U.S. patent application number 16/264735 was filed with the patent office on 2019-08-01 for therapeutic antigen-binding molecule with a fcrn-binding domain that promotes antigen clearance.
This patent application is currently assigned to Chugai Seiyaku Kabushiki Kaisha. The applicant listed for this patent is Chugai Seiyaku Kabushiki Kaisha. Invention is credited to Tomoyuki Igawa, Taichi Kuramochi, Atsuhiko Maeda, Futa Mimoto.
Application Number | 20190233525 16/264735 |
Document ID | / |
Family ID | 47996560 |
Filed Date | 2019-08-01 |
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United States Patent
Application |
20190233525 |
Kind Code |
A1 |
Igawa; Tomoyuki ; et
al. |
August 1, 2019 |
THERAPEUTIC ANTIGEN-BINDING MOLECULE WITH A FcRn-BINDING DOMAIN
THAT PROMOTES ANTIGEN CLEARANCE
Abstract
The present invention provides: a modified FcRn-binding domain
having an enhanced affinity for the Fc Receptor neonatal (FcRn) at
neutral pH; an antigen-binding molecule comprising said
FcRn-binding domain, which has low immunogenicity, high stability
and form only a few aggregates; a modified antigen-binding molecule
having an increased FcRn-binding activity at neutral or acidic pH
without an increased binding activity at neutral pH for a
pre-existing anti-drug antibody; use of the antigen-binding
molecules for improving antigen-binding molecule-mediated antigen
uptake into cells; use of the antigen-binding molecules for
reducing the plasma concentration of a specific antigen; use of the
modified FcRn-binding domain for increasing the total number of
antigens to which a single antigen-binding molecule can bind before
its degradation; use of the modified FcRn-binding domain for
improving pharmacokinetics of an antigen-binding molecule; methods
for decreasing the binding activity for a pre-existing anti-drug
antibody; and methods for producing said antigen-binding
molecules.
Inventors: |
Igawa; Tomoyuki; (Shizuoka,
JP) ; Maeda; Atsuhiko; (Shizuoka, JP) ;
Mimoto; Futa; (Shizuoka, JP) ; Kuramochi; Taichi;
(Shizuoka, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chugai Seiyaku Kabushiki Kaisha |
Tokyo |
|
JP |
|
|
Assignee: |
Chugai Seiyaku Kabushiki
Kaisha
Tokyo
JP
|
Family ID: |
47996560 |
Appl. No.: |
16/264735 |
Filed: |
February 1, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14347187 |
Jul 25, 2014 |
10253100 |
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PCT/JP2012/006218 |
Sep 28, 2012 |
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16264735 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2812 20130101;
C07K 2317/34 20130101; C07K 2317/94 20130101; C07K 2317/524
20130101; A61P 29/00 20180101; A61P 37/00 20180101; C07K 16/2866
20130101; A61K 2039/505 20130101; A61P 19/02 20180101; C07K
2317/526 20130101; A61P 37/06 20180101; C07K 2317/52 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 30, 2011 |
JP |
2011-218736 |
Mar 30, 2012 |
JP |
PCT/JP2012/058603 |
May 30, 2012 |
JP |
2012-123773 |
May 30, 2012 |
JP |
2012-123781 |
May 30, 2012 |
JP |
2012-123782 |
Jun 20, 2012 |
JP |
2012-139211 |
Aug 9, 2012 |
JP |
2012-177311 |
Claims
[0905] 1-61. (canceled)
62. An antigen-binding molecule comprising a modified FcRn-binding
domain, the amino acid sequence of which differs from the sequence
of a native human IgG FcRn-binding domain at one or more positions,
including an amino acid substitution at at least one of the
following positions (EU numbering): 238, 250, 252, 254, 255, 258,
286, 307, 308, 309, 311, 315, 428, 433, 434, and 436.
63. The antigen-binding molecule of claim 62, wherein the at least
one position that is substituted includes positions 252 and 434 and
one or more of the following positions: 238, 250, 254, 255, 256,
258, 286, 387, 307, 308, 309, 311, 315, 428, 433, and 436 (all
positions by EU numbering).
64. The antigen-binding molecule of claim 62, wherein at least one
of the following positions in the modified FcRn-binding domain is
occupied by the indicated amino acid (all positions by EU
numbering): aspartic acid at position 238; valine at position 250;
tyrosine at position 252; threonine at position 254; leucine at
position 255; glutamic acid at position 256; either aspartic acid
or isoleucine at position 258; glutamic acid at position 286;
glutamine at position 307; proline at position 308; glutamic acid
at position 309; either alanine or histidine at position 311;
aspartic acid at position 315; isoleucine at position 428; any one
of alanine, lysine, proline, arginine, and serine at position 433;
either tyrosine or tryptophan at position 434; any one of
isoleucine, leucine, valine, threonine, and phenylalanine at
position 436.
65. The antigen-binding molecule of claim 62, wherein the at least
one position that is substituted includes all members of a set of
positions selected from any of sets (a)-(g): (a) positions 252,
434, and 436; (b) positions 252, 307, 311 and 434; (c) positions
252, 315, and 434; (d) positions 252, 308, and 434; (e) positions
238, 252, and 434; (f) positions 252, 434, 307, 311, and 436; (g)
positions 252, 387, and 434 (all positions by EU numbering).
66. The antigen-binding molecule of claim 62, wherein the modified
FcRn-binding domain comprises one of the following combinations of
amino acid residues at the indicated positions: (a) tyrosine at
position 252, aspartic acid at position 315, and tyrosine at
position 434; (b) tyrosine at position 252, tyrosine at position
434, and isoleucine at position 436; (c) tyrosine at position 252,
tyrosine at position 434, and leucine at position 436; (d) tyrosine
at position 252, tyrosine at position 434, and valine at position
436; or (e) tyrosine at position 252, threonine at position 254,
tyrosine at position 434, and isoleucine at position 436 (all
positions by EU numbering).
67. The antigen-binding molecule of claim 62, wherein the at least
one position that is substituted includes all members of a set of
positions selected from any of sets (a)-(h): (a) positions 252,
434, 307, 311, and 286; (b) positions 252, 434, 307, 311, 286, and
254; (c) positions 252, 434, 307, 311, and 436; (d) positions 252,
434, 307, 311, 436, and 254; (e) positions 252, 434, 307, 311, 436,
and 250; (f) positions 252, 434, 308, and 250; (g) positions 252,
434, 308, 250, and 436; (h) positions 252, 434, 308, 250, 307, and
311 (all positions by EU numbering).
68. The antigen-binding molecule of claim 62, wherein the modified
FcRn-binding domain comprises one of the following combinations of
amino acid residues at the indicated positions: (a) tyrosine at
position 252, glutamic acid at position 286, glutamine at position
307, alanine at position 311, and tyrosine at position 434; (b)
tyrosine at position 252, threonine at position 254, glutamic acid
at position 286, glutamine at position 307, alanine at position
311, and tyrosine at position 434; (c) tyrosine at position 252,
glutamine at position 307, alanine at position 311, tyrosine at
position 434, and isoleucine at position 436; (d) tyrosine at
position 252, threonine at position 254, glutamic acid at position
286, glutamine at position 307, alanine at position 311, tyrosine
at position 434, and isoleucine at position 436; (e) valine at
position 250, tyrosine at position 252, threonine at position 254,
proline at position 308, tyrosine at position 434, and valine at
position 436; (f) valine at position 250, tyrosine at position 252,
glutamine at position 307, alanine at position 311, tyrosine at
position 434, and valine at position 436; (g) tyrosine at position
252, glutamine at position 307, alanine at position 311, tyrosine
at position 434, and valine at position 436; (h) valine at position
250, tyrosine at position 252, proline at position 308, and
tyrosine at position 434; or (i) valine at position 250, tyrosine
at position 252, glutamine at position 307, proline at position
308, alanine at position 311, and tyrosine at position 434 (all
positions by EU numbering).
69. The antigen-binding molecule of claim 62, wherein the at least
one position that is substituted includes all members of a set of
positions selected from any of sets (a)-(d): (a) positions 252,
434, 307, 311, 436, and 286; (b) positions 252, 434, 307, 311, 436,
250, and 308; (c) positions 252, 434, 307, 311, 436, 250, 286, and
308; (d) positions 252, 434, 307, 311, 436, 250, 286, 308, and 428
(all positions by EU numbering).
70. The antigen-binding molecule of claim 62, wherein the modified
FcRn-binding domain comprises one of the following combinations of
amino acid residues at the indicated positions: (a) tyrosine at
position 252, glutamic acid at position 286, glutamine at position
307, alanine at position 311, tyrosine at position 434, and valine
at position 436; (b) valine at position 250, tyrosine at position
252, glutamine at position 307, proline at position 308, alanine at
position 311, tyrosine at position 434, and valine at position 436;
(c) valine at position 250, tyrosine at position 252, glutamic acid
at position 286, glutamine at position 307, proline at position
308, alanine at position 311, tyrosine at position 434, and valine
at position 436; or (d) valine at position 250, tyrosine at
position 252, glutamic acid at position 286, glutamine at position
307, proline at position 308, alanine at position 311, tyrosine at
position 434, and valine at position 436 (all positions by EU
numbering).
71. The antigen-binding molecule of claim 62, wherein the at least
one position that is substituted includes all members of a set of
positions selected from any of sets (a)-(c): (a) positions 434,
307, and 311; (b) positions 434, 307, 309, and 311; (c) positions
434, 250, 252, and 436 (all positions by EU numbering).
72. The antigen-binding molecule of claim 62, wherein the modified
FcRn-binding domain comprises one of the following combinations of
amino acid residues at the indicated positions: (a) glutamine at
position 307, histidine at position 311, and tyrosine at position
434; (b) glutamine at position 307, glutamic acid at position 309,
alanine at position 311, and tyrosine at position 434; (c)
glutamine at position 307, glutamic acid at position 309, histidine
at position 311, and tyrosine at position 434; or (d) valine at
position 250, tyrosine at position 252, tyrosine at position 434,
and valine at position 436 (all positions by EU numbering).
73. The antigen-binding molecule of claim 62, wherein the
antigen-binding molecule comprises an antigen-binding domain whose
antigen-binding activity is lower at pH 5.8 than at pH 7.4.
74. The antigen-binding molecule of claim 62, wherein the
antigen-binding molecule comprises an antigen-binding domain whose
antigen-binding activity is lower at a first calcium concentration
that is between 0.1 .mu.M to 30 .mu.M than at a second calcium
concentration that is between 100 .mu.M to 10 mM.
75. The antigen-binding molecule of claim 62, wherein the
antigen-binding molecule has an FcRn receptor-binding affinity of
50-150 nM at pH 7.0, a melting temperature (Tm) above 63.0.degree.
C., and an Epibase score lower than 250.
76. The antigen-binding molecule of claim 62, wherein the
antigen-binding molecule has an FcRn receptor binding affinity of
15-50 nM at pH 7.0, a Tm above 60.degree. C., and an Epibase score
lower than 500.
77. The antigen-binding molecule of claim 62, wherein the
antigen-binding molecule has an FcRn receptor binding affinity
stronger than 15 nM at pH 7.0, a Tm higher than 57.5.degree. C.,
and an Epibase score lower than 500.
78. The antigen-binding molecule of claim 62, wherein the at least
one position that is substituted includes (a) one or more of
positions 238, 255, and 258; and (b) three or more additional
positions, wherein the three or more additional positions are a
combination of positions selected from any of the combinations
listed in any of Tables 4 to 7 (all positions by EU numbering).
79. The antigen-binding molecule of claim 62, wherein the amino
acid at position 257 (EU numbering) of the modified FcRn-binding
domain is not alanine, valine, isoleucine, leucine, or
threonine.
80. The antigen-binding molecule of claim 62, wherein the amino
acid at position 252 (EU numbering) of the modified FcRn-binding
domain is not tryptophan.
81. The antigen-binding molecule of claim 62, wherein a rheumatoid
factor (RF) antibody's binding affinity for the modified
FcRn-binding domain is the same or decreased compared to the
binding affinity of the RF antibody for a native human IgG
FcRn-binding domain of a control IgG antibody.
82. The antigen-binding molecule of claim 81, wherein the
difference between the sequence of the modified FcRn-binding domain
and the sequence of the native human IgG FcRn-binding domain also
includes a substitution at one or more of the following positions
(by EU numbering): 387, 422, 424, 426, 433, 436, 438 and 440.
83. The antigen-binding molecule of claim 82, wherein one or more
of the following positions (EU numbering) in the modified
FcRn-binding domain is occupied by the indicated amino acid:
arginine at position 387, any one of glutamic acid, arginine,
serine, aspartic acid, lysine, threonine and glutamine at position
422; any one of glutamic acid, arginine, lysine, and asparagine at
position 424; any one of aspartic acid, glutamine, alanine, and
tyrosine at position 426; aspartic acid at position 433; threonine
at position 436; any one of glutamic acid, arginine, serine, and
lysine at position 438; and any one of glutamic acid, aspartic acid
and glutamine at position 440.
84. The antigen-binding molecule of claim 62, wherein the modified
FcRn-binding domain differs from the native human IgG FcRn-binding
domain by substitutions comprising a combination of substitutions
selected from the combinations of three or more substitutions
listed in Tables 12 and 13.
85. The antigen-binding molecule of claim 62, wherein the modified
FcRn-binding domain differs from the native human IgG FcRn-binding
domain by substitutions comprising a combination of substitutions
selected from the combinations listed in Tables 14 and 15.
86. The antigen-binding molecule of claim 82, wherein the modified
FcRn-binding domain comprises any one of the following sets of
amino acids at the indicated positions (all positions by EU
numbering): (a) tyrosine at position 252, arginine at position 387,
tyrosine at position 434, and valine at position 436; or (b)
tyrosine at position 252, glutamic acid at position 422, tyrosine
at position 434, and valine at position 436; or (c) tyrosine at
position 252, arginine at position 422, tyrosine at position 434,
and valine at position 436; or (d) tyrosine at position 252, serine
at position 422, tyrosine at position 434, and valine at position
436; or (e) tyrosine at position 252, glutamic acid at position
424, tyrosine at position 434, and valine at position 436; or (f)
tyrosine at position 252, arginine at position 424, tyrosine at
position 434, and valine at position 436; or (g) tyrosine at
position 252, tyrosine at position 434, valine at position 436, and
glutamic acid at position 438; or (h) tyrosine at position 252,
tyrosine at position 434, valine at position 436, and arginine at
position 438; or (i) tyrosine at position 252, tyrosine at position
434, valine at position 436, and serine at position 438; or (j)
tyrosine at position 252, tyrosine at position 434, valine at
position 436, and glutamic acid at position 440.
87. The antigen-binding molecule of claim 62, wherein the
antigen-binding molecule is an antibody.
88. A composition comprising the antigen-binding molecule of claim
87, wherein at least 98% of the antigen-binding molecule in the
composition is in the form of antibody monomers containing two
heavy chains and two light chains.
89. A method for reducing plasma concentration of an antigen in a
subject, the method comprising administering to the subject the
antigen-binding molecule of claim 62, wherein the antigen-binding
molecule binds to the antigen.
Description
TECHNICAL FIELD
[0001] The present invention relates to: a modified FcRn-binding
domain having an enhanced affinity for the Fc Receptor neonatal
(FcRn) at neutral pH; an antigen-binding molecule comprising said
FcRn-binding domain, which has low immunogenicity, high stability
and form only a few aggregates; a modified antigen-binding molecule
having an increased FcRn-binding activity at neutral or acidic pH
without an increased binding activity at neutral pH for a
pre-existing anti-drug antibody; use of the antigen-binding
molecules for improving antigen-binding molecule-mediated antigen
uptake into cells; use of the antigen-binding molecules for
reducing the plasma concentration of a specific antigen; use of the
modified FcRn-binding domain for increasing the total number of
antigens to which a single antigen-binding molecule can bind before
its degradation; use of the modified FcRn-binding domain for
improving pharmacokinetics of an antigen-binding molecule; methods
for decreasing the binding activity for a pre-existing anti-drug
antibody; and methods for producing said antigen-binding
molecules.
BACKGROUND ART
[0002] Due to their high stability in plasma and few side effects,
an increasing number of antibodies are being used as
pharmaceuticals. A conventional antibody targeting a soluble
antigen binds the antigen in the plasma of the patient after
injection and then stably persists in the form of an
antibody-antigen complex until degradation. While a typical
antibody has generally a long half-life (1-3 weeks), an antigen has
a relatively short half-life of less than one day. An antigen in
complex with an antibody therefore has a significantly longer
half-life than the antigen alone. Consequently, the antigen
concentration tends to increase after the injection of a
conventional antibody. Such cases have been reported for antibodies
targeting various soluble antigens, such as IL-6 (J Immunotoxicol.
2005, 3, 131-9. (NPL 1)), beta amyloid (MAbs. 2010
September-October; 2(5):576-88 (NPL 2)), MCP-1 (ARTHRITIS &
RHEUMATISM 2006, 54,2387-92 (NPL 3)), hepcidin (AAPS J. 2010,
12(4):646-57. (NPL 4)) and sIL-6 receptor (Blood. 2008 Nov. 15;
112(10):3959-64. (NPL 5)). Reports have described an approximately
10 to 1000-fold increase (depending of the antigen) of total plasma
antigen concentration from the baseline upon antibody
administration.
[0003] As such an increase of the total plasma antigen
concentration is not desired, strategies for removing the antigen
by a therapeutic antibody have been developed. One of these
strategies is to dispose the antigen rapidly using a pH-dependent
antigen binding antibody that has increased binding affinity to the
neonatal Fc receptor for IgG (FcRn) (see e.g. PCT application no.
PCT/JP2011/001888 (PTL 1)). The FcRn is a protein found in the
membrane of many cells. An antibody with increased binding activity
to FcRn at neutral pH will bind FcRn on the cell surface, whereby
the receptor with the antibody is internalized into the cells in a
vesicle. As the pH in the interior of the vesicle is gradually
decreased, the antigen will dissociate from the pH-dependent
antigen binding antibody, owing to its low affinity in acidic pH.
The dissociated antigen is then degraded while the FcRn and bound
antibody are recycled back to the surface of the cells before
degradation. Accordingly, a pH-dependent antigen binding antibody
having increased binding activity to FcRn at neutral pH can be used
to remove an antigen from plasma and decrease its concentration in
plasma.
[0004] Previous studies have also demonstrated that Fc-engineering
to increase the binding affinity to FcRn at acidic pH can also
improve the endosomal recycling efficiency and the pharmacokinetics
of the antibody. For example, M252Y/S254T/T256E (YTE) variant (J
Biol Chem, 2006, 281:23514-23524. (NPL 6)), M428L/N434S (LS)
variant (Nat Biotechnol, 2010 28:157-159. (NPL 7)), T250Q/M428L (J
Immunol. 2006, 176(1):346-56. (NPL 8)) and N434H variant (Clinical
Pharmacology & Therapeutics (2011) 89(2):283-290. (NPL 9))
showed improvement in half-life relative to native IgG1.
[0005] However, such substitutions have also the risk of altering
properties of the antibody that are important for the development
of a therapeutic antibody such as the antibody's stability,
immunogenicity, aggregation behavior and binding affinity for
pre-existing antibodies (e.g. rheumatoid factor). It is therefore
the main objective of the present invention to provide a modified
FcRn-binding domain which not only enhances the clearance of an
antibody but also meets the criteria for developing a therapeutic
antigen-binding molecule. These developability criteria are in
particular high stability, low immunogenicity, low percentage of
aggregates, and low binding affinity for pre-existing anti-drug
antibodies (ADA).
[0006] Prior art documents related to the present invention are
shown below. All documents cited in this specification are
incorporated herein by reference.
CITATION LIST
Patent Literature
[0007] [PTL 1] PCT/JP2011/00188 (WO/2011/122011), ANTIGEN-BINDING
MOLECULES THAT PROMOTE ANTIGEN CLEARANCE
Non Patent Literature
[0007] [0008] [NPL 1] Martin P L, Cornacoff J, Prabhakar U, Lohr T,
Treacy G, Sutherland J E, Hersey S, Martin E; Reviews Preclinical
Safety and Immune-Modulating Effects of Therapeutic Monoclonal
Antibodies to Interleukin-6 and Tumor Necrosis Factor-alpha in
Cynomolgus Macaques; J Immunotoxicol. 2005, 3, 131-9 [0009] [NPL 2]
Davda J P, Hansen R J.; Properties of a general PK/PD model of
antibody-ligand interactions for therapeutic antibodies that bind
to soluble endogenous targets; MAbs. 2010 September-October;
2(5):576-88. [0010] [NPL 3] Haringman J J, Gerlag D M, Smeets T J,
Baeten D, van den Bosch F, Bresnihan B, Breedveld F C, Dinant H J,
Legay F, Gram H, Loetscher P, Schmouder R, Woodworth T, Tak P P.; A
randomized controlled trial with an anti-CCL2 (anti-monocyte
chemotactic protein 1) monoclonal antibody in patients with
rheumatoid arthritis; ARTHRITIS and RHEUMATISM 2006, 54,2387-92.
[0011] [NPL 4] Xiao J J, Krzyzanski W, Wang Y M, Li H, Rose M J, Ma
M, Wu Y, Hinkle B, Perez-Ruixo J J.; Pharmacokinetics of
anti-hepcidin monoclonal antibody Ab 12B9m and hepcidin in
cynomolgus monkeys; AAPS J. 2010, 12(4), 646-57.) [0012] [NPL 5]
Nishimoto N, Terao K, Mima T, Nakahara H, Takagi N, Kakehi T.;
Mechanisms and pathologic significances in increase in serum
interleukin-6 (IL-6) and soluble IL-6 receptor after administration
of an anti-IL-6 receptor antibody, tocilizumab, in patients with
rheumatoid arthritis and Castleman disease; Blood. 2008 Nov. 15;
112(10):3959-64. [0013] [NPL 6] J Biol Chem, 2006, 281:23514-23524
[0014] [NPL 7] Nat Biotechnol, 2010 28:157-159 [0015] [NPL 8] J
Immunol. 2006, 176(1):346-56 [0016] [NPL 9] Clinical Pharmacology
& Therapeutics (2011) 89(2):283-290
SUMMARY OF INVENTION
Technical Problem
[0017] The present invention was conceived in view of the
circumstances described above. An objective of the present
invention is to provide a modified FcRn-binding domain which has an
enhanced affinity for the FcRn at neutral pH; an antigen-binding
molecule comprising said FcRn-binding domain, wherein said
antigen-binding molecule has low immunogenicity, high stability and
forms only few aggregates; a modified antigen-binding molecule
having an increased FcRn-binding activity at neutral or acidic pH
without an increased binding activity at neutral pH for a
pre-existing anti-drug antibody; use of the antigen-binding
molecules for improving antigen-binding molecule-mediated antigen
uptake into cells; use of the antigen-binding molecule for reducing
the plasma concentration of a specific antigen; use of the modified
FcRn-binding domain for increasing the total number of antigens to
which a single antigen-binding molecule can bind before its
degradation; use of the modified FcRn-binding domain for improving
pharmacokinetics of an antigen-binding molecule; and methods for
producing said antigen-binding molecules.
Solution to Problem
[0018] The present inventors conducted dedicated studies on
modified FcRn-binding domains which have an enhanced affinity for
FcRn at neutral pH and on antigen-binding molecules comprising said
FcRn-binding domain which have low immunogenicity, high stability
and form only few aggregates. As a result, the present inventors
discovered that substitutions at specific positions of the
FcRn-binding domain increases the affinity for the FcRn at neutral
pH without substantially increasing the immunogenicity, without
substantially decreasing the stability and/or without substantially
increasing the ratio of high molecular weight species.
[0019] Furthermore, the present inventors conducted dedicated
studies on modified FcRn-binding domains with an enhanced affinity
for FcRn at neutral pH or acidic pH but without a significantly
increased binding activity for a pre-existing anti-drug antibody
and on antigen-binding molecules comprising such an FcRn-binding
domain. As a result, the present inventors discovered that
substitutions at specific positions of the FcRn-binding domain
decrease the affinity for a pre-existing anti-drug antibody at
neutral pH without substantially decreasing the FcRn-binding
activity.
[0020] Specifically, the present invention relates to:
[0021] [1] An antigen-binding molecule comprising a modified
FcRn-binding domain, wherein the modified FcRn-binding domain
comprises an amino acid substitution at one or more positions
selected from the group consisting of EU238, EU250, EU252, EU254,
EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428,
EU433, EU434, and EU436, wherein the numbers indicate the position
of the substitution according to the EU numbering.
[0022] [2] The antigen-binding molecule according to [1], wherein
the FcRn-binding domain has
[0023] a) an amino acid substitution of the amino acid at position
EU252 and EU434; and
[0024] b) an amino acid substitution at one or more positions
selected from the group consisting of EU238, EU250, EU252, EU254,
EU255, EU256, EU258, EU286, EU387, EU307, EU308, EU309, EU311,
EU315, EU428, EU433, EU434, and EU436.
[0025] [3] The antigen-binding molecule according to [1] or [2],
wherein the modified FcRn-binding domain comprises
[0026] at position EU238 an aspartic acid,
[0027] at position EU250 a valine,
[0028] at position EU252 a tyrosine,
[0029] at position EU254 a threonine,
[0030] at position EU255 a leucine,
[0031] at position EU256 a glutamic acid,
[0032] at position EU258 an aspartic acid or an isoleucine,
[0033] at position EU286 a glutamic acid,
[0034] at position EU307 a glutamine,
[0035] at position EU308 a proline,
[0036] at position EU309 a glutamic acid,
[0037] at position EU311 an alanine or a histidine,
[0038] at position EU315 an aspartic acid,
[0039] at position EU428 an isoleucine,
[0040] at position EU433 an alanine, a lysine, a proline, an
arginine, or a serine,
[0041] at position EU434 a tyrosine, or a tryptophan, and/or
[0042] at position EU436 an isoleucine, a leucine, a valine, a
threonine, or a phenylalanine.
[0043] [4] The antigen-binding molecule according to [2], wherein
the FcRn-binding domain comprises an amino acid substitution of an
amino acid at one or more position combinations selected from the
group consisting of
[0044] a) EU252, EU434, and EU436;
[0045] b) EU252, EU307, EU311 and EU434;
[0046] c) EU252, EU315, and EU434;
[0047] d) EU252, EU308, and EU434;
[0048] e) EU238, EU252, and EU434;
[0049] f) EU252, EU434, EU307, EU311, and EU436; and
[0050] g) EU252, EU387, and EU434.
[0051] [5] The antigen-binding molecule according to [4], wherein
the FcRn-binding domain comprises:
[0052] a) at position EU252 a tyrosine, at position EU315 an
aspartic acid, and at position EU434 a tyrosine; or
[0053] b) at position EU252 a tyrosine, at position EU434 a
tyrosine, and at position EU436 an isoleucine; or
[0054] c) at position EU252 a tyrosine, at position EU434 a
tyrosine, and at position EU436 a leucine; or
d) at position EU252 a tyrosine, at position EU434 a tyrosine, and
at position EU436 a valine; or e) at position EU252 a tyrosine, at
position EU254 a threonine, at position EU434 a tyrosine, and at
position EU436 an isoleucine. [6] The antigen-binding molecule
according to [2], wherein the FcRn-binding domain comprises an
amino acid substitution at three or more positions, wherein the
three or more positions are one of the combinations of the group
consisting of
[0055] a) EU252/EU434/EU307/EU311/EU286;
[0056] b) EU252/EU434/EU307/EU311/EU286/EU254;
[0057] c) EU252/EU434/EU307/EU311/EU436;
[0058] d) EU252/EU434/EU307/EU311/EU436/EU254;
[0059] e) EU252/EU434/EU307/EU311/EU436/EU250;
[0060] f) EU252/EU434/EU308/EU250;
[0061] g) EU252/EU434/EU308/EU250/EU436; and
[0062] h) EU252/EU434/EU308/EU250/EU307/EU311.
[0063] [7] The antigen-binding molecule according to [6], wherein
the FcRn-binding domain comprises:
[0064] a) at position EU252 a tyrosine, at position EU286 a
glutamic acid, at position EU307 a glutamine, at position EU311 an
alanine and at position EU434 a tyrosine; or
[0065] b) at position EU252 a tyrosine, at position EU254 a
threonine, at position EU286 a glutamic acid, at position EU307 a
glutamine, at position EU311 an alanine and at position EU434 a
tyrosine; or
[0066] c) at position EU252 a tyrosine, at position EU307 a
glutamine, at position EU311 an alanine, at position EU434 a
tyrosine and at position 436 an isoleucine; or
[0067] d) at position EU252 a tyrosine, at position EU254 a
threonine, at position EU286 a glutamic acid, at position EU307 a
glutamine, at position EU311 an alanine, at position EU434 a
tyrosine and at position EU436 an isoleucine; or
[0068] e) at position EU250 a valine, at position EU252 a tyrosine,
at position EU254 a threonine, at position EU308 a proline, at
position EU434 a tyrosine and at position EU436 a valine; or
[0069] f) at position EU250 a valine, at position EU252 a tyrosine,
at position EU307 a glutamine, at position EU311 an alanine, at
position EU434 a tyrosine and at position EU436 a valine; or
[0070] g) at position EU252 a tyrosine, at position EU307 a
glutamine, at position EU311 an alanine, at position EU434 a
tyrosine and at position EU436 a valine; or
[0071] h) at position EU250 a valine, at position EU252 a tyrosine,
at position EU308 a proline, and at position EU434 a tyrosine;
or
[0072] i) at position EU250 a valine, at position EU252 a tyrosine,
at position EU307 a glutamine, at position EU308 a proline, at
position EU311 an alanine, and at position EU434 a tyrosine.
[0073] [8] The antigen-binding molecule according to [2], wherein
the FcRn-binding domain comprises an amino acid substitution at
three or more positions wherein the three or more positions are one
of the combinations of the group consisting of
[0074] a) EU252 and EU434 and EU307 and EU311 and EU436 and
EU286;
[0075] b) EU252 and EU434 and EU307 and EU311 and EU436 and EU250
and EU308;
[0076] c) EU252 and EU434 and EU307 and EU311 and EU436 and EU250
and EU286 and EU308;
[0077] d) EU252 and EU434 and EU307 and EU311 and EU436 and EU250
and EU286 and EU308 and EU428.
[0078] [9] The antigen-binding molecule according to [8], wherein
the FcRn-binding domain comprises:
[0079] a) at position EU252 a tyrosine, at position EU286 a
glutamic acid, at position EU307 a glutamine, at position EU311 an
alanine, at position EU434 a tyrosine, and at position EU436 a
valine; or
[0080] b) at position EU250 a valine, at position EU252 a tyrosine,
at position EU307 a glutamine, at position EU308 proline, at
position EU311 an alanine, at position EU434 a tyrosine, and at
position EU436 a valine; or
[0081] c) at position EU250 a valine, at position EU252 a tyrosine,
at position EU286 a glutamic acid, at position EU307 a glutamine,
at position EU308 proline, at position EU311 an alanine, at
position EU434 a tyrosine, and at position EU436 a valine; or
[0082] d) at position EU250 a valine, at position EU252 a tyrosine,
at position EU286 a glutamic acid, at position EU307 a glutamine,
at position EU308 proline, at position EU311 an alanine, at
position EU434 a tyrosine, and at position EU436 a valine.
[0083] [10] The antigen-binding molecule according to [2], wherein
the FcRn-binding domain comprises an amino acid substitution at
three or more positions wherein the three or more positions are one
of the combinations of the group consisting of:
[0084] a) EU434 and EU307 and EU311;
[0085] b) EU434 and EU307 and EU309 and EU311; or
[0086] c) EU434 and EU250 and EU252 and EU436.
[0087] [11] The antigen-binding molecule according to [10], wherein
the FcRn-binding domain comprises:
[0088] a) at position EU307 a glutamine, at position EU311 a
histidine and at position EU434 a tyrosine; or
[0089] b) at position EU307 a glutamine, at position EU309 a
glutamic acid, at position EU311 an alanine and at position EU434 a
tyrosine; or
[0090] c) at position EU307 a glutamine, at position EU309 a
glutamic acid, at position EU311 an histidine and at position EU434
a tyrosine; or
[0091] d) at position EU250 a valine; at position EU252 a tyrosine,
at position EU434 a tyrosine and at position EU436 a valine.
[0092] [12] The antigen-binding molecule according to any one of
[1] to [11], wherein the ratio of high molecular weight species is
less than 2%.
[0093] [13] The antigen-binding molecule according to any one of
[1] to [12], wherein antigen-binding molecule comprises an
antigen-binding domain having
[0094] a) a lower binding activity for the antigen at pH 5.5-6.5
than at pH 7-8 or
[0095] b) a "calcium concentration-dependent binding" activity for
the antigen.
[0096] [14] The antigen-binding molecule according to any one of
[1] to [5], wherein the binding activity of said binding molecule
for the FcRn at pH 7 is 50-150 nM, Tm is higher than 63.0 degrees
C., and Epibase score is less than 250.
[0097] [15]. The antigen-binding molecule according to any one of
[1] to [3] and [6] to [7], and wherein the binding activity of said
binding molecule for FcRn at pH 7 is 15-50 nM, Tm is higher than 60
degrees C., and Epibase score is less than 500.
[0098] [16] The antigen-binding molecule according to any one of
[1] to [3] and [8] to [9], and wherein the binding activity of said
the binding molecule for FcRn at pH 7 is stronger than 15 nM, Tm is
higher than 57.5 degrees C., and Epibase score is less than
500.
[0099] [17] The antigen-binding molecule according to any one of
[1] to [3], wherein the FcRn-binding domain comprises an amino acid
substitution
[0100] a) at the positions EU238, EU255 and/or EU258, and
[0101] b) at three or more positions, wherein the three or more
positions are one of the combinations set forth in Tables 4 to
7.
[0102] [18] The antigen-binding molecule according to any one of
[1] to [17], wherein
[0103] a) at the position EU257 of the FcRn-binding domain the
amino acid is not an amino acid selected from the group consisting
of alanine, valine, isoleucine, leucine, and threonine, and/or
[0104] b) at the position EU252 of the FcRn-binding domain the
amino acid is not tryptophan.
[0105] [19] The antigen-binding molecule according to any one of
[1] to [18], wherein the antigen-binding molecule has a binding
activity for a pre-existing anti-drug antibody that is not
significantly increased as compared to the binding affinity of a
control antibody comprising an intact FcRn-binding domain.
[0106] [20] The antigen-binding molecule according to [19], wherein
the FcRn binding domain further comprises an amino acid
substitution at one or more positions selected from the group
consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and
EU440.
[0107] [21] The antigen-binding molecule according to [20], wherein
the FcRn binding domain comprises one or more amino acid
substitutions selected from the group consisting of
[0108] at position EU387 an arginine,
[0109] at position EU422 a glutamic acid, an arginine, or a serine,
an aspartic acid, a lysine, a threonine or a glutamine;
[0110] at position EU424 a glutamic acid or an arginine, a lysine,
or an asparagine;
[0111] at position EU426 an aspartic acid, a glutamine, an alanine,
or a tyrosine;
[0112] at position EU433 an aspartic acid;
[0113] at position EU436 a threonine;
[0114] at position EU438 a glutamic acid, an arginine, a serine, or
a lysine; and
[0115] at position EU440 a glutamic acid, aspartic acid or a
glutamine.
[0116] [22] The antigen-binding molecule according to any one of
[1] to [21], wherein the modified FcRn binding domain comprises
three or more substitutions, wherein the three or more
substitutions are one of the combinations set forth in Tables 12 to
13.
[0117] [23] The antigen-binding molecule according to any one of
[1] to [22], wherein the modified FcRn-binding domain comprises
three or more substitutions, wherein the three or more
substitutions are one of the combinations set forth in Tables 14 to
15.
[0118] [24] The antigen-binding molecule according to any one of
[20] to [23], wherein the FcRn-binding domain comprises:
[0119] a) at position EU252 a tyrosine, at position EU387 an
arginine, at position EU434 a tyrosine, and at position EU436 a
valine; or
[0120] b) at position EU252 a tyrosine, at position EU422 a
glutamic acid, at position EU434 a tyrosine, and at position EU436
a valine; or
[0121] c) at position EU252 a tyrosine, at position EU422 an
arginine, at position EU434 a tyrosine, and at position EU436 a
valine; or
[0122] d) at position EU252 a tyrosine, at position EU422 a serine,
at position EU434 a tyrosine, and at position EU436 a valine;
or
[0123] e) at position EU252 a tyrosine, at position EU424 a
glutamic acid, at position EU434 a tyrosine, and at position EU436
a valine; or
[0124] f) at position EU252 a tyrosine, at position EU424 an
arginine, at position EU434 a tyrosine, and at position EU436 a
valine; or
[0125] g) at position EU252 a tyrosine, at position EU434 a
tyrosine, at position EU436 a valine, and at position EU438 a
glutamic acid; or
[0126] h) at position EU252 a tyrosine, at position EU434 a
tyrosine, at position EU436 a valine, and at position EU438 an
arginine; or
[0127] i) at position EU252 a tyrosine, at position EU434 a
tyrosine, at position EU436 a valine, and at position EU438 a
serine; or
[0128] j) at position EU252 a tyrosine, at position EU434 a
tyrosine, at position EU436 a valine, and at position EU440 a
glutamic acid.
[0129] [25] The antigen-binding molecule according to any one of
[1] to [24], wherein the antigen-binding molecule is an
antibody.
[0130] [26] Use of the antigen-binding molecule according to any
one of [1] to [25] for improving antigen-binding molecule-mediated
antigen uptake into cells.
[0131] [27] Use of the antigen-binding molecule according to any
one of [1] to [25] for reducing the plasma concentration of a
specific antigen, wherein the antigen-binding molecule comprises an
antigen-binding domain which can bind said antigen.
[0132] [28] A method for improving the pharmacokinetics of an
antigen-binding molecule, comprising the step of introducing an
amino acid substitution into an FcRn-binding domain of said
antigen-binding molecules at one or more of the positions selected
from the group consisting of EU238, EU250, EU252, EU254, EU255,
EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433,
EU434, and EU436.
[0133] [29] A method for delaying the elimination of an
antigen-binding molecule in a subject, comprising the step of
introducing an amino acid substitution into an FcRn-binding domain
of said antigen-binding molecule at one or more of the positions
selected from the group consisting of EU238, EU250, EU252, EU254,
EU255, EU258 EU286, EU307, EU308, EU309, EU311, EU315, EU428,
EU433, EU434, and EU436.
[0134] [30] A method of prolonging the plasma retention time of an
antigen-binding molecule, comprising the step of introducing an
amino acid substitution into an FcRn-binding domain of said
antigen-binding molecule at one or more of the positions selected
from the group consisting of EU238, EU250, EU252, EU254, EU255,
EU258 EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433,
EU434, and EU436.
[0135] [31] A method for increasing the plasma antigen-elimination
rate of an antigen-binding molecule, comprising the step of
introducing an amino acid substitution into an FcRn-binding domain
of said antigen-binding molecule at one or more of the positions
selected from the group consisting of EU238, EU250, EU252, EU254,
EU255, EU258 EU286, EU307, EU308, EU309, EU311, EU315, EU428,
EU433, EU434, and EU436.
[0136] [32] A method for increasing the ability of an
antigen-binding molecule to eliminate plasma antigen, comprising
the step of introducing an amino acid substitution into an
FcRn-binding domain of said antigen-binding molecule at one or more
of the positions selected from the group consisting of EU238,
EU250, EU252, EU254, EU255, EU258 EU286, EU307, EU308, EU309,
EU311, EU315, EU428, EU433, EU434, and EU436.
[0137] [33] The method according to any one of [28] to [32],
wherein further an amino acid substitution at position EU256 into
the FcRn binding domain is introduced.
[0138] [34] The method according to any one of [28] to [33],
wherein the method further comprises a step of introducing into the
FcRn-binding domain an amino acid substitution at one or more
positions selected from the group consisting of EU387, EU422,
EU424, EU426, EU433, EU436, EU438 and EU440.
[0139] [35] A method for producing antigen-binding molecules
according to any one of [1] to [25], which comprises the steps
of
[0140] (a) selecting a parent FcRn-binding domain and altering the
parent FcRn by introducing an amino acid substitution at one or
more positions selected from the group consisting of EU238, EU250,
EU252, EU254, EU255, EU258 EU286, EU307, EU308, EU309, EU311,
EU315, EU428, EU433, EU434, and EU436;
[0141] (b) selecting an antigen-binding domain of an
antigen-binding molecule and altering at least one amino acid in
the antigen-binding domain in order to get a pH-dependent
antigen-binding domain or a calcium-ion dependent antigen-binding
domain;
[0142] (c) obtaining a gene encoding an antigen-binding molecule in
which the human FcRn-binding domain and the antigen-binding domain
prepared in (a) and (b) are linked and
[0143] (d) producing an antigen-binding molecule using the gene
prepared in (c).
[0144] [36] The method according to [35], wherein in step a)
further an amino acid substitution at position EU256 into the FcRn
binding domain is introduced.
[0145] [37] The method according to any one of [35] to [36],
wherein the method further comprises a step of introducing into the
FcRn-binding domain an amino acid substitution at one or more
positions selected from the group consisting of EU387, EU422,
EU424, EU426, EU433, EU436, EU438 and EU440.
[0146] [38] An antigen-binding molecule comprising a modified FcRn
binding domain, wherein the modified FcRn binding domain comprises
an amino acid substitution at one or more of the positions selected
from the group consisting of EU387, EU422, EU424, EU426, EU433,
EU436, EU438 and EU440, wherein the binding affinity of said
antigen-binding molecule for a pre-existing anti-drug antibody
(ADA) at a neutral pH is not significantly increased as compared to
the binding affinity of antigen-binding molecule comprising an
intact FcRn binding domain.
[0147] [39]. The antigen-binding molecule according to [38] wherein
the antigen-binding molecule further has an increased binding
affinity for an FcRn in the neutral or acidic pH ranges.
[0148] [40] The antigen-binding molecule according to [38] or [39],
wherein the amino acid substituting one or more of the positions
selected from the group consisting of EU387, EU422, EU424, EU426,
EU433, EU436, EU438 and EU440 is selected from the group consisting
of
[0149] a) at position EU387 an arginine;
[0150] b) at position EU422 a glutamic acid, an arginine, a serine,
aspartic acid, lysine, threonine, or glutamine;
[0151] c) at position EU424 a glutamic acid, an arginine, a lysine,
or asparagines;
[0152] d) at position EU426 an aspartic acid, a glutamine, an
alanine, or a tyrosine;
[0153] e) at position EU433 an aspartic acid
[0154] f) at position EU436 a threonine
[0155] g) at position EU438 a glutamic acid, an arginine, a serine,
or a lysine; and
[0156] h) at position EU440 a glutamic acid, an aspartic acid, or a
glutamine.
[0157] [41] The antigen-binding molecule according to any one of
[38] to [40], wherein the modified FcRn binding domain comprises an
amino acid substitution at one or more positions or one of the
combinations set forth in Table 10.
[0158] [42] The antigen-binding molecule according to any one of
[38] to [40], wherein the modified FcRn binding domain comprises
any one of the amino acid substitution or substitution combinations
set forth in Table 11.
[0159] [43] The antigen-binding molecule according to any one of
[39] to [42], wherein the modified FcRn binding domain further
comprises an amino acid substitution at one or more positions of
the FcRn binding domain selected from the group consisting of
EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307,
EU308, EU309, EU311, EU315, EU428, EU434, and EU436, wherein said
substitutions confer an increase in FcRn binding activity in the
neutral pH or acidic pH range.
[0160] [44] The antigen-binding molecule according to any one of
[39] to [43], wherein the modified FcRn binding domain comprises
amino acid substitutions at the FcRn binding domain positions
[0161] i) a) EU438/EU440 or b) EU424; and
[0162] ii) a) EU434, b) EU252/EU254/EU256; c) EU428/EU434; or d)
EU250/EU428.
[0163] [45] The antigen-binding molecule according to [44], wherein
the modified FcRn binding domain comprises amino acid
substitutions
[0164] i) a) EU438R/EU440E or b) EU424N; and
[0165] ii) a) M434H; b) M252Y/S254T/T256E; c) M428L/N434S; or d)
T250Q and M428L (EU numbering).
[0166] [46] The antigen-binding molecule according to [45], wherein
the modified FcRn binding domain comprises three or more amino acid
substitutions wherein the three or more substitutions are one of
the combinations set forth in Tables 13 and 15.
