asp Gly Ser Thr Asn Tyr Asn Pro Ser Val 50 55 60 Lys Gly Arg
Ile Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val Trp Gly
100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215
220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Glu His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro Ile 325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340
345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro
60447PRTArtificial SequenceAn artificially synthesized sequence
60Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Tyr His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 435 440 445 61447PRTArtificial SequenceAn
artificially synthesized sequence 61Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys
Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser
Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly
Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60
Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr
Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Asp 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Tyr Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Tyr His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445 62447PRTArtificial SequenceAn artificially synthesized sequence
62Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145
150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Asp 225 230 235 240 Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265
270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390
395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 445 63447PRTArtificial SequenceAn
artificially synthesized sequence 63Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys
Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser
Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly
Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60
Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr
Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440
445 64444PRTArtificial SequenceAn artificially synthesized sequence
64Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr
Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260
265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385
390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Leu 435 440 65464PRTArtificial sequenceAn artificially
synthesized sequence 65Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg
Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser
Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg
Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu
Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Pro Thr Ser Pro Lys
Val Phe 115 120 125 Pro Leu Ser Leu Cys Ser Thr Gln Pro Asp Gly Asn
Val Val Ile Ala 130 135 140 Cys Leu Val Gln Gly Phe Phe Pro Gln Glu
Pro Leu Ser Val Thr Trp 145 150 155 160 Ser Glu Ser Gly Gln Gly Val
Thr Ala Arg Asn Phe Pro Pro Ser Gln 165 170 175 Asp Ala Ser Gly Asp
Leu Tyr Thr Thr Ser Ser Gln Leu Thr Leu Pro 180 185 190 Ala Thr Gln
Cys Leu Ala Gly Lys Ser Val Thr Cys His Val Lys His 195 200 205 Tyr
Thr Asn Pro Ser Gln Asp Val Thr Val Pro Cys Pro Val Pro Ser 210 215
220 Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser
225 230 235 240 Cys Cys His Pro Arg Leu Ser Leu His Arg Pro Ala Leu
Glu Asp Leu 245 250 255 Leu Leu Gly Ser Glu Ala Asn Leu Thr Cys Thr
Leu Thr Gly Leu Arg 260 265 270 Asp Ala Ser Gly Val Thr Phe Thr Trp
Thr Pro Ser Ser Gly Lys Ser 275 280 285 Ala Val Gln Gly Pro Pro Glu
Arg Asp Leu Cys Gly Cys Tyr Ser Val 290 295 300 Ser Ser Val Leu Pro
Gly Cys Ala Glu Pro Trp Asn His Gly Lys Thr 305 310 315 320 Phe Thr
Cys Thr Ala Ala Tyr Pro Glu Ser Lys Thr Pro Leu Thr Ala 325 330 335
Thr Leu Ser Lys Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu 340
345 350 Pro Pro Pro Ser Glu Glu Leu Ala Leu Asn Glu Leu Val Thr Leu
Thr 355 360 365 Cys Leu Ala Arg Gly Phe Ser Pro Lys Asp Val Leu Val
Arg Trp Leu 370 375 380 Gln Gly Ser Gln Glu Leu Pro Arg Glu Lys Tyr
Leu Thr Trp Ala Ser 385 390 395 400 Arg Gln Glu Pro Ser Gln Gly Thr
Thr Thr Phe Ala Val Thr Ser Ile 405 410 415 Leu Arg Val Ala Ala Glu
Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys 420 425 430 Met Val Gly His
Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile 435 440 445 Asp Arg
Leu Ala Gly Lys Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 450 455 460
66451PRTArtificial sequenceAn artificially synthesized sequence
66Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp
Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Pro Arg Trp
Glu Thr Ala Ile Ser Ser Asp Ala Phe Asp Ile 100 105 110 Trp Gly Gln
Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135
140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260
265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385
390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro 450
67214PRTArtificial sequenceAn artificially synthesized sequence
67Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asp
Asp 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Asn Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln His Ser Ser Ser Pro Leu 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160 Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205 Phe Asn Arg Gly Glu Cys 210 68451PRTArtificial sequenceAn
artificially synthesized sequence 68Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly
Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Pro Arg Trp Glu Thr Ala Ile Ser Ser Asp
Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser
Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185
190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310
315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Tyr Pro
Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435
440 445 Leu Ser Pro 450 69543PRTArtificial sequenceAn artificially
synthesized sequence 69Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Leu Asp Pro
Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg
Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110 Val Ser Ser Ala Ser Thr Gln Ser Pro Ser Val Phe Pro Leu
Thr Arg 115 120 125 Cys Cys Lys Asn Ile Pro Ser Asn Ala Thr Ser Val
Thr Leu Gly Cys 130 135 140 Leu Ala Thr Gly Tyr Phe Pro Glu Pro Val
Met Val Thr Trp Asp Thr 145 150 155 160 Gly Ser Leu Asn Gly Thr Thr
Met Thr Leu Pro Ala Thr Thr Leu Thr 165 170 175 Leu Ser Gly His Tyr
Ala Thr Ile Ser Leu Leu Thr Val Ser Gly Ala 180 185 190 Trp Ala Lys
Gln Met Phe Thr Cys Arg Val Ala His Thr Pro Ser Ser 195 200 205 Thr
Asp Trp Val Asp Asn Lys Thr Phe Ser Val Cys Ser Arg Asp Phe 210 215
220 Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly
225 230 235 240 His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser
Gly Tyr Thr 245 250 255 Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp
Gly Gln Val Met Asp 260 265 270 Val Asp Leu Ser Thr Ala Ser Thr Thr
Gln Glu Gly Glu Leu Ala Ser 275 280 285 Thr Gln Ser Glu Leu Thr Leu
Ser Gln Lys His Trp Leu Ser Asp Arg 290 295 300 Thr Tyr Thr Cys Gln
Val Thr Tyr Gln Gly His Thr Phe Glu Asp Ser 305 310 315 320 Thr Lys
Lys Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu 325 330 335
Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile 340
345 350 Thr Cys Leu Val Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn
Leu 355 360 365 Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser
Thr Arg Lys 370 375 380 Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr Val
Thr Ser Thr Leu Pro 385 390 395 400 Val Gly Thr Arg Asp Trp Ile Glu
Gly Glu Thr Tyr Gln Cys Arg Val 405 410 415 Thr His Pro His Leu Pro
Arg Ala Leu Met Arg Ser Thr Thr Lys Thr 420 425 430 Ser Gly Pro Arg
Ala Ala Pro Glu Val Tyr Ala Phe Ala Thr Pro Glu 435 440 445 Trp Pro
Gly Ser Arg Asp Lys Arg Thr Leu Ala Cys Leu Ile Gln Asn 450 455 460
Phe Met Pro Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu Val Gln 465
470 475 480 Leu Pro Asp Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr
Lys Gly 485 490 495 Ser Gly Phe Phe Val Phe Ser Arg Leu Glu Val Thr
Arg Ala Glu Trp 500 505 510 Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala
Val His Glu Ala Ala Ser 515 520 525 Pro Ser Gln Thr Val Gln Arg Ala
Val Ser Val Asn Pro Gly Lys 530 535 540 70219PRTArtificial
sequenceAn artificially synthesized sequence 70Asp Val Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn
Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170
175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215 71443PRTArtificial sequenceAn artificially synthesized sequence
71Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1
5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr
His 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Ile Gly 35 40 45 Val Ile Asn Ser Ala Gly Asn Thr Tyr Tyr Ala
Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Val Ser Lys Thr Ser Thr
Thr Val Asp Leu Asn Leu Thr 65 70 75 80 Ser Leu Thr Thr Glu Asp Thr
Ala Thr Tyr Phe Cys Ala Arg Tyr Val 85 90 95 Phe Ser Ser Gly Ser
His Asp Ile Trp Gly Pro Gly Thr Leu Val Thr 100 105 110 Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135
140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260
265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385
390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 435 440 72217PRTArtificial sequenceAn artificially synthesized
sequence 72Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala
Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser
Ile Gly Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro Lys Glu Leu Ile 35 40 45 Tyr Gly Thr Ser Thr Leu Glu Ser
Gly Val Pro Ser Arg Phe Ile Gly 50 55 60 Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Gly Val Glu Cys 65 70 75 80 Ala Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Glu Asp Asn 85 90 95 Ile Asp
Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys Arg Thr 100 105 110