[0167] [47] The antigen-binding molecule according to any one of
[39] to [42] wherein the modified FcRn binding domain comprises
substitutions
[0168] a) at one or more of the positions selected from the group
consisting of EU387, EU422, EU424, EU438, EU440, EU433, or at two
or more positions wherein the two positions are one of the
combinations of the group consisting of EU422/EU424, and
EU438/EU440; and
[0169] b) two or more positions wherein the two positions are one
of the combinations set forth in Table 9.
[0170] [48] The antigen-binding molecule according to [47], wherein
the modified FcRn binding domain comprises three or more the amino
acid substitutions wherein the three or more the amino acid
substitutions are one of the combinations set forth in Tables 12 or
14.
[0171] [49] The antigen-binding molecule of any one of [39] to [48]
wherein said antigen-binding molecule comprises a pH-dependent
antigen-binding domain or a calcium ion-dependent antigen-binding
domain.
[0172] [50] A method for decreasing the binding activity for a
pre-existing ADA of an antigen-binding molecule comprising an FcRn
binding domain having an increased binding activity for an FcRn at
neutral or acidic pH and an increased binding activity for a
pre-existing ADA at a neutral pH, said method comprising the steps
of
[0173] a) providing an antigen-binding molecule with an FcRn
binding domain having an increased binding activity for FcRn at
neutral or acidic pH and an increased binding activity for a
pre-existing ADA at a neutral pH; and
[0174] b) substituting an amino acid in the FcRn binding domain at
one or more of the positions selected from the group consisting of
EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440 to yield
an antigen-binding molecule with a modified FcRn binding
domain.
[0175] [51] The method according to [50], wherein step b) comprises
substituting an amino acid at three or more positions wherein the
three or more positions are one of the combinations set forth in
Table 10.
[0176] [52] The method according to [50], wherein step b) comprises
introducing three or more the amino acid substitutions into the
FcRn-binding domain wherein the three or more the amino acid
substitutions are one of the combinations set forth in Table
11.
[0177] [53] A method for increasing the total number of antigens to
which a single antigen-binding molecule can bind without
significantly increasing the binding activity for a pre-existing
ADA at neutral pH as compared to a parent antibody, said method
comprising the steps of
[0178] a) providing an antigen-binding molecule comprising a parent
FcRn binding domain,
[0179] b) altering the parent FcRn binding domain of step a) by
substituting an amino acid in the amino acid sequence of the parent
FcRn binding domain at one or more of the positions selected from
the group consisting of EU238, EU250, EU252, EU254, EU255, EU256,
EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433,
EU434, and EU436; and
[0180] c) altering the modified FcRn-binding domain of step b) by
substituting an amino acid in the amino acid sequence of the parent
FcRn-binding domain at one or more positions selected from the
group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438
and EU440.
[0181] [54] A method for facilitating the extracellular release of
an antigen-free antigen-binding molecule taken up into cells in an
antigen-bound form without significantly increasing the binding
activity of said antigen-binding molecule for a pre-existing ADA at
neutral pH as compared to a parent antibody, comprising the steps
of
[0182] a) providing an antigen-binding molecule comprising a parent
FcRn-binding domain,
[0183] b) altering the parent FcRn binding domain by substituting
an amino acid in the amino acid sequence of the parent FcRn-binding
domain at one or more positions selected from the group consisting
of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307,
EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436, and
EU428; and
[0184] c) altering the modified FcRn-binding domain of step b) by
substituting an amino acid in the amino acid sequence of the parent
FcRn-binding domain at one or more positions selected from the
group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438
and EU440.
[0185] [55] A method for increasing the ability of an
antigen-binding molecule to eliminate plasma antigen without
significantly increasing the binding activity for pre-existing ADA
at neutral pH compared to parent antibody, said method comprising
the steps of
[0186] a) providing an antigen-binding molecule comprising a parent
FcRn-binding domain,
[0187] b) altering the parent FcRn binding domain by substituting
an amino acid in the amino acid sequence of the parent FcRn-binding
domain at one or more positions selected from the group consisting
of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307,
EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436, and
EU428; and
[0188] c) altering the modified FcRn-binding domain of step b) by
substituting an amino acid in the amino acid sequence of the parent
FcRn-binding domain at one or more positions selected from the
group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438
and EU440.
[0189] [56] A method for improving the pharmacokinetics of an
antigen-binding molecule without significantly increasing the
binding activity for a pre-existing ADA at neutral pH as compared
to a parent antibody, said method comprising the steps of
[0190] a) providing an antigen-binding molecule comprising a parent
FcRn-binding domain,
[0191] b) altering the parent FcRn-binding domain by substituting
an amino acid in the amino acid sequence of the parent FcRn-binding
domain at one or more positions selected from the group consisting
of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307,
EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436; and
[0192] c) altering the modified FcRn-binding domain of step b) by
substituting an amino acid in the amino acid sequence of the parent
FcRn-binding domain at one or more positions selected from the
group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438
and EU440.
[0193] [57] A method for reducing total or free antigen plasma
concentration without significantly increasing the binding activity
for a pre-existing ADA at neutral pH as compared to a parent
antibody, said method comprising the steps of
[0194] a) providing an antigen-binding molecule comprising a parent
FcRn-binding domain, wherein the antigen-binding molecule comprises
an antigen-binding domain which can bind said antigen,
[0195] b) altering the parent FcRn-binding domain by substituting
an amino acid in the amino acid sequence of the parent FcRn-binding
domain at one or more positions selected from the group consisting
of EU238, EU250, EU252, EU254, EU255, EU256, EU258 EU286, EU307,
EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436; and
[0196] c) altering the modified FcRn-binding domain of step b) by
substituting an amino acid in the amino acid sequence of the parent
FcRn-binding domain at one or more positions selected from the
group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438
and EU440.
[0197] [58] A method for producing an antigen-binding molecule
comprising an FcRn binding domain having an increased binding
activity for an FcRn at neutral or acidic pH and a decreased
binding activity for an pre-existing ADA at neutral pH, said method
comprising the steps of
[0198] (a) providing an FcRn binding domain having an increased
binding activity for an FcRn at neutral or acidic pH ranges and
pre-existing ADA at neutral pH ranges,
[0199] (b) substituting an amino acid at one or more of the
positions selected from the group consisting of EU387, EU422,
EU424, EU426, EU433, EU436, EU438 and EU440,
[0200] (c) selecting an antigen-binding domain of an
antigen-binding molecule and altering at least one amino acid in
the antigen-binding domain in order to get a pH-dependent
antigen-binding domain, or selecting an calcium-ion dependent
antigen-binding domain;
[0201] (d) obtaining a gene encoding an antigen-binding molecule in
which the human FcRn-binding domain and the antigen-binding domain
prepared in (a) and (b) are linked and
[0202] (e) producing an antigen-binding molecule using the gene
prepared in (c), wherein said antigen-binding molecule produced has
an increased binding activity for an FcRn at neutral or acidic pH
and a decreased binding activity for an endogenous ADA at neutral
pH as compared to a parent antigen-binding domain having an intact
FcRn binding domain.
[0203] [59] The method according to [58], wherein the FcRn binding
domain having an increased binding activity for FcRn and
pre-existing ADA at neutral or acidic pH ranges and for
pre-existing ADA in the neutral pH ranges comprises an amino acid
substitution at one or more positions selected from the group
consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258,
EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and
EU436.
[0204] [60] The method according to any one of [53] to [57],
wherein the amino acid substitution introduced in step a) are at
three or more positions wherein said three or more positions are
one of the combinations set forth in Tables 4 to 7.
[0205] [61] The method according to any one of [53] to [60],
wherein the amino acid substitutions introduced in step b) are at
three or more positions wherein said three or more positions are
one of the combinations set forth in Table 10.
BRIEF DESCRIPTION OF DRAWINGS
[0206] FIG. 1A shows a schematic depiction of antigen elimination
from plasma of an antibody of the prior art ("conventional
antibody") compared with pH-dependent antigen binding antibody with
enhanced FcRn, both binding a soluble antigen at neutral pH. The
conventional antibody binds to the antigen in the plasma and is
non-specifically taken up by cells into acidic endosomes. Under
acidic conditions of the endosomes, the conventional antibody binds
the FcRn inside a vesicle and is transported back to the surface of
the cell where it is again released. The antigen is bound to the
antigen-binding domain during the whole internalization and
recycling process. A pH-dependent antigen binding antibody with
enhanced FcRn binding at neutral pH binds to the FcRn on the
surface of the cell and is internalized rapidly into the cell and
therefore in a higher frequency than a conventional antibody. Under
the acidic condition in the endosomes, the antigen dissociates from
the modified antibody and is transferred to the lysosome where it
is proteolytically degraded. The antibody, still bound to the FcRn,
is recycled back to cell surface. There, the recycled free antibody
can bind to another antigen once again. By repeating this cycle of
FcRn-mediated uptake, antigen dissociation and degradation, and
antibody recycling, such pH-dependent antigen-binding antibody with
improved binding affinity to FcRn at neutral pH can deliver
significantly higher amount of antigen to the lysosome than a
conventional antibody and therefore can reduce the total antigen
concentration in plasma significantly more than a conventional
antibody.
[0207] FIG. 1B shows a schematic representation of the dissociation
of a soluble antigen from an IgG antibody with a pH-dependent
antigen-binding domain in the endosome. This results in increased
antigen elimination, and allows the antibody to bind to another
antigen in the plasma.
[0208] FIG. 2 shows the plot of hFcRn binding affinity (x axis) and
Tm of antibodies comprising Fc variants on they axis (Fc variants
F1-F599: open square; Fc variants F600-F1052: closed square).
[0209] FIG. 3 shows the plot of hFcRn binding affinity (x-axis) and
High Molecular Weight (HMW) portion (in %) (y axis) of antibodies
comprising Fc variants (Fc variants F1-F599: open square, Fc
variants F600-F1050: closed square)
[0210] FIG. 4 shows the plot of hFcRn binding affinity (x-axis) and
immunogenicity score (Epibase score) of antibodies comprising Fc
variants (Fc variants F1-F599: open square, Fc variants F600-F1052:
closed square).
[0211] FIG. 5 shows the plot of hFcRn binding affinity (x-axis) and
melting Temperature Tm (y axis) of antibodies comprising Fc
variants whose hFcRn binding affinity is stronger than 15 nM (Fc
variants of F1-F599 with Kd less than or equal to 15 nM: open
square, Fc variants of F600-F1052 with Kd less than or equal to 15
nM (Group 1): closed square).
[0212] FIG. 6 shows the plot of hFcRn binding affinity (x axis) and
HMW (in %) (y-axis) of antibodies comprising Fc variants whose
hFcRn binding affinity is stronger than 15 nM (Fc variants of
F1-F599 with Kd less than or equal to 15 nM: open square; Fc
variants of F600-F1052 with Kd less than or equal to 15 nM (Group
1): closed square).
[0213] FIG. 7 shows the plot of hFcRn binding affinity and
immunogenicity score of antibodies comprising Fc variants whose
hFcRn binding affinity is stronger than 15 nM (Fc variants of
F1-F599 with Kd less than or equal to 15 nM: open square; Fc
variants of F600-F1052 with Kd less than or equal to 15 nM (Group
1): closed square).
[0214] FIG. 8 shows the plot of hFcRn binding affinity and Tm of
antibodies comprising Fc variants whose hFcRn binding affinity is
between 15 nM and 50 nM (Fc variants of F1-F599 with Kd=15-50 nM,
open square; Fc variants of F600-F1052 with Kd=15-50 nM (Group 2):
closed square)
[0215] FIG. 9 shows the plot of hFcRn binding affinity and HMW (%)
of antibodies comprising Fc variants whose hFcRn binding affinity
is between 15 nM and 50 nM (Fc variants of F1-F599 with Kd=15-50
nM, open square; Fc variants of F600-F1052 with Kd=15-50 nM (Group
2): closed square).
[0216] FIG. 10 shows the plot of hFcRn binding affinity and
immunogenicity score of antibodies comprising Fc variants whose
hFcRn binding affinity is between 15 nM and 50 nM (Fc variants of
F1-F599 with Kd=15-50 nM, open square; Fc variants of F600-F1052
with Kd=15-50 nM (Group 2): closed square)
[0217] FIG. 11 shows the plot of hFcRn binding affinity and Tm of
antibodies comprising Fc variants whose hFcRn binding affinity is
between 50 nM and 150 nM (Fc variants of F1-F599 with Kd=50-150 nM,
open square; Fc variants of F600-F1052 with Kd=50-150 nM (Group 3):
closed square).
[0218] FIG. 12 shows the plot of hFcRn binding affinity and HMW (%)
of antibodies comprising Fc variants whose hFcRn binding affinity
is between 50 nM and 150 nM (Fc variants of F1-F599 with Kd=50-150
nM, open square; Fc variants of F600-F1052 with Kd=50-150 nM (Group
3): closed square).
[0219] FIG. 13 shows a plot of hFcRn binding affinity and
immunogenicity score of antibodies comprising Fc variants whose
hFcRn binding affinity is between 50 nM and 150 nM (Fc variants of
F1-F599 with Kd=50-150 nM: open square; Fc variants of F600-F1052
with Kd=50-150 nM (Group 3): closed square.
[0220] FIG. 14 shows the plot of hFcRn binding affinity and Tm of
antibodies comprising Fc variants whose hFcRn binding affinity is
between 150 nM and 700 nM (Fc variants of F1-F599 with Kd=150-700
nM, open square; Fc variants F600-F1052 with Kd=150-700 nM (Group
4): closed square).
[0221] FIG. 15 shows the plot of hFcRn binding affinity and HMW (%)
of antibodies comprising Fc variants whose hFcRn binding affinity
is between 150 nM and 700 nM (Fc variants of F1-F599 with
Kd=150-700 nM: open square; Fc variants of F600-F1052 with
Kd=150-700 nM (Group 4): closed square).
[0222] FIG. 16 shows the plot of hFcRn binding affinity and
immunogenicity score of antibodies comprising Fc variants whose
hFcRn binding affinity is between 150 nM and 700 nM (Fc variants of
F1-F599 with Kd=150-700 nM: open square; Fc variants of F600-F1052
with Kd=150-700 nM (Group 4): closed square).
[0223] FIG. 17 shows a graphical depiction of the plasma antigen
(hsIL-6R) concentration over time in a human FcRn transgenic mouse
after injection of Fv4-IgG1, Fv4-F652, Fv4-F890 and Fv4-F946 and in
a control mouse (no antibody injection).
[0224] FIG. 18 shows a graphical depiction of the plasma antibody
concentration over time in human FcRn transgenic mouse after
injection of Fv4-IgG1, Fv4-F652, Fv4-F890 and Fv4-F946.
[0225] FIG. 19 shows a graphical depiction of the plasma antigen
(hsIL-6R) concentration over time in human FcRn transgenic mouse of
control (no antibody injection) and after injection of Fv4-IgG1,
Fv4-F11 and Fv4-F652.
[0226] FIG. 20 shows a graphical depiction of the plasma antibody
concentration over time in human FcRn transgenic mouse after
injection of Fv4-IgG1, Fv4-F11 and Fv4-F652.
[0227] FIG. 21 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the humanized anti-IL-6 receptor
antibody Fv4-IgG1 (FIG. 21-1), an YTE variant (FIG. 21-2) and a LS
variant (FIG. 21-3) thereof.
[0228] FIG. 22 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 15
individual RA patients against the humanized anti-IL-6 receptor
antibody Fv4-IgG1 (FIG. 22-1), a Fv4-N434H (FIG. 22-2), Fv4-F11
(FIG. 22-3), Fv4-F68 (FIG. 22-4), Fv4-890 (FIG. 22-5) and Fv4-F947
(FIG. 22-6).
[0229] FIG. 23 shows the mean (FIG. 23-1), geomean (FIG. 23-2) and
median (FIG. 23-3) of the ECL response of the serum from fifteen
individual RA patients against Fv4-IgG1, Fv4-F11, Fv4-F68, Fv4-F890
and Fv4-F947 shown in FIG. 22.
[0230] FIG. 24 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 15
individual RA patients against the humanized anti-IL-6 receptor
antibody Fv4-IgG1 (FIG. 24-1) and of the variants Fv4-F890,
Fv4-F1058, Fv4-F1059, Fv4-F1060, Fv4-F1061, Fv4-F1062, Fv4-F1063,
Fv4-F1064, Fv4-F1065, Fv4-F1066, Fv4-F1067, Fv4-F1068, Fv4-F1069,
Fv4-F1070, Fv4-F1071, Fv4-F1072, and Fv4-F1073 (FIG. 24-2 to FIG.
24-18).
[0231] FIG. 25 shows the mean (FIG. 25-1), geomean (FIG. 25-2) and
median (FIG. 25-3) of the ECL response of the serum from fifteen
individual RA patients against Fv4-IgG1, variants Fv4-F890,
Fv4-F1058, Fv4-F1059, Fv4-F1060, Fv4-F1061, Fv4-F1062, Fv4-F1063,
Fv4-F1064, Fv4-F1065, Fv4-F1066, Fv4-F1067, Fv4-F1068, Fv4-F1069,
Fv4-F1070, Fv4-F1071, Fv4-F1072, and Fv4-F1073 shown in FIG.
24.
[0232] FIG. 26 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 15
individual RA patients against the variants Fv4-F1104 (FIG. 26-1),
Fv4-F1105 (FIG. 26-2), and Fv4-F1106 (FIG. 26-3).
[0233] FIG. 27 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 15
individual RA patients against the variants Fv4-F1107, Fv4-F1108,
Fv4-F1109, Fv4-F1110, Fv4-F1111, Fv4-F1112, Fv4-F1113, and
Fv4-F1114 (FIG. 27-1 to FIG. 27-8)
[0234] FIG. 28 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 15
individual RA patients against the variants Fv4-F1230 (FIG. 28-1),
Fv4-F1231 (FIG. 28-2), Fv4-F1232 (FIG. 28-3).
[0235] FIG. 29 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 15
individual RA patients against the variants Fv4-F947 (FIG. 29-1),
Fv4-F1119 (FIG. 29-2), Fv4-F1120 (FIG. 29-3), Fv4-F1121 (FIG.
29-4), Fv4-F1122 (FIG. 29-5), Fv4-F1123 (FIG. 29-6), and Fv4-F1124
(FIG. 29-7).
[0236] FIG. 30-1 to FIG. 30-4 show the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 15
individual RA patients against the variants Fv4-F939, Fv4-F1291,
Fv4-F1268, and Fv4-F1269. FIG. 30-5 to FIG. 30-9 show the graphical
depiction of the electrochemiluminescence (ECL) response of the
serum from 30 individual RA patients against the variants
Fv4-F1243, Fv4-F1245, Fv4-F1321, Fv4-F1340, and Fv4-F1323.
[0237] FIG. 31 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 15
individual RA patients against the variants Fv4-F890 (FIG. 31-1)
and Fv4-F1115 (=F890+S424N, FIG. 31-2).
[0238] FIG. 32 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 15 or 30
individual RA patients against the variants Fv4-YTE (FIG. 32-1),
Fv4-F1166 (=YTE+Q438R/S440E, FIG. 32-2), Fv4-F1167 (=YTE+S424N,
FIG. 32-3), Fv4-LS (FIG. 32-4), Fv4-F1170 (=LS+Q438R/S440E, FIG.
32-5), Fv4-F1171 (LS+S424N, FIG. 32-6), Fv4-N434H (FIG. 32-7),
Fv4-F1172 (=N434H+Q438R/S440E, FIG. 32-8), Fv4-F1173 (=N434H+S424N,
FIG. 32-9))
[0239] FIG. 33 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variants Fv4-LS (FIG. 33-1),
Fv4-F1380 (FIG. 33-2), Fv4-F1384 (FIG. 33-3), Fv4-F1385 (FIG.
33-4), Fv4-F1386 (LS+S426Y, FIG. 33-5), Fv4-F1388 (FIG. 33-6), and
Fv4-F1389 (LS+Y436T, FIG. 33-7).
[0240] FIG. 34 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F939.
[0241] FIG. 35 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1378
[0242] FIG. 36 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1379
[0243] FIG. 37 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1262
[0244] FIG. 38 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1138
[0245] FIG. 39 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1344
[0246] FIG. 40 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1349
[0247] FIG. 41 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1350
[0248] FIG. 42 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1351
[0249] FIG. 43 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1261
[0250] FIG. 44 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1263
[0251] FIG. 45 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1305
[0252] FIG. 46 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1306
[0253] FIG. 47 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1268
[0254] FIG. 48 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1269
[0255] FIG. 49 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1413
[0256] FIG. 50 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1416
[0257] FIG. 51 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1419
[0258] FIG. 52 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1420
[0259] FIG. 53 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1370
[0260] FIG. 54 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1371
[0261] FIG. 55 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1599
[0262] FIG. 56 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1600
[0263] FIG. 57 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1566
[0264] FIG. 58 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1448
[0265] FIG. 59 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1601
[0266] FIG. 60 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1602
[0267] FIG. 61 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1603
[0268] FIG. 62 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1531
[0269] FIG. 63 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1604
[0270] FIG. 64 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1605
[0271] FIG. 65 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1586
[0272] FIG. 66 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1592
[0273] FIG. 67 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1610
[0274] FIG. 68 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1611
[0275] FIG. 69 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1612
[0276] FIG. 70 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1613
[0277] FIG. 71 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1614
[0278] FIG. 72 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1615
[0279] FIG. 73 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1567
[0280] FIG. 74 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1572
[0281] FIG. 75 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1576
[0282] FIG. 76 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1578
[0283] FIG. 77 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1579
[0284] FIG. 78 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1641
[0285] FIG. 79 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1642
[0286] FIG. 80 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1643
[0287] FIG. 81 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1644
[0288] FIG. 82 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1645
[0289] FIG. 83 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1646
[0290] FIG. 84 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1647
[0291] FIG. 85 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1648
[0292] FIG. 86 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1649
[0293] FIG. 87 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1650
[0294] FIG. 88 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1651
[0295] FIG. 89 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1652
[0296] FIG. 90 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1653
[0297] FIG. 91 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1654
[0298] FIG. 92 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1655
[0299] FIG. 93 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1329
[0300] FIG. 94 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1331
[0301] FIG. 95 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1718
[0302] FIG. 96 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1719
[0303] FIG. 97 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1720
[0304] FIG. 98 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1721
[0305] FIG. 99 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1671
[0306] FIG. 100 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1670
[0307] FIG. 101 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1711
[0308] FIG. 102 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1712
[0309] FIG. 103 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1713
[0310] FIG. 104 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1722
[0311] FIG. 105 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1723
[0312] FIG. 106 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1724
[0313] FIG. 107 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1725
[0314] FIG. 108 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1675
[0315] FIG. 109 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1714
[0316] FIG. 110 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1715
[0317] FIG. 111 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1716
[0318] FIG. 112 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1717
[0319] FIG. 113 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1683
[0320] FIG. 114 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1756
[0321] FIG. 115 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1757
[0322] FIG. 116 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1758
[0323] FIG. 117 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1759
[0324] FIG. 118 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1681
[0325] FIG. 119 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1749
[0326] FIG. 120 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1750
[0327] FIG. 121 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1751
[0328] FIG. 122 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1760
[0329] FIG. 123 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1761
[0330] FIG. 124 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1762
[0331] FIG. 125 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1763
[0332] FIG. 126 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1752
[0333] FIG. 127 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1753
[0334] FIG. 128 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1754
[0335] FIG. 129 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1755
[0336] FIG. 130 shows the graphical depiction of the
electrochemiluminescence (ECL) response of the serum from 30
individual RA patients against the variant F1685
[0337] FIG. 131 shows a graphical depiction of the plasma antibody
concentration over time in human FcRn transgenic mouse after
injection of Fv4-IgG1, Fv4-F1243, and Fv4-F1245.
[0338] FIG. 132 shows a graphical depiction of the plasma antigen
(hsIL-6R) concentration over time in a human FcRn transgenic mouse
after injection of Fv4-IgG1, Fv4-F1243, and Fv4-F1245 and in a
control mouse (no antibody injection).
[0339] FIG. 133 shows a graphical depiction of the plasma antibody
concentration over time in human FcRn transgenic mouse after
injection of Fv4-IgG1, Fv4-F1389.
[0340] FIG. 134 shows a sensorgram of SPR analysis. hIgA binding of
anti-hIgA antibody was analyzed under different condition (pH,
Ca-concentration).
[0341] FIG. 135 shows a graphical depiction of the plasma antibody
concentration over time in human FcRn transgenic mouse after
injection of GA2-F760 and GA2-F1331.
[0342] FIG. 136 shows a graphical depiction of the plasma hIgA
concentration over time in human FcRn transgenic mouse after
injection of GA2-F760 and GA2-F1331.
[0343] FIG. 137. shows a graphical depiction of the plasma antibody
concentration over time in human FcRn transgenic mouse after
injection of 278-F760 and 278-F1331.
[0344] FIG. 138 shows a graphical depiction of the plasma hIgE
(Asp6) concentration over time in human FcRn transgenic mouse after
injection of 278-F760 and 278-F1331.
DESCRIPTION OF EMBODIMENTS
Detailed Description of the Invention
[0345] Before the present materials and methods are described, it
should be understood that these descriptions are merely
illustrative only and not intended to be limiting. It should also
be understood that the present invention is not limited to the
particular sizes, shapes, dimensions, materials, methodologies,
protocols, etc. described herein, as these may vary in accordance
with routine experimentation and/or optimization. The terminology
used in the description is for the purpose of describing the
particular versions or embodiments only, and is not intended to
limit the scope of the present invention that will be limited only
by the appended claims. Unless otherwise defined, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which the present
invention belongs. In case of conflict, the present specification,
including definitions, will control.
[0346] The disclosure of each publication, patent or patent
application mentioned in this specification is specifically
incorporated by reference herein in its entirety. However, nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue or prior
invention.
[0347] The words "a", "an", and "the" as used herein mean "at least
one" unless otherwise specifically indicated.
[0348] Studies described in the WO/2011/122011 have demonstrated
that antigen-binding molecules (e.g. anti-IL6 receptor antibody)
with increased binding to FcRn at pH7.4 are capable of eliminating
antigen from plasma and decreasing the total antigen concentration
in plasma, and that therefore, the efficiency of antigen
elimination can be improved by pH-dependent antigen binding (bind
to antigen within plasma at pH7.4 and dissociate the antigen within
acidic endosome at pH6.0) or by ionized calcium
concentration-dependent antigen binding (bind to antigen within
plasma at high ionized calcium concentration and dissociate the
antigen within the endosome at low ionized calcium concentration)
(see FIG. 1B). The mechanism of antigen elimination from plasma by
pH-dependent antigen binding antibody with improved binding
affinity to FcRn at neutral pH in comparison to the conventional
antibody is shown in FIG. 1A.
[0349] The present invention provides novel amino acid
substitutions in the FcRn-binding domain that increase FcRn binding
activity of an antigen-binding molecule in the acidic and neutral
pH ranges, wherein the FcRn-binding activity in the neutral pH
range is stronger than the one of an intact IgG or an
antigen-binding molecule comprising an intact FcRn-binding domain
(e.g. stronger than 3200 nM). The modified antigen-binding
molecules can reduce the total antigen concentration in plasma
after its administration more than a control antigen-binding
molecule comprising the same antigen-binding domain but an intact
human IgG FcRn-binding domain.
[0350] Fc receptors are proteins on the surface of immune cells
such as natural killer cells, macrophages, neutrophils and mast
cells. They bind to the Fc (Fragment, crystallizable) region of
antibodies that are attached to infected cells or invading
pathogens and stimulate phagocytic or cytotoxic cells to destroy
microbes, or infected cells by antibody-mediated phagocytosis or
antibody-dependent cell-mediated cytotoxicity.
[0351] There are several different types of Fc receptors, which are
classified based on the type of antibody that they recognize.
Herein, the term "FcRn" refers to the neonatal Fc receptor that
binds IgG, is similar in structure to MHC class I protein and that,
in humans, is encoded by the FCGRT gene.
[0352] The term "FcRn binding domain" as used herein refers to a
protein domain that directly or indirectly binds to the FcRn.
Preferably the FcRn is a mammalian FcRn, more preferably, a human
FcRn. An FcRn binding domain binding directly to an FcRn is an
antibody Fc region. Meanwhile, regions capable of binding to a
polypeptide such as albumin or IgG, which has human FcRn-binding
activity, can indirectly bind to human FcRn via albumin, IgG, or
such. Thus, such a human FcRn-binding region may be a region that
binds to a polypeptide having human FcRn-binding activity.
[0353] The term "Fc region" or "Fc region of an antigen-binding
molecule" as used herein refers to an FcRn-binding domain that
directly binds to FcRn, preferably to a mammalian FcRn, more
preferably to a human FcRn. In particular, an Fc region is an Fc
region of an antibody. Preferably, the Fc region is a mammalian Fc
region, more preferably a human Fc region. In particular, the Fc
region of the invention is an Fc region comprising the second and
third constant domain of a human immunoglobulin (CH2 and CH3), more
preferably the hinge, CH2 and CH3. Preferably, the immunoglobulin
is an IgG. Preferably, the Fc region is an Fc region of human
IgG1.
[0354] The present invention provides an antigen-binding molecule
having a modified FcRn-binding domain wherein said antigen-binding
molecule has an increased FcRn-binding activity in a neutral pH
range as compared to an antigen-binding molecule having an intact
FcRn-binding domain.
[0355] In particular, the present invention provides an
antigen-binding molecule having a modified FcRn-binding domain with
an amino acid substitution in an FcRn-binding domain at one or more
positions selected from the group consisting of EU238, EU250,
EU252, EU254, EU255, EU258 EU286, EU307, EU308, EU309, EU311,
EU315, EU428, EU433, EU434, and EU436. The antigen-binding molecule
of the present invention may also comprise substitutions at
additional positions. For example, the antigen-binding molecule may
comprise a substitution at position EU256 in addition to a
substitution at the one or more positions mentioned above.
Preferably, the amino acid at position EU256 is substituted with a
glutamic acid.
[0356] The term "binding affinity" or "binding activity" refers to
the strength of non-covalent interaction between two substances as
measured by the dissociation constant (KD) of the complex formed by
the two substances, unless expressly defined otherwise. A binding
protein (or "ligand") may, for example, have a KD of less than
10.sup.-5, 10.sup.-6, 10.sup.-7 or 10.sup.-8 M for a particular
target molecule, e.g. the FcRn. Higher affinity binding of a
binding ligand to a target a first pH range relative to a target at
a second pH range can be indicated by a smaller numerical value KD
for binding the target at the first pH range than the numerical
value KD for binding the target at the second pH range. Differences
in binding affinity can be at least 1.5, 2, 3, 4, 5, 10, 15, 20,
50, 70, 80, 100, 500, or 1000 fold. Binding affinity can be
determined by a variety of methods including surface plasmon
resonance, equilibrium dialysis, equilibrium binding, gel
filtration, ELISA, or spectroscopy (e.g., using a fluorescence
assay).
[0357] An increased binding affinity of an FcRn-binding domain for
FcRn at a pH range corresponds to a measured increase of the
FcRn-binding affinity as compared to the FcRn-binding affinity
measured for an intact FcRn-binding domain. Differences in binding
affinity of KD(intact)/KD(variant) is at least 1.5, 2, 3, 4, 5, 10,
15, 20, 50, 70, 80, 100, 500, or 1000 fold. An increased binding
affinity of an FcRn-binding domain for FcRn can be in the acidic or
neutral pH ranges.
[0358] The term "antigen-binding molecule comprising an intact FcRn
binding domain" refers to an antigen-binding molecule comprising an
unmodified FcRn-binding domain. The term "intact IgG FcRn-binding
domain" as used herein refers to an unmodified FcRn-binding domain
of a human IgG. In particular, the FcRn-binding domain is the
FcRn-binding domain of an intact human IgG. Preferably, an intact
FcRn-binding domain is an intact Fc region. The term "antibody
comprising an intact Fc region" refers to an antibody comprising an
unmodified Fc region. The antibody from which the unmodified Fc
region originates is preferably an IgG. More preferably, it is a
human IgG1, IgG2, IgG3 or IgG4, still more preferably, a human
IgG1. In a particularly preferred embodiment of the present
invention an antibody comprising an intact Fc region is an antibody
comprising an unmodified Fc region. An antibody comprising an
intact Fc region can be an intact human IgG.
[0359] The term "intact IgG" as used herein refers to an unmodified
IgG and is not limited to a specific class of IgG. This means that
human IgG1, IgG2, IgG3, IgG4 or their allotypic variants can be
used as "intact human IgG" as long as it can bind to the human FcRn
in the acidic pH range. Preferably, "intact IgG" is a human IgG1.
Preferably, an intact IgG is an IgG which comprises a wild type Fc
region.
[0360] In the context of the present invention, an increased
FcRn-binding activity of antigen-binding molecule in the neutral pH
ranges is preferably stronger than KD 3.2 micromolar. Preferably,
the increased FcRn-binding activity in the neutral pH range is
stronger than 700 nanomolar, more preferably stronger than 500
nanomolar and most preferably, stronger than 150 nanomolar.
[0361] An increased FcRn-binding activity of antigen-binding
molecule of the present invention in the acidic pH ranges is
generally an FcRn-binding activity in the range of about 2-fold to
about 100-fold stronger than the FcRn-binding activity of an intact
IgG. Preferably, the increased FcRn-binding activity of
antigen-binding molecule in the acidic pH ranges is at least
10-fold stronger than the FcRn-binding activity of an intact IgG.
More preferably, the increased FcRn-binding activity of an
antigen-binding molecule of the present invention in the acidic pH
range is at least 20-fold stronger than the FcRn-binding activity
of an intact IgG.
[0362] The terms "neutral pH range" and "neutral pH" as used
herein, typically refer to pH 6.7 to pH 10.0, preferably any pH
value within pH 7.0 to pH 8.0, examples of which include pH 7.0,
7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. A
particularly preferred acidic pH value is pH 7.4, which
approximates plasma (blood) pH in vivo.
[0363] The terms "acidic pH range" and "acidic pH" as used herein,
typically refer to pH 4.0 to pH 6.5, preferably to any pH value
within pH 5.5 to pH 6.5, examples of which include pH 5.5, 5.6,
5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. A particularly
preferred acidic pH value ranges from pH 5.8 to pH 6.0, which
approximates the pH in early endosome in vivo.
[0364] The amino acid positions referred to in this application,
such as e.g. "EU387" or "position 387", are, unless otherwise
indicated, numbered according to a scheme called the EU numbering
system (Kabat, E. A., T. T. Wu, H. M. Perry, K. S. Gottesman, C.
Foeler. 1991. Sequences of Proteins of Immunological Interest. No.
91-3242 U. S. Public Health Services, National Institutes of
Health, Bethesda) and refer to positions in an FcRn-binding domain,
in particular in an Fc region. In a similar fashion, substitutions
are indicated as for example "EU387R" or "EU440E", wherein the
number given after "EU" indicates the position of the substitution
according the EU numbering, and the letter after the number is the
substituted amino acid given in the one letter code. Substitutions
may also be written as (amino acid 1)-position-(amino acid2)
whereby the first amino acid is the substituted amino acid and the
second amino acid is the substituting amino acid at the specified
position.
[0365] The terms "substitution" and "substitution of an amino acid"
as used herein refer to a replacement of an amino acid in an amino
acid sequence with another one, wherein the latter is different
from the replaced amino acid. Methods for replacing an amino acid
are well known to the skilled in the art and include, but are not
limited to, mutations of the nucleotide sequence encoding the amino
acid sequence.
[0366] More particularly, a substitution of an amino acid in an
FcRn-binding domain refers to a replacement of an amino acid in
reference to the amino acid sequence of a parent FcRn-binding
domain. A modified FcRn-binding domain already having the desired
substitutions is also included in the FcRn-binding domain of the
present invention.
[0367] A parent FcRn-binding domain is an FcRn-binding domain
having at the position EU238 an proline, at position EU250 a
threonine, at position EU252 a methionine, at position EU254 a
serine, at position EU255 an arginine, at position EU256 a
threonine, at position EU258 a glutamic acid, at position EU286 an
asparagine, at position EU307 a threonine, at position EU308 a
valine, at position EU309 a leucine, at position EU311 a glutamine,
at position EU315 an asparagine, at position EU387 a proline, at
position EU422 a valine, at position EU424 a serine, at position
EU426 a serine, at position EU428 a methionine, at position EU433 a
histidine, at position EU434 an asparagine, at position EU436 a
tyrosine, at position EU438 a glutamine, and at position EU440 a
serine and no or low affinity for FcRn at neutral pH (weaker than
3200 nM). The parent FcRn-binding domain may comprise substitutions
at other positions but preferably, the parent FcRn-binding domain
is unmodified. Preferably, the parent FcRn binding domain is an Fc
region (parent Fc region). Preferably, the parent Fc region is
derived from a mammalian antibody; more preferably, the parent Fc
region is the Fc region of a human antibody. An Fc region of a
human antibody is herein referred to as a human Fc region.
[0368] A parent Fc region is, preferably an intact Fc region, more
preferably a human intact Fc region. Preferably, the parent Fc
region is the Fc region of an IgG, more preferably of a human IgG.
Even more preferably, a parent Fc region is a human Fc region
comprising the wild type hinge, wildtype CH2 and wildtype CH3
domain. In the context of the present invention, the term parent
antibody refers to an antibody comprising a parent Fc region.
[0369] Parent antigen-binding molecules include, but are not
limited to, receptor proteins (membrane-bound receptors and soluble
receptors), antibodies that recognize a membrane antigen such as
cell surface markers, and antibodies that recognize a soluble
antigen such as cytokines
[0370] The term "parent antigen-binding molecule" as used herein
refers to an antigen-binding molecule having a parent FcRn-binding
domain. The origin of "parent antigen-binding molecule" is not
limited and it may be obtained from any organism: of non-human
animals or human. Preferably, the organism is selected from the
group consisting of mouse, rat, guinea pig, hamster, gerbil, cat,
rabbit, dog, goat, sheep, cow, horse, camel, and non-human primate.
In another embodiment, "parent antigen-binding molecule" can also
be obtained from cynomolgus monkey, marmoset, rhesus monkey,
chimpanzee or human. The parent IgG may be a naturally occurring
IgG, or a variant or engineered version of a naturally occurring
IgG. Parent IgG may refer to the polypeptide itself, compositions
that comprise the parent IgG, or the amino acid sequence that
encodes it. It should be noted that "parent IgG" includes known
commercial, recombinantly produced IgG as outlined below.
Preferably, "parent IgG" is obtained from human IgG1 but not
limited to a specific subclass of IgG. This means that human IgG1,
IgG2, IgG3, or IgG4 can be appropriately used as "parent IgG". In
the a similar manner, any subclass of IgGs from any organisms
described hereinbefore can be preferably used as "parent IgG".
Example of variant or engineered version of a naturally occurring
IgG is described in Curr Opin Biotechnol. 2009 December; 20(6):
685-91, Curr Opin Immunol. 2008 August; 20(4): 460-70, Protein Eng
Des Sel. 2010 April; 23(4): 195-202, WO 2009/086320, WO
2008/092117, WO 2007/041635 and WO 2006/105338, but not limited
thereto.
[0371] An FcRn-binding domain or Fc region of the present invention
may comprise a substitution at two or more positions which is
herein referred to as a "combination" of substitutions. For example
an Fc region defined by the combination "EU424/EU434/EU436" is an
Fc region that comprises a substitution at the positions EU424,
EU434 and EU436.
[0372] The substituting amino acid (the amino acid with which the
amino acid in the parent FcRn-binding domain is substituted) may be
any amino acid unless specifically mentioned herein, including but
not limited to the group consisting of: alanine (Ala, A), arginine
(arg, R), asparagines (asn, N), aspartic acid (asp, D), cysteine,
(cys, C), glutamic acid (glu, E), glutamine (gln, Q), glycine (gly,
G), histidine (his, H), isoleucine (ile, I), leucine (leu, L),
lysine (lys, K), methionine (met, M), phenylalanine (phe, F),
proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan
(trp, W), tyrosine (tyr, Y), and valine (val, V). Preferably, the
substituting amino acid at any one of the positions EU387, EU422,
EU424, EU426, EU433, EU436, EU438 and EU440 is selected from the
group consisting of: alanine (Ala, A), arginine (arg, R), glutamic
acid (glu, E), glutamine (gln, Q), aspartic acid (asp, D), serine
(ser, S), threonine (thr, T), tyrosine (tyr, Y), and lysine (lys,
K).
[0373] In a preferred embodiment of the present invention, the
antigen-binding molecule of the present invention has a modified
FcRn-binding domain comprising an amino acid substitution with an
amino acid different from the substituted one
[0374] a) at position EU252 and EU434, and
[0375] b) at one or more positions selected from the group
consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258,
EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and
EU436.
[0376] The substituting amino acid may be any amino acid unless
specifically mentioned herein. Preferred substituting amino acids
for the positions EU238, EU250, EU252, EU254, EU255, EU258, EU286,
EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436
are shown in Table 1.
TABLE-US-00001 TABLE 1 Preferred substituting amino acids Position
(according to EU numbering scheme) Substituting amino acid EU238 an
aspartic acid EU250 a valine EU252 a tyrosine EU254 a threonine
EU255 a leucine EU256 a glutamic acid EU258 an aspartic acid or an
isoleucine EU286 a glutamic acid EU307 a glutamine EU308 a proline
EU309 a glutamic acid EU311 an alanine or a histidine EU315 an
aspartic acid EU428 an isoleucine EU433 an alanine, a lysine, a
proline, an arginine or a serine EU434 a tyrosine, or a tryptophan
EU436 an isoleucine, a leucine, a valine, a threonine, or a
phenylalanine.
[0377] Preferably, the modified FcRn-binding domain of the present
invention comprises at least one of amino acids substitutions set
forth in Table 1. It is possible to use the FcRn-binding domains
without any alteration as long as they already have at least one of
the above given amino acids at the specified position and said
FcRn-binding domain has human FcRn-binding activity in the acidic
and neutral pH ranges, whereby the FcRn-binding activity in the
neutral pH ranges is increased.
[0378] In a preferred embodiment, the modified antigen-binding
molecule of the present invention comprises a modification at three
or more positions in the FcRn-binding domain, wherein the three or
more positions are one of the combinations set forth in Tables 2, 4
to 7.
TABLE-US-00002 TABLE 2 Preferred combinations of positions for
substitutions in FcRn-binding domain a) EU252/EU434/EU436, b)
EU252/EU434/EU307/EU311, c) EU252/EU434/EU315, d)
EU252/EU434/EU308, e) EU252/EU434/EU238, f) EU252/EU434/EU436/
EU307/EU311, g) EU252/EU434/EU255 h) EU252/EU434/EU258 i)
EU252/EU434/EU433 j) EU252/EU434/EU255/EU258
[0379] In a more preferred embodiment, the antigen-binding molecule
of the present invention comprises three or more amino acid
substitutions in a FcRn-binding domain, wherein the three or more
substitutions are one of the combinations set forth in Tables 3,
12, 14, and 17 to 20.
TABLE-US-00003 TABLE 3 Preferred substitution combinations in
FcRn-binding domain 1 M252Y/N434Y/Y436V 2 M252Y/N434Y/Y436T 3
M252Y/N434Y/Y436F 4 M252Y/N434Y/Y436V 5 M252Y/N434Y/Y436V 6
M252Y/N434Y/Y436T 7 M252Y/N434Y/Y436T 8 M252Y/N434Y/Y436F 9
M252Y/N434Y/Y436F 10 M252Y/N434Y/Y436V 11 M252Y/N434Y/Y436V 12
M252Y/H433D/N434Y/Y436V 13 M252Y/H433D/N434Y/Y436V 14
M252Y/H433D/N434Y/Y436V 15 M252Y/H433D/N434Y/Y436V 16
M252Y/S254T/T256E/T307Q/Q311A/H433D/N434Y/Y436V 17
M252Y/S254T/T256E/V308P/H433D/N434Y/Y436V 18
M252Y/H433D/N434W/Y436V 19 M252Y/H433D/N434W/Y436V 20
M252Y/S254T/T256E/H433D/N434Y/Y436V 21
M252Y/S254T/T256E/H433D/N434Y/Y436V 22
M252Y/S254T/T256E/H433D/N434Y/Y436V 23
M252Y/S254T/T256E/H433D/N434Y/Y436V 24
M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V 25
M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V 26
M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V 27
M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V 28
M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V 29
M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V 30
M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V 31
M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V 32
M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V 33
M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V 34
M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V 35
M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V 36
M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V 37
M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V 38
M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V 39
M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V 40
M252Y/S254T/T256E/H433A/N434Y/Y436V 41
M252Y/S254T/T256E/H433K/N434Y/Y436V 42
M252Y/S254T/T256E/H433P/N434Y/Y436V 43
M252Y/S254T/T256E/H433R/N434Y/Y436V 44
M252Y/S254T/T256E/H433S/N434Y/Y436V 45
M252Y/S254T/T256E/H433A/N434Y/Y436V 46
M252Y/S254T/T256E/H433A/N434Y/Y436V 47
M252Y/S254T/T256E/H433A/N434Y/Y436V 48
M252Y/S254T/T256E/H433K/N434Y/Y436V 49
M252Y/S254T/T256E/H433K/N434Y/Y436V 50
M252Y/S254T/T256E/H433K/N434Y/Y436V 51
M252Y/S254T/T256E/H433P/N434Y/Y436V 52
M252Y/S254T/T256E/H433P/N434Y/Y436V 53
M252Y/S254T/T256E/H433P/N434Y/Y436V 54
M252Y/S254T/T256E/H433R/N434Y/Y436V 55
M252Y/S254T/T256E/H433R/N434Y/Y436V 56
M252Y/S254T/T256E/H433R/N434Y/Y436V 57
M252Y/S254T/T256E/H433S/N434Y/Y436V 58
M252Y/S254T/T256E/H433S/N434Y/Y436V 59
M252Y/S254T/T256E/H433S/N434Y/Y436V 60
L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V 61
L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V 62
P238D/M252Y/V308P/N434Y 63 P238D/M252W/N434Y 64
P238D/M252Y/M428F/N434Y
Stability, Immunogenicity and Aggregate Formation
[0380] Engineering an FcRn-binding domain by introducing
substitutions may reduce the stability of the antigen-binding
molecule (WO/2007/092772). Stability of a drug protein is critical
for manufacturing pharmaceuticals, because proteins with poor
stability tend to easily aggregate during storage. Therefore
reduced stability caused by substitutions in the Fc region would
make the development of a stable formulation difficult
(WO2007/092772).
[0381] In addition, purity of a drug protein with regard to monomer
species and high molecular weight species is also important for
pharmaceutical development. Wild type IgG1 after protein A
purification does not contain a significant amount of high
molecular weight species, but engineering an FcRn-binding domain by
introducing substitutions may result in a larger amount of high
molecular weight species. In such a case, high molecular weight
species need to be removed from bulk drug substances by a
purification process which may be difficult in the development of
the purification process.
[0382] Moreover, immunogenicity of a protein pharmaceutical in
human is important, since the presence of anti-drug antibodies
would result in clearance of the drug from the body and thus loss
of therapeutic efficacy (IDrugs 2009; 12:233-7.). When
substitutions are introduced into a wild type Fc domain (such as
IgG1 Fc domain), the modified sequence becomes a non-human
sequence. Such a modified sequence could be presented by MHC class
II and therefore could be immunogenic in human patients.
[0383] Proteins will not be developed as a drug if they comprise Fc
variants that exhibit poor stability and purity, and poor
immunogenicity would hinder clinical development. It is therefore
an objective of the present invention to improve the FcRn binding
affinity at pH7.4 without
[0384] losing significant stability;
[0385] increasing the amount of high molecular weight species
ratio, and
[0386] increasing the risk of immunogenicity (risk of anti-drug
antibody formation)
(Group 1)
[0387] Therefore, the present invention also provides an
antigen-binding molecule comprising an amino acid substitution in
the FcRn-binding domain at the positions EU252, EU434, EU307 and
EU311, having a binding activity for the FcRn at pH 7 of more than
15 nM, a melting temperature Tm of 57.5 degrees C. or higher, an
HMW of less than 2% and a low immunogenicity wherein a low
immunogenicity is equivalent to a score of less than 500 determined
with Epibase (Lonza).
[0388] Preferably, an antigen-binding molecule comprising an amino
acid substitution in the FcRn-binding domain at four or more
positions, wherein the four or more positions are one of the
combinations of the group consisting of
[0389] a) EU252/EU434/EU307/EU311/EU436, and
[0390] b) EU252/EU434/EU307/EU311/EU436 in combination with one or
more positions selected from the group consisting of EU286, EU308,
and EU428.
[0391] The preferred combinations are set forth in Table 4.
TABLE-US-00004 TABLE 4 a) EU252 and EU434 and EU307 and EU311and
EU436 and EU286; b) EU252 and EU434 and EU307 and EU311 and EU436
and EU308 c) EU252 and EU434 and EU307 and EU311 and EU436 and
EU286 and EU308 d) EU252 and EU434 and EU307 and EU311 and EU436
and EU428 e) EU252 and EU434 and EU307 and EU311 and EU436 and
EU308 and EU428 f) EU252 and EU434 and EU307 and EU311 and EU436
and EU250 and EU428 g) EU252 and EU434 and EU307 and EU311 and
EU436 and EU250 and EU308 h) EU252 and EU434 and EU307 and EU311
and EU436 and EU250 and EU286 and EU308 i) EU252 and EU434 and
EU307 and EU311 and EU436 and EU250 and EU286 and EU308 and
EU428
[0392] Particular preferred are combinations a), g), h) and i) of
Table 4.
[0393] In an even more preferred embodiment, the modified
FcRn-binding domain comprises:
[0394] a) at position EU252 a tyrosine, at position EU286 a
glutamic acid, at position EU307 a glutamine, at position EU311 an
alanine, at position EU434 a tyrosine, and at position EU436 a
valine; or
[0395] b) at position EU250 a valine, at position EU252 a tyrosine,
at position EU307 a glutamine, at position EU308 a proline, at
position EU311 an alanine, at position EU434 a tyrosine, and at
position EU436 a valine; or
[0396] c) at position EU250 a valine, at position EU252 a tyrosine,
at position EU286 a glutamic acid, at position EU307 a glutamine,
at position EU308 proline, at position EU311 an alanine, at
position EU434 a tyrosine, and at position EU436 a valine; or
[0397] d) at position EU250 a valine, at position EU252 a tyrosine,
at position EU286 a glutamic acid, at position EU307 a glutamine,
at position EU308 proline, at position EU311 an alanine, at
position EU434 a tyrosine, and at position EU436 a valine.
(Group 2)
[0398] The present invention also provides an antigen-binding
molecule comprising an amino acid substitution in the FcRn-binding
domain at three or more positions, wherein said three or more
positions are one of the combinations of the group consisting of a)
EU252/EU434/EU307/EU311; and b) EU252/EU434/EU308; wherein the
FcRn-binding activity of said antigen-binding molecule at neutral
pH is 15 to 50 nM, the Tm is higher than 60 degrees C., an HMW of
less than 2% and wherein the antigen-binding molecule has a low
immunogenicity whereby a low immunogenicity is equivalent to a
score of less than 500 determined with Epibase (Lonza).
[0399] In a preferred embodiment, the amino acid substitutions are
at four or more positions wherein the four or more positions are
one of the combinations set forth in Table 5.
TABLE-US-00005 TABLE 5 preferred combinations a)
EU252/EU434/EU307/EU311/EU286 b)
EU252/EU434/EU307/EU311/EU286/EU254 c)
EU252/EU434/EU307/EU311/EU436 d)
EU252/EU434/EU307/EU311/EU436/EU254 e)
EU252/EU434/EU307/EU311/EU436/EU250 f) EU252/EU434/EU308/EU250 g)
EU252/EU434/EU308/EU250/EU436/ h)
EU252/EU434/EU308/EU250/EU307/EU311
[0400] More preferred is an antigen-binding molecule comprising
four or more amino acid substitutions wherein the four or more
substitutions are one of the combinations of the group consisting
of:
[0401] a) at position EU252 a tyrosine, at position EU286 a
glutamic acid, at position EU307 a glutamine, at position EU311 an
alanine and at position EU434 a tyrosine;
[0402] b) at position EU252 a tyrosine, at position EU254 a
threonine, at position EU286 a glutamic acid, at position EU307 a
glutamine, at position EU311 an alanine and at position EU434 a
tyrosine;
[0403] c) at position EU252 a tyrosine, at position EU307 a
glutamine, at position EU311 an alanine, at position EU434 a
tyrosine and at position 436 an isoleucine;
[0404] d) at position EU252 a tyrosine, at position EU254 a
threonine, at position EU286 a glutamic acid, at position EU307 a
glutamine, at position EU311 an alanine, at position EU434 a
tyrosine and at position EU436 an isoleucine;
[0405] e) at position EU250 a valine, at position EU252 a tyrosine,
at position EU254 a threonine, at position EU308 a proline, at
position EU434 a tyrosine and at position EU436 a valine;
[0406] f) at position EU250 a valine, at position EU252 a tyrosine,
at position EU307 a glutamine, at position EU311 an alanine, at
position EU434 a tyrosine and at position EU436 a valine;
[0407] g) at position EU252 a tyrosine, at position EU307 a
glutamine, at position EU311 an alanine, at position EU434 a
tyrosine and at position EU436 a valine;
[0408] h) at position EU250 a valine, at position EU252 a tyrosine,
at position EU308 a proline, and at position EU434 a tyrosine;
and
[0409] i) at position EU250 a valine, at position EU252 a tyrosine,
at position 307 a glutamine, at position EU308 a proline, at
position EU311 an alanine, and at position EU434 a tyrosine.
(Group 3)
[0410] The present invention also provides an antigen-binding
molecule comprising an amino acid substitution in the FcRn-binding
domain
[0411] a) at the positions EU252/EU434; and
[0412] b) at position EU436 and/or at position EU254 and/or at
position EU315;
[0413] and having an FcRn-binding activity at pH 7 of 50 to 150 nM,
a Tm higher than 63 degrees C., an HMW of less than 2% and a very
low immunogenicity, wherein a very low immunogenicity is defined as
a score of less than 250 determined with Epibase (Lonza).
[0414] Preferably, the amino acid substitutions are at three or
more positions, wherein the three or more positions are one of the
combinations set forth in Table 6.
TABLE-US-00006 TABLE 6 preferred combinations a) EU252/EU315/EU434;
b) EU252/EU434/EU436 c) EU252/EU254/EU434/EU436
[0415] In a more preferred embodiment, the modified antigen-binding
molecule comprises three or more amino acid substitutions wherein
the three or more substitutions are one of the combinations of the
group consisting of:
[0416] a) at position EU252 a tyrosine, at position EU315 an
aspartic acid, and at position EU434 a tyrosine;
[0417] b) at position EU252 a tyrosine, at position EU434 a
tyrosine, and at position EU436 an isoleucine;
[0418] c) at position EU252 a tyrosine, at position EU434 a
tyrosine, and at position EU436 a leucine;
[0419] d) at position EU252 a tyrosine, at position EU434 a
tyrosine, and at position EU436 a valine; and
[0420] e) at position EU252 a tyrosine, at position EU254 a
threonine, at position EU434 a tyrosine, and at position EU436 an
isoleucine.
(Group 4)
[0421] The present invention further provides an antigen-binding
molecule that comprises an amino acid substitution in the
FcRn-binding domain at three or more positions, wherein the three
or more positions are one of the combinations set forth in Table 7.
Said modified antigen-binding molecules have a binding activity for
the FcRn at pH 7 of 150 to 700 nM, a Tm of higher than 66.5 degrees
C., an HMW of less than 2% and a very low immunogenicity, wherein a
very low immunogenicity is defined as a score of less than 250
determined with Epibase (Lonza).
TABLE-US-00007 TABLE 7 a) EU307/EU311/EU434 b)
EU307/EU309/EU311/EU434 c) EU307/EU309/EU311/EU434 d)
EU250/EU252/EU434/EU436
[0422] Preferably, the modified antigen-binding molecules comprise
three or more substitutions wherein the three or more substations
are one of the combinations of the group consisting of
[0423] a) at position EU307 a glutamine, at position EU311 a
histidine, and at position EU434 a tyrosine;
[0424] b) at position EU307 a glutamine, at position EU309 a
glutamic acid, at position EU311 an alanine, at position EU434 a
tyrosine;
[0425] c) at position EU307 a glutamine, at position EU309 a
glutamic acid, at position EU311 a histidine, at position EU434 a
tyrosine; or
[0426] d) at position EU250 a valine, at position EU252 a tyrosine,
at position EU434 a tyrosine, at position EU436 a valine.
Pre-Existing Anti-Drug Antibody
[0427] Substitutions of amino acids in an antibody can yield
negative consequences, for example an increase in the
immunogenicity of the therapeutic antibody which, in turn, can
result in a cytokine storm and/or production of anti-drug
antibodies (ADAs). Since ADAs can influence the efficacy and
pharmacokinetics of therapeutic antibodies and sometimes lead to
serious side effects, the clinical utility and efficacy of the
therapeutic antibodies can be limited. Many factors influence the
immunogenicity of therapeutic antibodies, and the presence of
effector T-cell epitopes is one of the factors. Likewise, the
presence of pre-existing antibodies against a therapeutic antibody
can also be problematic. An example of such pre-existing antibody
is the rheumatoid factor (RF), an auto-antibody (an antibody
directed against a self protein) against the Fc portion of an
antibody (i.e. IgG). The rheumatoid factor is found in particular
in patients suffering of systemic lupus erythematosus (SLE) or
rheumatoid arthritis. In arthritis patients, RF and IgG join to
form immune complexes that contribute to the disease process.
Recently, it was reported that a humanized anti-CD4 IgG1 antibody
having an Asn434His mutation elicited significant rheumatoid factor
binding (Clin Pharmacol Ther. 2011 February; 89(2):283-90 (NPL 9)).
Detailed studies have confirmed that the Asn434His mutation in the
human IgG1 increased the binding of rheumatoid factor to the Fc
region of the antibody compared to the parent human IgG1.
[0428] RF is a polyclonal auto-antibody against human IgG, and the
epitope of the RF in the sequence of the human IgG varies among the
clones, but the RF epitope seems to be located in the CH2/CH3
interface region as well as CH3 domain which could overlap with the
FcRn binding epitope. Therefore, mutations to increase the binding
affinity to FcRn at neutral pH might also increase the binding
affinity to specific clone of RF.
[0429] Accordingly, it is preferable to increase the FcRn binding
affinity at neutral and/or acidic pH without also increasing the
binding affinity of the therapeutic antibody for a pre-existing
antibody in the plasma at neutral pH.
[0430] Therefore, present invention also provides antigen-binding
molecules comprising a modified FcRn-binding domain (preferably a
modified Fc region), whereby the binding activity for a
pre-existing ADA at a neutral pH is not significantly increased as
compared to the binding affinity of an antigen-binding molecule
comprising a wild type Fc region. The modified FcRn-binding domain
(modified Fc region) preferably comprises an amino acid
substitution at one or more of the positions selected from the
group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438
and EU440.
[0431] The above described substitutions are preferably introduced
in an FcRn-binding domain or Fc region of an antigen-binding
molecule that has increased affinity for the FcRn at neutral or
acidic pH whereby said modified FcRn-binding domain or Fc region
has an increased binding activity for a pre-existing ADA at neutral
pH. The effect of the substitutions is a decrease of the binding
activity for the pre-existing ADA. Therefore, in a preferred
embodiment, a modified FcRn-binding domain or a modified Fc region
of the present invention has a decreased binding activity to a
pre-existing ADA as compared to an FcRn binding domain or an Fc
region that has an increased binding activity to the FcRn at
neutral or acidic pH, and an increased binding activity to the
pre-existing anti-drug antibody in the neutral pH ranges.
Preferably, an antigen-binding molecule having an increased binding
activity at neutral pH for the FcRn and a pre-existing ADA are the
antigen-binding molecules comprising an amino acid substitution at
one or more positions selected from the group consisting of EU238,
EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309,
EU311, EU315, EU428, EU433, EU434, and EU436 as described above. It
may also comprise a substitution at position EU256 in addition to a
substitution at the one or more positions mentioned above.
Preferably, the amino acid at position EU256 is substituted with a
glutamic acid.
[0432] Therefore, the present invention also provides
antigen-binding molecules comprising a modified Fc region which has
an increased affinity for FcRn at neutral or acidic pH whereby the
affinity for a pre-existing anti-drug antibody (ADA) at a neutral
pH is not significantly increased compared to the binding affinity
of antigen-binding molecule comprising a wild type Fc region. In a
preferred embodiment, the present invention provides an
antigen-binding molecule comprising a modified Fc region with an
increased affinity for FcRn at neutral or acidic pH which comprises
an amino acid substitution at one or more of the positions selected
from the group consisting of EU387, EU422, EU424, EU426, EU433,
EU436, EU438 and EU440.
[0433] Preferably, the antigen-binding molecule comprising a
modified Fc region with an increased affinity for FcRn at neutral
or acidic pH, whereby the binding activity at neutral pH for a
pre-existing ADA is not significantly increased as compared to a
control antigen-binding molecule, wherein the modified Fc region
comprises an amino acid substitution at one or more of the
positions selected from the substitutions as shown in Table 8.
TABLE-US-00008 TABLE 8 Substituting amino acids: position
Substitutions with EU387 R EU422 E R S D K T Q EU424 E R K N EU426
D Q A Y EU433 D EU436 T EU438 E R S K EU440 E D Q
[0434] The term "anti-drug antibody" and "ADA" as used herein
refers to an endogenous antibody that has binding affinity for an
epitope located on a therapeutic antibody and is thus capable of
binding said therapeutic antibody. The term "pre-existing anti-drug
antibody" and "pre-existing ADA" as used herein refers to an
anti-drug antibody that is present and detectable in the blood of a
patient prior to the administration of the therapeutic antibody to
the patient. Preferably, the pre-existing ADA is a human antibody.
In a particularly preferred embodiment, the pre-existing ADA is the
rheumatoid factor, a polyclonal or monoclonal autoantibody against
the Fc region of human IgG antibody. The epitopes of rheumatoid
factor are located in the CH2/CH3 interface region as well as the
CH3 domain but can vary among clones.
[0435] An antigen-binding molecule comprising an FcRn-binding
domain region (or an Fc region) that has an increased affinity for
FcRn at neutral or acidic pH and for a pre-existing anti-drug
antibody at neutral pH is an antigen-binding molecule comprising an
FcRn-binding domain (or an Fc region) that was modified to increase
the binding affinity of the FcRn-binding domain (or Fc region) of
an antigen-binding molecule for FcRn as compared to an antibody
comprising an intact FcRn-binding domain (or intact Fc region).
Modifications contemplated include, but are not limited to,
substitutions of the amino acids in the amino acid sequence of the
Fc portion of an antigen-binding domain. The antigen-binding
molecule comprising an FcRn-binding domain or an Fc region, which
has an increased binding activity for a) a pre-existing ADA in a
neutral pH range and for FcRn at neutral (in case of an
antigen-binding molecule of interest having an increased
FcRn-binding activity at a neutral pH) or acidic pH (in case of an
antigen-binding molecule of interest having an increased
FcRn-binding activity at an acidic pH) is referred herein as
"Reference Antibody". A "Reference Antibody" is preferably the
modified antigen-binding molecule before substituting an amino acid
at one or more positions selected from the group consisting of
EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440, more
preferably before introducing any one of the substitutions set for
in Table 8. A "Reference Antibody" may be an antigen-binding
molecule comprising an amino acid substitution in an FcRn-binding
domain at one or more positions selected from the group consisting
of EU238, EU250, EU252, EU254, EU255, EU256, EU258 EU286, EU307,
EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436.
[0436] An example for a "Reference Antibody" having an increased
FcRn-binding activity in the neutral pH ranges is an
antigen-binding molecule comprising an Fc region with increased
affinity for FcRn in the neutral pH ranges and having increased
affinity for a pre-existing ADA at neutral pH comprising an amino
acid substitution in the Fc region at
[0437] a) positions EU252 and EU434; and
[0438] b) one or more positions selected from the group consisting
of EU238, EU250, EU254, EU255, EU256, EU258, EU286, EU307, EU308,
EU309, EU311, EU315, EU428, EU433, and EU436.
[0439] More preferably, the antigen-binding molecule comprising an
Fc region with increased affinity for FcRn in the neutral pH ranges
and having increased affinity for a pre-existing ADA at neutral pH
ranges comprises one of the combinations set forth in Table 9.
TABLE-US-00009 TABLE 9 Preferred combinations of substitutions of a
Reference Antibody having an increased FcRn-binding activity in the
neutral pH ranges. 1 M252Y/N434Y/ 2 M252Y/N434Y/Y436V 3
M252Y/N434Y/Y436F 4 M252Y/N434Y/Y436V 5
M252Y/S254T/T256E/T307Q/Q311A/N434Y/Y436V 6
M252Y/S254T/T256E/V308P/N434Y/Y436V 7 M252Y/N434W/Y436V 8
M252Y/S254T/T256E/N434Y/Y436V 9 M252Y/S254T/T256E/N286E/N434Y/Y436V
10 M252Y/S254T/R255L/T256E/N434Y/Y436V 11
M252Y/S254T/R255L/T256E/N434Y/Y436V 12
M252Y/S254T/R255L/T256E/E258D/N434Y/Y436V 13
M252Y/S254T/R255L/T256E/E258I/N434Y/Y436V 14
M252Y/S254T/T256E/H433A/N434Y/Y436V 15
M252Y/S254T/T256E/H433K/N434Y/Y436V 16
M252Y/S254T/T256E/H433P/N434Y/Y436V 17
M252Y/S254T/T256E/H433R/N434Y/Y436V 18
M252Y/S254T/T256E/H433S/N434Y/Y436V 19
M252Y/S254T/T256E/H433A/N434Y/Y436V 20
L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V 21
L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V 22
EU238D/EU252Y/EU434Y/EU436V 23 EU252Y/EU434Y/EU436V 24
EU250V/EU252Y/EU434Y/EU436V/EU307Q/EU308P/EU311A 25
EU252Y/EU434Y/EU436V/EU235R/EU239K 26 EU252Y/EU434Y 27
EU252Y/EU434Y/EU436V
[0440] An example for a "Reference Antibody" having an increased
FcRn-binding activity in the acidic pH ranges is an antigen-binding
molecule comprising an Fc region with increased affinity for FcRn
in the acidic pH ranges and having increased affinity for a
pre-existing ADA at neutral pH ranges preferably comprise a
substitution
[0441] i) at position EU434, or
[0442] ii) at two or more positions, wherein the two or more
positions are one of the combinations of the group consisting of a)
EU252/EU254/EU256; b) EU428/EU434; and c) EU250/EU428.
[0443] Preferably, the antigen-binding molecule comprising an Fc
region with increased affinity at acidic pH ranges and having
increased affinity for a pre-existing ADA at neutral pH
comprises
[0444] i) the substitutions M434H; or
[0445] ii) one of the combinations of the group consisting of a)
M252Y/S254T/T256E; b) M428L/N434S; and c) T250Q and M428L (EU
numbering).
[0446] Preferably, the antigen-binding molecule comprising an Fc
region which comprises one of the following substitutions or
combinations a) M252Y/S254T/T256E, b) M428L/N434S or c) T250Q and
M428L or d) M434H (EU numbering) has an increased binding activity
to the FcRn at acidic pH without increasing the binding activity in
the neutral pH ranges.
[0447] The binding activity of an Fc region of antigen-binding
molecule for a pre-existing anti-drug antibody is expressed in the
present application as an electrochemiluminescence (ECL) response
at neutral pH; however, there are other suitable methods for
determining the binding activity for a pre-existing ADA known to
the skilled in the art. An ECL assay is for example described in
Moxness et al (Clin Chem, 2005, 51:1983-85) and in the EXAMPLES of
the present invention. Conditions used in the assay for determining
the binding activity for a pre-existing ADA can be appropriately
selected by those skilled in the art, and thus are not particularly
limited.
[0448] An increased or higher binding affinity for a pre-existing
ADA is increased as compared to the binding affinity for the
pre-existing ADA of a Control Antigen-binding Molecule.
[0449] The term "Control Antigen-binding Molecule" as used herein
refers to an antigen-binding molecule comprising an intact human Fc
region, preferably an antibody or antibody derivative comprising an
intact human Fc region.
[0450] The binding affinity for a pre-existing ADA may be assessed
at any temperature from 10 degrees Celsius to 50 degrees Celsius.
Preferably, a temperature at from 15 degrees Celsius to 40 degrees
Celsius is employed in order to determine the binding affinity
between human Fc region and human pre-existing ADA. More
preferably, any temperature at from 20 degrees Celsius to 35
degrees Celsius, like any one of 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, and 35 degrees C. is employed in order
to determine the binding affinity between human Fc region and human
pre-existing ADA. Preferably, the temperature is between 20 and 25
degrees C., more preferably at 25 degrees C. In a preferred
embodiment, the interaction between human pre-existing ADA and
human Fc region is measured at pH 7.4 (or pH7.0) and at 25 degrees
C.
[0451] In the context of the present invention, the term "an
increased binding affinity for a pre-existing ADA" refers to a
measured increase in binding affinity (i.e., KD) of an
antigen-binding molecule of the present invention for a
pre-existing ADA as compared to the binding affinity measured of a
Control Antigen-binding Molecule for the pre-existing ADA. Such an
increase in binding affinity for a pre-existing ADA can be observed
in an individual patient or in a patient group.
[0452] The terms "patients" and "patient" as used herein, are not
particularly limited and include all human beings who suffer from a
disease and to whom in the course of a treatment a therapeutic
antigen-binding molecule is administered. Preferably, a patient is
a person suffering from an autoimmune disease. More preferably, a
patient is a person suffering from an arthritic disease or systemic
lupus erythematosus (SLE). Arthritic diseases include in particular
rheumatoid arthritis.
[0453] In the context of the present invention, a significant
increase of the binding activity for a pre-existing ADA in an
individual patient corresponds to a measured increase of at least
10%, at least 20%, at least 30%, at least 40%, at least 50%, at
least 60% of the binding activity for a pre-existing ADA of a
therapeutic antigen-binding molecule (i.e. a therapeutic antibody)
comprising a modified Fc region in a patient as compared to the
binding affinity for the pre-existing ADA of a Control
Antigen-binding Molecule. Preferably the increase is at least 20%,
more preferably the increase is at least 30%, even more preferably,
it is at least 40% and most preferably the increase is at least 50%
of the binding activity of a antigen-binding molecule comprising a
modified Fc region as compared to the binding affinity for the
pre-existing ADA of a control antigen-binding molecule.
Alternatively, a significant increase in the binding activity of an
antigen-binding molecule for a pre-existing ADA in a patient is
preferably an ECL response to the antigen-binding molecule of more
than 250, preferably to an ECL of at least 500, more preferably to
an ECL of at least 1000, most preferably to an ECL of at least
2000. More preferably, the increase is an increase as compared with
the ECL response of a Control Antigen-binding Molecule of less than
500 (preferably of less than 250). Preferred ranges between an the
binding activity for a pre-existing ADA of the Control
Antigen-binding Molecule and that of an antigen-binding molecule
with a modified Fc region are in particular ECL responses from less
than 250 to at least 250, from less than 250 to at least 500, from
less than 500 to 500 or more, from less than 500 to 1000 or more,
and from less than 500 to at least 2000.
[0454] The increase in the binding activity for a pre-existing ADA
may also correspond to a measured increase in the portion of
patients in a patient population having an ECL response of at least
500 (preferably at least 250) to an the antigen-binding molecule
with an increased binding activity to a) the FcRn at neutral or
acidic pH and b) an pre-existing ADA at neutral pH as compared to
the portion of patients having an ECL response of at least 500
(preferably at least 250) at neutral pH to a control
antigen-binding molecule. A "significant" increase in the portion
of patients in a patient population is preferably an increase of at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%
patients having a ECL response of the therapeutic antigen-binding
molecule comprising a modified Fc region to the rheumatoid factor
at neutral pH of 500 or less (preferably of 250 or more) compared
to the portion of patients having an ECL response to a Control
Antigen-binding Molecule. Preferably the increase is at least 20%,
more preferably at least 30%, even more preferably, it is at least
40% and most preferably it is 50% or more.
[0455] In the context of the present invention, a decrease in the
binding affinity for a pre-existing ADA refers to a measured
decrease in binding activity (i.e., KD or ECL response) as compared
to the binding activity measured for a Reference Antibody, Such a
decrease of binding affinity for a pre-existing ADA can be observed
in an individual patient or in a patient group. The decrease of the
affinity of a therapeutic antigen-binding molecule for a
pre-existing ADA at neutral pH in an individual patient refers to a
measured decrease at neutral pH in the binding activity as compared
to the binding activity measured for a Reference Antibody for the
pre-existing ADA at neutral pH in said patient. Preferably, a
significant decrease in an individual patient is a measured
decrease at neutral pH of at least 10%, at least 20%, at least 30%,
at least 40%, at least 50% in the binding activity of the modified
antigen-binding molecule for a pre-existing ADA as compared to the
binding activity of a Reference Antibody for a pre-existing ADA at
neutral pH. More preferably, the decrease is at least 30%, even
more preferably, it is 40% and most preferably it is 50% or more as
compared to a Reference antibody.
[0456] Alternatively, the significant decrease in an individual
patient of a modified antigen-binding molecule's binding activity
for a pre-existing ADA may be measured as a decrease of the ECL
response of said antigen-binding molecule as compared with the ECL
response of a Reference Antibody from an ECL response of 500 or
more, (preferably, from an ECL of 1000 or more, most preferably
from an ECL of 2000 or more), to less than 500, preferably of less
than 250. Preferred decreases are from an ECL response of 500 or
more to an ECL response of less than 500, more preferably from at
least 250 to less than 250, even more preferably from at least 500
to less than 250. Preferred ranges are, in particular, from at
least 250 to less than 250, from at least 500 to less than 250,
from at least 1000 to less than 250, from at least 2000 to less
than 250, from at least 500 to less than 500, from at least 1000 to
less than 500, and from at least 2000 to less than 500.
[0457] The decrease may also be a decrease in the percentage of
patients in a patient population that has an increased binding of
their pre-existing ADA to the modified antigen-binding molecule in
neutral pH ranges. In other words, the decrease may be measured as
a decrease of the percentage of people having an ECL response of
their pre-existing ADA to a modified antigen-binding molecule as
compared to the ECL response to a Reference Antibody. Preferably, a
decrease may be a decrease of at least 10%, at least 20%, at least
30%, at least 40%, at least 50% in the portion of patients in a
patient population in which the therapeutic antigen-binding
molecule has an increased binding activity to a pre-existing ADA as
compared to the portion of patients having an increased binding
activity of the Reference antibody to the pre-existing ADA, wherein
the increased binding is expressed as an ECL response of 500 or
more, preferably 250 or more. Preferably the decrease is at least
20%, more preferably the decrease is at least 30%, even more
preferably, it is 40% and most preferably it is 50% or more.
[0458] In a preferred embodiment, a therapeutic antigen-binding
molecule of the present invention has low binding activity for a
pre-existing ADA at a neutral pH. In particular, the binding
activity of a modified antigen-binding molecule of the present
invention for a pre-existing ADA at a neutral pH is preferably
significantly decreased compared to the binding activity of a
Reference Antibody for a pre-existing ADA at neutral pH. More
preferably, the binding activity of a modified antigen-binding
molecule of the present invention for a pre-existing ADA at a
neutral pH is not significantly increased as compared to the
binding affinity of a Control Antigen-binding Molecule (has about
the same binding activity for a pre-existing ADA as Control
Antigen-binding Molecule). A low binding activity or baseline
affinity for a pre-existing ADA is preferably an ECL response of
less than 500 in a individual patient. Preferably, a ECL response
is less than 250. In a patient population, a low binding activity
for a pre-existing ADA is an ECL response of less than 500 in 90%
of the patients in the patient population, more preferably in 95%
of the patients, most preferably in 98% of the patients.
[0459] In a more preferred embodiment, the antigen-binding molecule
comprising a modified FcRn-binding domain with an increased
affinity for FcRn at neutral or acidic pH, wherein the binding
activity at neutral pH for a pre-existing ADA is not significantly
increased as compared to a control antigen-binding molecule,
whereby the modified FcRn-binding domain of the present invention
comprises a substitution at one or more of the positions or
combinations set forth in Table 10.
TABLE-US-00010 TABLE 10 Positions and combinations of positions for
substitutions in FcRn-binding domain: 1) EU387 2) EU422 3) EU424 4)
EU426 5) EU436 6) EU438 7) EU440 8) EU438 and EU440 9) EU422 and
EU424 10) EU433
[0460] In a more preferred embodiment, the antigen-binding molecule
of the present invention comprises a modified FcRn-binding domain
having one or more of the substitutions or combinations set forth
in Table 11.
TABLE-US-00011 TABLE 11 Substitutions and combinations of
substitutions in FcRn-binding domain: 1 EU387R 2 EU422E 3 EU422R 4
EU422S 5 EU424E 6 EU424R 7 EU438E 8 EU438R 9 EU438S 10 EU440E 11
EU422E/EU424R 12 EU422S/EU424R 13 EU438R/EU440E 14 EU422D 15 EU422K
16 EU422T 17 EU422Q 18 EU438K 19 EU440D 20 EU440Q 21 EU438R/EU440D
22 EU438K/EU440E 23 EU438K/EU440D 24 EU424N 25 EU426D 26 EU426A 27
EU426Q 28 EU426Y 29 EU436F 30 EU436T 31 EU433D
[0461] In a preferred embodiment, the antigen-binding molecule
comprising a modified FcRn-binding domain with a) an increased
affinity for FcRn at neutral or acidic pH b) a binding affinity for
a pre-existing ADA at neutral pH which is not significantly
increased compared to a Control Antigen-binding Molecule, said
antigen-binding molecule comprises any one of the combinations of
substitutions set forth in Table 12.
[0462] Also preferably, an antigen-binding molecule having an
increased FcRn binding activity at neutral pH ranges and a binding
affinity for a pre-existing ADA at neutral pH that is not
significantly increased as compared to an antigen-binding molecule
comprising a wild type Fc region comprises an amino acid
substitution in an FcRn-binding domain at a) one or more of the
positions selected from the group consisting: EU387, EU422, EU424,
EU438, EU440, EU433, or b) at two or more positions, wherein the
two or more positions are the combination EU422/EU424; or
EU438/EU440. More preferably, the substitutions are selected from
among the substitutions set forth in Table 11.
[0463] Even more preferably, an FcRn-binding domain of an
antigen-binding molecule having an increased binding activity for
the FcRn at neutral pH ranges and a binding affinity for a
pre-existing ADA at neutral pH that is not significantly increased
as compared to an antigen-binding molecule comprising a wild type
Fc region comprising any one of the substitution combinations set
forth in Table 12. In particular, preferred modified
antigen-binding molecules having an increased FcRn-binding activity
in neutral pH ranges whereby the binding affinity at neutral pH for
a pre-existing ADA is not significantly increased comprises three
or more substitutions in the FcRn-binding domain, wherein the three
or more substitutions are any one of the combinations no. (2) to
(26) and (28) to (59) set forth in Table 12.
TABLE-US-00012 TABLE 12 Combinations of substitutions in Fc region
that increase the FcRn-binding activity in the neutral pH ranges
without significantly increasing the binding activity for a
pre-existing ADA (positions given according to the EU numbering
scheme). 1 M252Y/N434Y/Y436T 2 M252Y/N434Y/Y436V/Q438K/S440E 3
M252Y/N434Y/Y436V/Q438R/S440E 4 M252Y/N434Y/Y436T/Q438K/S440E 5
M252Y/N434Y/Y436T/Q438R/S440E 6 M252Y/N434Y/Y436F/Q438K/S440E 7
M252Y/N434Y/Y436F/Q438R/S440E 8 M252Y/N434Y/Y436V/Q438R/S440D 9
M252Y/N434Y/Y436V/Q438K/S440D 10
M252Y/H433D/N434Y/Y436V/Q438R/S440D 11
M252Y/H433D/N434Y/Y436V/Q438K/S440E 12
M252Y/H433D/N434Y/Y436V/Q438R/S440E 13
M252Y/H433D/N434Y/Y436V/Q438K/S440D 14
M252Y/S254T/T256E/T307Q/Q311A/H433D/N434Y/Y436V/Q438K/S440E 15
M252Y/S254T/T256E/V308P/H433D/N434Y/Y436V/Q438K/S440E 16
M252Y/H433D/N434W/Y436V/Q438R/S440E 17
M252Y/H433D/N434W/Y436V/Q438K/S440E 18
M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438K/S440E 19
M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438R/S440E 20
M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438K/S440D 21
M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438R/S440D 22
M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V/Q438K/S440E 23
M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V/Q438R/S440E 24
M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V/Q438K/S440D 25
M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V/Q438R/S440D 26
M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V/Q438K/S440E 27
M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V/Q438R/S440E 28
M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V/Q438K/S440D 29
M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V/Q438R/S440D 30
M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V/Q438R/S440E 31
M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V/Q438R/S440E 32
M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V/Q438K/S440E 33
M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V/Q438K/S440E 34
M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V/Q438K/S440D 35
M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V/Q438K/S440D 36
M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V/Q438R/S440D 37
M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V/Q438R/S440D 38
M252Y/S254T/T256E/H433A/N434Y/Y436V/Q438R/S440E 39
M252Y/S254T/T256E/H433K/N434Y/Y436V/Q438R/S440E 40
M252Y/S254T/T256E/H433P/N434Y/Y436V/Q438R/S440E 41
M252Y/S254T/T256E/H433R/N434Y/Y436V/Q438R/S440E 42
M252Y/S254T/T256E/H433S/N434Y/Y436V/Q438R/S440E 43
M252Y/S254T/T256E/H433A/N434Y/Y436V/Q438K/S440E 44
M252Y/S254T/T256E/H433A/N434Y/Y436V/Q438R/S440D 45
M252Y/S254T/T256E/H433A/N434Y/Y436V/Q438K/S440D 46
M252Y/S254T/T256E/H433K/N434Y/Y436V/Q438K/S440E 47
M252Y/S254T/T256E/H433K/N434Y/Y436V/Q438R/S440D 48
M252Y/S254T/T256E/H433K/N434Y/Y436V/Q438K/S440D 49
M252Y/S254T/T256E/H433P/N434Y/Y436V/Q438K/S440E 50
M252Y/S254T/T256E/H433P/N434Y/Y436V/Q438R/S440D 51
M252Y/S254T/T256E/H433P/N434Y/Y436V/Q438K/S440D 52
M252Y/S254T/T256E/H433R/N434Y/Y436V/Q438K/S440E 53
M252Y/S254T/T256E/H433R/N434Y/Y436V/Q438R/S440D 54
M252Y/S254T/T256E/H433R/N434Y/Y436V/Q438K/S440D 55
M252Y/S254T/T256E/H433S/N434Y/Y436V/Q438K/S440E 56
M252Y/S254T/T256E/H433S/N434Y/Y436V/Q438R/S440D 57
M252Y/S254T/T256E/H433S/N434Y/Y436V/Q438K/S440D 58
L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440E 59
L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E 60
EU238D/EU252Y/EU434Y/EU436V/EU387R 61
EU238D/EU252Y/EU434Y/EU436V/EU422E 62
EU238D/EU252Y/EU434Y/EU436V/EU422R 63
EU238D/EU252Y/EU434Y/EU436V/EU422S 64
EU238D/EU252Y/EU434Y/EU436V/EU424E, 65
EU238D/EU252Y/EU434Y/EU436V/EU424R 66
EU238D/EU252Y/EU434Y/EU436V/EU438E 67
EU238D/EU252Y/EU434Y/EU436V/EU438R 68
EU238D/EU252Y/EU434Y/EU436V/EU438S, 69
EU238D/EU252Y/EU434Y/EU436V/EU440E 70 EU252Y/EU387R/EU434Y/EU436V
71 EU252Y/EU422E/EU434Y/EU436V 72 EU252Y/EU422R/EU434Y/EU436V 73
EU252Y/EU422S/EU434Y/EU436V 74 EU252Y/EU424E/EU434Y/EU436V 75
EU252Y/EU424R/EU434Y/EU436V 76 EU252Y/EU434Y/EU436V/EU438E 77
EU252Y/EU434Y/EU436V/EU438R 78 EU252Y/EU434Y/EU436V/EU438S 79
EU252Y/EU434Y/EU436V/EU440E 80 EU252Y/EU422E/EU424R/EU434Y/EU436V
81 EU252Y/EU422S/EU424R/EU434Y/EU436V 82
EU252Y/EU434Y/EU436V/EU438R/EU440E 83 EU252Y/EU422D/EU434Y/EU436V
84 EU252Y/EU422K/EU434Y/EU436V 85 EU252Y/EU422T/EU434Y/EU436V 86
EU252Y/EU422Q/EU434Y/EU436V 87 EU252Y/EU434Y/EU436V/EU438K 88
EU252Y/EU434Y/EU436V/EU440D 89 EU252Y/EU434Y/EU436V/EU440Q 90
EU252Y/EU434Y/EU436V/EU438R/EU440D 91
EU252Y/EU434Y/EU436V/EU438K/EU440E 92
EU252Y/EU434Y/EU436V/EU438K/EU440D 93
EU250V/EU252Y/EU422E/EU434Y/EU436V/EU307Q/EU308P/EU311A 94
EU250V/EU252Y/EU424R/EU434Y/EU436V/EU307Q/EU308P/EU311A 95
EU250V/EU252Y/EU422E/EU424R/EU434Y/EU436V/EU307Q/EU308P/EU311A 96
EU250V/EU252Y/EU434Y/EU436V/EU438R/EU307Q/EU308P/EU311A 97
EU250V/EU252Y/EU434Y/EU436V/EU440E/EU307Q/EU308P/EU311A 98
EU250V/EU252Y/EU434Y/EU436V/EU438R/EU440E/EU307Q/EU308P/EU311A 99
EU252Y/EU433D/EU434Y/EU436V/EU438R/EU440E/EU235R/EU239K 100
EU252Y/EU433D/EU434Y/EU436V/EU438K/EU440D/EU235R/EU239K 101
EU252Y/EU434Y/EU436V/EU438R/EU440E/EU235R/EU239K 102
EU252Y/EU434Y/EU436V/EU438K/EU440E/EU235R/EU239K 103
EU252Y/EU433D/EU434Y/EU436V/EU438K/EU440E/EU235R/EU239K 104
EU252Y/EU424N/EU434Y 105 EU252Y/EU424N/EU434Y/EU436V
[0464] The present invention also provides an antigen-binding
molecule having an increased binding activity for the FcRn at
acidic pH ranges and a binding affinity for a pre-existing ADA at
neutral pH that is not significantly increased as compared to a
Control Antigen-binding Molecule, comprising an amino acid
substitution at a) position EU424 or b) position EU438/EU440.
[0465] More preferably, the substitutions are selected among a)
EU424N and EU438R/EU440E.
[0466] Preferably, an FcRn-binding domain of an antigen-binding
molecule that has an increased binding activity for the FcRn at
acidic pH ranges and a binding affinity for a pre-existing ADA at
neutral pH that is not significantly increased as compared to a
Control Antigen-binding Molecule, comprises one of the substitution
combinations set forth in Table 13. More preferably, the
antigen-binding molecule having an increased FcRn-binding activity
in the acidic pH ranges whereby the binding affinity for a
pre-existing ADA at neutral pH that is not significantly increased
as compared to a Control Antigen-binding Molecule, comprises any
one of the substitution combinations no. (13) to (28) set forth in
Table 13.
TABLE-US-00013 TABLE 13 Combinations of substitutions in Fc region
that increase the FcRn-binding activity in the acidic pH ranges
without significantly increasing the binding activity for a
pre-existing ADA (positions given according to the EU numbering
scheme). 1 EU252Y/EU254T/EU256E/EU438R/EU440E 2
EU252Y/EU254T/EU256E/EU424N 3 EU428L/EU434S/EU438R/EU440E 4
EU424N/EU428L/EU434S 5 EU426D/EU428L/EU434S 6 EU426A/EU428L/EU434S
7 EU426Q/EU428L/EU434S 8 EU426Y/EU428L/EU434S 9
EU428L/EU434S/EU436F 10 EU428L/EU434S/EU436T 11
EU434H/EU438R/EU440E 12 EU424N/EU434H 13 N434Y/Y436V/Q438R/S440E 14
N434Y/Y436V/Q438R/S440D 15 N434Y/Y436V/Q438K/S440E 16
N434Y/Y436V/Q438K/S440D 17 H433D/N434Y/Y436V/Q438R/S440E 18
H433D/N434Y/Y436V/Q438R/S440D 19 H433D/N434Y/Y436V/Q438K/S440E 20
H433D/N434Y/Y436V/Q438K/S440D 21 N434Y/Y436T/Q438R/S440E 22
N434Y/Y436T/Q438R/S440D 23 N434Y/Y436T/Q438K/S440E 24
N434Y/Y436T/Q438K/S440D 25 H433D/N434Y/Y436T/Q438R/S440E 26
H433D/N434Y/Y436T/Q438R/S440D 27 H433D/N434Y/Y436T/Q438K/S440E 28
H433D/N434Y/Y436T/Q438K/S440D
[0467] In addition to a substitution at any one of the positions
EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440, the Fc
region of the present invention may also comprise further
substitution of an amino acid at one or more of the following
positions: EU248, EU249, EU250, EU251, EU252, EU253, EU254, EU255,
EU256, EU257, EU305, EU306, EU307, EU308, EU309, EU310, EU311,
EU312, EU313, EU314, EU342, EU343, EU344, EU345, EU346, EU347,
EU348, EU349, EU350, EU351, EU352, EU380, EU381, EU382, EU383,
EU384, EU385, EU386, EU388, EU414, EU415, EU416, EU417, EU418,
EU419, EU420, EU421, EU423, EU425, EU427, EU428, EU429, EU430,
EU431, EU432, EU433, EU434, EU435, EU436, EU437, EU441, EU442,
EU443, and EU444.