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115
120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 215 7332PRTArtificial sequenceAn
artificially synthesized sequence 73Val Asp Asp Ala Pro Gly Asn Ser
Gln Gln Ala Thr Pro Lys Asp Asn 1 5 10 15 Glu Ile Ser Thr Phe His
Asn Leu Gly Asn Val His Ser Pro Leu Lys 20 25 30 74443PRTArtificial
sequenceAn artificially synthesized sequence 74Gln Ser Leu Glu Glu
Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu
Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr His 20 25 30 Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40
45 Val Ile Asn Ser Ala Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly
50 55 60 Arg Phe Thr Val Ser Lys Thr Ser Thr Thr Val Asp Leu Asn
Leu Thr 65 70 75 80 Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys
Ala Arg Tyr Val 85 90 95 Phe Ser Ser Gly Ser His Asp Ile Trp Gly
Pro Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170
175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280
285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Tyr Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405
410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
75543PRTArtificial sequenceAn artificially synthesized sequence
75Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser
Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser
Ala Ser Thr Gln Ser Pro Ser Val Phe Pro Leu Thr Arg 115 120 125 Cys
Cys Lys Asn Ile Pro Ser Asp Ala Thr Ser Val Thr Leu Gly Cys 130 135
140 Leu Ala Thr Gly Tyr Phe Pro Glu Pro Val Met Val Thr Trp Asp Thr
145 150 155 160 Gly Ser Leu Asp Gly Thr Thr Met Thr Leu Pro Ala Thr
Thr Leu Thr 165 170 175 Leu Ser Gly His Tyr Ala Thr Ile Ser Leu Leu
Thr Val Ser Gly Ala 180 185 190 Trp Ala Lys Gln Met Phe Thr Cys Arg
Val Ala His Thr Pro Ser Ser 195 200 205 Thr Asp Trp Val Asp Asp Lys
Thr Phe Ser Val Cys Ser Arg Asp Phe 210 215 220 Thr Pro Pro Thr Val
Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly 225 230 235 240 His Phe
Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr 245 250 255
Pro Gly Thr Ile Asp Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp 260
265 270 Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala
Ser 275 280 285 Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu
Ser Asp Arg 290 295 300 Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His
Thr Phe Glu Asp Ser 305 310 315 320 Thr Lys Lys Cys Ala Asp Ser Asn
Pro Arg Gly Val Ser Ala Tyr Leu 325 330 335 Ser Arg Pro Ser Pro Phe
Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile 340 345 350 Thr Cys Leu Val
Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asp Leu 355 360 365 Thr Trp
Ser Arg Ala Ser Gly Lys Pro Val Asp His Ser Thr Arg Lys 370 375 380
Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu Pro 385
390 395 400 Val Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys
Arg Val 405 410 415 Thr His Pro His Leu Pro Arg Ala Leu Met Arg Ser
Thr Thr Lys Thr 420 425 430 Ser Gly Pro Arg Ala Ala Pro Glu Val Tyr
Ala Phe Ala Thr Pro Glu 435 440 445 Trp Pro Gly Ser Arg Asp Lys Arg
Thr Leu Ala Cys Leu Ile Gln Asn 450 455 460 Phe Met Pro Glu Asp Ile
Ser Val Gln Trp Leu His Asn Glu Val Gln 465 470 475 480 Leu Pro Asp
Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly 485 490 495 Ser
Gly Phe Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu Trp 500 505
510 Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala Val His Glu Ala Ala Ser
515 520 525 Pro Ser Gln Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly
Lys 530 535 540 76443PRTArtificial sequenceAn artificially
synthesized sequence 76Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val
Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly
Phe Ser Leu Ser Ser Tyr His 20 25 30 Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Val Ile Asn Ser Ala
Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr
Val Ser Lys Thr Ser Thr Thr Val Asp Leu Asn Leu Thr 65 70 75 80 Ser
Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Tyr Val 85 90
95 Phe Ser Ser Gly Ser His Asp Ile Trp Gly Pro Gly Thr Leu Val Thr
100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215
220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser
Asn Asp Ala Tyr Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340
345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro 435 440 77443PRTArtificial sequenceAn
artificially synthesized sequence 77Gln Ser Leu Glu Glu Ser Gly Gly
Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr
Ala Ser Gly Phe Ser Leu Ser Ser Tyr His 20 25 30 Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Val Ile
Asn Ser Ala Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60
Arg Phe Thr Val Ser Lys Thr Ser Thr Thr Val Asp Leu Asn Leu Thr 65
70 75 80 Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg
Tyr Val 85 90 95 Phe Ser Ser Gly Ser His Asp Ile Trp Gly Pro Gly
Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185