[0468] Substituting an Fc region at any one of these positions may
reduce the binding affinity for a pre-existing ADA, in particular
for the rheumatoid factor, without negatively affecting the binding
affinity for FcRn.
[0469] Furthermore, the methods of the present invention may
further comprise the step of substituting the Fc region of the
antigen-binding molecule as described above at one or more of the
following positions:
[0470] EU248, EU249, EU250, EU251, EU252, EU253, EU254, EU255,
EU256, EU257, EU305, EU306, EU307, EU308, EU309, EU310, EU311,
EU312, EU313, EU314, EU342, EU343, EU344, EU345, EU346, EU347,
EU348, EU349, EU350, EU351, EU352, EU380, EU381, EU382, EU383,
EU384, EU385, EU386, EU388, EU414, EU415, EU416, EU417, EU418,
EU419, EU420, EU421, EU423, EU425, EU427, EU428, EU429, EU430,
EU431, EU432, EU433, EU434, EU435, EU436, EU437, EU441, EU442,
EU443, and EU444.
Weak or No Binding Activity for an Effector Receptor or a
Complement Protein
[0471] Binding to Fc gamma receptors or complement proteins may
also cause undesired effects (e.g. inappropriate platelet
activation). A modified antigen-binding molecule that does not bind
effector receptors such as Fc gamma RIIa receptor is safer and/or
more effective. Therefore, in a preferred embodiment, the modified
antigen-binding molecules of the present invention additionally
have a weak binding activity for an effector receptor or do not
bind to an effector receptor. Examples of an effector receptor
include but are not limited to activating Fc gamma receptors, in
particular Fc gamma receptor I, Fc gamma receptor II and Fc gamma
receptor III. Fc gamma receptor I includes Fc gamma receptor Ia, Fc
gamma receptor Ib, and Fc gamma receptor Ic, and subtypes thereof.
Fc gamma receptor II includes Fc gamma receptor IIa (which has two
allotypes R131 and H131) and Fc gamma receptor IIb. Fc gamma
receptor III includes Fc gamma receptor Ma (which has two
allotypes: V158 and F158) and Fc gamma receptor Mb (which has two
allotypes: Fc gamma IIIb-NA1 and Fc gamma IIIb-NA2). Antibodies
that have a weak binding activity for effector receptors or do not
bind to them are for examples antibodies comprising a silent Fc
region or antibodies without an Fc region (e.g. Fab, F(ab)'2, scFv,
sc(Fv)2, diabodies).
[0472] Examples for Fc regions having a weak or no binding activity
for effector receptors are e.g. described in Strohl et al. (Current
Opinion in Biotechnology (2009) 20(6), 685-691). In particular it
describes for example deglycosylated Fc regions (N297A, N297Q), and
examples of a silent Fc region, which are Fc regions engineered for
silenced (or immunosuppressive) effector functionality
(IgG1-L234A/L235A, IgG1-H268Q/A330S/P331S, IgG1-C226S/C229S,
IgG1-C226S/C229S/E233P/L234V/L235A, IgG1-L234F/L235E/P331S,
IgG2-V234A/G237A, IgG2-H268Q/V309L/A330S/A331S,
IgG4-L235A/G237A/E318A, IgG4-L236E). WO2008/092117 discloses
antibodies comprising silent Fc regions that comprise the
substitutions G236R/L328R, L235G/G236R, N325A/L328R, or N325L/L328R
(positions according to the EU numbering system). Furthermore, WO
2000/042072 discloses antibodies comprising silent Fc regions which
comprise substitutions at one or more of the positions EU233,
EU234, EU235, and EU237. WO 2009/011941 discloses antibodies
comprising silent Fc regions which comprise deletion of residues
from EU231 to EU238. Davis et al (Journal of Rheumatology (2007)
34(11): 2204-2210) discloses antibodies comprising silent Fc
regions which comprise the substitutions C220S/C226S/C229S/P238S.
Shields et al (Journal of Biological Chemistry (2001) 276 (9),
6591-6604) discloses antibodies comprising silent Fc regions which
comprise the substitution D265A.
[0473] The term "weak binding for effector receptors" refers to a
binding activity that is 95% or less, preferably 90% or less, 85%
or less, 80% or less, 75% or less, more preferably 70% or less, 65%
or less, 60% or less, 55% 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, 9% or less, 8% or less, 7% or less, 6% or less,
5% or less, 4% or less, 3% or less, 2% or less, 1% or less of the
binding activity of an intact IgG (or an antibody comprising an
intact Fc region) for the effector receptor. The binding activity
to an Fc gamma R preferably reduced by a factor of at least about
10 fold or more, about 50-fold or more, about 100-fold or more as
compared with the binding activity of an intact IgG (or an antibody
comprising an intact Fc region) for the effector receptor.
[0474] A silent Fc region is a modified Fc region comprising one or
more amino acid substitutions, insertions, additions and/or
deletions which reduce the binding for an effector receptor as
compared to an intact Fc region. The binding activity for an
effector receptor may be so much reduced that the Fc region does
not bind an effector receptor anymore. Examples of a silent Fc
region include but are not limited to Fc regions which comprise an
amino acid substitution at one or more of the positions selected
from the group consisting of: EU234, EU235, EU236, EU237, EU238,
EU239, EU265, EU266, EU267, EU269, EU270, EU271, EU295, EU296,
EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331, and
EU332.
[0475] In particular, a silent Fc region has a substitution at one
or more the positions selected from the group consisting of EU234,
EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269,
EU270, EU271, EU295, EU296, EU297, EU298, EU300, EU324, EU325,
EU327, EU328, EU329, EU331, and EU332 with an amino acid selected
from the list below. Preferably, a silent Fc region has a
substitution at one or more positions selected from the group
consisting of EU235, EU237, EU238, EU239, EU270, EU298, EU325, and
EU329 with an amino acid selected from the list below.
[0476] The amino acid at position EU234 is preferably replaced with
one of an amino acid selected from the group consisting of: Ala,
Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, and
Thr.
[0477] The amino acid at position EU235 is preferably replaced with
one of an amino acid selected from the group consisting of: Ala,
Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val and
Arg.
[0478] The amino acid at position EU236 is preferably replaced with
one of an amino acid selected from the group consisting of: Arg,
Asn, Gln, His, Leu, Lys, Met, Phe, Pro and Tyr.
[0479] The amino acid at position EU237 is preferably replaced with
one of an amino acid selected from the group consisting of: Ala,
Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val,
Tyr and Arg.
[0480] The amino acid at position EU238 is preferably replaced with
one of an amino acid selected from the group consisting of: Ala,
Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp and Arg.
[0481] The amino acid at position EU239 is preferably replaced with
one of an amino acid selected from the group consisting of: Gln,
His, Lys, Phe, Pro, Trp, Tyr and Arg.
[0482] The amino acid at position EU265 is preferably replaced with
one of an amino acid selected from the group consisting of: Ala,
Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp,
Tyr and Val.
[0483] The amino acid at position EU266 is preferably replaced with
one of an amino acid selected from the group consisting of: Ala,
Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp and
Tyr.
[0484] The amino acid at position EU267 is preferably replaced with
one of an amino acid selected from the group consisting of: Arg,
His, Lys, Phe, Pro, Trp and Tyr.
[0485] The amino acid at position EU269 is preferably replaced with
one of an amino acid selected from the group consisting of: Ala,
Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,
Trp, Tyr and Val.
[0486] The amino acid at position EU270 is preferably replaced with
one of an amino acid selected from the group consisting of: Ala,
Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,
Trp, Tyr and Val.
[0487] The amino acid at position EU271 is preferably replaced with
one of an amino acid selected from the group consisting of: Arg,
His, Phe, Ser, Thr, Trp and Tyr.
[0488] The amino acid at position EU295 is preferably replaced with
one of an amino acid selected from the group consisting of: Arg,
Asn, Asp, Gly, His, Phe, Ser, Trp and Tyr.
[0489] The amino acid at position EU296 is preferably replaced with
one of an amino acid selected from the group consisting of: Arg,
Gly, Lys and Pro.
[0490] The amino acid at position EU297 is preferably replaced with
Ala,
[0491] The amino acid at position EU298 is preferably replaced with
one of an amino acid selected from the group consisting of: Arg,
Gly, Lys, Pro, Trp and Tyr.
[0492] The amino acid at position EU300 is preferably replaced with
one of an amino acid selected from the group consisting of: Arg,
Lys and Pro.
[0493] The amino acid at position EU324 is preferably replaced with
Lys or Pro.
[0494] The amino acid at position EU325 is preferably replaced with
one of an amino acid selected from the group consisting of: Ala,
Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr, and Val.
[0495] The amino acid at position EU327 is preferably replaced with
one of an amino acid selected from the group consisting of: Arg,
Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and
Val.
[0496] The amino acid at position EU328 is preferably replaced with
one of an amino acid selected from the group consisting of: Arg,
Asn, Gly, His, Lys and Pro.
[0497] The amino acid at position EU329 is preferably replaced with
one of an amino acid selected from the group consisting of: Asn,
Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp,
Tyr, Val and Arg.
[0498] The amino acid at position EU330 is preferably replaced with
Pro or Ser.
[0499] The amino acid at position EU331 is preferably replaced with
one of an amino acid selected from the group consisting of: Arg,
Gly and Lys.
[0500] The amino acid at position EU332 is preferably replaced with
one of an amino acid selected from the group consisting of: Arg,
Lys and Pro.
[0501] Preferably, a silent Fc region comprises a substitution at
position EU235 with Lys or Arg, EU237 with Lys or Arg, EU238 with
Lys or Arg, EU239 with Lys or Arg, EU270 with Phe, EU298 with Gly,
EU325 with Gly or EU329 with Lys or Arg. More preferably, a silent
Fc region comprises a substitution at position EU235 with arginine
and at position EU239 with lysine. More preferably, it comprises
the substitutions L235R/S239K.
[0502] Furthermore, the modified antigen-binding molecules of the
present invention are preferably deglycosylated. More preferably,
the modified antigen-binding molecule of the present invention
comprises a mutation at a heavy chain glycosylation site to prevent
glycosylation at the site such as e.g. described in WO2005/03175.
Thus, in a preferred embodiment of the present invention, the
modified aglycosyl antigen-binding molecules are prepared by
modifying the heavy chain glycosylation site, i.e., introducing the
substitution N297Q or N297A (position according to EU numbering
system), and expressing the protein in an appropriate host cell.
For introducing a substitution a method as described in the
EXAMPLES can be used.
[0503] In a specific embodiment of the present invention, the
modified antigen-binding molecules of the present invention thereof
have a weak binding activity for a complement protein or do not
bind to a complement protein. Preferably, the complement protein is
C1q. A weak binding activity for a complement protein is preferably
a binding activity for a complement protein which is reduced by a
factor of about 10 fold or more, about 50-fold or more, about
100-fold or more as compared to the binding activity for a
complement protein of an intact IgG or an antibody comprising an
intact Fc region. The binding activity of an Fc region for a
complement protein can be reduced by modification of the amino acid
sequence such as amino acid substitutions, insertions, additions
and/or deletions
[0504] In a preferred embodiment of the present invention, the
antigen-binding molecule has an increased FcRn-binding affinity in
the acidic or neutral pH and has a weak or no binding activity for
an effector receptor and/or a complement protein. Preferably, such
an antigen-binding molecule comprises a substitution in the
FcRn-binding domain at
[0505] a) one or more positions selected from the group consisting
of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307,
EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436, and
[0506] b) at one or more positions selected from the group
consisting of: EU234, EU235, EU236, EU237, EU238, EU239, EU265,
EU266, EU267, EU269, EU270, EU271, EU295, EU296, EU297 EU298,
EU300, EU324, EU325, EU327, EU328, EU329, EU331, and EU332
(according to the EU numbering system). More preferably, the
modified antigen-binding molecule of the present invention having a
reduced or no binding activity for effector receptors and/or
complement proteins comprises one or more substitutions in the Fc
regions selected from the group consisting of a substitution at
position EU235 with Lys or Arg, at position EU237 with Lys or Arg,
at position EU238 with Lys or Arg, at position EU239 with Lys or
Arg, at position EU270 with Phe, EU298 with Gly, at position EU325
with Gly and at position EU329 with Lys or Arg. Even more
preferably, it comprises a substitution in the Fc region at
position EU235 with Arg and at position EU239 with Lys. And even
more preferably, it comprises the substitution combination
L235R/S239K in the Fc region.
[0507] Preferably, such antigen-binding molecules have also no
significantly increased binding activity for a pre-existing ADA.
Therefore, the antigen-binding molecule of the present invention
having a reduced or no binding activity for effector receptor(s)
and/or complement proteins further comprises an amino acid
substitutions at c) one or more positions selected from the group
consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and
EU440. In a more preferred embodiment of the present invention, the
modified antigen-binding molecules comprise three or more amino
acid substitutions in the FcRn-binding domain, wherein the three or
more substitutions are one of the combinations set forth in Tables
14 and 15.
TABLE-US-00014 TABLE 14 Substitution combinations that increase
FcRn-binding activity at neutral pH without significantly
increasing the binding activity for a pre-existing ADA, and
reducing the binding activity for effector receptor(s) and/or
complement proteins 1 L235R/S239K/M252Y/N434Y/Y436T 2
L235R/S239K/M252Y/N434Y/Y436V/Q438K/S440E 3
L235R/S239K/M252Y/N434Y/Y436V/Q438R/S440E 4
L235R/S239K/M252Y/N434Y/Y436T/Q438K/S440E 5
L235R/S239K/M252Y/N434Y/Y436T/Q438R/S440E 6
L235R/S239K/M252Y/N434Y/Y436F/Q438K/S440E 7
L235R/S239K/M252Y/N434Y/Y436F/Q438R/S440E 8
L235R/S239K/M252Y/N434Y/Y436V/Q438R/S440D 9
L235R/S239K/M252Y/N434Y/Y436V/Q438K/S440D 10
L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438R/S440D 11
L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438K/S440E 12
L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438R/S440E 13
L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438K/S440D 14
L235R/S239K/M252Y/S254T/T256E/T307Q/Q311A/H433D/N434Y/Y436V/Q438K/S440E
15
L235R/S239K/M252Y/S254T/T256E/V308P/H433D/N434Y/Y436V/Q438K/S440E
16 L235R/S239K/M252Y/H433D/N434W/Y436V/Q438R/S440E 17
L235R/S239K/M252Y/H433D/N434W/Y436V/Q438K/S440E 18
L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438K/S440E 19
L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438R/S440E 20
L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438K/S440D 21
L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438R/S440D 22
L235R/S239K/M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V/Q438K/S440E
23
L235R/S239K/M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V/Q438R/S440E
24
L235R/S239K/M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V/Q438K/S440D
25
L235R/S239K/M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V/Q438R/S440D
26
L235R/S239K/M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V/Q438K/S440E
27
L235R/S239K/M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V/Q438R/S440E
28
L235R/S239K/M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V/Q438K/S440D
29
L235R/S239K/M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V/Q438R/S440D
30
L235R/S239K/M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V/Q438R/S440E
31
L235R/S239K/M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V/Q438R/S440E
32
L235R/S239K/M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V/Q438K/S440E
33
L235R/S239K/M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V/Q438K/S440E
34
L235R/S239K/M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V/Q438K/S440D
35
L235R/S239K/M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V/Q438K/S440D
36
L235R/S239K/M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V/Q438R/S440D
37
L235R/S239K/M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V/Q438R/S440D
38 L235R/S239K/M252Y/S254T/T256E/H433A/N434Y/Y436V/Q438R/S440E 39
L235R/S239K/M252Y/S254T/T256E/H433K/N434Y/Y436V/Q438R/S440E 40
L235R/S239K/M252Y/S254T/T256E/H433P/N434Y/Y436V/Q438R/S440E 41
L235R/S239K/M252Y/S254T/T256E/H433R/N434Y/Y436V/Q438R/S440E 42
L235R/S239K/M252Y/S254T/T256E/H433S/N434Y/Y436V/Q438R/S440E 43
L235R/S239K/M252Y/S254T/T256E/H433A/N434Y/Y436V/Q438K/S440E 44
L235R/S239K/M252Y/S254T/T256E/H433A/N434Y/Y436V/Q438R/S440D 45
L235R/S239K/M252Y/S254T/T256E/H433A/N434Y/Y436V/Q438K/S440D 46
L235R/S239K/M252Y/S254T/T256E/H433K/N434Y/Y436V/Q438K/S440E 47
L235R/S239K/M252Y/S254T/T256E/H433K/N434Y/Y436V/Q438R/S440D 48
L235R/S239K/M252Y/S254T/T256E/H433K/N434Y/Y436V/Q438K/S440D 49
L235R/S239K/M252Y/S254T/T256E/H433P/N434Y/Y436V/Q438K/S440E 50
L235R/S239K/M252Y/S254T/T256E/H433P/N434Y/Y436V/Q438R/S440D 51
L235R/S239K/M252Y/S254T/T256E/H433P/N434Y/Y436V/Q438K/S440D 52
L235R/S239K/M252Y/S254T/T256E/H433R/N434Y/Y436V/Q438K/S440E 53
L235R/S239K/M252Y/S254T/T256E/H433R/N434Y/Y436V/Q438R/S440D 54
L235R/S239K/M252Y/S254T/T256E/H433R/N434Y/Y436V/Q438K/S440D 55
L235R/S239K/M252Y/S254T/T256E/H433S/N434Y/Y436V/Q438K/S440E 56
L235R/S239K/M252Y/S254T/T256E/H433S/N434Y/Y436V/Q438R/S440D 57
L235R/S239K/M252Y/S254T/T256E/H433S/N434Y/Y436V/Q438K/S440D 58
L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440E 59
L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E
TABLE-US-00015 TABLE 15 Substitution combinations that increase
FcRn-binding activity at acidic pH without significantly increasing
the binding activity for a pre-existing ADA, and reducing the
binding activity for effector receptor(s) and/or complement
proteins 1 L235R/S239K/N434Y/Y436V/Q438R/S440E 2
L235R/S239K/N434Y/Y436V/Q438R/S440D 3
L235R/S239K/N434Y/Y436V/Q438K/S440E 4
L235R/S239K/N434Y/Y436V/Q438K/S440D 5
L235R/S239K/H433D/N434Y/Y436V/Q438R/S440E 6
L235R/S239K/H433D/N434Y/Y436V/Q438R/S440D 7
L235R/S239K/H433D/N434Y/Y436V/Q438K/S440E 8
L235R/S239K/H433D/N434Y/Y436V/Q438K/S440D 9
L235R/S239K/N434Y/Y436T/Q438R/S440E 10
L235R/S239K/N434Y/Y436T/Q438R/S440D 11
L235R/S239K/N434Y/Y436T/Q438K/S440E 12
L235R/S239K/N434Y/Y436T/Q438K/S440D 13
L235R/S239K/H433D/N434Y/Y436T/Q438R/S440E 14
L235R/S239K/H433D/N434Y/Y436T/Q438R/S440D 15
L235R/S239K/H433D/N434Y/Y436T/Q438K/S440E 16
L235R/S239K/H433D/N434Y/Y436T/Q438K/S440D
Further Modifications
[0508] Furthermore, the antigen-binding molecule of the present
invention comprises in addition to the modifications described
above, at the position EU257 of the FcRn-binding domain not an
amino acid selected from the group consisting of: alanine, valine,
isoleucine, leucine, and threonine,
[0509] and/or at the position EU252 of the FcRn-binding domain not
a tryptophan. In other words, the preferred antigen-binding
molecule of the present invention comprises in addition to any of
the modifications described above at positions EU257 an alanine, a
valine, an isoleucine, a leucine, a threonine, an arginine, an
asparagine, an aspartic acid, a cysteine, a glutamic acid, a
glutamine, a glycine, a histidine, a lysine, a methione, a
phenylalanine, a proline, a serine, a tryptophan, or a tyrosine,
and at position EU252 an arginine, an asparagine, an aspartic acid,
a cysteine, a glutamic acid, a glutamine, a glycine, a histidine, a
lysine, a methione, a phenylalanine, a proline, a serine, or a
tyrosine
[0510] Also preferred is a modified FcRn-binding domain which
comprises in addition to the substitutions at any one of the herein
mentioned positions or combinations of positions, at position EU239
a lysine and/or at position EU270 a phenylalanine.
Antigen-Binding Molecule
[0511] The antigen-binding molecules of the present invention are
not particularly limited, as long as they include an
antigen-binding domain having a binding activity specific to a
target antigen and an FcRn-binding domain of the present invention.
Preferred antigen-binding domains comprise, for example, domains
having an antigen-binding region of an antibody. The
antigen-binding region of an antibody comprises, for example, CDRs.
The antigen-binding region of an antibody may contain all six CDRs
from the whole antibody, or one, two, or more CDRs. The
antigen-binding region of antibody comprise amino acid deletions,
substitutions, additions, and/or insertions, or it may comprise a
portion of CDR.
[0512] On the other hand, antigen-binding molecules of the present
invention include antigen-binding molecules that have an
antagonistic activity (antagonistic antigen-binding molecules),
antigen-binding molecules that have an agonistic activity
(agonistic antigen-binding molecule), and molecules having
cytotoxicity. In a preferred embodiment, the antigen-binding
molecules are antagonistic antigen-binding molecules, in
particular, antagonistic antigen-binding molecules that recognize
an antigen such as a receptor or cytokine.
[0513] The antigen-binding molecule of the present invention is
preferably an antibody. The antibodies preferred in the context of
the present invention include, for example, IgG antibodies. When
the antibody to be used is an IgG antibody, the type of IgG is not
particularly limited; thus, the IgG may belong to any isotype
(subclass) such as IgG1, IgG2, IgG3, or IgG4. For a human IgG1,
IgG2, IgG3, or IgG4 constant region, gene polymorphisms (allotypes)
are described in "Sequences of proteins of immunological interest,
NIH Publication No. 91-3242". These allotypes can also be used for
constant region in this application. Especially, for human IgG1,
both of the amino acids Asp-Glu-Leu (DEL) and Glu-Glu-Met (EEM) can
be used for residues in 356-358 in EU numbering. Similarly, for
human immunoglobulin kappa constant region, gene polymorphisms
(allotypes) are described in "Sequences of proteins of
immunological interest, NIH Publication No. 91-3242". These
allotypes can also be used for constant region in this application.
Furthermore, the antigen-binding molecules of the present invention
may include antibody constant region, and amino acid mutations may
be introduced into the constant region. Amino acid mutations to be
introduced include, for example, those potentiate or impair the
binding to Fcgamma receptor (Proc Natl Acad Sci USA. 2006 Mar. 14;
103(11): 4005-10), but are not limited to these examples.
Alternatively, it is also possible to alter the pH-dependent
binding by selecting an appropriate constant region such as of IgG2
(WO09125825).
[0514] When an antigen-binding molecule of the present invention is
an antibody, the antibody may be derived from any animal, such as a
mouse, human, rat, rabbit, goat, or camel. Preferably, the antibody
is a human antibody. Furthermore, the antibody may be an altered
antibody, for example, a chimeric antibody, and in particular, an
altered antibody that comprises an amino acid substitution in the
sequence of a humanized antibody, etc. The category of antibodies
contemplated by the present invention also include bispecific
antibodies, antibody modification products linked with various
molecules, and polypeptides that comprise antibody fragments
(particularly immunogenic and/or immunoreactive antibody
fragments). In a preferred embodiment, the antigen-binding molecule
is a monoclonal antibody.
[0515] "Chimeric antibodies" are antibodies prepared by combining
sequences derived from different animals. Specifically, a chimeric
antibody includes, for example, antibodies having heavy and light
chain variable (V) regions from a mouse antibody and heavy and
light chain constant (C) regions from a human antibody. Methods for
generating chimeric antibodies are known. In the case of a
human-mouse chimeric antibody, for example, a DNA encoding an
antibody V region may be linked to a DNA encoding a human antibody
C region; this can be inserted into an expression vector and
introduced into a host to produce the chimeric antibody.
[0516] "Humanized antibodies", also referred to as reshaped human
antibodies, are known in the art as antibodies in which
complementarity determining regions (CDRs) of an antibody derived
from a nonhuman mammal, for example, a mouse, are transplanted into
the CDRs of a human antibody. Methods for identifying CDRs are
known (Kabat et al., Sequence of Proteins of Immunological Interest
(1987), National Institute of Health, Bethesda, Md.; Chothia et
al., Nature (1989) 342: 877). General genetic recombination
technologies suitable for this purpose are also known (see European
Patent Application EP 125023; and WO 96/02576). Humanized
antibodies can be produced by known methods, for example, the CDR
of a mouse antibody can be determined, and a DNA encoding an
antibody in which the CDR is linked to the framework region (FR) of
a human antibody is obtained. Humanized antibodies can then be
produced using a system that uses conventional expression vectors.
Such DNAs can be synthesized by PCR, using as primers several
oligonucleotides prepared to have portions that overlap with the
end regions of both the CDR and FR (see the method described in WO
98/13388). Human antibody FRs linked via CDRs are selected such
that the CDRs form a suitable antigen binding site. If required,
amino acids in the FRs of an antibody variable region may be
altered so that the CDRs of the reshaped human antibody can form a
suitable antigen binding site (Sato et al., Cancer Res. (1993) 53:
10.01-6). Amino acid residues in the FRs that can be altered
include portions that directly bind to an antigen via non-covalent
bonds (Amit et al., Science (1986) 233: 747-53), portions that
influence or have an effect on the CDR structure (Chothia et al.,
J. Mol. Biol. (1987) 196: 901-17), and portions involved in VH-VL
interactions (EP 239400).
[0517] When the antigen-binding molecules of the present invention
are chimeric antibodies or humanized antibodies, the constant
regions of these antibodies are preferably derived from human
antibodies. For example, C-gamma1, C-gamma2, C-gamma3, and C-gamma4
can be used for the H chain, while C-kappa and C-lambda can be used
for the L chain. Moreover, if required, amino acid mutations may be
introduced into the human antibody C region to enhance or lower the
binding to Fc-gamma receptor or to improve antibody stability or
productivity. A chimeric antibody of the present invention
preferably includes a variable region of an antibody derived from a
nonhuman mammal and a constant region derived from a human
antibody. Meanwhile, a humanized antibody preferably includes CDRs
of an antibody derived from a nonhuman mammal and FRs and C regions
derived from a human antibody. The constant regions derived from
human antibodies preferably include a human FcRn-binding region.
Such antibodies include, for example, IgGs (IgG1, IgG2, IgG3, and
IgG4). The constant regions used for the humanized antibodies of
the present invention may be constant regions of antibodies of any
isotype. A constant region derived from human IgG1 is preferably
used, though it is not limited thereto. The FRs derived from a
human antibody, which are used for the humanized antibodies, are
not particularly limited either, and may be derived from an
antibody of any isotype.
[0518] The term "bispecific antibody" as used herein refers to an
antibody that has, in the same antibody molecule, variable regions
that recognize different epitopes. A bispecific antibody may be an
antibody that recognizes two or more different antigens, or an
antibody that recognizes two or more different epitopes on a same
antigen.
[0519] Furthermore, polypeptides including antibody fragments may
be, for example, scFv-Fc (WO 2005/037989), dAb-Fc, and Fc fusion
proteins. Antibody fragments in such polypeptides can be for
example Fab fragments, F(ab')2 fragments, scFvs (Nat Biotechnol.
2005 September; 23(9): 1126-36), domain antibodies (dAbs) (WO
2004/058821, WO 2003/002609), Fc region can be used as a human
FcRn-binding domain when a molecule includes an Fc region.
Alternatively, an FcRn-binding domain may be fused to these
molecules.
[0520] Further, antigen-binding molecules that are applicable to
the present invention can be or can comprise antibody-like
molecules (e.g. a fusion protein of an Fc region of the present
invention with an antibody-like molecule). An antibody-like
molecule (scaffold molecule, peptide molecule) is a molecule that
can exhibit functions by binding to a target molecule (Current
Opinion in Biotechnology (2006) 17: 653-658; Current Opinion in
Biotechnology (2007) 18: 1-10; Current Opinion in Structural
Biology (1997) 7: 463-469; Protein Science (2006) 15: 14-27), and
includes, for example, DARPins (WO 2002/020565), Affibody (WO
1995/001937), Avimer (WO 2004/044011; WO 2005/040229), and Adnectin
(WO 2002/032925). If these antibody-like molecules can bind to
target molecules in a pH-dependent or calcium-dependent manner
and/or have human FcRn-binding activity in the neutral pH range, it
is possible to facilitate antigen uptake into cells by
antigen-binding molecules, facilitate the reduction of plasma
antigen concentration by administering antigen-binding molecules,
and improve pharmacokinetics of the antigen-binding molecules, and
increase the number of antigens to which a single antigen-binding
molecule can bind.
[0521] Furthermore, the antigen-binding molecule can be a protein
resulting from fusion between an FcRn-binding domain of the present
invention and a receptor protein that binds to a target including a
ligand, and includes, for example, TNFR-Fc fusion proteins, IL1R-Fc
fusion proteins, VEGFR-Fc fusion proteins, and CTLA4-Fc fusion
proteins (Nat Med. 2003, January; 9(1): 47-52; BioDrugs. (2006)
20(3): 151-60). If these receptor-FcRn-binding domain fusion
proteins bind to a target molecule including a ligand in a
pH-dependent or calcium-dependent manner in addition to having
FcRn-binding activity in the neutral pH range, it is possible to
facilitate antigen uptake into cells by antigen-binding molecules,
facilitate the reduction of plasma antigen concentration by
administering antigen-binding molecules, and improve
pharmacokinetics of the antigen-binding molecules, and increase the
number of antigens to which a single antigen-binding molecule can
bind. A receptor protein is appropriately designed and modified so
as to include a binding domain of the receptor protein to a target
including a ligand. As referred to the examples hereinbefore (i.e.
TNFR-Fc fusion proteins, IL1R-Fc fusion proteins, VEGFR-Fc fusion
proteins and CTLA4-Fc fusion proteins) a soluble receptor molecule
comprising an extracellular domain of those receptor proteins that
is required for binding to those targets including ligands is
particularly preferred. Such designed and modified receptor
molecules are referred to as artificial receptors in the present
invention. Methods for designing and modifying a receptor molecule
to construct an artificial receptor molecule are known and indeed
conventional in the art.
[0522] Furthermore, the antibodies of the present invention can
have modified sugar chains. Antibodies with modified sugar chains
include, for example, antibodies with modified glycosylation (WO
99/54342), antibodies that are deficient in fucose that is added to
the sugar chain (WO 00/61739; WO 02/31140; WO 2006/067847;
WO2006/067913), and antibodies having sugar chains with bisecting
GlcNAc (WO 02/79255).
[0523] According to the Journal of Immunology (2009) 182:
7663-7671, the human FcRn-binding activity of intact human IgG1 in
the acidic pH range (pH 6.0) is KD 1.7 micromolar (microM), while
in the neutral pH range the activity is almost undetectable. Thus,
in a preferred embodiment, the antigen-binding molecule of the
present invention includes antigen-binding molecules whose human
FcRn-binding activity in the acidic pH range is stronger than KD
1.7 micromolar and is identical or stronger in the neutral pH range
than that of intact human IgG. In a more preferred embodiment its
binding activity for a pre-existing ADA in the neutral pH ranges is
not significantly increased compared to intact IgG1. The above KD
values are determined by the method described in the Journal of
Immunology (2009) 182: 7663-7671 (by immobilizing the
antigen-binding molecule onto a chip and loading human FcRn as an
analyte).
[0524] Dissociation constant (KD) can be used as a value of human
FcRn-binding activity. However, the human FcRn-binding activity of
intact human IgG has little human FcRn-binding activity in the
neutral pH range (pH 7.4). Accordingly, it is often difficult to
calculate the activity as KD. Methods for assessing whether the
human FcRn-binding activity is higher than that of intact human IgG
at pH 7.4 include assessment methods by comparing the intensities
of Biacore response after loading analytes at the same
concentration. Specifically, when the response after loading a
human FcRn chip immobilized with an antigen-binding molecule at pH
7.4 is stronger than the response after loading human FcRn onto a
chip immobilized with intact human IgG at pH 7.4, the human
FcRn-binding activity of the antigen-binding molecule is judged to
be higher than that of intact human IgG at pH 7.4.
[0525] In the context of the present invention, pH 7.0 can be used
as the neutral pH range. Using pH 7.0 as a neutral pH can
facilitate weak interaction between human FcRn and FcRn-binding
domain. As a temperature employed in the assay condition, a binding
affinity may be assessed at any temperature from 10 degrees Celsius
to 50 degrees Celsius. Preferably, a temperature ranging from 15
degrees Celsius to 40 degrees Celsius is employed in order to
determine the binding affinity between human FcRn-binding domain
and human FcRn. More preferably, any temperature ranging from 20
degrees Celsius to 35 degrees Celsius, like any one of 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 degrees C.
is also employed in order to determine the binding affinity between
human FcRn-binding domain and human FcRn. A temperature at 25
degrees C. described in EXAMPLE 5 of WO2011/122011 is one example
for the embodiment of this invention. In a preferred embodiment, an
interaction between human FcRn and FcRn-binding domain can be
measured at pH 7.0 and at 25 degrees C. as described in EXAMPLE 5
of WO2011/122011. Binding affinity of antigen-binding molecule to
human FcRn can be measured by Biacore as described in EXAMPLE 5 of
WO2011/122011.
[0526] Preferably the binding affinity at neutral pH ranges is
measured at pH 7.4, which is close to in vivo plasma (blood) pH. pH
7.0 can be used as an alternative to pH 7.4 when it is difficult to
assess the binding affinity between human FcRn-binding domain and
human FcRn due its low affinity at pH 7.4. Preferably the binding
affinity at acidic pH ranges is measured at pH 6.0, which is close
to the pH in early endosome in vivo. As a temperature employed in
the assay condition, a binding affinity between human FcRn-binding
domain and human FcRn may be assessed at any temperature from 10
degrees C. to 50 degrees C. Preferably, a temperature from 15
degrees C. to 40 degrees C. is employed in order to determine the
binding affinity between human FcRn-binding domain and human FcRn.
More preferably, any temperature at from 20 degrees C. to 35
degrees C., like any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, and 35 degrees C. is also employed in order to
determine the binding affinity between human FcRn-binding domain
and human FcRn. A temperature at 25 degrees C. is described for
example in Example 5 of WO2011/122011 and in the EXMAPLES of this
invention.
[0527] An intact human IgG1, IgG2, IgG3 or IgG4 is preferably used
as the reference intact human IgG to be compared with the
antigen-binding molecules for their human FcRn binding activity or
in vivo activity. Preferably, an antigen-binding molecule that
comprises the same antigen-binding domain as the antigen-binding
molecule of the interest and an intact human IgG Fc region as a
human FcRn-binding domain is used as reference. More preferably, an
intact human IgG1 is used as reference intact human IgG for
comparing its human FcRn binding activity or in vivo activity with
the human FcRn binding activity or in vivo activity of an
antigen-binding molecule of the present invention.
[0528] Conditions used in the assay for the antigen-binding or
human FcRn-binding activity other than pH can be appropriately
selected by those skilled in the art, and the conditions are not
particularly limited. For example, the conditions of using MES
buffer at 37 degrees C. as described in WO 2009/125825 may be used
to determine the activity. In another embodiment, Na-phosphate
buffer at 25 degrees C. as described in Example 4 or 5 of
WO2011/122011may be used to determine the activity. Meanwhile, the
antigen-binding activity and human FcRn-binding activity of
antigen-binding molecule can be determined by methods known to
those skilled in the art, for example, using Biacore (GE
Healthcare) or such. When the antigen is a soluble antigen, the
activity of an antigen-binding molecule to bind to the soluble
antigen can be determined by loading the antigen as an analyte onto
a chip immobilized with the antigen-binding molecule.
Alternatively, when the antigen is a membrane-type antigen, the
activity of the antigen-binding molecule to bind to the
membrane-type antigen can be determined by loading the
antigen-binding molecule as an analyte onto an antigen-immobilized
chip. The human FcRn-binding activity of an antigen-binding
molecule can be determined by loading human FcRn or the
antigen-binding molecule as an analyte onto a chip immobilized with
the antigen-binding molecule or human FcRn, respectively.
[0529] The present invention provides an antigen-binding molecule
of the present invention that comprises an antigen-binding domain
and a human Fc region having an increased FcRn-binding activity in
the neutral pH ranges. Preferably, its binding activity for a
pre-existing ADA in the neutral pH ranges is not significantly
increased. The FcRn-binding activity of such antigen-binding
molecule in the neutral pH ranges is preferably stronger than KD
3.2 micromolar. More preferably, the FcRn-binding activity in the
neutral pH range is stronger than 700 nanomolar, even more
preferably stronger than 500 nanomolar and most preferably,
stronger than 150 nanomolar. Preferably, the antigen-binding
molecule has an increased human FcRn-binding activity in the
neutral pH ranges and an antigen-binding activity that is lower in
the acidic pH range than in the neutral pH range or that is lower
at a low calcium concentration than at a high calcium concentration
condition. Preferably, binding activity of such an antigen-binding
molecule for a pre-existing ADA in the neutral pH ranges is not
significantly increased. The present invention also provides an
antigen-binding molecule of the present invention that comprises an
antigen-binding domain and a human FcRn-binding domain, wherein its
human FcRn-binding activity is increased in the neutral pH ranges,
further wherein the human FcRn-binding activity in the neutral pH
ranges is 28-fold stronger than that of an intact human IgG, more
preferably, the human FcRn-binding activity in the neutral pH
ranges is 38-fold stronger than that of an intact human IgG.
Preferably, binding activity of such an antigen-binding molecule
for a pre-existing ADA in the neutral pH ranges is not
significantly increased. The antigen-binding molecule of the
present invention with an increased FcRn-binding activity in the
neutral pH ranges. Preferably a binding activity for a pre-existing
ADA in the neutral pH ranges that is not significantly increased
preferably, has human FcRn-binding activity at pH 7.0 and at 25
degrees C. which is 28-fold stronger, preferably 38-fold stronger,
than intact human IgG than intact human IgG. Alternatively, the
human FcRn-binding activity of the antigen-binding molecule with an
increased FcRn binding activity at pH 7.0 and at 25 degrees C. is
preferably stronger than KD 3.2 micromolar. More preferably, the
FcRn-binding activity in at pH 7.0 and at 25 degrees Celsius is
stronger than 700 nanomolar, more preferably stronger than 500
nanomolar and most preferably, stronger than 150 nanomolar.
[0530] The present invention provides an antigen-binding molecule
of the present invention, comprising an antigen-binding domain and
a human Fc region of the present invention, with an increased
FcRn-binding activity in the acidic pH ranges and a binding
activity for a pre-existing ADA in the neutral pH ranges that is
not significantly increased. The present invention also provides an
antigen-binding molecule of the present invention comprising an
antigen-binding domain and a human FcRn-binding domain having an
increased human FcRn-binding activity in the acidic pH range and a
binding activity for a pre-existing ADA in the neutral pH ranges
that is not significantly increased as compared to the binding
activity for a pre-existing ADA of an intact IgG, wherein the human
FcRn-binding activity in the acidic pH ranges is in the range of
about 2-fold to about 100-fold stronger than the human FcRn-binding
activity of an intact human IgG. Preferably, the human FcRn-binding
activity of antigen-binding molecule of the present invention in
the acidic pH ranges is at least 10-fold stronger than the
FcRn-binding activity of an intact human IgG, more preferably, the
human FcRn-binding activity in the acidic pH ranges is at least
20-fold stronger than that of an intact human IgG. The
antigen-binding molecule of the present invention with an increased
FcRn-binding activity in the acidic pH ranges whereby its binding
activity for a pre-existing ADA in the neutral pH ranges is not
significantly increased has human FcRn-binding activity at pH 6.0
and at 25 degrees C. which is 10-fold stronger, preferably 20-fold
stronger, than intact human IgG.
[0531] The antigen-binding molecules of the present invention may
have an increased FcRn-binding activity in the neutral pH ranges as
well as an antigen-binding activity in the acidic pH range that is
lower than the antigen-binding activity in the neutral pH range or
an antigen-binding activity at a low calcium concentration that is
lower than the antigen-binding activity at a high calcium
concentration condition. Specific examples of such antigen-binding
molecules include those that have a higher binding activity for
human FcRn at pH 7.4 than an intact Ig, and whose antigen-binding
activity is lower at pH 5.8 than at pH 7.4 which are presumed to be
the in vivo pH of the early endosome and plasma, respectively. An
antigen-binding molecule whose antigen-binding activity is lower at
pH 5.8 than at pH 7.4 can also be referred to as an antigen-binding
molecule whose antigen-binding activity is stronger at pH 7.4 than
at pH 5.8. The value of KD (pH 5.8)/KD (pH 7.4), which is a ratio
of dissociation constant (KD) against an antigen at pH 5.8 and pH
7.4, is 1.5, 2, 3, 4, 5, 10, 15, 20, 50, 70, 80, 100, 500, 1000 or
10,000 preferably 2 or greater, more preferably 10 or greater, and
still more preferably 40 or greater. The upper limit of the KD (pH
5.8)/KD (pH 7.4) value is not particularly limited, and may be any
value, for example, 400, 1,000, or 10,000, as long as production is
possible using the technologies of those skilled in the art.
[0532] Also preferred are antigen-binding molecules of the present
invention that have an increased FcRn-binding activity in the
acidic pH ranges, as well as a lower antigen-binding activity in
the acidic pH range than that in the neutral pH range or a lower
antigen-binding activity at a low calcium concentration than that
at a high calcium concentration. Preferably, binding activity of
such an antigen-binding molecule for a pre-existing ADA in the
neutral pH ranges is not significantly increased. Specific examples
of such antigen-binding molecules include those that have a higher
binding activity for human FcRn at pH 5.8 to pH 6.0 than an IgG,
which is presumed to be the in vivo pH of the early endosome and
whose antigen-binding activity is lower at pH 5.8 than at pH 7.4.
An antigen-binding molecule whose antigen-binding activity is lower
at pH 5.8 than at pH 7.4 can also be referred to as an
antigen-binding molecule whose antigen-binding activity is weaker
at pH 5.8 than at pH 7.4. Preferably, an antigen-binding molecule
having an increased binding activity for FcRn in the acidic pH
ranges has stronger FcRn-binding activity than intact human IgG in
the neutral pH range.
[0533] The modified FcRn-binding domains of the present invention
are applicable to any antigen-binding molecules, regardless of the
type of target antigen.
[0534] An antigen-binding molecule of the present invention may
have other properties. For example, it may be an agonistic or
antagonistic antigen-binding molecule, provided that it has a) the
requisite increased human FcRn-binding activity neutral pH ranges,
or b) an increased human FcRn-binding activity for in the acidic
ranges and its binding activity for a pre-existing ADA is not
significantly increased. Preferably, the antigen-binding activity
of such an antigen-binding molecule is lower in the acidic pH range
than in the neutral pH range. Preferred antigen-binding molecules
of the present invention include, for example, antagonistic
antigen-binding molecules. Such an antagonistic antigen-binding
molecule is typically an antigen-binding molecule that inhibits
receptor-mediated intracellular signaling by blocking the binding
between ligand (agonist) and receptor.
[0535] Meanwhile, an antigen-binding molecule of the present
invention may recognize any antigen. Specific antigens recognized
by an antigen-binding molecule of the present invention include,
for example, the above-described receptor proteins (membrane-bound
receptors and soluble receptors), membrane antigens such as
cell-surface markers, and soluble antigens such as cytokines. Such
antigens include, for example, the antigens described below.
[0536] Antigen-binding molecules of the present invention
comprising an antigen-binding domain can utilize a difference of pH
as an environmental difference between plasma and endosome for
differential binding affinity of an antigen binding molecule to an
antigen at plasma and endosome (strong binding at plasma and weak
binding at endosome). Since environmental difference between plasma
and endosome is not limited to a difference of pH, pH dependent
binding property on binding of an antigen-binding molecule to an
antigen can be substituted by utilizing other factors whose
concentration is different within the plasma and the endosome, such
as for example the ionized calcium concentration. Such factor may
also be used to generate an antibody that binds to the antigen
within plasma but dissociates the antigen within endosome.
Therefore, the present invention also includes an antigen-binding
molecule comprising a human FcRn-binding domain, whose human
FcRn-binding activity is increased in the neutral pH ranges and
whose antigen-binding activity in the endosome is lower as compared
to the plasma. Preferably, the binding activity of these
antigen-binding molecules in the neutral pH ranges for a
pre-existing ADA is not significantly increased. The human
FcRn-binding activity of such an antigen-binding molecule is in the
plasma stronger than that of intact human IgG, and further the
antigen-binding domain of such an antigen-binding molecule has a
lower affinity for the antigen inside the endosome than in the
plasma. Preferably, the antigen-binding domain is an
antigen-binding domain whose antigen-binding activity in the acidic
pH range is lower than that in the neutral pH range (pH-dependent
antigen-binding domain) or an antigen-binding domain whose
antigen-binding activity is lower with a low calcium concentration
than under a high calcium concentration condition
(calcium-concentration-dependent antigen-binding domain). The
present invention also includes an antigen-binding molecule with a
human FcRn-binding domain, which has an increased human
FcRn-binding activity in the acidic pH ranges, and said
antigen-binding molecule further comprises an antigen-binding
domain which has a lower affinity for the antigen inside the
endosome than in the plasma, such that the human FcRn-binding
activity of the antigen-binding molecule in the endosome is
stronger than that of intact human IgG, and the antigen-binding
activity of the antigen-binding molecule in the endosome is
stronger than in the plasma. Preferably, the binding activity of
these antigen-binding molecules in the neutral pH ranges for a
pre-existing ADA is not significantly increased. Preferably, the
antigen-binding domain is an antigen-binding domain whose
antigen-binding activity in the acidic pH range is lower than that
in the neutral pH range (pH-dependent antigen-binding domain) or an
antigen-binding domain whose antigen-binding activity is lower with
a low calcium concentration than under a high calcium concentration
condition (calcium-concentration-dependent antigen-binding
domain).
[0537] The antigen-binding molecules of the present invention
facilitate antigen uptake into cells, in particular when the
antigen-binding molecules of the present invention comprising an
antigen-binding domain that is a pH-dependent antigen-binding
domain or a calcium-concentration-dependent antigen-binding domain.
The antigen-binding molecules are easily dissociated from the
antigen in the endosome, and then released to the outside of the
cell by binding to human FcRn. The antigen-binding molecules of the
present invention are presumed to bind easily to an antigen in the
plasma again. Thus, for example, when the antigen-binding molecule
of the present invention is a neutralizing antigen-binding
molecule, reduction of the plasma antigen concentration can be
facilitated by administering the molecule.
Antigen-Binding Domain
[0538] Preferably, the antigen-binding domain of the
antigen-binding molecule has a decreased affinity for the antigen
at an acidic pH or at low calcium ion concentration. More
preferably, the antigen-binding domain is a pH-dependent
antigen-binding domain or a ionized calcium concentration dependent
antigen-binding domain described herein.
A) pH-Dependent Antigen-Binding Domain,
[0539] Furthermore, the antigen-binding molecule of the present
invention comprises preferably a pH-dependent antigen-binding
domain whose antigen-binding activity in the acidic pH range is
lower than that in the neutral pH range. Said antigen-binding
molecule has preferably a lower antigen-binding activity in the
acidic pH range than in the neutral pH range. The binding activity
ratio is not limited, provided that the antigen-binding activity is
lower in the acidic pH range than in the neutral pH range. In a
preferred embodiment, the antigen-binding molecules of the present
invention include antigen-binding molecules whose antigen-binding
activity at pH 7.4 is twice or higher than that at pH 5.8,
preferably the antigen-binding activity at pH 7.4 is ten times or
higher than that at pH 5.8. In a still more preferred embodiment,
the antigen-binding molecules of the present invention include
antigen-binding molecules whose antigen-binding activity at pH 7.4
is 40 times or higher than that at pH 5.8.
[0540] Specific examples of antigen-binding molecules of the
present invention include the embodiments described in WO
2009/125825. In a preferred embodiment, the antigen-binding
molecule of the present invention comprising a pH-dependent
antigen-binding domain has an antigen-binding activity at pH 5.8
that is lower than that at pH 7.4, wherein the value of KD
(pH5.8)/KD (pH7.4), which is a ratio of KD for the antigen at pH
5.8 and that at pH 7.4, is preferably 2 or greater, more preferably
10 or greater, and still more preferably 40 or greater. The upper
limit of the KD (pH5.8)/KD (pH7.4) value is not particularly
limited, and may be any value, for example, 400, 1,000, or 10,000,
as long as production is possible using the technologies of those
skilled in the art.
[0541] In another preferred embodiment, the antigen-binding
molecule of the present invention whose antigen-binding activity at
pH 5.8 is lower than that at pH 7.4, has a value of KD (pH5.8)/KD
(pH7.4), which is a ratio of the KD for the antigen at pH 5.8 and
the KD for the antigen at pH 7.4, that is 2 or greater, more
preferably 5 or greater, even more preferably 10 or greater, and
still more preferably 30 or greater. The upper limit of the KD
(pH5.8)/KD (pH7.4) value is not particularly limited, and may be
any value, for example, 50, 100, or 200, provided that the
production is possible using the technologies of those skilled in
the art.
[0542] Conditions other than the pH at which the antigen-binding
activity, binding activity for a pre-existing ADA and human
FcRn-binding activity are measured can be appropriately selected by
those skilled in the art, and such conditions are not particularly
limited; however, the measurements can be carried out, for example,
under conditions of MES buffer and at 37 degrees C., as described
in the Examples. Furthermore, the antigen-binding activity of an
antigen-binding molecule can be determined by methods known to
those skilled in the art, for example, using Biacore T100 (GE
Healthcare) or the like, as described in the Examples.
[0543] Methods for reducing (impairing) the antigen-binding
activity of an antigen-binding molecule in the acidic pH range to
less than that the antigen-binding activity in the neutral pH range
(methods for conferring the pH-dependent binding ability) are not
particularly limited and suitable methods are known to the skilled
in the art. WO 2009/125825, for example, describes methods for
reducing (impairing) the antigen-binding activity in the acidic pH
range to less than that in the neutral pH range by substituting
histidine for an amino acid in the antigen-binding domain or
inserting histidine into the antigen-binding domain. It is further
known that an antibody can be conferred with a pH-dependent
antigen-binding activity by substituting histidine for an amino
acid in the antibody (FEBS Letter (1992) 309(1): 85-88). Other
suitable methods include methods for substituting non-natural amino
acids for amino acids in the antigen-binding domain or inserting
non-natural amino acids into the antigen-binding domain. It is
known that pKa can be artificially adjusted by using non-natural
amino acids (Angew. Chem. Int. Ed. 2005, 44, 34; Chem Soc Rev. 2004
Sep. 10, 33 (7): 422-30; Amino Acids. (1999) 16(3-4): 345-79). Any
non-natural amino acid may be used in context of the present
invention. In fact, it is possible to use non-natural amino acids
known to those skilled in the art.
[0544] In a preferred embodiment, the antigen-binding molecule of
the present invention comprising an antigen-binding domain with an
antigen-binding activity that is lower in the acidic pH range than
that in the neutral pH range, includes antigen-binding molecules in
which at least one amino acid in the antigen-binding molecule is
replaced with histidine or a non-natural amino acid, and/or in
which at least one histidine or a non-natural amino acid has been
inserted. The site into which the histidine or non-natural amino
acid mutation is introduced is not particularly limited and may be
any site deemed suitable by those of skilled in the art, provided
that the resultant antigen-binding activity in the acidic pH range
is weaker than that in the neutral pH range (the KD (in the acidic
pH range)/KD (in the neutral pH range) value is greater or the kd
(in the acidic pH range)/kd (in the neutral pH range) value is
greater) as compared to before substitution. Examples include
variable regions and CDRs of an antibody in the case the
antigen-binding molecule is an antibody. The number of amino acids
to be replaced with histidine or non-natural amino acid and the
number of amino acids to be inserted can be appropriately
determined by those skilled in the art. One amino acid may be
replaced with histidine or non-natural amino acid, or one amino
acid may be inserted, or two or more amino acids may be replaced
with histidine or non-natural amino acids, or two or more amino
acids may be inserted. Moreover, apart from the substitutions of
histidine or non-natural amino acid or insertion of histidine or of
non-natural amino acid, deletion, addition, insertion, and/or
substitution and such of other amino acids may also be
simultaneously carried out. Substitutions of histidine or
non-natural amino acid or insertion of histidine or of non-natural
amino acid may be carried out at random using a method such as
histidine scanning, which uses histidine instead of alanine in
alanine scanning which is known to those skilled in the art.
Antigen-binding molecules whose KD (pH5.8)/KD (pH7.4) or kd
(pH5.8)/kd (pH7.4) is increased as compared to before mutation can
be selected from antigen-binding molecules into which histidine or
non-natural amino acid mutation has been introduced at random.
[0545] Preferably, the binding activity of the antigen-binding
domain at neutral pH (i.e. pH7.4) is maintained. When the
antigen-binding activity of an antigen-binding molecule before
histidine or non-natural amino acid mutation is set as 100%, the
antigen-binding activity of the antigen-binding molecule at pH7.4
after histidine or non-natural amino acid mutation 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 at
pH 7.4 after histidine or non-natural amino acid mutation may be
stronger than the antigen-binding activity at pH 7.4 before
histidine or non-natural amino acid mutation. When the
antigen-binding activity of the antigen-binding molecule is
decreased due to substitution or insertion of histidine or
non-natural amino acid, the antigen-binding activity may be
adjusted by introducing substitution, deletion, addition, and/or
insertion and such of one or more amino acids into the
antigen-binding molecule so that the antigen-binding activity
becomes equivalent to that before histidine substitution or
insertion.
[0546] In the context of present invention, when the
antigen-binding molecule is an antibody, possible sites of
histidine or non-natural amino acid substitution include, for
example, CDR sequences and sequences responsible for the CDR
structure of an antibody, including, for example, the sites
described in WO 2009/125825.
[0547] Furthermore, the present invention provides antigen-binding
molecules having substitution of histidine or a non-natural amino
acid for at least one amino acid at one of the following sites
[0548] Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61,
H62, H63, H64, H65, H99, H100b, and H102
[0549] Light chain: L24, L27, L28, L32, L53, L54, L56, L90, L92,
and L94 H32, H61, L53, L90, and L94 of these alteration sites, are
presumed to be highly general alteration sites. The amino acid
positions are shown according to Kabat numbering (Kabat et al.,
Sequences of Immunological Interest. 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)). The Kabat
numbering system is generally used when referring to a residue in
the variable domain (approximately residues 1-107 of the light
chain and residues 1-113 of the heavy chain). Specifically
preferred combinations of sites for histidine or non-natural amino
acid substitutions include, for example, the combination of H27,
H31, and H35; the combination of H27, H31, H32, H35, H58, H62, and
H102; the combination of L32 and L53; and the combination of L28,
L32, and L53. Furthermore, preferred combinations of substitutions
sites in the heavy and light chains include, for example, the
combination of H27, H31, L32, and L53.
[0550] When the antigen is an IL-6 receptor (for example, human
IL-6 receptor), preferred alteration sites include but are not
particularly limited to the following:
[0551] Heavy chain: H27, H31, H32, H35, H50, H58, H61, H62, H63,
H64, H65, H100b, and H102
[0552] Light chain: L24, L27, L28, L32, L53, L56, L90, L92, and
L94
[0553] Specifically preferred combinations of sites for histidine
or non-natural amino acid substitution include, for example, the
combination of H27, H31, and H35; the combination of H27, H31, H32,
H35, H58, H62, and H102; the combination of L32 and L53; and the
combination of L28, L32, and L53. Furthermore, preferred
combinations of substitution sites in the heavy and light chains
include, for example, the combination of H27, H31, L32, and
L53.
[0554] Histidine or non-natural amino acids can be substituted at
one or more of the positions mentioned above.
[0555] Alternatively, the antigen-binding molecule of the present
invention may comprise an antibody constant region that was altered
so that the antigen-binding activity at pH 5.8 is lower than that
at pH 7.4. Methods for altering antibody constant regions contained
in the antigen-binding molecules are known and indeed conventional
to the skilled in the art. Specific examples of antibody constant
regions after alteration include the constant regions described in
the Examples in WO 2009/125825 (SEQ ID NOs: 11, 12, 13, and
14).
[0556] Meanwhile, methods for altering an antibody constant region
include, for example, methods for assessing various constant region
isotypes (IgG1, IgG2, IgG3, and IgG4) and selecting isotypes that
reduce the antigen-binding activity in the acidic pH range
(increase the dissociation rate in the acidic pH range) are known.
Such methods also include methods for reducing the antigen-binding
activity in the acidic pH range (increasing the dissociation rate
in the acidic pH range) by introducing amino acid substitutions
into the amino acid sequences of wild-type isotypes (amino acid
sequences of wild type IgG1, IgG2, IgG3, or IgG4). The sequence of
hinge region in the antibody constant region is considerably
different among isotypes (IgG1, IgG2, IgG3, and IgG4), and the
difference in the hinge region amino acid sequence has a great
impact on the antigen-binding activity. Thus, it is possible to
select an appropriate isotype to reduce the antigen-binding
activity in the acidic pH range (increase the dissociation rate in
the acidic pH range) depending on the type of antigen or epitope.
Furthermore, since the difference in the hinge region amino acid
sequence has a great impact on the antigen-binding activity,
preferred amino acid substitution sites in the amino acid sequences
of wild type isotypes are presumed to be within the hinge
region.
[0557] The above-described methods can be used to produce
antigen-binding molecules whose antigen-binding activity in the
acidic pH range is reduced (weakened) to less than that in the
neutral pH range (antigen-binding molecules that bind in a
pH-dependent manner) by amino acid substitution or insertion from
antigen-binding molecules that do not have such property. Other
methods include methods for directly obtaining antigen-binding
molecules having the above-described property. For example,
antibodies having a desired property of interest may be directly
selected by screening using the pH-dependent antigen binding as an
indicator from antibodies obtained by immunizing animals (mice,
rats, hamsters, rabbits, human immunoglobulin-transgenic mice,
human immunoglobulin-transgenic rats, human
immunoglobulin-transgenic rabbits, llamas, camels, etc.) with an
antigen. Antibodies can be generated by hybridoma technology or
B-cell cloning technology (Bernasconi et al, Science (2002) 298,
2199-2202; WO2008/081008) which are methods known to those skilled
in the art, but not limited thereto. Alternatively, antibodies that
have the property of interest may be directly selected by screening
using the pH-dependent antigen binding as an indicator from a
library of presenting antigen-binding domain in vitro. Such library
includes human naive library, immunized library from non-human
animal and human, semi-synthetic library and synthetic library
which are libraries known to those skilled in the art (Methods Mol
Biol. 2002; 178: 87-100; J Immunol Methods. 2004 June; 289(1-2):
65-80; and Expert Opin Biol Ther. 2007 May; 7(5): 763-79), but not
limited thereto. However, the methods are not particularly limited
to these examples.
B) Ionized Calcium-Dependent Antigen-Binding Domain
[0558] In another preferred embodiment, the antigen-binding
molecule of the present invention comprises a calcium-ion dependent
antigen-binding domain. The antigen-binding activity of such an
antigen-binding molecule depends of the calcium concentration,
whereby the antigen-binding activity at a low calcium concentration
is lower than that at a high calcium concentration.
[0559] Preferably, the antigen-binding activity includes the
antigen-binding activity at an ionized calcium concentration of 0.5
to 10 micromolar. More preferable ionized calcium concentrations
include the ionized calcium concentration in the early endosome in
vivo. Specifically, the antigen-binding activity includes the
activity at 1 to 5 micromolar. Meanwhile, the antigen-binding
activity of an antigen-binding molecule at a high calcium
concentration is not particularly limited, provided that it is the
antigen-binding activity at an ionized calcium concentration of 100
micromolar to 10 mM. Preferably, the antigen-binding activity
includes the antigen-binding activity at an ionized calcium
concentration of 200 micromolar to 5 mM. Preferably, a low calcium
concentration is an ionized calcium concentration of 0.1 to 30
micromolar, and a high calcium concentration is an ionized calcium
concentration of 100 micromolar to 10 mM.
[0560] Preferably, the low calcium concentration is an
intraendosomal concentration of ionized calcium, and the high
calcium concentration is a plasma concentration of ionized calcium.
More specifically, the antigen-binding molecules comprising said
calcium-dependent antigen-binding domain include antigen-binding
molecules whose antigen-binding activity at the ionized calcium
concentration in the early endosome in vivo (a low calcium
concentration of such as 1 to 5 micromolar) is lower than that at
the ionized calcium concentration in plasma in vivo (a high calcium
concentration of such as 0.5 to 2.5 mM).
[0561] With respect to the antigen-binding activity of an
antigen-binding molecule whose antigen-binding activity at a low
calcium concentration is lower than that at a high calcium
concentration, there is no limitation on this difference in the
antigen-binding activity, provided that the antigen-binding
activity at a low calcium concentration is lower than that at a
high calcium concentration. It is even acceptable that the
antigen-binding activity of an antigen-binding molecule is only
slightly lower under a low calcium concentration condition.
[0562] In a preferred embodiment, for an antigen-binding molecule
of the present invention whose antigen-binding activity at a low
calcium concentration (low Ca) is lower than that at a high calcium
concentration (high Cal), the value of KD (low Ca)/KD (high Ca),
which is the KD ratio between low and high calcium concentration,
is 2 or more, preferably the value of KD (low Ca)/KD (high Ca) is
10 or more, and more preferably the value of KD (low Ca)/KD (high
Ca) is 40 or more. The upper limit of the KD (low Ca)/KD (high Ca)
value is not particularly limited, and may be any value such as
400, 1,000, and 10,000 provided that it can be produced by
techniques known to those skilled in the art.
[0563] In another preferred embodiment, for an antigen-binding
molecule comprising a calcium-dependent antigen-binding domain
whose antigen-binding activity at a low calcium concentration is
lower than that at a high calcium concentration, the value of kd
(low Ca)/kd (high Ca), which is the ratio of kd for an antigen
between a low calcium concentration condition and pH 7.4, is 2 or
more, preferably the value of kd (low Ca)/kd (high Ca) is 5 or
more, more preferably the value of kd (low Ca)/kd (high Ca) is 10
or more, and still more preferably the value of kd (low Ca)/kd
(high Ca) is 30 or more. The upper limit of kd (low Ca)/kd (high
Ca) value is not particularly limited, and may be any value such as
50, 100, and 200 as long as it can be produced by techniques known
to those skilled in the art.
[0564] The antigen-binding activity of an antigen-binding molecule
can be determined by methods known to those skilled in the art.
Appropriate conditions besides ionized calcium concentration can be
selected by those skilled in the art. The antigen-binding activity
of an antigen-binding molecule can be assessed by using KD
(dissociation constant), apparent KD (apparent dissociation
constant), dissociation rate kd (dissociation rate), apparent kd
(apparent dissociation: apparent dissociation rate), or the like.
They can be determined by methods known to those skilled in the
art, for example, using Biacore (GE Healthcare), Scatchard plot,
FACS, or such.
[0565] Antigen-binding molecules to be screened by the screening
method of the present invention may be any antigen-binding
molecules. It is possible to screen, for example, antigen-binding
molecules having a natural sequence or antigen-binding molecules
having an amino acid sequence with a substitution. Antigen-binding
molecules comprising a calcium-ion dependent antigen-binding domain
to be screened by the screening method of the present invention may
be prepared by any methods. It is possible to use, for example,
preexisting antibodies, preexisting libraries (phage libraries,
etc.), and antibodies and libraries prepared from B cells of
immunized animals or hybridomas prepared by immunizing animals,
antibodies or libraries obtained by introducing amino acids capable
of chelating calcium (for example, aspartic acid or glutamic acid)
or non-natural amino acid mutations into such antibodies or
libraries (libraries with high content of non-natural amino acids
or amino acids capable of chelating calcium (for example, aspartic
acid or glutamic acid), libraries introduced with non-natural amino
acid mutations or mutations with amino acids capable of chelating
calcium (for example, aspartic acid or glutamic acid) at specific
sites, or such), or the like.
[0566] An antigen-binding molecule whose antigen-binding activity
under a low calcium concentration condition is lower than that
under a high calcium concentration condition can be readily
screened, identified and isolated using methods conventional in the
art (see e.g. PCT application no. PCT/JP2011/077619. Examples of
such screening methods include the step of assaying for an
antigen-binding molecule having at least one function selected
from:
[0567] (i) the function to promote uptake of an antigen into
cells;
[0568] (ii) the function to bind to an antigen two or more
times;
[0569] (iii) the function to promote reduction of the plasma
antigen concentration; and (iv) the function of superior plasma
retention.
[0570] Specifically, the present invention provides methods of
screening for an antigen-binding molecule comprising a calcium-ion
dependent antigen-binding domain, which comprises the steps of:
[0571] (a) determining the antigen-binding activity of an
antigen-binding molecule under a low calcium concentration
condition;
[0572] (b) determining the antigen-binding activity of the
antigen-binding molecule under a high calcium concentration
condition; and
[0573] (c) selecting an antigen-binding molecule whose
antigen-binding activity under the low calcium concentration
condition is lower than that under the high calcium concentration
condition.
[0574] A method for producing an antigen-binding molecule with a
calcium-ion dependent antigen-binding domain is for example a
method comprising the steps of:
[0575] (a) determining the antigen-binding activity of an
antigen-binding molecule under a low calcium concentration
condition;
[0576] (b) determining the antigen-binding activity of the
antigen-binding molecule under a high calcium concentration
condition; and
[0577] (c) selecting an antigen-binding molecule whose
antigen-binding activity under the low calcium concentration
condition is lower than that under the high calcium concentration
condition.
[0578] Another method of producing an antigen-binding molecule with
a calcium-ion dependent antigen-binding domain is the method
comprising the steps of:
[0579] (a) contacting an antigen with an antigen-binding molecule
or a library of antigen-binding molecules under a high calcium
concentration condition;
[0580] (b) obtaining an antigen-binding molecule that bound to the
antigen in step (a);
[0581] (c) allowing the antigen-binding molecule obtained in step
(b) to stand under a low calcium concentration condition;
[0582] (d) obtaining an antigen-binding molecule whose
antigen-binding activity in step (c) is lower than the activity for
the selection in step (b);
[0583] (e) obtaining a gene encoding the antigen-binding molecule
obtained in step (d); and
[0584] (f) producing the antigen-binding molecule using the gene
obtained in step (e).
[0585] Steps (a) to (e) may be repeated two or more times. Thus,
the present invention provides the methods further comprising the
step of repeating steps (a) to (e) two or more times in the
above-described methods. The number of repetitions of steps (a) to
(e) is not particularly limited; however, the number is generally
ten or less.
[0586] Antigen-binding molecules that are used in the production
methods of the present invention may be prepared by any
conventional method. For example, it is possible to use
pre-existing antibodies, pre-existing libraries (phage libraries
and the like), antibodies and libraries that are prepared from
hybridomas obtained by immunizing animals or from B cells of
immunized animals, antibodies and libraries prepared by introducing
histidine or non-natural amino acid mutations into the
above-described antibodies and libraries (libraries with high
content of histidine or non-natural amino acid, libraries
introduced with histidine or non-natural amino acid at specific
sites, and the like), and such.
[0587] Further methods to screen such calcium-ion dependent
antigen-binding molecules or calcium-ion dependent antigen-binding
domains are described in the PCT application no.
PCT/JP2011/077619.
Antigens
[0588] Antigens that are recognized by antigen-binding molecules of
the present invention, such as the antibodies of the present
invention, are not particularly limited. Such antigen-binding
molecules of the present invention may recognize any antigen.
Specific examples of an antigen that is recognized by the
antigen-binding molecule of the present invention include but are
not limited to: 17-IA, 4-1 BB, 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, ADAM8, ADAM9, ADAMTS, ADAMTS4,
ADAMTS5, Addressins, adiponectin, ADP ribosyl cyclase-1, aFGF, AGE,
ALCAM, ALK, ALK-1, ALK-7, allergen, alpha1-antichemotrypsin,
alpha1-antitrypsin, alpha-synuclein, alpha-V/beta-1 antagonist,
aminin, amylin, amyloid beta, amyloid immunoglobulin heavy chain
variable region. amyloid immunoglobulin light chain variable
region, Androgen, ANG, angiotensinogen, Angiopoietin ligand-2,
anti-Id, antithrombinIII, Anthrax, APAF-1, APE, APJ, apo A1, apo
serum amyloid A, Apo-SAA, APP, APRIL, AR, ARC, ART, Artemin,
ASPARTIC, Atrial natriuretic factor, Atrial natriuretic peptide,
atrial natriuretic peptides A, atrial natriuretic peptides B,
atrial natriuretic peptides C, av/b3 integrin, Axl, B7-1, B7-2,
B7-H, BACE, BACE-1, Bacillus anthracis protective antigen, Bad,
BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF,
b-ECGF, beta-2-microglobulin, betalactamase, bFGF, BID, Bik, BIM,
BLC, BL-CAM, BLK, B-lymphocyte Stimulator (BIyS), 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), BMPR, BMPR-IA (ALK-3), BMPR-IB
(ALK-6), BMPR-II (BRK-3), BMPs, BOK, Bombesin, Bone-derived
neurotrophic factor, bovine growth hormone, BPDE, BPDE-DNA, BRK-2,
BTC, B-lymphocyte cell adhesion molecule, C10, C1-inhibitor, C1q,
C3, C3a, C4, C5, C5a (complement 5a), CA125, CAD-8, Cadherin-3,
Calcitonin, cAMP, Carbonic anhydrase-IX, carcinoembryonic antigen
(CEA), carcinoma-associated antigen, Cardiotrophin-1, 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/I-309, CCL11/Eotaxin, CCL12/MCP-5,
CCL13/MCP-4, CCL14/HCC-1, CCL15/HCC-2, CCL16/HCC-4, CCL17/TARC,
CCL18/PARC, CCL19/ELC, CCL2/MCP-1, CCL20/MIP-3-alpha, CCL21/SLC,
CCL22/MDC, CCL23/MPIF-1, CCL24/Eotaxin-2, CCL25/TECK,
CCL26/Eotaxin-3, CCL27/CTACK, CCL28/MEC, CCL3/M1P-1-alpha,
CCL3L1/LD-78-beta, CCL4/MIP-1-beta, CCL5/RANTES, CCL6/C10,
CCL7/MCP-3, CCL8/MCP-2, CCL9/10/MTP-1-gamma, CCR, CCR1, CCR10,
CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD10, CD105,
CD11a, CD11b, CD11c, CD123, CD13, CD137, CD138, CD14, CD140a,
CD146, CD147, CD148, CD15, CD152, CD16, CD164, CD18, CD19, CD2,
CD20, CD21, CD22, CD23, CD25, CD26, CD27L, CD28, CD29, CD3, CD30,
CD30L, CD32, CD33 (p67 proteins), CD34, CD37, CD38, CD3E, CD4,
CD40, CD40L, CD44, CD45, CD46, CD49a, CD49b, CD5, CD51, CD52, CD54,
CD55, CD56, CD6, CD61, CD64, CD66e, CD7, CD70, CD74, CD8, CD80
(B7-1), CD89, CD95, CD105, CD158a, CEA, CEACAM5, CFTR, cGMP, CGRP
receptor, CINC, CKb8-1, Claudin18, CLC, Clostridium botulinum
toxin, Clostridium difficile toxin, Clostridium perfringens toxin,
c-Met, CMV, CMV UL, CNTF, CNTN-1, complement factor 3 (C3),
complement factor D, corticosteroid-binding globulin, Colony
stimulating factor-1 receptor, COX, C-Ret, CRG-2, CRTH2, CT-1,
CTACK, CTGF, CTLA-4, CX3CL1/Fractalkine, CX3CR1, CXCL,
CXCL1/Gro-alpha, CXCL10, CXCL11/I-TAC, CXCL12/SDF-1-alpha/beta,
CXCL13/BCA-1, CXCL14/BRAK, CXCL15/Lungkine. CXCL16, CXCL16,
CXCL2/Gro-beta CXCL3/Gro-gamma, CXCL3, CXCL4/PF4, CXCL5/ENA-78,
CXCL6/GCP-2, CXCL7/NAP-2, CXCL8/IL-8, CXCL9/Mig, CXCL1O/IP-10,
CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cystatin C,
cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN,
Decay accelerating factor, Delta-like protein ligand 4,
des(1-3)-IGF-1 (brain IGF-1), Dhh, DHICA oxidase, Dickkopf-1,
digoxin, Dipeptidyl peptidase IV, DK1, DNAM-1, Dnase, Dpp,
DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR
(ErbB-1), EGF like domain containing protein 7, Elastase, elastin,
EMA, EMMPRIN, ENA, ENA-78, Endosialin, endothelin receptor,
endotoxin, Enkephalinase, eNOS, Eot, Eotaxin, Eotaxin-2, eotaxini,
EpCAM, Ephrin B2/EphB4, Epha2 tyrosine kinase receptor, epidermal
growth factor receptor (EGFR), ErbB2 receptor, ErbB3 tyrosine
kinase receptor, ERCC, erythropoietin (EPO), Erythropoietin
receptor, E-selectin, ET-1, Exodus-2, F protein of RSV, F10, F11,
F12, F13, F5, F9, Factor Ia, Factor IX, Factor Xa, Factor VII,
factor VIII, Factor VIIIc, Fas, FcalphaR, FcepsilonRI, FcgammaIIb,
FcgammaRI, FcgammaRIIa, FcgammaRIIIa, FcgammaRIIIb, FcRn, FEN-1,
Ferritin, FGF, FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8,
FGF-acidic, FGF-basic, FGFR, FGFR-3, Fibrin, fibroblast activation
protein (FAP), fibroblast growth factor, fibroblast growth
factor-10, fibronectin, FL, FLIP, Flt-3, FLT3 ligand, Folate
receptor, follicle stimulating hormone (FSH), Fractalkine (CX3C),
free heavy chain, free light chain, FZD1, FZD10, FZD2, FZD3, FZD4,
FZD5, FZD6, FZD7, FZD8, FZD9, G250, Gas 6, GCP-2, GCSF, G-CSF,
G-CSF receptor, GD2, GD3, GDF, GDF-1, GDF-15 (MIC-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, GDNF, Gelsolin, GFAP,
GF-CSF, GFR-alpha1, GFR-alpha2, GFR-alpha3, GF-beta1, gH envelope
glycoprotein, GITR, Glucagon, Glucagon receptor, Glucagon-like
peptide 1 receptor, Glut 4, Glutamate carboxypeptidase II,
glycoprotein hormone receptors, glycoprotein IIb/IIIa (GP
IIb/IIIa), Glypican-3, GM-CSF, GM-CSF receptor, gp130, gp140, gp72,
granulocyte-CSF (G-CSF), GRO/MGSA, Growth hormone releasing factor,
GRO-beta, GRO-gamma, H. pylori, Hapten (NP-cap or NIP-cap), HB-EGF,
HCC, HCC 1, HCMV gB envelope glycoprotein, HCMV UL, Hemopoietic
growth factor (HGF), Hep B gp120, heparanase, heparin cofactor II,
hepatic growth factor, Bacillus anthracis protective antigen,
Hepatitis C virus E2 glycoprotein, Hepatitis E, Hepcidin, Her1,
Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex
virus (HSV) gB glycoprotein, HGF, HGFA, High molecular weight
melanoma-associated antigen (HMW-MAA), HIV envelope proteins such
as GP120, HIV MIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM,
HMGB-1, HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human cardiac
myosin, human cytomegalovirus (HCMV), human growth hormone (hGH),
human serum albumin, human tissue-type plasminogen activator
(t-PA), Huntingtin, HVEM, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS,
IFN-alpha, IFN-beta, IFN-gamma, IgA, IgA receptor, IgE, IGF, IGF
binding proteins, IGF-1, IGF-1 R, IGF-2, IGFBP, IGFR, IL, IL-1,
IL-10, IL-10 receptors, IL-11, IL-11 receptors, IL-12, IL-12
receptors, IL-13, IL-13 receptors, IL-15, IL-15 receptors, IL-16,
IL-16 receptors, IL-17, IL-17 receptors, IL-18 (IGIF), IL-18
receptors, IL-1alpha, IL-1beta, IL-1 receptors, IL-2, IL-2
receptors, IL-20, IL-20 receptors, IL-21, IL-21 receptors, IL-23,
IL-23 receptors, IL-2 receptors, IL-3, IL-3 receptors, IL-31, IL-31
receptors, IL-3 receptors, IL-4, IL-4 receptors IL-5, IL-5
receptors, IL-6, IL-6 receptors, IL-7, IL-7 receptors, IL-8, IL-8
receptors, IL-9, IL-9 receptors, immunoglobulin immune complex,
immunoglobulins, INF-alpha, INF-alpha receptors, INF-beta, INF-beta
receptors, INF-gamma, INF-gamma receptors, IFN type-I, IFN type-I
receptor, influenza, inhibin, Inhibin alpha, Inhibin beta, iNOS,
insulin, Insulin A-chain, Insulin B-chain, Insulin-like growth
factor 1, insulin-like growth factor 2, insulin-like growth factor
binding proteins, integrin, integrin alpha2, integrin alpha3,
integrin alpha4, integrin alpha4/beta1, integrin alpha-V/beta-3,
integrin alpha-V/beta-6, integrin alpha4/beta7, integrin
alpha5/beta1, integrin alpha5/beta3, integrin alpha5/beta6,
integrin alpha-delta (alphaV), integrin alpha-theta, integrin
beta1, integrin beta2, integrin beta3 (GPIIb-IIIa), IP-10, I-TAC,
JE, kalliklein, Kallikrein 11, Kallikrein 12, Kallikrein 14,
Kallikrein 15, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein
L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, kallistatin, KC,
KDR, Keratinocyte Growth Factor (KGF), Keratinocyte Growth Factor-2
(KGF-2), KGF, killer immunoglobulin-like receptor, kit ligand (KL),
Kit tyrosine kinase, laminin 5, LAMP, LAPP (Amylin, islet-amyloid
polypeptide), LAP (TGF-1), latency associated peptide, Latent
TGF-1, Latent TGF-1 bp1, LBP, LDGF, LDL, LDL receptor, LECT2,
Lefty, Leptin, leutinizing hormone (LH), Lewis-Y antigen, Lewis-Y
related antigen, LFA-1, LFA-3, LFA-3 receptors, Lfo, LIF, LIGHT,
lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1,
Lung surfactant, Luteinizing hormone, Lymphotactin, Lymphotoxin
Beta Receptor, Lysosphingolipid receptor, Mac-1, macrophage-CSF
(M-CSF), MAdCAM, MAG, MAP2, MARC, maspin, MCAM, MCK-2, MCP, MCP-1,
MCP-2, MCP-3, MCP-4, MCP-I (MCAF), M-CSF, MDC, MDC (67 a.a.), MDC
(69 a.a.), megsin, Mer, MET tyrosine kinase receptor family,
METALLOPROTEASES, Membrane glycoprotein OX2, Mesothelin, MGDF
receptor, MGMT, MHC (HLA-DR), microbial protein, MIF, MIG, MIP,
MIP-1 alpha, MIP-1 beta, MIP-3 alpha, MIP-3 beta, MIP-4, 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, monocyte attractant protein,
monocyte colony inhibitory factor, mouse gonadotropin-associated
peptide, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, mucin (Mud),
Muellerian-inhibiting substance, Mug, MuSK, Myelin associated
glycoprotein, myeloid progenitor inhibitor factor-1 (MPIF-I), NAIP,
Nanobody, NAP, NAP-2, NCA 90, NCAD, N-Cadherin, NCAM, Neprilysin,
Neural cell adhesion molecule, neroserpin, Neuronal growth factor
(NGF), Neurotrophin-3, Neurotrophin-4, Neurotrophin-6, Neuropilin
1, Neurturin, NGF-beta, NGFR, NKG20, N-methionyl human growth
hormone, nNOS, NO, Nogo-A, Nogo receptor, non-structural protein
type 3 (NS3) from the hepatitis C virus, NOS, Npn, NRG-3, NT, NT-3,
NT-4, NTN, OB, OGG1, Oncostatin M, OP-2, OPG, OPN, OSM, OSM
receptors, osteoinductive factors, osteopontin, OX40L, OX40R,
oxidized LDL, p150, p95, PADPr, parathyroid hormone, PARC, PARP,
PBR, PBSF, PCAD, P-Cadherin, PCNA, PCSK9, PDGF, PDGF receptor,
PDGF-AA, PDGF-AB, PDGF-BB, PDGF-D, PDK-1, PECAM, PEDF, PEM, PF-4,
PGE, PGF, PGI2, PGJ2, PIGF, PIN, PLA2, Placenta growth factor,
placental alkaline phosphatase (PLAP), placental lactogen,
plasminogen activator inhibitor-1, platelet-growth factor, plgR,
PLP, poly glycol chains of different size (e.g. PEG-20, PEG-30,
PEG40), PP14, prekallikrein, prion protein, procalcitonin,
Programmed cell death protein 1, proinsulin, prolactin, Proprotein
convertase PC9, prorelaxin, prostate specific membrane antigen
(PSMA), Protein A, Protein C, Protein D, Protein S, Protein Z, PS,
PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PTN, P-selectin glycoprotein
ligand-1, R51, RAGE, RANK, RANKL, RANTES, relaxin, Relaxin A-chain,
Relaxin B-chain, renin, respiratory syncytial virus (RSV) F, Ret,
reticulon 4, Rheumatoid factors, RLI P76, RPA2, RPK-1, RSK, RSV
Fgp, 5100, RON-8, SCF/KL, SCGF, Sclerostin, SDF-1, SDF1 alpha, SDF1
beta, SERINE, Serum Amyloid P, Serum albumin, sFRP-3, Shh, Shiga
like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD,
SPARC, sphingosine 1-phosphate receptor 1, Staphylococcal
lipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF),
streptokinase, superoxide dismutase, syndecan-1, TACE, TACI, TAG-72
(tumor-associated glycoprotein-72), TARC, TB, TCA-3, T-cell
receptor alpha/beta, TdT, TECK, TEM1, TEMS, TEM7, TEM8, Tenascin,
TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF,
TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RII, TGF-beta
RIIb, TGF-beta RIII, TGF-beta RI (ALK-5), TGF-beta1, TGF-beta2,
TGF-beta3, TGF-beta4, TGF-beta5, TGF-I, Thrombin, thrombopoietin
(TPO), Thymic stromal lymphoprotein receptor, Thymus Ck-1, thyroid
stimulating hormone (TSH), thyroxine, thyroxine-binding globulin,
Tie, TIMP, TIQ, Tissue Factor, tissue factor protease inhibitor,
tissue factor protein, TMEFF2, Tmpo, TMPRSS2, TNF receptor I, TNF
receptor II, TNF-alpha, TNF-beta, 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), TNFRSF12A,
TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM
ATAR/HveA/LIGHT R/TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA),
TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ/TRADE), TNFRSF19L (RELT),
TNFRSF1A (TNF R1 CD120a/p55-60), TNFRSF1B (TNF RII CD120b/p75-80),
TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3
Apo-3/LARD/TR-3/TRAMP/WSL-1), 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 (4-1 BB CD137/ILA), TNFRST23
(DcTRAIL R1 TNFRH1), 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 (4-1 BB Ligand CD137 Ligand), TNF-alpha, TNF-beta, TNIL-I,
toxic metabolite, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1,
TRAIL-R2, TRANCE, transferrin receptor, transforming growth factors
(TGF) such as TGF-alpha and TGF-beta, Transmembrane glycoprotein
NMB, Transthyretin, TRF, Trk, TROP-2, Trophoblast glycoprotein,
TSG, TSLP, Tumor Necrosis Factor (TNF), tumor-associated antigen CA
125, tumor-associated antigen expressing Lewis Y related
carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VAP-1,
vascular endothelial growth factor (VEGF), vaspin, VCAM, VCAM-1,
VECAD, VE-Cadherin, VE-Cadherin-2, VEFGR-1 (flt-1), VEFGR-2, VEGF
receptor (VEGFR), VEGFR-3 (flt-4), VEGI, VIM, Viral antigens,
VitB12 receptor, Vitronectin receptor, VLA, VLA-1, VLA-4, VNR
integrin, von Willebrand Factor (vWF), WIF-1, WNT1, WNT10A, WNT10B,
WNT11, WNT16, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B,
WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1,
XCL2/SCM-1-beta, XCL1/Lymphotactin, XCR1, XEDAR, XIAP, XPD, HMGB1,
IgA, Aa, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17/IL-17R,
IL-20/IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F,
SOD1, Chromogranin A, Chromogranin B, tau, VAP1, High molecular
weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4,
Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s,
C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8,
C9, factor B, factor D, factor H, properdin, sclerostin,
fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V,
factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa,
factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa,
factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI,
antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen,
plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2, Syndecan-3,
Syndecan-4, LPA, S1P.
[0589] Antigen binding molecules described in present invention are
capable of reducing total antigen concentration of the
above-described antigens in plasma. Antigen binding molecules
described in present invention are also capable of eliminating
virus, bacteria, and fungus from plasma by binding to structural
components of virus, bacteria and fungus. Particularly, F protein
of RSV, Staphylococcal lipoteichoic acid, Clostridium difficile
toxin, Shiga like toxin II, Bacillus anthracis protective antigen
and Hepatitis C virus E2 glycoprotein can be used as a structural
components of virus, bacteria and fungus.
Use
[0590] The present invention also provides many uses of the
antigen-binding molecules of the present invention as described
above.
[0591] Thus, the present invention provides the use of the modified
antigen-binding molecules of the present invention for improving
the antigen-binding molecule-mediated antigen uptake into cells.
Furthermore, the present invention also provides methods for
improving antigen-binding molecule-mediated antigen uptake into
cells comprising altering an antigen-binding molecule comprising a
parent FcRn-binding domain, by substituting an amino acid in the
parent FcRn-binding domain at one or more positions selected from
the group consisting of EU238, EU250, EU252, EU254, EU255, EU258,
EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and
EU436 and thereby increasing the FcRn-binding activity at neutral
pH as compared to an antigen-binding molecule having an intact
FcRn-binding domain.
[0592] Herein, the term "antigen uptake into cells" mediated by an
antigen-binding molecule means that antigens are taken up into
cells by endocytosis. Meanwhile, herein, the term "facilitate the
uptake into cells" means that the rate of intracellular uptake of
antigen-binding molecule bound to an antigen in plasma is enhanced,
and/or the quantity of recycling of uptaken antigen to the plasma
is reduced. This means that the rate of uptake into cells is
facilitated as compared to the antigen-binding molecule before the
modification of the FcRn-binding domain and thus before increasing
the human FcRn-binding activity of the antigen-binding molecule in
the neutral pH range, or before increasing the human FcRn-binding
activity and reducing the antigen-binding activity (binding
ability) of the antigen-binding molecule in the acidic pH range to
less than its antigen-binding activity in the neutral pH range. The
rate is improved preferably as compared to intact IgG, and more
preferably as compared to intact human IgG. Thus, in the present
invention, whether antigen uptake into cells is facilitated by an
antigen-binding molecule can be assessed based on an increase in
the rate of antigen uptake into cells. The rate of antigen uptake
into cells can be calculated, for example, by monitoring over time
reduction in the antigen concentration in the culture medium
containing human FcRn-expressing cells after adding the antigen and
antigen-binding molecule to the medium, or monitoring over time the
amount of antigen uptake into human FcRn-expressing cells. Using
methods of the present invention for facilitating the rate of
antigen-binding molecule-mediated antigen uptake into cells, for
example, the rate of antigen elimination from the plasma can be
enhanced by administering antigen-binding molecules of the present
invention. Thus, whether antigen-binding molecule-mediated antigen
uptake into cells is facilitated can also be assessed, for example,
by testing whether the rate of antigen elimination from the plasma
is accelerated or whether the total antigen concentration in plasma
is reduced by administering an antigen-binding molecule of the
present invention.