190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Arg Gly Gly Pro
Lys Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310
315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser 340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 78545PRTHomo
sapiens 78Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val
Arg Ser 1 5 10 15 Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val
Pro Glu Thr Pro 20 25 30 Val Pro Gly Ser Asp Leu Gln Val Cys Leu
Pro Lys Gly Pro Thr Cys 35 40 45 Cys Ser Arg Lys Met Glu Glu Lys
Tyr Gln Leu Thr Ala Arg Leu Asn 50 55 60 Met Glu Gln Leu Leu Gln
Ser Ala Ser Met Glu Leu Lys Phe Leu Ile 65 70 75 80 Ile Gln Asn Ala
Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg 85 90 95 His Ala
Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser 100 105 110
Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val 115
120 125 Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val
Asn 130 135 140 Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln
Leu Met Asn 145 150 155 160 Pro Gly Leu Pro Asp Ser Ala Leu Asp Ile
Asn Glu Cys Leu Arg Gly 165 170 175 Ala Arg Arg Asp Leu Lys Val Phe
Gly Asn Phe Pro Lys Leu Ile Met 180 185 190 Thr Gln Val Ser Lys Ser
Leu Gln Val Thr Arg Ile Phe Leu Gln Ala 195 200 205 Leu Asn Leu Gly
Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe 210 215 220 Ser Lys
Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr 225 230 235
240 Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val
245 250 255 Val Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys
Tyr Trp 260 265 270 Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn
Gly Met Tyr Arg 275 280 285 Ile Tyr Asp Met Glu Asn Val Leu Leu Gly
Leu Phe Ser Thr Ile His 290 295 300 Asp Ser Ile Gln Tyr Val Gln Lys
Asn Ala Gly Lys Leu Thr Thr Thr 305 310 315 320 Ile Gly Lys Leu Cys
Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala 325 330 335 Tyr Tyr Pro
Glu Asp Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala 340 345 350 His
Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile 355 360
365 Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr
370 375 380 Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys
Trp Asn 385 390 395 400 Gly Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys
Ala Ala Arg Asn Gly 405 410 415 Met Lys Asn Gln Phe Asn Leu His Glu
Leu Lys Met Lys Gly Pro Glu 420 425 430 Pro Val Val Ser Gln Ile Ile
Asp Lys Leu Lys His Ile Asn Gln Leu 435 440 445 Leu Arg Thr Met Ser
Met Pro Lys Gly Arg Val Leu Asp Lys Asn Leu 450 455 460 Asp Glu Glu
Gly Phe Glu Ala Gly Asp Cys Gly Asp Asp Glu Asp Glu 465 470 475 480
Cys Ile Gly Gly Ala Gly Asp Gly Met Ile Lys Val Lys Asn Gln Leu 485
490 495 Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala
Pro 500 505 510 Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile
Ser Thr Phe 515 520 525 His Asn Leu Gly Asn Val His Ser Pro Leu Lys
His His His His His 530 535 540 His 545 79433PRTArtificial
sequenceAn artificially synthesized sequence 79Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His
Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40
45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met
Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser
Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130
135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr
Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe
Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser
Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr
Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys
Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys
Glu Ala Asn Glu Val Glu Asp Val Phe Ile Phe Pro 225 230 235 240 Pro
Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250
255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser
260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln
Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser
Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu
Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala
Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340 345 350 Gln Met Ala
Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe
Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375
380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr
385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp
Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly
Leu His Asn His His 420 425 430 Thr 80328PRTArtificial sequenceAn
artificially synthesized sequence 80Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65
70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 81325PRTArtificial
sequenceAn artificially synthesized sequence 81Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170
175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295
300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320 Leu Ser Leu Ser Leu 325
* * * * *
References