[0593] Herein, the term "total antigen concentration in plasma"
means 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. Various methods to measure "total antigen
concentration in plasma" and "free antigen concentration in plasma"
are well known in the art as described hereinafter.
[0594] The present invention also provides use of the
antigen-binding molecule of the present invention for increasing
the total number of antigens to which a single antigen-binding
molecule can bind before its degradation. The present invention
also provides methods for increasing the number of antigens to
which a single antigen-binding molecule can bind, by using an
antigen-binding molecule of the present invention. Specifically,
the present invention provides methods for increasing the total
number of antigens to which a single antigen-binding molecule can
bind, by substituting an amino acid in the parent FcRn-binding
domain of said antigen-binding molecule at one or more positions
selected from the group consisting of EU238, EU250, EU252, EU254,
EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428,
EU433, EU434, and EU436 and thereby increasing the FcRn-binding
activity at neutral pH as compared to an antigen-binding molecule
having an intact FcRn-binding domain.
[0595] A "conventional antibody" can usually bind only one or two
antigens before it is degraded in the endosome. An antigen-binding
molecule of the present invention can increase the number of cycles
achieved until the antigen-binding molecule is degraded, whereby
each cycle consists of: binding of an antigen to the
antigen-binding molecule in plasma, intracellular uptake of the
antigen-binding molecule bound to the antigen, and dissociation
from the antigen in the endosome, followed by return of the
antigen-binding molecule to the plasma. This means that the number
of cycles is increased as compared to the antigen-binding molecule
before the modification of the FcRn-binding domain and thus before
increasing the human FcRn-binding activity of the antigen-binding
molecule in the neutral pH or acidic range, or before increasing
the human FcRn-binding activity and reducing the antigen-binding
activity (binding ability) of the antigen-binding molecule in the
acidic pH range to less than its antigen-binding activity in the
neutral pH range. Thus, whether the number of cycles is increased
can be assessed by testing whether the above-described
"intracellular uptake is facilitated" or whether the
"pharmacokinetics is improved" as described below.
[0596] The present invention also provides for the use of the
antigen-binding molecules of the present invention for improving
the antigen-removal from the blood in mammals, i.e. in humans. In
particular, the present invention provides the use of the
antigen-binding molecule of the present invention for reducing the
plasma concentration of a specific antigen, wherein the
antigen-binding molecule comprises an antigen-binding domain which
can bind said antigen. The present invention also provides a method
for reducing the plasma concentration of a specific antigen,
wherein the antigen-binding molecule comprises an antigen-binding
domain which can bind said antigen, by substituting an amino acid
in a parent FcRn-binding domain at one or more positions selected
from the group consisting of EU238, EU250, EU252, EU254, EU255,
EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433,
EU434, and EU436 and thereby increasing the FcRn-binding activity
at neutral pH as compared to an antigen-binding molecule having an
intact FcRn-binding domain.
[0597] The present invention also provides the use of the
antigen-binding molecules of the present invention for facilitating
the extracellular release of antigen-free antigen-binding molecule
taken up into cells in an antigen-bound form. More specifically,
the present invention provides methods for facilitating the
extracellular release of antigen-free antigen-binding molecule
taken up into cells in an antigen-bound form without significantly
increasing the binding activity for a pre-existing ADA at neutral
pH compared to parent antibody, by substituting an amino acid in
the parent FcRn-binding domain at one or more positions selected
from the group consisting of EU238, EU250, EU252, EU254, EU255,
EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433,
EU434, and EU436 and thereby increasing the FcRn-binding activity
at neutral pH as compared to an antigen-binding molecule having an
intact FcRn-binding domain
[0598] Herein, the "extracellular release of antigen-free
antigen-binding molecule taken up into cells in an antigen-bound
form" does not necessarily mean that all of the antigen-binding
molecules bound to antigen taken up into cells are released in an
antigen-free form outside of the cell. It is acceptable that the
proportion of antigen-binding molecules released in an antigen-free
form to the outside of the cell is increased as compared to before
the modification of the FcRn-binding domain and thus before
reducing the antigen-binding activity of the antigen-binding
molecule in the acidic pH range to less than that in the neutral pH
range and increasing the human FcRn-binding activity in the neutral
pH range. The antigen-binding molecule released to the outside of
the cell preferably retains the antigen-binding activity.
[0599] The present invention also provides the use of an
FcRn-binding domain of the present invention for increasing the
ability of the antigen-binding molecule to eliminate plasma
antigen. In the present invention, "methods for increasing the
ability to eliminate plasma antigen" is synonymous to "methods for
augmenting the ability of an antigen-binding molecule to eliminate
antigen from plasma". More specifically, the present invention
provides methods for increasing the ability of an antigen-binding
molecule to eliminate plasma antigen by substituting an amino acid
in the parent FcRn-binding domain at one or more positions selected
from the group consisting of EU238, EU250, EU252, EU254, EU255,
EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433,
EU434, and EU436 and thereby increasing the FcRn-binding activity
at neutral and/or acidic pH as compared to an antigen-binding
molecule having an intact FcRn-binding domain.
[0600] Herein, the term "ability to eliminate plasma antigen" means
the ability to remove antigen from the plasma when antigen-binding
molecules are administered or secreted in vivo. Thus, "increase in
the ability of antigen-binding molecule to eliminate plasma
antigen" herein means that the rate of antigen elimination from the
plasma is accelerated upon administration of the antigen-binding
molecule as compared to before the modification of the FcRn-binding
domain and thus before increasing the human FcRn-binding activity
of the antigen-binding molecule in the neutral pH range or before
increasing the human FcRn-binding activity and simultaneously
reducing its antigen-binding activity in the acidic pH range to
less than that in the neutral pH range. The increase in the
activity of an antigen-binding molecule to eliminate antigen from
the plasma can be assessed, for example, by administering a soluble
antigen and an antigen-binding molecule in vivo, and measuring the
concentration of the soluble antigen in plasma after
administration. When the concentration of soluble antigen in plasma
after administration of the soluble antigen and modified
antigen-binding molecule is reduced, the ability of antigen-binding
molecule to eliminate plasma antigen can be judged to be increased.
A form of soluble antigen can be antigen-binding molecule bound
antigen or antigen-binding molecule non-bound antigen whose
concentration can be determined as "antigen-binding molecule bound
antigen concentration in plasma" and "antigen-binding molecule
non-bound antigen concentration in plasma" respectively. The latter
is synonymous to "free antigen concentration in plasma". Since
"total antigen concentration in plasma" means 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, the
concentration of soluble antigen can be determined as "total
antigen concentration in plasma". Various methods for measuring
"total antigen concentration in plasma" or "free antigen
concentration in plasma" are well known in the art as described
hereinafter.
[0601] The present invention also provides the use of the
FcRn-binding domain of the present invention for improving the
pharmacokinetics of antigen-binding molecules. More specifically,
the present invention provides methods for improving the
pharmacokinetics of the antigen-binding molecule by substituting an
amino acid in the parent FcRn-binding domain at one or more
positions selected from the group consisting of EU238, EU250,
EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311,
EU315, EU428, EU433, EU434, and EU436 and thereby increasing the
FcRn-binding activity at neutral and/or acidic pH as compared to an
antigen-binding molecule having an intact FcRn-binding domain.
[0602] Herein, the terms "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 used herein as synonyms.
[0603] Improving the pharmacokinetics particularly encompasses:
[0604] (1) a delayed elimination: prolonging the time between
administration and elimination of the antigen-binding molecules
from plasma as compared to a Control Antigen-binding Molecule (e.g.
antigen-binding molecules having an intact FcRn-binding domain);
and/or
[0605] (2) prolonging the plasma retention time of the
antigen-binding molecules, preferably in a form in which the
antibody or antibody derivative can bind to its antigen after
administration of the Antigen-binding molecules as compared to the
plasma retention time of a Control Antigen-binding Molecule (e.g.
antigen-binding molecules having an intact FcRn-binding domain);
and/or
[0606] (3) shortening the period during which the antigen is free
(not bound to an antigen-binding molecule in the body) between
administration and elimination of the antigen-binding molecules as
compared to a Control Antigen-binding Molecule (prolonging of the
period between administration and elimination during which the
antigen-binding molecules is bound to its antigen in the body of a
subject as compared to a control antigen-binding molecules (e.g.
antigen-binding molecules having an intact FcRn-binding domain);
and/or
[0607] (4) increasing the ratio of antigen bound to an
antigen-binding molecules vs. total antigen in the body as compared
to the ratio of antigen bound to a Control Antigen-binding Molecule
(e.g. antigen-binding molecules having an intact FcRn-binding
domain) before degradation of the antibody (increasing the number
of binding events of the antigen-binding molecules with its antigen
between administration and degradation of the antibody or antibody
derivative as compared to the number of binding events of a control
antigen-binding molecules between administration and
degradation).
[0608] (5) reducing plasma total or free antigen concentration
after the administration of the antigen-binding molecules compared
to the plasma total or free antigen concentration after the
administration of a Control Antigen-binding Molecule (e.g.
antigen-binding molecules having an intact FcRn-binding
domain).
[0609] The present invention also provides a method for delaying
the elimination of an antigen-binding molecule in a subject,
comprising the step of introducing a modification into a
FcRn-binding domain of said antigen-binding molecule at one or more
of the positions selected from the group consisting of EU238,
EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309,
EU311, EU315, EU428, EU433, EU434, and EU436.
[0610] The term "improvement of pharmacokinetics" as used herein
refers not only to prolongation of the period between
administration of the antigen-binding molecule to a subject
(humans, or non-human animals such as mice, rats, monkeys, rabbits,
and dogs) and elimination from the plasma (for example, until the
antigen-binding molecule is degraded intracellularly or the like
and cannot return to the plasma) to, but also to the 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 from
administration until degradation of the antigen-binding
molecule.
[0611] Therefore, the present invention also provides a method of
prolonging the plasma retention time of an antigen-binding
molecule, comprising the step of introducing a modification into a
FcRn-binding domain of said antigen-binding molecule at one or more
of the positions selected from the group consisting of EU238,
EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309,
EU311, EU315, EU428, EU433, EU434, and EU436. Intact human IgG can
bind to FcRn from non-human animals. For example, administration to
mice is preferably used to confirm the property of the
antigen-binding molecule of the invention since intact human IgG
can bind to mouse FcRn stronger than to human FcRn (Int Immunol.
2001 December; 13(12): 1551-9). 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.
[0612] Specifically, "improvement of pharmacokinetics" also
includes prolongation of the period between administration and
degradation of the antigen-binding molecule during which it is not
bound to an antigen (the antigen-free form of antigen-binding
molecule). The antigen-binding molecule in plasma cannot bind to a
new antigen when the antigen-binding molecule has already bound to
an antigen. Thus, the longer the period during which the
antigen-binding molecule is not bound to an antigen, the longer is
the period during which it has the potential to bind to a new
antigen (the higher the chance of binding to another antigen). In
other words, more antigens are bound during a shorter period of
time. Therefore, the plasma concentration of the antigen-free form
of antigen-binding molecule can be increased and the total period
during which antigen is bound to the antigen-binding molecule can
be prolonged by accelerating the antigen elimination from the
plasma by administration of the modified antigen-binding
molecule.
[0613] Specifically, herein "improvement of the pharmacokinetics of
antigen-binding molecule" 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 during
which the antigen is bound to the antigen-binding molecule after
administration of the modified antigen-binding molecule, and
acceleration of antigen-binding molecule-mediated antigen
elimination from the plasma.
[0614] 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 ni yoru 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
non-compartmental 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): 406-17.
[0615] Herein, the term "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 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.
[0616] The present invention also provides the use of the
antigen-binding molecules of the present invention for reducing
total or free antigen plasma concentration of a specific antigen,
wherein the antigen-binding molecule comprises an antigen-binding
domain which can bind said antigen. More specifically, the present
invention provides methods for reducing total or free antigen
plasma concentration, said method comprising the steps of:
[0617] a) providing an antigen-binding molecule comprising a parent
FcRn-binding domain, wherein the antigen-binding molecule comprises
an antigen-binding domain which can bind said antigen,
[0618] b) substituting an amino acid in the parent FcRn-binding
domain at one or more positions selected from the group consisting
of EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308,
EU309, EU311, EU315, EU428, EU433, EU434, and EU436 and thereby
increasing the FcRn-binding activity at neutral pH as compared to
an antigen-binding molecule having an intact FcRn-binding
domain.
[0619] Moreover, the present invention provides, comprising the
step of introducing a modification into an FcRn-binding domain of
said antigen-binding molecule at one or more of the positions
selected from the group consisting of EU238, EU250, EU252, EU254,
EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428,
EU433, EU434, and EU436. The term "antigen-elimination rate" as
used herein refers to the number of antigens that an
antigen-binding molecule can remove from the plasma in the time
between administration and elimination (i.e. degradation) of the
antibody or antibody derivative.
[0620] 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 described 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.
[0621] 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. Herein, the term
"plasma antigen concentration" means either "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.
[0622] Total antigen concentration in plasma can be lowered by
administration of 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 compared to the administration
of a reference antigen-binding molecule comprising the intact human
IgG Fc region as a human FcRn-binding domain or compared to when
antigen-binding domain molecule of the present invention is not
administered.
[0623] Molar antigen/antigen-binding molecule ratio can be
calculated as shown below;
[0624] value A: Molar antigen concentration at each time point
[0625] value B: Molar antigen-binding molecule concentration at
each time point
[0626] value C: Molar antigen concentration per molar
antigen-binding molecule concentration (molar
antigen/antigen-binding molecule ratio) at each time point
C=A/B.
[0627] 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.
[0628] Molar antigen/antigen-binding molecule ratio can be lowered
by administration of antigen-binding molecule of 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 the
administration of a reference antigen-binding molecule comprising
the intact human IgG Fc region as a human FcRn-binding domain.
[0629] Herein, an intact human IgG1, IgG2, IgG3 or IgG4 is
preferably used as the intact human IgG for a purpose of a
reference intact human IgG to be compared with the antigen-binding
molecules for their human FcRn binding activity or in vivo
activity. Preferably, a reference antigen-binding molecule
comprising the same antigen-binding domain as an antigen-binding
molecule of the interest and intact human IgG Fc region as a human
FcRn-binding domain can be appropriately used. More preferably, an
intact human IgG1 is used for a purpose of a reference intact human
IgG to be compared with the antigen-binding molecules for their
human FcRn binding activity or in vivo activity.
[0630] Reduction of total antigen concentration in plasma or molar
antigen/antibody ratio can be assessed as described in Examples 6,
8, and 13 of WO2011/122011. More specifically, using human FcRn
transgenic mouse line 32 or line 276 (Jackson Laboratories, Methods
Mol Biol. (2010) 602: 93-104.), they can be assessed by either
antigen-antibody co-injection model or steady-state antigen
infusion model when the antigen-binding molecule of interest does
not cross-react with the mouse counterpart antigen. When
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 the co-injection model, a mixture of
antigen-binding molecule and antigen is administered to the mouse.
In the steady-state antigen infusion model, an infusion pump
containing an antigen solution is implanted to the mouse to achieve
a constant plasma antigen concentration, and then the
antigen-binding molecule is injected to the mouse. The same dosage
is used for all administered test antigen-binding molecules. Total
antigen concentration in plasma, free antigen concentration in
plasma and plasma antigen-binding molecule concentration is
measured at an appropriate time point using methods known to those
skilled in the art.
[0631] 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.
[0632] Total or free antigen concentration in plasma and molar
antigen/antigen-binding molecule ratio can be measured at 15 min,
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 min, 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.
[0633] More specifically, those antigen-binding molecules having a
long term effect on activity for eliminating antigen in plasma as
described in the present invention have human FcRn-binding activity
at pH 7.0 and 25 degrees C. within a range of 28-fold to 440-fold
stronger than intact human IgG1 or KD within a range of 3.0
micromolar to 0.2 micromolar. Preferably, the KD is within a range
of 700 nanomolar to 0.2 nanomolar, more preferably, the KD is
within a range of 500nanomolar to 3.5 nanomolar, more preferably,
within a range of 150 nanomolar to 3.5 nanomolar. 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 long term effect of the antigen-binding
molecule of the present invention on activity for eliminating
antigen in plasma. 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.
[0634] Still more specifically, those antigen-binding molecules
having a short term effect on for eliminating antigen in plasma as
described in the present invention have human FcRn-binding activity
at pH 7.0 and at 25 degrees Celsius 440-fold stronger than intact
human IgG or KD stronger than 0.2 micromolar, preferably stronger
than 700nanomolar, more preferably stronger than 500nanomolar, most
preferably, stronger than 150 nanomolar. 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 min, 1, 2, 4, 8, 12, or 24 hours after
administration of an antigen-binding molecule in order to evaluate
the short term effect of the antigen-binding molecule of the
present invention on activity for eliminating antigen in
plasma.
[0635] 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.
[0636] In the context of the present invention, improvement of
pharmacokinetics 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).
[0637] Herein the term "reducing the antigen-binding activity of an
antigen-binding molecule in the acidic pH range to less than that
in the neutral pH range" means that the antigen-binding activity of
the antigen-binding molecule at pH 4.0 to pH 6.5 is impaired as
compared to its antigen-binding activity at pH 6.7 to pH 10.0.
Preferably, the above phrase means that the antigen-binding
activity of an antigen-binding molecule at pH 5.5 to pH 6.5 is
impaired as compared to that at pH 7.0 to pH 8.0, more preferably
means that its antigen-binding activity at the early endosomal pH
is impaired as compared to its antigen-binding activity at the
plasma pH in vivo. Specifically, the antigen-binding activity of an
antigen-binding molecule at pH 5.8 to pH 6.0 is impaired as
compared to the antigen-binding activity of the antigen-binding
molecule at pH 7.4.
[0638] Herein the term "reducing the antigen-binding activity of an
antigen-binding molecule in the neutral pH range to less than that
in the acidic pH range" means that the antigen-binding activity of
the antigen-binding molecule at pH 6.7 to pH 10.0 is impaired as
compared to its antigen-binding activity at pH 4.0 to pH 6.5.
Preferably, the above phrase means that the antigen-binding
activity of an antigen-binding molecule at pH 7.0 to pH 8.0 is
impaired as compared to that at pH 5.5 to pH 6.5, more preferably
means that its antigen-binding activity at the plasma pH in vivo is
impaired as compared to its antigen-binding activity at the early
endosomal pH. Specifically, the antigen-binding activity of an
antigen-binding molecule at pH 7.4 is impaired as compared to the
antigen-binding activity of the antigen-binding molecule at pH 5.8
to pH 6.0.
[0639] Meanwhile, herein the expression "reducing the
antigen-binding activity of an antigen-binding molecule in the
acidic pH range to less than that in the neutral pH range" is also
expressed as "increasing the antigen-binding activity of an
antigen-binding molecule in the neutral pH range to more than that
in the acidic pH range". Specifically, in the present invention, it
is possible to increase the ratio of antigen binding activity of an
antigen-binding molecule between acidic and neutral pH ranges. For
example, the value of KD (pH 5.8)/KD (pH 7.4) is increased in an
embodiment described below. The ratio of antigen-binding activity
of an antigen-binding molecule between acidic and neutral pH ranges
can be increased, for example, by reducing its antigen-binding
activity in the acidic pH range, increasing its antigen-binding
activity in the neutral pH range, or both. The expression "reducing
the antigen-binding activity of an antigen-binding molecule in the
neutral pH range to less than that in the acidic pH range" is also
expressed as "increasing the antigen-binding activity of an
antigen-binding molecule in the acidic pH range to more than that
in the neutral pH range". Specifically, in the present invention,
it is possible to increase the ratio of antigen binding activity of
an antigen-binding molecule between acidic and neutral pH ranges.
For example, the value of KD (pH7.4)/KD (pH 5.8) is increased in an
embodiment described below. The ratio of antigen-binding activity
of an antigen-binding molecule between acidic and neutral pH ranges
can be increased, for example, by reducing its antigen-binding
activity in the neutral pH range, increasing its antigen-binding
activity in the acidic pH range, or both.
[0640] The term "reducing the antigen-binding activity (binding
ability) at low calcium-ion concentrations to less than its
antigen-binding activity at high calcium-ion concentration" as used
herein refers to decreasing the binding affinity of the
antigen-binding domain for the antigen at a low calcium-ion
concentration compared with the binding affinity for the antigen of
said antigen-binding domain at a high calcium-ion concentration.
The low calcium concentration is preferably 0.5 to 10 micromolar,
more preferably 0.1 to 30 micromolar of ionized calcium, and the
high calcium concentration is 100 micromolar to 10 mM, more
preferably 200 micromolar to 5 mM of ionized calcium.
[0641] Herein, the expression "impairing the antigen-binding
activity in the acidic pH range as compared to that in the neutral
pH range" is sometimes used instead of "reducing the
antigen-binding activity in the acidic pH range to less than that
in the neutral pH range".
[0642] Herein, the human FcRn-binding activity in the acidic pH
range means the human FcRn-binding activity at pH 4.0 to pH 6.5,
preferably the human FcRn-binding activity at pH 5.5 to pH 6.5, and
particularly preferably the human FcRn-binding activity at pH 5.8
to pH 6.0, which is comparable to the in vivo early endosomal pH.
Meanwhile, herein the human FcRn-binding activity in the neutral pH
range means the human FcRn-binding activity at pH 6.7 to pH 10.0,
preferably the human FcRn-binding activity at pH 7.0 to pH 8.0, and
particularly preferably the human FcRn-binding activity at pH 7.4,
which is comparable to the in vivo plasma pH.
[0643] Although the antigen-binding molecule and uses of the
present invention are not limited to any particular theory, the
relationship between the reduction (impairment) of the
antigen-binding ability of antigen-binding molecule in the acidic
pH range to less than that in the neutral pH range and/or the
increase (enhancement) of the human FcRn-binding activity in the
neutral pH range and the increase in the number of antigens to
which a single antigen-binding molecule can bind, due to
facilitation of uptake of antigen-binding molecules into cells, and
the enhancement of antigen elimination from the plasma can be
explained as follows.
[0644] For example, when the antigen-binding molecule is an
antibody that binds to a membrane antigen, the antibody
administered into the body binds to the antigen and then is taken
up via internalization into endosomes in the cells together with
the antigen while the antibody is kept bound to the antigen. Then,
the antibody translocates to lysosomes while the antibody is kept
bound to the antigen, and the antibody is degraded by the lysosome
together with the antigen. The internalization-mediated elimination
from the plasma is called antigen-dependent elimination, and such
elimination has been reported with numerous antibody molecules
(Drug Discov Today. 2006 January; 11(1-2): 81-8). When a single
molecule of IgG antibody binds to antigens in a divalent manner,
the single antibody molecule is internalized while the antibody is
kept bound to the two antigen molecules, and degraded in the
lysosome. Accordingly, in the case of typical antibodies, one
molecule of IgG antibody cannot bind to three or more molecules of
antigen. For example, a single IgG antibody molecule having a
neutralizing activity cannot neutralize three or more antigen
molecules.
[0645] The relatively prolonged retention (slow elimination) of IgG
molecules in the plasma is due to the function of human FcRn which
is known as a salvage receptor of IgG molecules. When taken up into
endosomes via pinocytosis, IgG molecules bind to human FcRn
expressed in the endosomes under the acidic condition in the
endosomes. While IgG molecules that did not bind to human FcRn
transfer to lysosomes where they are degraded, IgG molecules that
are bound to human FcRn translocate to the cell surface and return
again in the plasma by dissociating from human FcRn under the
neutral condition in the plasma.
[0646] Alternatively, when the antigen-binding molecule is an
antibody that binds to a soluble antigen, the antibody administered
into the body binds to the antigen and then is taken up into cells
while the antibody is kept bound to the antigen. Many antibodies
taken up into cells are released to the outside of the cell via
FcRn. However, since the antibodies are released to the outside of
the cell, with the antibodies kept bound to antigens, the
antibodies cannot bind to antigens again. Thus, similar to
antibodies that bind to membrane antigens, in the case of typical
antibodies, one molecule of IgG antibody cannot bind to three or
more antigen molecules.
[0647] pH-dependent antigen-binding antibodies that strongly bind
to an antigen under the neutral conditions in plasma but dissociate
from the antigen under acidic conditions in the endosome (i.e.,
antibodies that bind under neutral conditions but dissociate under
acidic conditions) can dissociate from the antigen in the endosome.
Such pH-dependent antigen-binding antibodies can bind to antigens
again when they are recycled to the plasma by FcRn after antigen
dissociation; thus, each antibody can repeatedly bind to a number
of antigens. Furthermore, the antigen bound to the antigen-binding
molecule is dissociated in the endosome and not recycled to the
plasma. This facilitates the antigen-binding molecule-mediated
antigen uptake into cells. Thus, the administration of an
antigen-binding molecule can enhance the antigen elimination and
thereby reduces the plasma antigen concentration.
[0648] A calcium concentration-dependent antigen-binding antibody,
which strongly binds to an antigen under a high calcium
concentration condition in plasma, and dissociates from the antigen
under a low calcium concentration condition in the endosome, can
dissociate from the antigen within the endosome. The calcium
concentration-dependent antigen-binding antibody can bind to an
antigen again when recycled to plasma via FcRn after antigen
dissociation. Thus, such a single antibody can repeatedly bind to
multiple antigens. Meanwhile, an antigen bound to the
antigen-binding molecule is not recycled to plasma because the
antigen dissociates in the endosome, and thus, the antigen-binding
molecule promotes uptake of the antigen into cells. The
administration of the antigen-binding molecule promotes the
elimination of an antigen, and this allows a decrease in the
antigen concentration in plasma.
[0649] The antigen-binding molecule-mediated antigen uptake into
cells can be further facilitated by conferring the human
FcRn-binding activity under neutral conditions (pH 7.4) to an
antibody that binds to an antigen in a pH-dependent manner (binds
under neutral conditions but dissociates under acidic conditions).
Thus, the administration of an antigen-binding molecule can enhance
the antigen elimination and thereby reduces the plasma antigen
concentration. Normally, both antibody and antigen-antibody complex
are taken up into cells by non-specific endocytosis, and then
transported to the cell surface by binding to FcRn under acidic
conditions in the endosome. The antibody and antigen-antibody
complex are recycled to the plasma via dissociation from FcRn under
the neutral condition on cell surface. Thus, when an antibody that
exhibits sufficient pH dependency in antigen binding (binds under
neutral conditions but dissociates under acidic conditions) binds
to the antigen in the plasma and then is dissociated from the bound
antigen in the endosome, the antigen elimination rate is presumed
to be equal to the rate of antigen uptake into cells via
non-specific endocytosis. On the other hand, when the pH dependency
is insufficient, the antigen that did not dissociate in the
endosome is also recycled to the plasma. Meanwhile, when the pH
dependency is sufficient, the rate-determining step in the antigen
elimination is the uptake into cells by non-specific endocytosis.
Some of FcRn is presumed to be localized on the cell surface
because FcRn transports antibodies from the endosome to the cell
surface.
[0650] The present inventors presumed that IgG-type
immunoglobulins, which are one of antigen-binding molecules,
typically have little FcRn-binding ability in the neutral pH range,
but those that exhibit FcRn-binding ability in the neutral pH range
could bind to FcRn on the cell surface and thus are taken up into
cells in an FcRn-dependent manner by binding to cell-surface FcRn.
The rate of FcRn-mediated uptake into cells is more rapid than the
rate of uptake into cells by non-specific endocytosis. Thus, the
rate of antigen elimination can be further accelerated by
conferring FcRn-binding ability in the neutral pH range.
Specifically, an antigen-binding molecule having FcRn-binding
ability in the neutral pH range transports an antigen into cells
more rapidly than the typical (intact human) IgG-type
immunoglobulin, and then the antigen-binding molecule is
dissociated from the antigen in the endosome. The antigen-binding
molecule is recycled to the cell surface or plasma, and again binds
to another antigen and is taken up into cells via FcRn. The rate of
this cycle can be accelerated by improving FcRn-binding ability in
the neutral pH range, thereby accelerating the rate of antigen
elimination from the plasma. Furthermore, the efficiency can be
further improved by reducing the antigen-binding activity of an
antigen-binding molecule in the acidic pH range to less than that
in the neutral pH range. In addition, the number of antigens to
which a single antigen-binding molecule can bind is presumed to
increase with an increasing number of cycles achieved by a single
antigen-binding molecule. The antigen-binding molecule of the
present invention comprises an antigen-binding domain and an
FcRn-binding domain. Since the FcRn-binding domain does not affect
antigen binding, or in view of the mechanism described above,
facilitation of the antigen-binding molecule-mediated antigen
uptake into cells can be expected regardless of the type of
antigen, and as a result increases the antigen elimination rate by
reducing the antigen-binding activity of an antigen-binding
molecule in the acidic pH range (binding ability) to less than that
in the neutral pH range and/or increasing its FcRn-binding activity
at the plasma pH.
[0651] In all forgoing uses the antigen-binding molecules of the
present invention may also comprise a substitution at position
EU256 in addition to a substitution at the mentioned one or more
positions. Preferably, the amino acid at position EU256 is
substituted with a glutamic acid. Furthermore, all foregoing
methods of use may also comprise a substitution at position EU256
in addition to a substitution at the one or more positions selected
from the group consisting of EU238, EU250, EU252, EU254, EU255,
EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433,
EU434, and EU436, whereby the amino acid at position EU256 is
preferably substituted with a glutamic acid.
[0652] In a preferred embodiment of all foregoing uses and methods,
the FcRn-binding region is an Fc region in more preferably, it is a
human Fc region.
[0653] Moreover, the substitution in the amino acid sequence of the
parent FcRn-binding domain for increasing the FcRn-binding activity
at neutral or acidic pH are preferably at position EU252 and EU434
and one at one or more positions selected from the group consisting
of EU238, EU250, EU254, EU255, EU256, EU258, EU286, EU307, EU308,
EU309, EU311, EU315, EU428, EU433, and EU436. More preferably, the
substitutions are at three or more positions wherein said three or
more positions are one of the combinations set forth in Tables 2,
and 4 to 7. Even more preferably, the substitutions are one of the
combinations set forth in Table 3
[0654] Furthermore, the foregoing methods of use may additionally
comprise a step introducing an amino acid substitution at one or
more positions selected from the group consisting of EU387, EU422,
EU424, EU426, EU433, EU436, EU438 and EU440, and thereby decreasing
an increased binding activity for a pre-existing ADA. Preferably,
the substitutions are combinations selected from Table 11.
[0655] In addition, the forgoing methods of use may also comprise
an additional step of introducing in an amino acid substitution in
an FcRn-binding domain at one or more positions selected from the
group consisting of EU234, EU235, EU236, EU237, EU238, EU239,
EU265, EU266, EU267, EU269, EU270, EU271, EU295, EU296, EU297
EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331, and EU332
(according to the EU numbering system). Preferably, the
substitutions L235R/S239K are introduced. Preferably, the
substitutions are combinations selected from Table 14.
[0656] The antigen-binding molecule of all foregoing uses and
method of uses may comprise a pH-dependent antigen-binding domain
or a calcium ion-dependent antigen-binding domain. The antigen
uptake into cells mediated by the antigen-binding molecules of the
present invention is further improved by reducing the
antigen-binding activity (binding ability) in the acidic pH range
of the above-described antigen-binding molecule to less than its
antigen-binding activity in the neutral pH range. Also preferred is
the further improvement of the antigen uptake into cells by
reducing the antigen-binding activity (binding ability) of the
antigen-binding molecule of the present invention at low
calcium-ion concentration (i.e. at 0.5 to 10 micromolar.) to less
than its antigen-binding activity at high calcium-ion concentration
(i.e. 100 micromolar to 10 mM), for example by replacing the
antigen-binding domain of the antigen-binding molecule with an
ionized calcium-concentration dependent antigen-binding domain.
Alternatively, the parent antigen-binding molecule already
comprises an ionized calcium-concentration dependent
antigen-binding domain. Methods for reducing the antigen-binding
activity (binding ability) in the acidic pH range by altering at
least one amino acid in the antigen-binding domain of the
above-described antigen-binding molecule are described above.
Preferably, the antigen-binding domain is altered by substituting
histidine for at least one amino acid or inserting at least one
histidine into the antigen-binding domain of the above-described
antigen-binding molecule which facilitates antigen uptake into
cells.
[0657] Clearance of modified antigen-binding molecule that has an
increased FcRn-binding activity at neutral or acidic pH can be
decreased when these modifications increase the antigen-binding
molecule's affinity for a pre-existing ADA, e.g. the rheumatoid
factor. This means by further modifying such an antibody and
thereby decreasing the affinity for a pre-existing ADA, the number
of cycles can be increased compared to the antigen-binding molecule
before the second modification, and thus before the affinity for a
pre-existing ADA was decreased.
[0658] Those antigen-binding molecules of the present invention
whose affinity for a pre-existing anti-drug antibody is not
significantly increased at a neutral pH as compared to a wild type
Fc region, in particular those that comprise in the FcRn binding
domain an amino acid substitution at one or more positions selected
from the group consisting of EU387, EU422, EU424, EU426, EU433,
EU436, EU438 and EU440, are particularly useful as therapeutic
antibodies for treating human patients suffering from an autoimmune
disease, transplant rejections (graft-vs.-host disease), other
inflammatory diseases and allergic diseases.
[0659] An autoimmune disease is an illness that occurs when body
tissue is attacked by its own immune system. Examples of autoimmune
diseases contemplated herein include systemic lupus erythematosus,
lupus nephritis, pemphigoid, pemphigus, dermatomyositis, autoimmune
hepatitis, Sjogren syndrome, Hashimoto thyroiditis, rheumatoid
arthritis, juvenile (type 1) diabetes, polymyositis, scleroderma,
Addison disease, Coeliac disease, Guillain-Barre syndrome, dilated
cardiomyopathy, mixed connective tissue disease, Wegener's
granulomatosis, anti-phospholipid antibody syndrome, vitiligo,
pernicious anemia, glomerulonephritis, and pulmonary fibrosis.
Myasthenia gravis, Graves' disease, idiopathic thrombocytopenia
purpura, hemolytic anemia, diabetes mellitus, inflammatory bowel
disease, Crohn's disease, ulcerative colitis, multiple sclerosis,
psoriasis, and drug-induced autoimmune diseases, e.g., drug-induced
lupus. Preferably, the autoimmune disease is systemic lupus
erythematosus or Lupus nephritis. Transplant rejection include
graft-versus-host disease is a process in which a transplant
recipient's immune system attacks the transplanted organ or tissue.
Other inflammatory and allergic diseases include atherosclerosis
and hay fever.
[0660] An increased binding affinity for a pre-existing ADA can
reduce the clinical utility and efficacy of a therapeutic antibody.
As such the utility of a therapeutic antibody can be limited by the
pre-existing ADAs, since these ADA can influence their efficacy and
pharmacokinetics (e.g. degradation rate). Sometimes this binding
can lead to serious side effects. Furthermore, the present
invention provides a method for decreasing the binding activity at
neutral pH for a pre-existing ADA of antigen-binding domain
comprising an Fc region with an increased binding activity for FcRn
at neutral or acidic pH and an increased binding activity for a
pre-existing ADA at neutral pH. The present invention also provides
the use of the antigen-binding molecules of the present invention
comprising a modified FcRn-binding domain for decreasing the
binding activity for a pre-existing ADA at neutral pH of an
antigen-binding molecule that has an increased affinity for FcRn at
neutral or acidic pH and an increased binding activity for a
pre-existing ADA.
[0661] In particular, the present invention provides a method for
decreasing the binding activity at neutral pH for a pre-existing
ADA of an Fc region of an antigen-binding molecule that has an
increased binding activity for FcRn at neutral or acidic pH, said
method comprising
[0662] a) providing a Fc region having an increased binding
activity for FcRn at neutral or acidic pH and for a pre-existing
ADA at neutral pH and
[0663] b) substituting an amino acid at one or more of the
positions selected from the group consisting of: EU387, EU422,
EU424, EU426, EU433, EU436, EU438 and EU440. The preferred Fc
region in step a) is a human Fc region. Preferably, the Fc region
having an increased binding activity for FcRn at neutral or acidic
pH ranges and for pre-existing ADA in the neutral pH ranges
comprises a substitution of an amino acid at one or more positions
selected from the group consisting of: EU238, EU250, EU252, EU254,
EU255, EU256, EU258 EU286, EU307, EU308, EU309, EU311, EU315,
EU428, EU433, EU434, and EU436. More preferably, it comprises a
substitution at the positions at any one of the combinations of
positions selected from Table 2 and 4 to 7. Even more preferably,
it comprises any one of the substitutions or combinations of
substitutions set forth in any one of Tables 3 and 17 to 20.
[0664] Preferably, step b) comprises substituting an amino acid at
any one of the positions in Table 8. More preferably, step b)
comprises introducing one of the substitutions or combinations
selected from Table 11.
[0665] Also preferably, the antigen-binding molecule comprises
additionally a pH-dependent antigen-binding domain or a calcium
ion-dependent antigen-binding domain.
[0666] Moreover, the present invention provides a method for
decreasing the binding activity for a pre-existing ADA of an
antigen-binding molecule that comprises an Fc region having an
increased binding activity for FcRn at neutral pH, said method
comprising the steps of:
[0667] a) providing an antigen-binding molecule comprising a Fc
region with an increased binding activity for FcRn at acidic pH and
for a pre-existing ADA at a neutral pH and
[0668] b) substituting an amino acid in the Fc region at one or
more of the positions selected from the group consisting of: EU387,
EU422, EU424, EU426, EU433, EU436, EU438 and EU440.
[0669] The preferred Fc region in step a) is a human Fc region.
Preferably, the Fc region having an increased binding activity for
FcRn at neutral pH ranges and for pre-existing ADA in the neutral
pH ranges comprises a substitution of an amino acid at one or more
positions selected from the group consisting of: EU238, EU250,
EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311,
EU315, EU428, EU433, EU434, and EU436. It may also comprise a
substitution at position EU256 in addition to a substitution at the
one or more positions mentioned above, whereby the amino acid at
position EU256 is preferably substituted with a glutamic acid. More
preferably, it comprises a substitution at the positions of any one
of the position combinations selected from Tables 2 and 4 to 7.
Even more preferably, it comprises any one of the substitutions or
substitution combinations selected from any one of Table 3 and 17
to 20.
[0670] Preferably, step b) comprises substituting an amino acid at
any one of the positions in Table 10. More preferably, the
positions are selected from the group consisting of a) EU387, b)
EU422, c) EU424, d) EU438, e) EU440, f) EU422/EU424, and g)
EU438/EU440. Even more preferably, step b) comprises introducing
one of the substitutions or combinations selected from Table
11.
[0671] Moreover, the present invention provides a method for
decreasing the binding activity for a pre-existing ADA of an
antigen-binding molecule that comprises an Fc region having an
increased binding activity for FcRn at acidic pH, said method
comprising the steps of:
[0672] a) providing an antigen-binding molecule comprising a Fc
region with an increased binding activity for FcRn at acidic pH and
for a pre-existing ADA at a neutral pH and
[0673] b) substituting an amino acid in the Fc region at one or
more of the positions selected from the group consisting of: EU387,
EU422, EU424, EU426, EU433, EU436, EU438 and EU440.
[0674] The preferred Fc region in step a) is a human Fc region.
Preferably, the Fc region having an increased binding activity for
FcRn at acidic pH and for pre-existing ADA in the neutral pH ranges
comprises an amino acid substitution comprise a substitution
[0675] i) at position EU434, or
[0676] ii) at two or more positions, wherein the two or more
positions are one of the combinations of the group consisting of a)
EU252/EU254/EU256; b) EU428/EU434; and c) EU250/EU428. Preferably,
the Fc region comprises i) the substitution M434H; or ii) one of
the combinations of the group consisting of a) M252Y/S254T/T256E;
b) M428L/N434S; and c) T250Q and M428L (EU numbering).
[0677] In a preferred embodiment, in step b) an amino acid is
substituted at a) position EU424 or b) the positions EU438/EU440.
More preferably, the substitutions are a) EU424N or b) the
combination EU438R/EU440E.
[0678] In a further preferred embodiment, the methods for
decreasing the binding activity for a pre-existing ADA further
comprises the step c) confirming that said antigen-binding molecule
with a modified Fc domain has a decreased binding activity for an
endogenous ADA as compared the binding activity for the original
antigen-binding molecule as set forth in step a) comprised of an
intact Fc domain.
[0679] Also preferably, the antigen-binding molecule comprises
additionally a pH-dependent antigen-binding domain or a Calcium
ion-dependent antigen-binding domain.
[0680] The present invention provides the use of an antigen-binding
molecule of the present invention for increasing antigen removal
from blood of a mammal, preferably a human patient suffering from
an autoimmune disease.
[0681] The present invention further provides a method for
increasing the total number of antigens to which a single
antigen-binding molecule can bind without significantly increasing
the binding activity for a pre-existing ADA at neutral pH as
compared to a parent antibody, said method comprising the steps
of
[0682] a) providing an antigen-binding molecule comprising a parent
FcRn binding domain,
[0683] b) altering the parent FcRn binding domain of step a) by
substituting an amino acid in the amino acid sequence of the parent
FcRn binding domain at one or more of the positions selected from
the group consisting of EU238, EU250, EU252, EU254, EU255, EU256,
EU258 EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433,
EU434, and EU436; and
[0684] c) altering the modified FcRn-binding domain of step b) by
substituting an amino acid in the amino acid sequence of the parent
FcRn-binding domain at one or more positions selected from the
group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438
and EU440.
[0685] The present invention further provides a method for
facilitating the extracellular release of an antigen-free
antigen-binding molecule taken up into cells in an antigen-bound
form without significantly increasing the binding activity of said
antigen-binding molecule for a pre-existing ADA at neutral pH as
compared to a parent antibody, comprising the steps of
[0686] a) providing an antigen-binding molecule comprising a parent
FcRn-binding domain,
[0687] b) altering the parent FcRn binding domain by substituting
an amino acid in the amino acid sequence of the parent FcRn-binding
domain at one or more positions selected from the group consisting
of EU238, EU250, EU252, EU254, EU255, EU256, EU258 EU286, EU307,
EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436, and
EU428; and
[0688] c) altering the modified FcRn-binding domain of step b) by
substituting an amino acid in the amino acid sequence of the parent
FcRn-binding domain at one or more positions selected from the
group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438
and EU440.
[0689] The present invention further provides a method for
increasing the ability of an antigen-binding molecule to eliminate
plasma antigen without significantly increasing the binding
activity for pre-existing ADA at neutral pH compared to parent
antibody, said method comprising the steps of
[0690] a) providing an antigen-binding molecule comprising a parent
FcRn-binding domain,
[0691] b) altering the parent FcRn binding domain by substituting
an amino acid in the amino acid sequence of the parent FcRn-binding
domain at one or more positions selected from the group consisting
of EU238, EU250, EU252, EU254, EU255, EU256, EU258 EU286, EU307,
EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436, and
EU428; and
[0692] c) altering the modified FcRn-binding domain of step b) by
substituting an amino acid in the amino acid sequence of the parent
FcRn-binding domain at one or more positions selected from the
group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438
and EU440.
[0693] The present invention further provides a method for
improving the pharmacokinetics of an antigen-binding molecule
without significantly increasing the binding activity for a
pre-existing ADA at neutral pH as compared to a parent antibody,
said method comprising the steps of
[0694] a) providing an antigen-binding molecule comprising a parent
FcRn-binding domain,
[0695] b) altering the parent FcRn-binding domain by substituting
an amino acid in the amino acid sequence of the parent FcRn-binding
domain at one or more positions selected from the group consisting
of EU238, EU250, EU252, EU254, EU255, EU256, EU258 EU286, EU307,
EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436; and
[0696] c) altering the modified FcRn-binding domain of step b) by
substituting an amino acid in the amino acid sequence of the parent
FcRn-binding domain at one or more positions selected from the
group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438
and EU440.
[0697] The present invention further provides a method for reducing
total or free antigen plasma concentration without significantly
increasing the binding activity for a pre-existing ADA at neutral
pH as compared to a parent antibody, said method comprising the
steps of
[0698] a) providing an antigen-binding molecule comprising a parent
FcRn-binding domain, wherein the antigen-binding molecule comprises
an antigen-binding domain which can bind said antigen,
[0699] b) altering the parent FcRn-binding domain by substituting
an amino acid in the amino acid sequence of the parent FcRn-binding
domain at one or more positions selected from the group consisting
of EU238, EU250, EU252, EU254, EU255, EU256, EU258 EU286, EU307,
EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436; and
[0700] c) altering the modified FcRn-binding domain of step b) by
substituting an amino acid in the amino acid sequence of the parent
FcRn-binding domain at one or more positions selected from the
group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438
and EU440.
[0701] The preferred Fc region in step a) in the foregoing methods
of use is a human Fc region. In a preferred embodiment, the amino
acid substitution at one or more positions in step b) is a
substitution at one or more positions selected from the group
consisting of: EU238, EU250, EU252, EU254, EU255, EU258, EU286,
EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436,
whereby the Fc region of step b) has an increased binding activity
for FcRn and a pre-existing ADA in the neutral pH ranges. It may
also comprise a substitution at position EU256 in addition to a
substitution at the one or more positions mentioned above, whereby
the amino acid at position EU256 is preferably substituted with a
glutamic acid. More preferably, it comprises a substitution at the
positions of any one of the position combinations selected from
Tables 2 and 4 to 7. Even more preferably, it comprises any one of
the substitutions or substitution combinations selected from any
one of Table 3 and 17 to 20.
[0702] Preferably, step c) comprises substituting an amino acid at
any one of the positions in Table 10. More preferably, the
positions are selected from the group consisting of a) EU387, b)
EU422, c) EU424, d) EU438, e) EU440, f) EU422/EU424, and g)
EU438/EU440. Even more preferably, step c) comprises introducing
one of the substitutions or combinations selected from Table
11.
[0703] In another preferred embodiment, the amino acid substitution
at one or more positions in step b) is a substitution
[0704] i) at position EU434, or
[0705] ii) at two or more positions, wherein the two or more
positions are one of the combinations of the group consisting of a)
EU252/EU254/EU256; b) EU428/EU434; and c) EU250/EU428, whereby the
Fc region of step b) has an increased FcRn-binding activity in the
acidic ranges and an increased binding activity for a pre-existing
ADA in the neutral pH ranges. Preferably, the Fc region comprises
i) the substitution M434H; or
[0706] ii) one of the combinations of the group consisting of a)
M252Y/S254T/T256E; b) M428L/N434S; and c) T250Q and M428L (EU
numbering). In a preferred embodiment, in step c) an amino acid is
substituted at a) position EU424 or b) the positions EU438/EU440.
More preferably, the substitutions are a) EU424N or b) the
combination EU438R/EU440E.
Pharmaceutical Composition
[0707] The present invention also relates to pharmaceutical
compositions that include antigen-binding molecules of the present
invention, or antigen-binding molecules produced by the production
methods of the present invention. The antigen-binding molecules of
the present invention and antigen-binding molecules produced by the
production methods of the present invention have greater activity
to reduce plasma antigen concentration by administration as
compared to typical antigen-binding molecules, and are therefore
useful as pharmaceutical compositions. The pharmaceutical
composition of the present invention may include pharmaceutically
acceptable carriers. In the present invention, pharmaceutical
compositions ordinarily refer to agents for treating or preventing,
or testing and diagnosing diseases.
[0708] 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
may be formulated by mixing in the form of unit dose required in
the generally approved medicine manufacturing practice by
appropriately combining with pharmaceutically 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 may be
readily and routinely adjusted to obtain an appropriate amount in a
pre-determined range.
[0709] 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).
[0710] 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.
[0711] The pharmaceutical compositions of the present invention are
preferably administered parenterally. For example, the compositions
may be in the dosage form for injections, transnasal
administration, transpulmonary administration, or transdermal
administration. Such compositions may be administered systemically
or locally by intravenous injection, intramuscular injection,
intraperitoneal injection, subcutaneous injection, or such.
[0712] 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
may be, for example, from 0.0001 to 1,000 mg/kg for each
administration. Alternatively, the dose may be, for example, from
0.001 to 100,000 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.
[0713] 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.
Production Methods
[0714] The present invention provides methods for producing
antigen-binding molecules of the present invention. In particular,
the present invention provides a method for producing
antigen-binding molecules having an FcRn-binding domain with an
increased binding activity for FcRn at neutral pH as compared to an
antigen-binding molecule comprising a wild type Fc region.
[0715] The present invention provides a method for producing an
antigen-binding molecule, which comprises the steps of:
[0716] (a) selecting a parent FcRn-binding domain and altering the
parent FcRn with a substitution of an amino acid in the amino acid
sequence with another amino acid at one or more positions selected
from the group consisting of EU252, EU434, EU436, EU315, EU311,
EU308, EU307, EU286, EU254, EU250, EU238, EU387, EU422, EU424,
EU428, EU438 and EU440;
[0717] (b) selecting an antigen-binding domain of an
antigen-binding molecule and altering at least one amino acid in
the antigen-binding domain in order to get a pH-dependent
antigen-binding domain or a calcium-ion dependent antigen-binding
domain;
[0718] (c) obtaining a gene encoding an antigen-binding molecule in
which the human FcRn-binding domain and the antigen-binding domain
prepared in (a) and (b) are linked; and
[0719] (d) producing an antigen-binding molecule using the gene
prepared in (c).
[0720] Preferably, the selected antigen-binding molecule comprises
an antigen-binding domain that has a lower binding activity for the
antigen at a pH 5.5-6.5 than at pH 7-8 or has a calcium dependent
antigen binding activity. Preferably, the FcRn-binding domain of
step a) is a FcRn-binding domain of the present invention. More
preferably, the FcRn-binding domain comprises an amino acid
substations at three or more positions, wherein said three or more
positions are one of the combinations set forth in Tables 2, and 4
to 7. Even more preferably, the FcRn-binding domain comprises three
or more substitutions wherein said three or more substitutions are
one of the combinations set forth in Tables 3, 17 to 20.
[0721] Steps (a) may comprise substituting an amino acid
substitution at one or more positions selected from the group
consisting of EU238, EU250, EU252, EU254, EU255, EU258, EU286,
EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436
and selecting a FcRn-binding domain that has stronger human
FcRn-binding activity in the neutral pH range than KD 3.2
micromolar.
[0722] Step (b) may comprise selecting an antigen-binding domain
and altering at least one amino acid in the antigen-binding domain
as described above in order to get a pH-dependent antigen-binding
domain, or selecting a calcium-ion dependent antigen-binding
domain. Altering an amino acid is preferably substituting histidine
for at least one amino acid or inserting at least one histidine.
Meanwhile, the site where the at least one histidine mutation is
introduced is not particularly limited, and thus it may be
introduced at any position as long as the histidine mutation
reduces the antigen-binding activity in the acidic pH range to less
than that in the neutral pH range. Such histidine mutations may be
introduced at a single site or two or more sites. Steps a) and b)
may be repeated twice or more times. The number of times of
repeating steps (a) and (b) is not particularly limited; however,
the number is typically ten times or less.
[0723] A linker operably linking the FcRn-binding domain and the
antigen-binding domain prepared in (a) and (b) is not limited to
any form. The human FcRn-binding domain and the antigen-binding
domain can be linked by either covalent or non-covalent forces. In
particular, the linker can be a peptide linker or a chemical linker
or a binding pair like a combination of biotin and streptavidin.
Modification of a polypeptide including the human FcRn-binding
domain and the antigen-binding domain is known in the art. In
another embodiment, the human FcRn-binding domain and the
antigen-binding domain of the present invention can be linked by
forming a fusion protein between the human FcRn-binding domain and
the antigen-binding domain. In order to construct fusion protein
between the human FcRn-binding domain and the antigen-binding
domain, genes encoding the human FcRn-binding domain and the
antigen-binding domain can be operationally linked so as to form in
frame fusion polypeptide. Appropriately, a linker comprising
peptide consisting of several amino acids can be inserted between
the human FcRn-binding domain and the antigen-binding domain.
Various flexible linkers like the linker whose sequence consists of
(GGGGS)n (SEQ ID NO: 11) is known in the art.
[0724] The present invention further provides a method for
producing an antigen-binding molecule comprising an FcRn-binding
domain with an increased binding activity for FcRn at neutral or
acidic pH without a significantly increased binding activity for a
pre-existing ADA at neutral pH compared to an antigen-binding
molecule comprising a wild type Fc region.
[0725] Preferably, the methods for producing an antigen-binding
molecule comprising a Fc region with an increased binding activity
for FcRn at neutral or acidic pH and a decreased binding activity
for an pre-existing ADA at neutral pH, comprises the steps of:
[0726] (a) providing a Fc region having an increased binding
activity for FcRn in the neutral or acidic pH ranges and an
increased binding activity for a pre-existing ADA in the neutral pH
ranges,
[0727] b) substituting an amino acid in the amino acid sequence of
the Fc region at one or more of the positions selected from the
group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438
and EU440.
[0728] (c) altering at least one amino acid in the antigen-binding
domain of an antigen-binding molecule and selecting an
antigen-binding molecule that has stronger antigen-binding activity
in the neutral pH range than in the acidic pH range;
[0729] (d) obtaining a gene encoding an antigen-binding molecule in
which the human FcRn-binding domain prepared in (b) and the
antigen-binding domain prepared in (c) are linked and
[0730] (e) producing an antigen-binding molecule using the gene
prepared in (d).
[0731] The preferred Fc region in step a) is a human Fc region.
Preferably, the Fc region having an increased binding activity for
FcRn and pre-existing ADA at neutral or acidic pH ranges and for
pre-existing ADA in the neutral pH ranges comprises a substitution
of an amino acid at one or more positions selected from the group
consisting of: EU238, EU250, EU252, EU254, EU255, EU258, EU286,
EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436.
More preferably, it comprises substitutions at the positions of any
one of the position combinations selected from Tables 2 and 4 to 7.
Even more preferably, it comprises any one of the substitutions or
combinations of substitutions set forth in any one of Tables 3 and
17 to 20. Preferably, step a) includes providing a nucleotide
sequence encoding an Fc region having an increased binding activity
for FcRn and pre-existing ADA at neutral or acidic pH ranges.
Preferably, the substitutions in step b) amino acid substitutions
at one or more positions or a position combination as set forth in
Table 10. More preferably, the substitutions of step b) are one of
the substitution combinations set forth in Table 11. The amino acid
at one or more of the positions selected from the group consisting
of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440 in
step b) is preferably substituted by replacing one or more
nucleotides in the nucleotide sequence.
[0732] Steps (b) and (c) may be carried out in either order.
Furthermore, step c) may comprise altering at least one amino acid
in the antigen-binding domain as described above in order to get a
pH-dependent antigen-binding domain, or selecting a calcium-ion
dependent antigen-binding domain. In step (c), altering an amino
acid is preferably substituting histidine for at least one amino
acid or inserting at least one histidine. Meanwhile, the site where
the at least one histidine mutation is introduced is not
particularly limited, and thus it may be introduced at any position
as long as the histidine mutation reduces the antigen-binding
activity in the acidic pH range to less than that in the neutral pH
range. Such histidine mutations may be introduced at a single site
or two or more sites. Steps b) and c) may be repeated twice or more
times. The number of times of repeating steps (b) and (c) is not
particularly limited; however, the number is typically ten times or
less.
[0733] A linker operably linking the FcRn-binding domain and the
antigen-binding domain prepared in (b) and (c) is not limited to
any form. The human FcRn-binding domain and the antigen-binding
domain can be linked by either covalent or non-covalent forces. In
particular, the linker can be a peptide linker or a chemical linker
or a binding pair like a combination of biotin and streptavidin.
Modification of a polypeptide including the human FcRn-binding
domain and the antigen-binding domain is known in the art. In
another embodiment, the human FcRn-binding domain and the
antigen-binding domain of the present invention can be linked by
forming a fusion protein between the human FcRn-binding domain and
the antigen-binding domain. In order to construct the fusion
protein between the human FcRn-binding domain and the
antigen-binding domain, genes encoding the human FcRn-binding
domain and the antigen-binding domain can be operationally linked
so as to form in frame fusion polypeptide. Appropriately, a linker
comprising peptide consisting of several amino acids can be
inserted between the human FcRn-binding domain and the
antigen-binding domain. Various flexible linkers like the linker
whose sequence consists of (GGGGS)n (SEQ ID NO: 11) is known in the
art.
[0734] Thus, the production methods of the present invention may
further comprise the steps of altering the above-described amino
acids and substituting or inserting histidine. In the production
methods of the present invention, non-natural amino acids may be
used instead of histidine. Therefore, the present invention can
also be understood by replacing the above-mentioned histidine with
non-natural amino acids.
[0735] Step a) of the production methods of the present invention
may also comprise a substitution at position EU256 in addition to a
substitution at the one or more positions mentioned above, whereby
the amino acid at position EU256 is preferably substituted with a
glutamic acid.
[0736] Furthermore, the production methods of the present invention
may further comprise a step comprising substituting an amino acid
in the amino acid sequence of the Fc region at one or more of the
positions selected from the group consisting of EU234, EU235,
EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270,
EU271, EU295, EU296, EU297 EU298, EU300, EU324, EU325, EU327,
EU328, EU329, EU331, and EU332 (according to the EU numbering
system). Preferably, the substitutions L235R/S239K are
introduced.
[0737] Parent FcRn-binding domains and antigen-binding molecules
comprising them that are used in the production methods of the
present invention may be prepared by any method. For example, it is
possible to use pre-existing antibodies, pre-existing libraries
(phage libraries and the like), antibodies and libraries that are
prepared from hybridomas obtained by immunizing animals or from B
cells of immunized animals, antibodies and libraries prepared by
introducing random amino acid alterations into the above-described
antibodies and libraries, antibodies and libraries prepared by
introducing histidine or non-natural amino acid mutations into the
above-described antibodies and libraries (libraries with high
content of histidine or non-natural amino acid, libraries
introduced with histidine or non-natural amino acid at specific
sites, and the like), and such.
[0738] The antigen-binding activity and human FcRn binding activity
of an antigen-binding molecule can be determined by methods known
to those skilled in the art. Conditions except for pH can be
appropriately determined by those skilled in the art.
[0739] In the above-described production methods, the antigen and
antigen-binding molecule may bind to each other in any state, and
the human FcRn and antigen-binding molecule may bind to each other
in any state. The state is not particularly limited; for example,
the antigen or human FcRn may be contacted with an immobilized
antigen-binding molecule to bind the antigen-binding molecule.
Alternatively, the antigen-binding molecule may be contacted with
an immobilized antigen or human FcRn to bind the antigen-binding
molecule. Alternatively, the antigen-binding molecule may be
contacted with the antigen or human FcRn in a solution to bind the
antigen-binding molecule.
[0740] The antigen-binding molecules produced by the
above-described methods may be any antigen-binding molecule of the
present invention; and preferred antigen-binding molecules include,
for example, those having an antigen-binding domain which is an
ionized calcium-concentration dependent antigen-binding domain or
an antigen-binding domain with histidine substitution for amino
acid(s) or insertion of at least one histidine, and said
antigen-binding molecule further comprising a human FcRn-binding
domain, which comprise an amino acid substitution at one or more
positions selected from the group consisting of EU238, EU250,
EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311,
EU315, EU428, EU433, EU434, and EU436 (EU numbering). The
antigen-binding molecule of the present invention may also comprise
a substitution at position EU256 in addition to a substitution at
the one or more positions mentioned above. Preferably, the amino
acid at position EU256 is substituted with a glutamic acid. More
preferably, the FcRn-binding domain comprises an amino acid
substations at three or more positions, wherein said three or more
positions are one of the combinations set forth in Tables 2, and 4
to 7. Even more preferably, the FcRn-binding domain comprises three
or more substitutions wherein said three or more substitutions are
one of the combinations set forth in Tables 3, 17 to 20.
[0741] Further preferred antigen-binding molecules include for
example, those having an antigen-binding domain which is an ionized
calcium-concentration dependent antigen-binding domain or an
antigen-binding domain with histidine substitution for amino
acid(s) or insertion of at least one histidine, and said
antigen-binding molecule further comprising a human Fc region with
substitutions of the amino acid at one or more of the positions
selected from the group consisting of: EU387, EU422, EU424, EU426,
EU433, EU436, EU438 and EU440. More preferably, the FcRn-binding
domain contains substitutions of an amino acid in the human
FcRn-binding domain at three or more positions wherein said three
or more positions are one of the combinations set forth in Tables 9
and 10.
[0742] A more preferred antigen-binding molecule includes those
having those having an antigen-binding domain which is an ionized
calcium-concentration dependent antigen-binding domain or an
antigen-binding domain with histidine substitution for amino
acid(s) or insertion of at least one histidine, and said
antigen-binding molecule further comprising a human Fc region with
substitutions of the amino acid
[0743] a) at one or more of the positions selected from the group
consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258,
EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and
EU436, and
[0744] b) at one or more of the positions selected from the group
consisting of: EU387, EU422, EU424, EU426, EU433, EU436, EU438 and
EU440 (EU numbering).
[0745] Preferably, the amino acid at position EU256 is substituted
with a glutamic acid.
[0746] More preferably, the antigen-binding molecules comprise a
substitution combination set forth in Tables 11 to 13.
[0747] An antibody having a desired activity may be selected by
screening from a number of antibodies obtained from the antibody
libraries or hybridomas described below.
[0748] When altering amino acids in an antigen-binding molecule, it
is possible to use a known sequence for the amino acid sequence of
an antigen-binding molecule before alteration or the amino acid
sequence of an antigen-binding molecule newly identified by methods
known to those skilled in the art. For example, when the
antigen-binding molecule is an antibody, it can be obtained from
antibody libraries or by cloning an antibody-encoding gene from
monoclonal antibody-producing hybridomas.
[0749] Regarding antibody libraries, many antibody libraries are
already known, and methods for producing antibody libraries are
also known; therefore, those skilled in the art can appropriately
obtain antibody libraries. For example, regarding phage libraries,
one can refer to the literature such as Clackson et al., Nature
(1991) 352: 624-8; Marks et al., J. Mol. Biol. (1991) 222: 581-97;
Waterhouses et al., Nucleic Acids Res. (1993) 21: 2265-6; Griffiths
et al., EMBO J. (1994) 13: 324.0-60; Vaughan et al., Nature
Biotechnology (1996) 14: 309-14; and Japanese Patent Kohyo
Publication No. (JP-A) H20-504970 (unexamined Japanese national
phase publication corresponding to a non-Japanese international
publication). In addition, it is possible to use known methods,
such as methods using eukaryotic cells as libraries (WO 95/15393)
and ribosome display methods. Furthermore, technologies to obtain
human antibodies by panning using human antibody libraries are also
known. For example, variable regions of human antibodies can be
expressed on the surface of phages as single chain antibodies
(scFvs) using phage display methods, and phages that bind to
antigens can be selected. Genetic analysis of the selected phages
can determine the DNA sequences encoding the variable regions of
human antibodies that bind to the antigens. Once the DNA sequences
of scFvs that bind to the antigens is revealed, suitable expression
vectors can be produced based on these sequences to obtain human
antibodies. These methods are already well known, and one can refer
to WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172,
WO 95/01438, and WO 95/15388.
[0750] As for methods for obtaining genes encoding antibodies from
hybridomas, known technologies may be basically used, which involve
the use of desired antigens or cells expressing the desired
antigens as sensitizing antigens, using these to perform
immunizations according to conventional immunization methods,
fusing the resulting immune cells with known parent cells by
conventional cell fusion methods, screening monoclonal antibody
producing cells (hybridomas) by conventional screening methods,
synthesizing cDNAs of antibody variable regions (V regions) from
mRNAs of the obtained hybridomas using reverse transcriptase, and
linking them with DNAs encoding the desired antibody constant
regions (C regions).
[0751] More specifically, sensitizing antigens to obtain the
above-described antigen-binding molecule genes encoding the H
chains and L chains may include, for example, both complete
antigens with immunogenicity and incomplete antigens including
haptens and the like with no immunogenicity; however they are not
limited to these examples. For example, it is possible to use whole
proteins and partial peptides of proteins of interest. In addition,
it is known that substances comprising polysaccharides, nucleic
acids, lipids, and such can be antigens. Thus, the antigens of the
antigen-binding molecules of the present invention are not
particularly limited. The antigens can be prepared by methods known
to those skilled in the art, for example, by baculovirus-based
methods (for example, WO 98/46777) and such. Hybridomas can be
produced, for example, by the method of Milstein et al. (G. Kohler
and C. Milstein, Methods Enzymol. (1981) 73: 3-46) and such. When
the immunogenicity of an antigen is low, immunization may be
performed after linking the antigen with a macromolecule having
immunogenicity, such as albumin. Alternatively, if necessary,
antigens may be converted into soluble antigens by linking them
with other molecules. When transmembrane molecules such as membrane
antigens (for example, receptors) are used as antigens, portions of
the extracellular regions of the membrane antigens can be used as a
fragment, or cells expressing transmembrane molecules on their cell
surface may be used as immunogens.
[0752] Antigen-binding molecule-producing cells can be obtained by
immunizing animals using appropriate sensitizing antigens described
above. Alternatively, antigen-binding molecule-producing cells can
be prepared by in vitro immunization of lymphocytes that can
produce antigen-binding molecules. Various mammals can be used for
immunization; such commonly used animals include rodents,
lagomorphas, and primates. Such animals include, for example,
rodents such as mice, rats, and hamsters; lagomorphas such as
rabbits; and primates including monkeys such as cynomolgus monkeys,
rhesus monkeys, baboons, and chimpanzees. In addition, transgenic
animals carrying human antibody gene repertoires are also known,
and human antibodies can be obtained by using these animals (see WO
96/34096; Mendez et al., Nat. Genet. (1997) 15: 146-56). Instead of
using such transgenic animals, for example, desired human
antibodies having binding activity against antigens can be obtained
by in vitro sensitization of human lymphocytes with desired
antigens or cells expressing the desired antigens, and then fusing
the sensitized lymphocytes with human myeloma cells such as U266
(see Japanese Patent Application Kokoku Publication No. (JP-B)
H01-59878 (examined, approved Japanese patent application published
for opposition)). Furthermore, desired human antibodies can be
obtained by immunizing transgenic animals carrying a complete
repertoire of human antibody genes, with desired antigens (see WO
93/12227, WO 92/03918, WO 94/02602, WO 96/34096, and WO
96/33735).
[0753] Animal immunization can be carried out by appropriately
diluting and suspending a sensitizing antigen in phosphate buffered
saline (PBS), physiological saline, or such, and mixing it with an
adjuvant to emulsify, if necessary. This is then intraperitoneally
or subcutaneously injected into animals. Then, the sensitizing
antigen mixed with Freund's incomplete adjuvant is preferably
administered several times every four to 21 days. Antibody
production can be confirmed by measuring the titer of the antibody
of interest in animal sera using conventional methods.
[0754] Antigen-binding molecule-producing cells obtained from
lymphocytes or animals immunized with a desired antigen can be
fused with myeloma cells to generate hybridomas using conventional
fusing agents (for example, polyethylene glycol) (Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) 59-103). When required, hybridoma cells can be cultured and
grown, and the binding specificity of the antigen-binding molecule
produced from these hybridomas can be measured using known analysis
methods, such as immunoprecipitation, radioimmunoassay (RIA), and
enzyme-linked immunosorbent assay (ELISA). Thereafter, if
necessary, hybridomas producing antigen-binding molecules of
interest whose specificity, affinity, or activity has been
determined can be subcloned by methods such as limiting
dilution.
[0755] Next, genes encoding the selected antigen-binding molecules
can be cloned from hybridomas or antigen-binding molecule-producing
cells (sensitized lymphocytes, and such) using probes that can
specifically bind to the antigen-binding molecules (for example,
oligonucleotides complementary to sequences encoding the antibody
constant regions). It is also possible to clone the genes from mRNA
using RT-PCR. Immunoglobulins are classified into five different
classes, IgA, IgD, IgE, IgG, and IgM. These classes are further
divided into several subclasses (isotypes) (for example, IgG-1,
IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2; and such). H chains and L
chains used in the present invention to produce antigen-binding
molecules are not particularly limited and may originate from
antibodies belonging to any of these classes or subclasses;
however, IgG is particularly preferred.
[0756] Herein, it is possible to alter H-chain-encoding genes and
L-chain-encoding genes using genetic engineering technologies.
Genetically altered antibodies, such as chimeric antibodies and
humanized antibodies, which have been artificially altered for the
purpose of decreasing heterologous immunogenicity and such against
humans, can be appropriately produced for antibodies such as mouse
antibodies, rat antibodies, rabbit antibodies, hamster antibodies,
sheep antibodies, and camel antibodies. Chimeric antibodies are
antibodies including H-chain and L-chain variable regions of
nonhuman mammal antibody, such as mouse antibody, and the H-chain
and L-chain constant regions of human antibody. Chimeric antibodies
can be obtained by ligating a DNA encoding a variable region of a
mouse antibody to a DNA encoding a constant region of a human
antibody, inserting this into an expression vector, and introducing
the vector into a host to produce the antibody. A humanized
antibody, which is also called a reshaped human antibody, can be
synthesized by PCR using several oligonucleotides produced so that
they have overlapping portions at the ends of DNA sequences
designed to link the complementarity determining regions (CDRs) of
an antibody of a nonhuman mammal such as a mouse. The resulting DNA
can be ligated to a DNA encoding a human antibody constant region.
The ligated DNA can be inserted into an expression vector, and the
vector can be introduced into a host to produce the antibody (see
EP 239400 and WO 96/02576). Human antibody FRs that are ligated via
the CDR are selected when the CDR forms a favorable antigen-binding
site. If necessary, amino acids in the framework region of an
antibody variable region may be replaced such that the CDR of the
reshaped human antibody forms an appropriate antigen-binding site
(K. Sato et al., Cancer Res. (1993) 53: 10.01-10.06).
[0757] In addition to the humanization described above, antibodies
may be altered to improve their biological properties, for example,
the binding to the antigen. In the present invention, such
alterations can be achieved by methods such as site-directed
mutagenesis (see for example, Kunkel (1910.0) Proc. Natl. Acad.
Sci. USA 82: 488), PCR mutagenesis, and cassette mutagenesis. In
general, mutant antibodies whose biological properties have been
improved show amino acid sequence homology and/or similarity of 70%
or higher, more preferably 80% or higher, and even more preferably
90% or higher (for example, 95% or higher, 97%, 98%, or 99%), when
compared to the amino acid sequence of the original antibody
variable region. Herein, sequence homology and/or similarity is
defined as the ratio of amino acid residues that are homologous
(same residue) or similar (amino acid residues classified into the
same group based on the general properties of amino acid side
chains) to the original antibody residues, after the sequence
homology value has been maximized by sequence alignment and gap
introduction, if necessary. In general, natural amino acid residues
are classified into groups based on the characteristics of their
side chains as follows:
[0758] (1) hydrophobic: alanine, isoleucine, valine, methionine,
and leucine;
[0759] (2) neutral hydrophilic: asparagine, glutamine, cysteine,
threonine, and serine;
[0760] (3) acidic: aspartic acid and glutamic acid;
[0761] (4) basic: arginine, histidine, and lysine;
[0762] (5) residues that affect the orientation of the chain:
glycine, and proline; and
[0763] (6) aromatic: tyrosine, tryptophan, and phenylalanine.
[0764] Furthermore, the present invention provides genes encoding
the FcRn-binding domain of the present invention and the
antigen-binding molecules of the present invention. The genes
encoding the antigen-binding molecules of the present invention may
be any genes, and may be DNAs, RNAs, nucleic acid analogs, or the
like.
[0765] Furthermore, the present invention also provides host cells
carrying the genes described above. The host cells are not
particularly limited and include, for example, E. coli and various
animal cells. The host cells may be used, for example, as a
production system to produce and express the antibodies of the
present invention. In vitro and in vivo production systems are
available for polypeptide production systems. Such in vitro
production systems include, for example, production systems using
eukaryotic cells or prokaryotic cells.
[0766] Eukaryotic cells that can be used as host cells include, for
example, animal cells, plant cells, and fungal cells. Animal cells
include: mammalian cells, for example, CHO(Chinese hamster ovary
cell line), COS (Monkey kidney cell line), myeloma (Sp2/O, NS0
etc), BHK (baby hamster kidney cell line) Hela, Vero, HEK293 (human
embryonic kidney cell line with sheared adenovirus (Ad)5 DNA),
PER.C6 cell (human embryonic retinal cell line transformed with the
Adenovirus Type 5 (Ad5) E1A and E1B genes) 293, etc (see Current
Protocols in Protein Science (May, 2001, Unit 5.9, Table 5.9.1)),
amphibian cells such as Xenopus laevis oocytes (Valle et al.,
Nature (1981) 291: 338-340); and insect cells such as Sf9, Sf21,
and Tn5.CHO-DG44, CHO-DX11B, COS7 cells, HEK293 cells, and BHK
cells are preferably used to express the antibodies of the present
invention. Among animal cells, CHO cells are particularly
preferable for large-scale expression. Vectors can be introduced
into host cells, for example, by calcium phosphate methods,
DEAE-dextran methods, methods using cationic liposome DOTAP
(Boehringer-Mannheim), electroporation methods, and lipofection
methods.
[0767] Regarding plant cells, for example, Nicotiana
tabacum-derived cells and duckweed (Lemna minor) are known as a
protein production system. Calluses can be cultured from these
cells to produce the antigen-binding molecules of the present
invention. Regarding fungal cells, known protein expression systems
are those using yeast cells, for example, cells of genus
Saccharomyces (such as Saccharomyces cerevisiae and Saccharomyces
pombe); and cells of filamentous fungi, for example, genus
Aspergillus (such as Aspergillus niger). These cells can be used as
a host to produce the antigen-binding molecules of the present
invention.
[0768] Bacterial cells can be used in the prokaryotic production
systems. Regarding bacterial cells, production systems using
Bacillus subtilis are known in addition to the production systems
using E. coli described above. Such systems can be used in
producing the antigen-binding molecules of the present
invention.
[0769] Genes obtained by the production methods of the present
invention are typically carried by (inserted into) appropriate
vectors, and then introduced into host cells. The vectors are not
particularly limited as long as they stably retain the inserted
nucleic acids. For example, when E. coli is used as the host,
preferred cloning vectors include pBluescript vector (Stratagene);
however, various commercially available vectors may be used. When
using vectors to produce the antigen-binding molecules of the
present invention, expression vectors are particularly useful. The
expression vectors are not particularly limited as long as the
vectors express the antigen-binding molecules in vitro, in E. coli,
in culture cells, or in a body of an organism. For example, pBEST
vector (Promega) is preferred for in vitro expression; pET vector
(Invitrogen) is preferred for E. coli; pME18S-FL3 vector (GenBank
Accession No. AB009864) is preferred for culture cells; and pME18S
vector (Mol Cell Biol. (1988) 8: 466-472) is preferred for bodies
of organisms. In addition, EBNA1 protein may be co-expressed to
increase the number of copies of the gene of interest. In this
case, a vector that includes OriP as a initiation site of
replication is used (Biotechnol Bioeng. 2001 Oct. 20;
75(2):197-203, Biotechnol Bioeng. 2005 Sep. 20; 91(6):670-7.) DNAs
of the present invention can be inserted into the vectors by
conventional methods, for example, by ligation using restriction
enzyme sites (Current protocols in Molecular Biology, edit. Ausubel
et al., (1987) Publish. John Wiley & Sons, Section
11.4-11.11).
[0770] The above host cells are not particularly limited, and
various host cells may be used depending on the purpose. Examples
of cells for expressing the antigen-binding molecules include
bacterial cells (such as those of Streptococcus, Staphylococcus, E.
coli, Streptomyces, and Bacillus subtilis), eukaryotic cells (such
as those of yeast and Aspergillus), insect cells (such as
Drosophila S2 and Spodoptera SF9), animal cells (such as CHO, COS,
HeLa, C127, 3T3, BHK, HEK293, and Bowes melanoma cells), and plant
cells. Vectors can be introduced into a host cell by known methods,
for example, calcium phosphate precipitation methods,
electroporation methods (Current protocols in Molecular Biology
edit. Ausubel et al. (1987) Publish. John Wiley & Sons, Section
9.1-9.9), lipofection methods, and microinjection methods.
[0771] The host cells can be cultured by known methods. For
example, when using animal cells as a host, DMEM, MEM, RPMI1640, or
IMDM may be used as the culture medium. They may be used with serum
supplements such as FBS or fetal calf serum (FCS). The cells may be
cultured in serum-free cultures. The preferred pH is about 6 to 8
during the course of culturing. Incubation is carried out typically
at 30 to 40 degrees C. for about 15 to 200 hours. Medium is
exchanged, aerated, or agitated, as necessary.
[0772] Appropriate secretion signals may be incorporated to
polypeptides of interest so that the antigen-binding molecules
expressed in the host cell are secreted into the lumen of the
endoplasmic reticulum, into the periplasmic space, or into the
extracellular environment. These signals may be endogenous to the
antigen-binding molecules of interest or may be heterologous
signals.
[0773] On the other hand, for example, production systems using
animals or plants may be used as systems for producing polypeptides
in vivo. A polynucleotide of interest is introduced into an animal
or plant and the polypeptide is produced in the body of the animal
or plant, and then collected. The "hosts" of the present invention
include such animals and plants.
[0774] The production system using animals include those using
mammals or insects. It is possible to use mammals such as goats,
pigs, sheep, mice, and bovines (Vicki Glaser SPECTRUM Biotechnology
Applications (1993)). The mammals may be transgenic animals.
[0775] For example, a polynucleotide encoding an antigen-binding
molecule of the present invention is prepared as a fusion gene with
a gene encoding a polypeptide specifically produced in milk, such
as the goat beta-casein. Next, goat embryos are injected with
polynucleotide fragments containing the fusion gene, and then
transplanted to female goats. Desired antigen-binding molecules can
be obtained from milk produced by the transgenic goats, which are
born from the goats that received the embryos, or from their
offspring. Hormones may be administered as appropriate to increase
the volume of milk containing the antigen-binding molecule produced
by the transgenic goats (Ebert et al., Bio/Technology (1994) 12:
699-702).
[0776] Insects such as silkworms may be used to produce the
antigen-binding molecules of the present invention. When silkworms
are used, baculoviruses carrying a polynucleotide encoding an
antigen-binding molecule of interest can be used to infect
silkworms, and the antigen-binding molecule of interest can be
obtained from their body fluids.
[0777] Furthermore, when plants are used to produce the
antigen-binding molecules of the present invention, for example,
tobacco may be used. When tobacco is used, a polynucleotide
encoding an antigen-binding molecule of interest is inserted into a
plant expression vector, for example, pMON 530, and then the vector
is introduced into bacteria, such as Agrobacterium tumefaciens. The
bacteria are then allowed to infect tobacco such as Nicotiana
tabacum, and the desired antigen-binding molecules can be collected
from their leaves (Ma et al., Eur. J. Immunol. (1994) 24: 131-138).
Alternatively, it is possible to infect duckweed (Lemna minor) with
similar bacteria. After cloning, the desired antigen-binding
molecules can be obtained from the duckweed cells (Cox K M et al.,
Nat. Biotechnol. 2006 December; 24(12): 1591-1597).
[0778] The thus obtained antigen-binding molecules may be isolated
from the inside or outside (such as the medium and milk) of host
cells, and purified as substantially pure and homogenous
antigen-binding molecules. The methods for isolating and purifying
antigen-binding molecules are not particularly limited, and
isolation and purification methods usually used for polypeptide
purification can be used. Antigen-binding molecules may be isolated
and purified, by appropriately selecting and combining, for
example, chromatographic columns, filtration, ultrafiltration,
salting out, solvent precipitation, solvent extraction,
distillation, immunoprecipitation, SDS-polyacrylamide gel
electrophoresis, isoelectric focusing, dialysis, and
recrystallization.
[0779] Examples of chromatography techniques include, but are not
limited to, affinity chromatography, ion exchange chromatography,
hydrophobic chromatography, gel filtration, reverse-phase
chromatography, and adsorption chromatography (Strategies for
Protein Purification and Characterization: A Laboratory Course
Manual. Ed Daniel R. Marshak et al., (1996) Cold Spring Harbor
Laboratory Press). Such chromatographic methods can be conducted
using liquid phase chromatography such as HPLC and FPLC. Columns
used for affinity chromatography include, protein A columns and
protein G columns. Columns using protein A include, for example,
Hyper D, POROS, and Sepharose F. F. (Pharmacia).
[0780] If necessary, an antigen-binding molecule can be modified
arbitrarily, and peptides can be partially deleted by allowing an
appropriate protein modification enzyme to act before or after
purification of the antigen-binding molecule. Such protein
modification enzymes include, for example, trypsin, chymotrypsin,
lysyl endopeptidases, protein kinases, and glucosidases.
EXAMPLES
[0781] While the present invention is herein described in detail
and with reference to specific embodiments thereof, it is to be
understood that the foregoing description is exemplary and
explanatory in nature and is intended to illustrate the present
invention and its preferred embodiments. Through routine
experimentation, one skilled in the art will readily recognize that
various changes and modifications can be made therein without
departing from the spirit and scope of the present invention, the
metes and bounds of which are defined by the appended claims.
[Example 1] Construction of New Neutral pH FcRn Binding Affinity
Improved Fc Variants
[0782] Fc regions of the antigen-binding molecule (antibody) which
interacts with FcRn (Nat Rev Immunol. 2007 September; 7(9):715-25)
were engineered to have an improved binding affinity to FcRn at
neutral pH in order to enhance the antigen elimination from plasma.
The mechanism of antigen elimination from plasma by pH-dependent
antigen binding antibody with improved binding affinity to FcRn at
neutral pH in comparison to the conventional antibody is shown in
FIG. 1A.
[0783] The Examples 1-17 of WO2011/122011 disclose mutations (amino
acid substitutions) that improve the binding affinity to FcRn at
neutral pH and describes Fc variants F1 to F599 (Table 16) that
were generated with the focus on improving the binding affinity to
FcRn at neutral pH of antibodies. However, for the pharmaceutical
development of antibodies comprising such Fc variants, not only
their pharmacological property (i.e improved FcRn binding) but also
stability, purity and immunogenicity should be considered.
Antibodies which exhibit poor stability and purity are not suitable
as a drug, and poor immunogenicity would hinder their clinical
development.
1-1. Design and Generation of Fc Variants with Improved Binding
Affinity to hFcRn at Neutral pH
[0784] Various Fc variants with improved binding affinity to hFcRn
at neutral pH while maintaining high stability, high purity and low
immunogenicity risk were designed. Mutations (amino acid
substitutions) introduced into Fc region of wild type IgG1 are
shown for each Fc variants in Table 16 (IgG1-F1 to F1434). The
amino acid substitutions were introduced into VH3-IgG1 (SEQ ID NO:
1) to generate Fc variants by the method known to those skilled in
the art described in Reference Example 1 of WO2011/122011.
TABLE-US-00016 TABLE 16 Fc variants hFcRn Immuno- Variant KD Tm HMW
genicity name (M) (.degree. C.) (%) score Mutation IgG 8.8E-5 nd nd
nd none M73 1.4E-05 nd nd nd (see WO209/125825) F1 8.1E-07 67.6
12.64 91 N434W F2 3.2E-06 64.7 2.96 39 M252Y/S254T/T256E F3 2.5E-06
68.5 1.43 112 N434Y F4 5.8E-06 68.5 0.58 38 N434S F5 6.8E-06 69.2
0.29 16 N434A F7 5.6E-06 65.5 0.60 131 M252Y F8 4.2E-06 64.3 0.97
84 M252W F9 1.4E-07 62.6 0.72 152 M252Y/S254T/T256E/N434Y F10
6.9E-08 61.7 0.94 130 M252Y/S254T/T256E/N434W F11 3.1E-07 64.6 0.73
243 M252Y/N434Y F12 1.7E-07 63.8 10.11 222 M252Y/N434W F13 3.2E-07
63.7 1.21 196 M252W/N434Y F14 1.8E-07 62.9 17.73 175 M252W/N434W
F19 4.6E-07 63.8 2.88 228 P257L/N434Y F20 4.6E-07 54.8 1.96 327
V308F/N434Y F21 3.0E-08 56.6 0.81 424 M252Y/V308P/N434Y F22 2.0E-06
69.8 1.26 116 M428L/N434S F25 9.2E-09 54.9 9.95 311
M252Y/S254T/T256E/V308P/N434W F26 1.0E-06 68.4 0.31 2 I332V F27
7.4E-06 69.6 0.85 113 G237M F29 1.4E-06 67.1 0.79 114 I332V/N434Y
F31 2.8E-06 56.3 0.97 328 G237M/V308F F32 8.0E-07 67.7 11.90 122
S254T/N434W F33 2.3E-06 68.4 0.49 144 S254T/N434Y F34 2.8E-07 64.6
10.22 93 T256E/N434W F35 8.4E-07 66.1 1.15 115 T256E/N434Y F36
3.6E-07 64.9 14.88 118 S254T/T256E/N434W F37 1.1E-06 65.6 0.68 140
S254T/T256E/N434Y F38 1.0E-07 64.1 11.74 193 M252Y/S254T/N434W F39
3.0E-07 64.8 0.84 214 M252Y/S254T/N434Y F40 8.2E-08 61.4 11.54 130
M252Y/T256E/N434W F41 1.5E-07 61.7 0.77 152 M252Y/T256E/N434Y F42
1.0E-06 63.2 0.81 56 M252Y/S254T/T256E/N434A F43 1.7E-06 64.3 0.63
147 M252Y/N434A F44 1.1E-06 64.5 1.81 100 M252W/N434A F47 2.4E-07
62.4 7.75 153 M252Y/T256Q/N434W F48 3.2E-07 64.2 0.82 175
M252Y/T256Q/N434Y F49 5.1E-07 61.2 16.63 173 M252F/T256D/N434W F50
1.2E-06 63.7 1.20 195 M252F/T256D/N434Y F51 8.1E-06 69.1 2.64 68
N434F/Y436H F52 3.1E-06 69.7 1.07 68 H433K/N434F/Y436H F53 1.0E-06
66.5 8.01 92 I332V/N434W F54 8.4E-08 64.2 8.27 271 V308P/N434W F56
9.4E-07 66.7 1.22 169 I332V/M428L/N434Y F57 1.1E-05 70.4 1.76 0
G385D/Q386P/N389S F58 7.7E-07 67.9 6.43 91 G385D/Q386P/N389S/N434W
F59 2.4E-06 68.7 2.12 112 G385D/Q386P/N389S/N434Y F60 1.1E-05 69
2.17 14 G385H F61 9.7E-07 67.6 10.94 104 G385H/N434W F62 1.9E-06
68.5 0.98 126 G385H/N434Y F63 2.5E-06 68.5 1.18 95 N434F F64
5.3E-06 69 0.78 24 N434H F65 2.9E-07 62.9 4.38 135
M252Y/S254T/T256E/N434F F66 4.3E-07 63.3 2.96 63
M252Y/S254T/T256E/N434H F67 6.3E-07 63.1 1.08 227 M252Y/N434F F68
9.3E-07 63.6 0.89 155 M252Y/N434H F69 5.1E-07 67.9 7.45 167
M428L/N434W F70 1.5E-06 69 1.82 167 M428L/N434Y F71 8.3E-08 59.7
1.49 207 M252Y/S254T/T256E/M428L/N434W F72 2.0E-07 62.6 2.46 207
M252Y/S254T/T256E/M428L/N434Y F73 1.7E-07 63.4 1.86 298
M252Y/M428L/N434W F74 4.6E-07 64.6 1.52 298 M252Y/M428L/N434Y F75
1.4E-06 62.6 0.98 226 M252Y/M428L/N434A F76 1.0E-06 62.4 1.08 134
M252Y/S254T/T256E/M428L/N434A F77 9.9E-07 66.7 1.62 170
T256E/M428L/N434Y F78 7.8E-07 67.9 10.76 199 S254T/M428L/N434W F79
5.9E-06 67.7 0.96 44 S254T/T256E/N434A F80 2.7E-06 63.8 0.86 79
M252Y/T256Q/N434A F81 1.6E-06 63.3 1.85 56 M252Y/T256E/N434A F82
1.1E-06 67.4 10.30 120 T256Q/N434W F83 2.6E-06 65.4 1.37 142
T256Q/N434Y F84 2.8E-07 62.5 13.96 139 M252W/T256Q/N434W F85
5.5E-07 59.9 1.61 161 M252W/T256Q/N434Y F86 1.5E-06 67.6 10.35 118
S254T/T256Q/N434W F87 4.3E-06 68.6 1.36 139 S254T/T256Q/N434Y F88
1.9E-07 63.5 10.67 128 M252Y/S254T/T256Q/N434W F89 3.6E-07 64 1.38
149 M252Y/S254T/T256Q/N434Y F90 1.9E-08 64 10.23 311
M252Y/T256E/V308P/N434W F91 4.8E-08 53.7 1.63 479
M252Y/V308P/M428L/N434Y F92 1.1E-08 56.2 11.80 388
M252Y/S254T/T256E/V308P/M428L/N434W F93 7.4E-07 62.2 12.74 252
M252W/M428L/N434W F94 3.7E-07 63.4 11.86 283 P257L/M428L/N434Y F95
2.6E-07 53.2 1.36 212 M252Y/S254T/T256E/M428L/N434F F99 6.2E-07
62.9 1.27 63 M252Y/T256E/N434H F101 1.1E-07 60.7 4.54 179
M252W/T256Q/P257L/N434Y F103 4.4E-08 48.3 1.21 541
P238A/M252Y/V308P/N434Y F104 3.7E-08 48.6 1.03 424
M252Y/D265A/V308P/N434Y F105 7.5E-08 55.1 1.06 441
M252Y/T307A/V308P/N434Y F106 3.7E-08 53.9 1.31 415
M252Y/V303A/V308P/N434Y F107 3.4E-08 53.2 1.85 562
M252Y/V308P/D376A/N434Y F108 4.1E-08 53 0.98 414
M252Y/V305A/V308P/N434Y F109 3.2E-08 56.8 1.13 454
M252Y/V308P/Q311A/N434Y F111 3.2E-08 56.3 1.36 446
M252Y/V308P/K317A/N434Y F112 6.4E-08 48.4 2.11 510
M252Y/V308P/E380A/N434Y F113 3.2E-08 54.6 0.71 466
M252Y/V308P/E382A/N434Y F114 3.8E-08 57.1 0.97 467
M252Y/V308P/S424A/N434Y F115 6.6E-06 70 0.53 228 T307A/N434A F116
8.7E-06 64.2 0.91 102 E380A/N434A F118 1.4E-05 71.2 0.96 77 M428L
F119 5.4E-06 65.3 1.23 112 T250Q/M428L F120 6.3E-08 54.2 3.54 464
P257L/V308P/M428L/N434Y F121 1.5E-08 49.2 2.37 388
M252Y/T256E/V308P/M428L/N434W F122 1.2E-07 61.4 1.71 207
M252Y/T256E/M428L/N434W F123 3.0E-08 49.3 1.79 332
M252Y/T256E/V308P/N434Y F124 2.9E-07 62.7 1.49 207
M252Y/T256E/M428L/N434Y F125 2.4E-08 49.1 1.35 388
M252Y/S254T/T256E/V308P/M428L/N434Y F128 1.7E-07 63.8 7.11 283
P257L/M428L/N434W F129 2.2E-07 64.8 3.28 244 P257A/M428L/N434Y F131
3.0E-06 61.6 7.07 229 P257G/M428L/N434Y F132 2.1E-07 61.3 8.23 288
P257I/M428L/N434Y F133 4.1E-07 62.7 6.78 254 P257M/M428L/N434Y F134
2.7E-07 57.6 8.30 268 P257N/M428L/N434Y F135 7.5E-07 63.3 8.81 251
P257S/M428L/N434Y F136 3.8E-07 62 10.86 251 P257T/M428L/N434Y F137
4.6E-07 65 13.28 283 P257V/M428L/N434Y F139 1.5E-08 55 5.08 356
M252W/V308P/N434W F140 3.6E-08 62.4 3.51 483
S239K/M252Y/V308P/N434Y F141 3.5E-08 60.2 8.80 438
M252Y/S298G/V308P/N434Y F142 3.7E-08 60.8 1.35 454
M252Y/D270F/V308P/N434Y F143 2.0E-07 61.1 6.55 444
M252Y/V308A/N434Y F145 5.3E-08 48 17.47 458 M252Y/V308F/N434Y F147
2.4E-07 65.4 7.78 419 M252Y/V308I/N434Y F149 1.9E-07 58.9 1.49 430
M252Y/V308L/N434Y F150 2.0E-07 56.3 1.64 447 M252Y/V308M/N434Y F154
1.8E-07 59.3 1.05 436 M252Y/V308T/N434Y F157 1.5E-07 53.3 1.24 425
P257A/V308P/M428L/N434Y F158 5.9E-08 45.7 6.38 431
P257T/V308P/M428L/N434Y F159 4.4E-08 53.3 9.24 464
P257V/V308P/M428L/N434Y F160 8.5E-07 63 1.96 240 M252W/M428I/N434Y
F162 1.6E-07 66.9 1.11 216 M252W/M428Y/N434Y F163 4.2E-07 66.8 1.22
208 M252W/M428F/N434Y F164 3.7E-07 57.6 1.50 313 P238A/M252W/N434Y
F165 2.9E-07 58.1 1.53 196 M252W/D265A/N434Y F166 1.5E-07 64.6 1.37
342 M252W/T307Q/N434Y F167 2.9E-07 60.7 2.02 429 M252W/V303A/N434Y
F168 3.2E-07 59.8 2.33 335 M252W/D376A/N434Y F169 2.9E-07 61.5 1.18
377 M252W/V305A/N434Y F170 1.7E-07 63.3 1.46 335 M252W/Q311A/N434Y
F171 1.9E-07 55.3 1.58 249 M252W/D312A/N434Y F172 2.2E-07 62.7 1.18
218 M252W/K317A/N434Y F173 7.7E-07 58.1 1.40 283 M252W/E380A/N434Y
F174 3.4E-07 61.9 5.58 238 M252W/E382A/N434Y F175 2.7E-07 63.6 0.88
239 M252W/S424A/N434Y F176 2.9E-07 68.8 0.98 255 S239K/M252W/N434Y
F177 2.8E-07 66.7 5.20 210 M252W/S298G/N434Y F178 2.7E-07 67.1 2.39
226 M252W/D270F/N434Y F179 3.1E-07 66.8 1.32 286 M252W/N325G/N434Y
F182 6.6E-08 62.8 4.26 244 P257A/M428L/N434W F183 2.2E-07 59.6
10.28 251 P257T/M428L/N434W F184 2.7E-07 63.3 11.21 283
P257V/M428L/N434W F185 2.6E-07 62.1 0.98 198 M252W/I332V/N434Y F188
3.0E-06 59.2 3.09 282 P257I/Q311I F189 1.9E-07 65.3 1.35 456
M252Y/T307A/N434Y F190 1.1E-07 65.5 0.98 389 M252Y/T307Q/N434Y F191
1.6E-07 62.4 0.91 495 P257L/T307A/M428L/N434Y F192 1.1E-07 63.2
0.82 456 P257A/T307A/M428L/N434Y F193 8.5E-08 58.6 7.10 463
P257T/T307A/M428L/N434Y F194 1.2E-07 62.2 0.61 495
P257V/T307A/M428L/N434Y F195 5.6E-08 63.2 1.22 429
P257L/T307Q/M428L/N434Y F196 3.5E-08 64.3 0.73 390
P257A/T307Q/M428L/N434Y F197 3.3E-08 60.9 8.30 396
P257T/T307Q/M428L/N434Y F198 4.8E-08 63.3 3.41 429
P257V/T307Q/M428L/N434Y F201 2.1E-07 61.9 0.74 331
M252Y/T307D/N434Y F203 2.4E-07 60.3 6.07 415 M252Y/T307F/N434Y F204
2.1E-07 63.3 0.70 397 M252Y/T307G/N434Y F205 2.0E-07 62.6 0.70 369
M252Y/T307H/N434Y F206 2.3E-07 61.7 3.45 392 M252Y/T307I/N434Y F207
9.4E-07 64.6 0.62 379 M252Y/T307K/N434Y F208 3.9E-07 60.8 6.14 416
M252Y/T307L/N434Y F209 1.3E-07 62.1 1.19 416 M252Y/T307M/N434Y F210
2.9E-07 63.4 0.72 398 M252Y/T307N/N434Y F211 2.4E-07 69 0.60 390
M252Y/T307P/N434Y F212 6.8E-07 65.6 0.99 414 M252Y/T307R/N434Y F213
2.3E-07 64.5 0.94 423 M252Y/T307S/N434Y F214 1.7E-07 63.4 1.28 415
M252Y/T307V/N434Y F215 9.6E-08 59.4 3.41 392 M252Y/T307W/N434Y F216
2.3E-07 61.3 1.03 430 M252Y/T307Y/N434Y F217 2.3E-07 62.3 1.08 268
M252Y/K334L/N434Y F218 2.6E-07 64.2 0.91 257 M252Y/G385H/N434Y F219
2.5E-07 62.6 0.87 266 M252Y/T289H/N434Y F220 2.5E-07 63.3 0.92 318
M252Y/Q311H/N434Y F221 3.1E-07 58.6 1.13 282 M252Y/D312H/N434Y F222
3.4E-07 62.2 0.98 243 M252Y/N315H/N434Y F223 2.7E-07 64.4 1.44 243
M252Y/K360H/N434Y F225 1.5E-06 61.5 0.92 265 M252Y/L314R/N434Y F226
5.4E-07 61 0.94 265 M252Y/L314K/N434Y F227 1.2E-07 62.5 0.94 306
M252Y/N286E/N434Y F228 2.3E-07 65.3 0.69 330 M252Y/L309E/N434Y F229
5.1E-07 61.2 0.83 241 M252Y/R255E/N434Y F230 2.5E-07 66.2 0.66 243
M252Y/P387E/N434Y F236 8.9E-07 67.7 2.23 229 K248I/M428L/N434Y F237
2.3E-07 64.3 1.02 270 M252Y/M428A/N434Y F238 7.4E-07 63.9 0.97 255
M252Y/M428D/N434Y F240 7.2E-07 66.9 0.69 255 M252Y/M428F/N434Y F241
1.5E-06 62.5 1.41 278 M252Y/M428G/N434Y F242 8.5E-07 68.7 0.73 220
M252Y/M428H/N434Y F243 1.8E-07 63.8 0.81 286 M252Y/M428I/N434Y F244
1.3E-06 58.1 1.87 253 M252Y/M428K/N434Y F245 4.7E-07 63.1 0.82 279
M252Y/M428N/N434Y F246 1.1E-06 54.8 8.29 286 M252Y/M428P/N434Y F247
4.4E-07 59.9 1.00 275 M252Y/M428Q/N434Y F249 6.4E-07 64.2 0.72 273
M252Y/M428S/N434Y F250 2.9E-07 62.5 0.74 271 M252Y/M428T/N434Y F251
1.9E-07 63.4 1.20 289 M252Y/M428V/N434Y F252 1.0E-06 68.4 0.83 255
M252Y/M428W/N434Y F253 7.1E-07 67.2 0.79 263 M252Y/M428Y/N434Y F254
7.5E-08 69 1.30 362 M252W/T307Q/M428Y/N434Y F255 1.1E-07 66.5 1.23
355 M252W/Q311A/M428Y/N434Y F256 5.4E-08 67.7 1.27 403
M252W/T307Q/Q311A/M428Y/N434Y F257 5.0E-07 67.9 0.60 475
M252Y/T307A/M428Y/N434Y F258 3.2E-07 68.2 0.63 409
M252Y/T307Q/M428Y/N434Y F259 2.8E-07 68.1 1.15 273
M252Y/D270F/N434Y F260 1.3E-07 64.4 0.78 496
M252Y/T307A/Q311A/N434Y F261 8.4E-08 64.9 0.82 430
M252Y/T307Q/Q311A/N434Y F262 1.9E-07 64.1 0.82 448
M252Y/T307A/Q311H/N434Y F263 1.1E-07 64.6 0.80 358
M252Y/T307Q/Q311H/N434Y F264 2.8E-07 62.9 1.15 285
M252Y/E382A/N434Y F265 6.8E-07 65.1 0.57 305
M252Y/E382A/M428Y/N434Y F266 4.7E-07 65.9 0.77 517
M252Y/T307A/E382A/M428Y/N434Y F267 3.2E-07 66.4 0.81 450
M252Y/T307Q/E382A/M428Y/N434Y F268 6.3E-07 61.4 0.81 371
P238A/M252Y/M428F/N434Y F269 5.2E-07 65.2 0.65 435
M252Y/V305A/M428F/N434Y F270 6.6E-07 70.3 0.41 344
M252Y/N325G/M428F/N434Y F271 6.9E-07 63.3 0.87 393
M252Y/D376A/M428F/N434Y F272 6.8E-07 60 1.15 341
M252Y/E380A/M428F/N434Y F273 6.5E-07 65.3 0.69 297
M252Y/E382A/M428F/N434Y F274 7.6E-07 58.4 2.46 392
M252Y/E380A/E382A/M428F/N434Y F275 4.2E-08 61.3 0.69 525
S239K/M252Y/V308P/E382A/N434Y F276 4.1E-08 59.2 0.74 496
M252Y/D270F/V308P/E382A/N434Y F277 1.3E-07 65.5 0.57 503
S239K/M252Y/V308P/M428Y/N434Y F278 3.0E-08 55 0.62 411
M252Y/T307Q/V308P/E382A/N434Y F279 6.1E-08 53.5 0.67 462
M252Y/V308P/Q311H/E382A/N434Y F280 4.1E-08 65.5 0.58 513
S239K/M252Y/D270F/V308P/N434Y F281 9.2E-08 57.2 0.71 477
M252Y/V308P/E382A/M428F/N434Y F282 2.9E-08 49.2 0.82 521
M252Y/V308P/E382A/M428L/N434Y F283 1.0E-07 57 0.49 485
M252Y/V308P/E382A/M428Y/N434Y F284 1.0E-07 59.3 0.79 444
M252Y/V308P/M428Y/N434Y F285 9.9E-08 59.4 0.60 436
M252Y/V308P/M428F/N434Y F286 1.2E-07 63.6 0.64 544
S239K/M252Y/V308P/E382A/M428Y/N434Y F287 1.0E-07 47 2.92 573
M252Y/V308P/E380A/E382A/M428F/N434Y F288 1.9E-07 60.9 1.04 193
M252Y/T256E/E382A/N434Y
F289 4.8E-07 65.2 1.13 171 M252Y/T256E/M428Y/N434Y F290 4.6E-07
63.2 0.98 213 M252Y/T256E/E382A/M428Y/N434Y F292 2.3E-08 60.8 1.16
568 S239K/M252Y/V308P/E382A/M428I/N434Y F293 5.3E-08 46.3 3.40 604
M252Y/V308P/E380A/E382A/M428I/N434Y F294 1.1E-07 65 0.64 495
S239K/M252Y/V308P/M428F/N434Y F295 6.8E-07 65.5 1.58 451
S239K/M252Y/E380A/E382A/M428F/N434Y F296 4.9E-07 66.6 0.76 401
M252Y/Q311A/M428Y/N434Y F297 5.1E-07 59.1 0.81 315
M252Y/D312A/M428Y/N434Y F298 4.8E-07 58.5 1.09 464
M252Y/Q311A/D312A/M428Y/N434Y F299 9.4E-08 64.8 0.71 532
S239K/M252Y/V308P/Q311A/M428Y/N434Y F300 8.3E-08 56.6 1.39 555
S239K/M252Y/V308P/D312A/M428Y/N434Y F301 7.2E-08 56 1.08 627
S239K/M252Y/V308P/Q311A/D312A/M428Y/N434Y F302 1.9E-07 67.2 0.77
298 M252Y/T256E/T307P/N434Y F303 6.7E-07 71.8 0.59 409
M252Y/T307P/M428Y/N434Y F304 1.6E-08 58.9 1.10 397
M252W/V308P/M428Y/N434Y F305 2.7E-08 48.9 0.76 374
M252Y/T256E/V308P/E382A/N434Y F306 3.6E-08 53.5 0.93 419
M252W/V308P/E382A/N434Y F307 3.6E-08 60 1.76 478
S239K/M252W/V308P/E382A/N434Y F308 1.9E-08 63.2 1.19 497
S239K/M252W/V308P/E382A/M428Y/N434Y F310 9.4E-08 59.8 2.44 521
S239K/M252W/V308P/E382A/M428I/N434Y F311 2.8E-08 64.1 0.98 448
S239K/M252W/V308P/M428F/N434Y F312 4.5E-07 64.3 1.33 404
S239K/M252W/E380A/E382A/M428F/N434Y F313 6.5E-07 77.9 0.77 468
S239K/M252Y/T307P/M428Y/N434Y F314 3.2E-07 56.1 1.27 372
M252Y/T256E/Q311A/D312A/M428Y/N434Y F315 6.8E-07 72.8 0.80 322
S239K/M252Y/M428Y/N434Y F316 7.0E-07 75.3 1.07 351
S239K/M252Y/D270F/M428Y/N434Y F317 1.1E-07 68.5 0.84 532
S239K/M252Y/D270F/V308P/M428Y/N434Y F318 1.8E-08 62 0.96 526
S239K/M252Y/V308P/M428I/N434Y F320 2.0E-08 63.9 1.00 657
S239K/M252Y/V308P/N325G/E382A/M428I/N434Y F321 3.2E-08 65.6 0.80
602 S239K/M252Y/D270F/V308P/N325G/N434Y F322 9.2E-08 61.8 0.87 448
S239K/M252Y/D270F/T307P/V308P/N434Y F323 2.7E-08 63.1 1.10 421
S239K/M252Y/T256E/D270F/V308P/N434Y F324 2.8E-08 63 1.07 458
S239K/M252Y/D270F/T307Q/V308P/N434Y F325 2.1E-08 62.4 0.84 473
S239K/M252Y/D270F/T307Q/V308P/Q311A/N434Y F326 7.5E-08 73.2 0.90
518 S239K/M252Y/D270F/T307Q/Q311A/N434Y F327 6.5E-08 70.4 1.19 427
S239K/M252Y/T256E/D270F/T307Q/Q311A/N434Y F328 1.9E-08 62.3 0.76
556 S239K/M252Y/D270F/V308P/M428I/N434Y F329 1.2E-08 64.2 0.97 575
S239K/M252Y/D270F/N286E/V308P/N434Y F330 3.6E-08 65.5 0.75 414
S239K/M252Y/D270F/V308P/L309E/N434Y F331 3.0E-08 63.8 0.77 513
S239K/M252Y/D270F/V308P/P387E/N434Y F333 7.4E-08 75.3 1.00 418
S239K/M252Y/D270F/T307Q/L309E/Q311A/N434Y F334 1.9E-08 65.2 1.25
645 S239K/M252Y/D270F/V308P/N325G/M428I/N434Y F335 1.5E-08 63.2
1.15 464 S239K/M252Y/T256E/D270F/V308P/M428I/N434Y F336 1.4E-08
64.7 0.95 516 S239K/M252Y/D270F/T307Q/V308P/Q311A/M428I/N434Y F337
5.6E-08 72.9 1.20 562 S239K/M252Y/D270F/T307Q/Q311A/M428I/N434Y
F338 7.7E-09 63.4 1.03 618
S239K/M252Y/D270F/N286E/V308P/M428I/N434Y F339 1.9E-08 65.2 1.09
457 S239K/M252Y/D270F/V308P/L309E/M428I/N434Y F343 3.2E-08 60.2
1.28 568 S239K/M252Y/D270F/V308P/M428L/N434Y F344 3.0E-08 56.3 0.92
538 S239K/M252Y/V308P/M428L/N434Y F349 1.5E-07 57.8 0.97 530
S239K/M252Y/V308P/L309P/M428L/N434Y F350 1.7E-07 58.2 1.01 538
S239K/M252Y/V308P/L309R/M428L/N434Y F352 6.0E-07 67.7 1.12 496
S239K/M252Y/L309P/M428L/N434Y F353 1.1E-06 73.1 1.01 504
S239K/M252Y/L309R/M428L/N434Y F354 2.8E-08 57.5 0.86 483
S239K/M252Y/T307Q/V308P/M428L/N434Y F356 3.4E-08 67.2 0.85 414
S239K/M252Y/D270F/V308P/L309E/P387E/N434Y F357 1.6E-08 62.7 1.16
554 S239K/M252Y/T256E/D270F/V308P/N325G/M428I/N434Y F358 1.0E-07
70.8 0.79 448 S239K/M252Y/T307Q/N434Y F359 4.2E-07 63.5 7.84 417
P257V/T307Q/M428I/N434Y F360 1.3E-06 63.9 4.93 419
P257V/T307Q/M428V/N434Y F362 5.4E-08 70.1 13.44 518
P257V/T307Q/N325G/M428L/N434Y F363 4.1E-08 63.3 15.02 470
P257V/T307Q/Q311A/M428L/N434Y F364 3.5E-08 68.9 3.25 559
P257V/T307Q/Q311A/N325G/M428L/N434Y F365 5.1E-08 60.2 3.22 458
P257V/V305A/T307Q/M428L/N434Y F367 1.5E-08 59.4 1.25 500
S239K/M252Y/E258H/D270F/T307Q/V308P/Q311A/N434Y F368 2.0E-08 64
0.97 687 S239K/M252Y/D270F/V308P/N325G/E382A/M428I/N434Y F369
7.5E-08 62.8 4.07 408 M252Y/P257V/T307Q/M428I/N434Y F372 1.3E-08
65.4 1.09 456 S239K/M252W/V308P/M428Y/N434Y F373 1.1E-08 64.5 1.89
485 S239K/M252W/V308P/Q311A/M428Y/N434Y F374 1.2E-08 63 1.30 399
S239K/M252W/T256E/V308P/M428Y/N434Y F375 5.5E-09 63.5 1.18 518
S239K/M252W/N286E/V308P/M428Y/N434Y F376 9.6E-09 61.9 14.59 483
S239K/M252Y/T256E/D270F/N286E/V308P/N434Y F377 1.3E-07 77.7 0.95
421 S239K/M252W/T307P/M428Y/N434Y F379 9.0E-09 62.2 54.64 428
S239K/M252W/T256E/V308P/Q311A/M428Y/N434Y F380 5.6E-09 60.8 0.97
461 S239K/M252W/T256E/N286E/V308P/M428Y/N434Y F381 1.1E-07 61.7
7.47 536 P257V/T307A/Q311A/M428L/N434Y F382 8.7E-08 58.6 3.55 489
P257V/V305A/T307A/M428L/N434Y F386 3.2E-08 56.7 2.02 325
M252Y/V308P/L309E/N434Y F387 1.5E-07 57.9 1.64 330
M252Y/V308P/L309D/N434Y F388 7.0E-08 57.4 1.12 401
M252Y/V308P/L309A/N434Y F389 1.7E-08 59.2 1.19 298
M252W/V308P/L309E/M428Y/N434Y F390 6.8E-08 60.2 1.29 302
M252W/V308P/L309D/M428Y/N434Y F391 3.6E-08 59.4 0.91 374
M252W/V308P/L309A/M428Y/N434Y F392 6.9E-09 60.3 0.92 588
S239K/M252Y/N286E/V308P/M428I/N434Y F393 1.2E-08 61.1 0.83 545
S239K/M252Y/N286E/V308P/N434Y F394 5.3E-08 70.3 0.99 532
S239K/M252Y/T307Q/Q311A/M428I/N434Y F395 2.4E-08 60.1 1.46 391
S239K/M252Y/T256E/V308P/N434Y F396 2.0E-08 71.7 1.53 624
S239K/M252Y/D270F/N286E/T307Q/Q311A/M428I/N434Y F397 4.5E-08 75.1
1.23 562 S239K/M252Y/D270F/T307Q/Q311A/P387E/M428I/N434Y F398
4.4E-09 63.3 1.14 578
S239K/M252Y/D270F/N286E/T307Q/V308P/Q311A/M428I/N434Y F399 6.5E-09
63.2 1.02 563 S239K/M252Y/D270F/N286E/T307Q/V308P/M428I/N434Y F400
6.1E-09 63.4 1.22 647
S239K/M252Y/D270F/N286E/V308P/Q311A/M428I/N434Y F401 6.9E-09 65.3
1.11 618 S239K/M252Y/D270F/N286E/V308P/P387E/M428I/N434Y F402
2.3E-08 63.7 11.07 429 P257V/T307Q/M428L/N434W F403 5.1E-08 61.9
11.06 495 P257V/T307A/M428L/N434W F404 9.4E-08 66.3 2.14 342
P257A/T307Q/L309P/M428L/N434Y F405 1.7E-07 68.1 6.73 381
P257V/T307Q/L309P/M428L/N434Y F406 1.5E-07 66.7 1.14 342
P257A/T307Q/L309R/M428L/N434Y F407 1.6E-07 64.3 7.87 381
P257V/T307Q/L309R/M428L/N434Y F408 2.5E-07 63.2 8.96 345
P257V/N286E/M428L/N434Y F409 2.0E-07 64.4 10.17 283
P257V/P387E/M428L/N434Y F410 2.2E-07 62.1 10.56 408
P257V/T307H/M428L/N434Y F411 1.3E-07 62.4 10.50 438
P257V/T307N/M428L/N434Y F412 8.8E-08 60.4 8.29 437
P257V/T307G/M428L/N434Y F413 1.2E-07 66.9 7.29 430
P257V/T307P/M428L/N434Y F414 1.1E-07 63.6 9.61 463
P257V/T307S/M428L/N434Y F415 5.6E-08 61.1 8.69 558
P257V/N286E/T307A/M428L/N434Y F416 9.4E-08 62.5 6.73 495
P257V/T307A/P387E/M428L/N434Y F418 6.2E-07 81.7 0.58 558
S239K/M252Y/T307P/N325G/M428Y/N434Y F419 1.6E-07 64.2 5.96 448
M252Y/T307A/Q311H/K360H/N434Y F420 1.5E-07 65.9 1.10 448
M252Y/T307A/Q311H/P387E/N434Y F421 1.3E-07 63.4 0.56 474
M252Y/T307A/Q311H/M428A/N434Y F422 1.8E-07 62.4 0.46 489
M252Y/T307A/Q311H/E382A/N434Y F423 8.4E-08 58.2 1.22 405
M252Y/T307W/Q311H/N434Y F424 9.4E-08 57.9 1.25 484
S239K/P257A/V308P/M428L/N434Y F425 8.0E-08 54.7 1.36 326
P257A/V308P/L309E/M428L/N434Y F426 8.4E-08 65.3 3.50 374
P257V/T307Q/N434Y F427 1.1E-07 63.1 3.79 411
M252Y/P257V/T307Q/M428V/N434Y F428 8.0E-08 63.9 10.27 420
M252Y/P257V/T307Q/M428L/N434Y F429 3.7E-08 63.5 10.07 365
M252Y/P257V/T307Q/N434Y F430 8.1E-08 64.7 2.99 385
M252Y/P257V/T307Q/M428Y/N434Y F431 6.5E-08 64.9 4.77 377
M252Y/P257V/T307Q/M428F/N434Y F432 9.2E-07 67.1 6.21 550
P257V/T307Q/Q311A/N325G/M428V/N434Y F433 6.0E-08 68.4 2.56 504
P257V/T307Q/Q311A/N325G/N434Y F434 2.0E-08 69.3 1.66 524
P257V/T307Q/Q311A/N325G/M428Y/N434Y F435 2.5E-08 69.2 1.85 516
P257V/T307Q/Q311A/N325G/M428F/N434Y F436 2.5E-07 63.4 4.67 380
P257A/T307Q/M428V/N434Y F437 5.7E-08 65.3 0.75 335
P257A/T307Q/N434Y F438 3.6E-08 68.6 0.74 354
P257A/T307Q/M428Y/N434Y F439 4.0E-08 67.9 3.44 346
P257A/T307Q/M428F/N434Y F440 1.5E-08 68.7 9.15 622
P257V/N286E/T307Q/Q311A/N325G/M428L/N434Y F441 1.8E-07 64 2.87 383
P257A/Q311A/M428L/N434Y F442 2.0E-07 63.5 1.91 318
P257A/Q311H/M428L/N434Y F443 5.5E-08 64.5 8.58 431
P257A/T307Q/Q311A/M428L/N434Y F444 1.4E-07 62.8 1.96 497
P257A/T307A/Q311A/M428L/N434Y F445 6.2E-08 64.4 1.22 359
P257A/T307Q/Q311H/M428L/N434Y F446 1.1E-07 62.6 1.00 448
P257A/T307A/Q311H/M428L/N434Y F447 1.4E-08 63.9 1.20 452
P257A/N286E/T307Q/M428L/N434Y F448 5.3E-08 61.9 1.05 519
P257A/N286E/T307A/M428L/N434Y F449 5.7E-07 81.9 0.64 587
S239K/M252Y/D270F/T307P/N325G/M428Y/N434Y F450 5.2E-07 82.5 0.67
501 S239K/M252Y/T307P/L309E/N325G/M428Y/N434Y F451 1.0E-07 60.4
4.14 463 P257S/T307A/M428L/N434Y F452 1.4E-07 60.8 4.31 466
P257M/T307A/M428L/N434Y F453 7.8E-08 55.5 7.22 480
P257N/T307A/M428L/N434Y F454 9.6E-08 60.4 5.16 500
P257I/T307A/M428L/N434Y F455 2.7E-08 66.3 2.85 393
P257V/T307Q/M428Y/N434Y F456 3.4E-08 66.2 2.45 385
P257V/T307Q/M428F/N434Y F457 4.0E-08 61.2 5.82 523
S239K/P257V/V308P/M428L/N434Y F458 1.5E-08 57.1 7.48 499
P257V/T307Q/V308P/N325G/M428L/N434Y F459 1.3E-08 56.5 5.57 514
P257V/T307Q/V308P/Q311A/N325G/M428L/N434Y F460 4.7E-08 55.5 8.56
570 P257V/T307A/V308P/N325G/M428L/N434Y F462 8.5E-08 57.2 1.10 514
P257A/V308P/N325G/M428L/N434Y F463 1.3E-07 53 0.92 442
P257A/T307A/V308P/M428L/N434Y F464 5.5E-08 54.3 1.14 370
P257A/T307Q/V308P/M428L/N434Y F465 2.1E-08 69.2 9.95 581
P257V/N286E/T307Q/N325G/M428L/N434Y F466 3.5E-07 63.6 0.35 167
T256E/P257V/N434Y F467 5.7E-07 60.5 0.52 142 T256E/P257T/N434Y F468
5.7E-08 55.8 0.71 490 S239K/P257T/V308P/M428L/N434Y F469 5.6E-08
48.6 1.49 521 P257T/V308P/N325G/M428L/N434Y F470 5.4E-08 48.2 1.81
467 T256E/P257T/V308P/N325G/M428L/N434Y F471 6.6E-08 48.4 1.43 563
P257T/V308P/N325G/E382A/M428L/N434Y F472 5.4E-08 48.8 5.04 521
P257T/V308P/N325G/P387E/M428L/N434Y F473 4.5E-07 48.7 2.13 513
P257T/V308P/L309P/N325G/M428L/N434Y F474 3.5E-07 48.4 1.04 521
P257T/V308P/L309R/N325G/M428L/N434Y F475 4.3E-08 61.9 3.67 368
T256E/P257V/T307Q/M428L/N434Y F476 5.5E-08 61.8 4.57 471
P257V/T307Q/E382A/M428L/N434Y F477 4.3E-08 62.8 2.35 429
P257V/T307Q/P387E/M428L/N434Y F480 3.9E-08 53.8 2.30 409
P257L/V308P/N434Y F481 5.6E-08 59.2 1.26 341 P257T/T307Q/N434Y F482
7.0E-08 66.4 1.32 463 P257V/T307Q/N325G/N434Y F483 5.7E-08 62.1
1.42 415 P257V/T307Q/Q311A/N434Y F484 6.2E-08 58.6 1.84 403
P257V/V305A/T307Q/N434Y F485 9.7E-08 60.4 2.64 503
P257V/N286E/T307A/N434Y F486 3.4E-07 60.4 0.76 381
P257V/T307Q/L309R/Q311H/M428L/N434Y F488 3.5E-08 56.4 3.69 553
P257V/V308P/N325G/M428L/N434Y F490 7.5E-08 59.4 2.29 519
S239K/P257V/V308P/Q311H/M428L/N434Y F492 9.8E-08 61.7 3.29 579
P257V/V305A/T307A/N325G/M428L/N434Y F493 4.9E-07 84 0.52 456
S239K/D270F/T307P/N325G/M428Y/N434Y F497 3.1E-06 58.2 4.34 453
P257T/T307A/M428V/N434Y F498 1.3E-06 62.2 6.16 234
P257A/M428V/N434Y F499 5.2E-07 61.3 2.03 447
P257A/T307A/M428V/N434Y F500 4.3E-08 61 2.81 396
P257S/T307Q/M428L/N434Y F506 1.9E-07 57.1 8.70 454
P257V/N297A/T307Q/M428L/N434Y F507 5.1E-08 59.9 4.51 499
P257V/N286A/T307Q/M428L/N434Y F508 1.1E-07 65.1 3.93 429
P257V/T307Q/N315A/M428L/N434Y F509 5.8E-08 62.8 3.94 447
P257V/T307Q/N384A/M428L/N434Y F510 5.3E-08 62.7 4.35 429
P257V/T307Q/N389A/M428L/N434Y F511 4.2E-07 63 2.28 228 P257V/N434Y
F512 5.8E-07 60 3.41 195 P257T/N434Y F517 3.1E-07 61.4 2.82 290
P257V/N286E/N434Y F518 4.2E-07 58.5 4.05 258 P257T/N286E/N434Y F519
2.6E-08 61.3 2.45 436 P257V/N286E/T307Q/N434Y F521 1.1E-08 63.5
1.51 456 P257V/N286E/T307Q/M428Y/N434Y F523 2.6E-08 62.6 1.39 422
P257V/V305A/T307Q/M428Y/N434Y F526 1.9E-08 62.2 1.60 361
P257T/T307Q/M428Y/N434Y F527 9.4E-09 60.6 0.94 463
P257V/T307Q/V308P/N325G/M428Y/N434Y F529 2.5E-08 61.7 1.82 353
P257T/T307Q/M428F/N434Y F533 1.2E-08 64.8 1.01 409
P257A/N286E/T307Q/M428F/N434Y F534 1.2E-08 65.6 0.84 417
P257A/N286E/T307Q/M428Y/N434Y F535 3.9E-08 63.3 4.36 449
T250A/P257V/T307Q/M428L/N434Y F538 9.9E-08 66.7 2.57 484
T250F/P257V/T307Q/M428L/N434Y F541 6.0E-08 65.9 3.53 484
T250I/P257V/T307Q/M428L/N434Y F544 3.1E-08 64.5 4.00 484
T250M/P257V/T307Q/M428L/N434Y F549 5.4E-08 61.8 5.82 415
T250S/P257V/T307Q/M428L/N434Y F550 5.9E-08 66.6 3.19 484
T250V/P257V/T307Q/M428L/N434Y F551 1.2E-07 65.1 3.72 484
T250W/P257V/T307Q/M428L/N434Y F552 1.1E-07 65.4 2.98 484
T250Y/P257V/T307Q/M428L/N434Y F553 1.7E-07 64.1 1.52 382
M252Y/Q311A/N434Y F554 2.8E-08 62.8 1.39 454
S239K/M252Y/S254T/V308P/N434Y F556 1.5E-06 66.5 0.96 318
M252Y/T307Q/Q311A F559 8.0E-08 62.8 1.20 277
M252Y/S254T/N286E/N434Y F560 2.8E-08 56.9 1.15 395
M252Y/S254T/V308P/N434Y F561 1.4E-07 65.5 1.23 427
M252Y/S254T/T307A/N434Y F562 8.3E-08 65.7 1.21 360
M252Y/S254T/T307Q/N434Y F563 1.3E-07 64.2 1.02 353
M252Y/S254T/Q311A/N434Y F564 1.9E-07 63.7 1.02 289
M252Y/S254T/Q311H/N434Y F565 9.2E-08 65 1.00 467
M252Y/S254T/T307A/Q311A/N434Y F566 6.1E-08 64.9 1.24 401
M252Y/S254T/T307Q/Q311A/N434Y F567 2.2E-07 64.2 1.52 257
M252Y/S254T/M428I/N434Y F568 1.1E-07 61.6 0.99 356
M252Y/T256E/T307A/Q311H/N434Y F569 2.0E-07 64.4 0.96 379
M252Y/T256Q/T307A/Q311H/N434Y F570 1.3E-07 64.5 1.04 419
M252Y/S254T/T307A/Q311H/N434Y F571 8.1E-08 62.4 1.03 510
M252Y/N286E/T307A/Q311H/N434Y F572 1.0E-07 63.6 1.33 491
M252Y/T307A/Q311H/M428I/N434Y F576 1.6E-06 62.7 0.99 154
M252Y/T256E/T307Q/Q311H F577 1.3E-06 64.4 1.44 447
M252Y/N286E/T307A/Q311A F578 5.7E-07 64.3 1.33 380
M252Y/N286E/T307Q/Q311A F580 8.6E-07 63.9 1.06 308
M252Y/N286E/T307Q/Q311H F581 7.2E-08 59.8 1.22 214
M252Y/T256E/N286E/N434Y F582 7.5E-07 64.5 1.10 371
S239K/M252Y/V308P F583 7.8E-07 63 1.01 412 S239K/M252Y/V308P/E382A
F584 6.3E-07 61.8 1.45 279 S239K/M252Y/T256E/V308P F585 2.9E-07
62.7 1.15 433 S239K/M252Y/N286E/V308P F586 1.4E-07 62 1.21 498
S239K/M252Y/N286E/V308P/M428I F587 1.9E-07 61.9 1.47 361
M252Y/N286E/M428L/N434Y F592 2.0E-07 62.9 0.99 256
M252Y/S254T/E382A/N434Y F593 3.1E-08 60.8 1.27 497
S239K/M252Y/S254T/V308P/M428I/N434Y F594 1.6E-08 59.3 1.32 434
S239K/M252Y/T256E/V308P/M428I/N434Y F595 1.8E-07 69.4 0.91 345
S239K/M252Y/M428I/N434Y F596 4.0E-07 56.6 1.05 357
M252Y/D312A/E382A/M428Y/N434Y F597 2.2E-07 64.6 0.78 283
M252Y/E382A/P387E/N434Y F598 1.4E-07 58.1 1.06 296
M252Y/D312A/P387E/N434Y F599 5.2E-07 68.9 0.76 263
M252Y/P387E/M428Y/N434Y
F600 2.8E-07 62.5 0.89 217 M252Y/T256Q/E382A/N434Y F601 9.6E-09
54.7 0.96 486 M252Y/N286E/V308P/N434Y F611 2.8E-07 69.5 0.96 358
M252Y/V305T/T307P/V308I/L309A/N434Y F612 3.6E-07 70.8 0.92 360
M252Y/T307P/V308I/L309A/N434Y F617 7.4E-07 71.6 1.41 149
S239K/N434W F618 6.4E-07 60.7 2.70 386 S239K/V308F/N434Y F619
3.1E-07 70 0.67 302 S239K/M252Y/N434Y F620 2.1E-07 69.6 1.18 273
S239K/M252Y/S254T/N434Y F621 1.5E-07 70 0.76 506
S239K/M252Y/T307A/Q311H/N434Y F622 3.5E-07 69.9 0.74 234
S239K/M252Y/T256Q/N434Y F623 1.8E-07 68 2.39 234 S239K/M252W/N434W
F624 1.4E-08 69.1 1.99 511 S239K/P257A/N286E/T307Q/M428L/N434Y F625
7.6E-08 69.7 1.85 449 S239K/P257A/T307Q/M428L/N434Y F626 1.3E-06
62.7 0.81 181 V308P F629 3.9E-08 52 0.56 481
M252Y/V279L/V308P/N434Y F630 3.7E-08 58.5 0.87 540
S239K/M252Y/V279L/V308P/N434Y F633 2.4E-08 53.7 0.65 446
M252Y/V282D/V308P/N434Y F634 3.2E-08 59.5 0.88 505
S239K/M252Y/V282D/V308P/N434Y F636 4.8E-08 57.1 0.91 520
S239K/M252Y/V284K/V308P/N434Y F637 1.5E-07 54.9 1.05 455
M252Y/K288S/V308P/N434Y F638 1.4E-07 60.9 0.90 514
S239K/M252Y/K288S/V308P/N434Y F639 2.7E-08 56.2 0.92 424
M252Y/V308P/G385R/N434Y F640 3.6E-08 62.1 0.83 483
S239K/M252Y/V308P/G385R/N434Y F641 3.0E-08 56.3 0.83 438
M252Y/V308P/Q386K/N434Y F642 3.0E-08 62.2 0.87 497
S239K/M252Y/V308P/Q386K/N434Y F643 3.2E-08 62.4 0.73 479
L235G/G236R/S239K/M252Y/V308P/N434Y F644 3.0E-08 62.1 0.80 518
G236R/S239K/M252Y/V308P/N434Y F645 3.3E-08 54.6 1.25 551
S239K/M252Y/V308P/L328R/N434Y F646 3.8E-08 48.5 3.58 509
S239K/M252Y/N297A/V308P/N434Y F647 2.9E-08 43 8.59 502
P238D/M252Y/V308P/N434Y F649 1.2E-07 68.3 0.90 364
S239K/M252Y/N286E/N434Y F650 1.7E-07 68.1 1.19 210
S239K/M252Y/T256E/N434Y F651 1.8E-07 69.4 0.69 441
S239K/M252Y/Q311A/N434Y F652 2.4E-07 54.8 0.95 322
P238D/M252Y/N434Y F654 3.2E-08 62.2 0.80 493
L235K/S239K/M252Y/V308P/N434Y F655 3.4E-08 62.5 0.50 489
L235R/S239K/M252Y/V308P/N434Y F656 3.3E-08 60 0.74 482
G237K/S239K/M252Y/V308P/N434Y F657 3.2E-08 61.3 0.54 485
G237R/S239K/M252Y/V308P/N434Y F658 3.2E-08 48.4 6.06 531
P238K/S239K/M252Y/V308P/N434Y F659 3.0E-08 47.2 9.54 547
P238R/S239K/M252Y/V308P/N434Y F660 3.1E-08 60.3 0.82 564
S239K/M252Y/V308P/P329K/N434Y F661 3.4E-08 61 0.66 541
S239K/M252Y/V308P/P329R/N434Y F663 6.4E-09 60.7 0.81 506
S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y F664 3.9E-08 54.4 0.90
494 M252Y/N286A/V308P/N434Y F665 2.0E-08 56.9 1.09 438
M252Y/N286D/V308P/N434Y F666 2.1E-08 52.7 1.06 494
M252Y/N286F/V308P/N434Y F667 3.0E-08 56.1 0.59 486
M252Y/N286G/V308P/N434Y F668 4.0E-08 56.2 0.55 486
M252Y/N286H/V308P/N434Y F670 2.1E-07 52.1 0.13 486
M252Y/N286K/V308P/N434Y F671 2.2E-08 49.5 0.67 494
M252Y/N286L/V308P/N434Y F672 2.4E-08 53.4 0.87 494
M252Y/N286M/V308P/N434Y F673 2.3E-08 48.2 5.02 486
M252Y/N286P/V308P/N434Y F674 3.2E-08 53.3 0.39 486
M252Y/N286Q/V308P/N434Y F675 5.1E-08 49.3 0.67 494
M252Y/N286R/V308P/N434Y F676 3.2E-08 55.6 0.64 494
M252Y/N286S/V308P/N434Y F677 4.7E-08 57.1 0.92 486
M252Y/N286T/V308P/N434Y F679 1.7E-08 48.7 4.00 486
M252Y/N286W/V308P/N434Y F680 1.5E-08 56 0.80 494
M252Y/N286Y/V308P/N434Y F681 4.9E-08 54.4 0.95 455
M252Y/K288A/V308P/N434Y F682 8.2E-08 54.5 0.98 424
M252Y/K288D/V308P/N434Y F683 5.0E-08 56.2 1.17 432
M252Y/K288E/V308P/N434Y F684 5.1E-08 56.6 1.27 458
M252Y/K288F/V308P/N434Y F685 5.3E-08 48.5 1.14 432
M252Y/K288G/V308P/N434Y F686 4.6E-08 56.3 1.00 455
M252Y/K288H/V308P/N434Y F687 4.9E-08 59.1 1.13 463
M252Y/K288I/V308P/N434Y F688 2.8E-08 56.5 1.07 466
M252Y/K288L/V308P/N434Y F689 4.1E-08 56.4 0.95 455
M252Y/K288M/V308P/N434Y F690 1.0E-07 54.9 0.98 455
M252Y/K288N/V308P/N434Y F692 3.9E-08 56 1.18 440
M252Y/K288Q/V308P/N434Y F693 3.6E-08 56.2 0.82 478
M252Y/K288R/V308P/N434Y F694 4.7E-08 57.5 0.88 466
M252Y/K288V/V308P/N434Y F695 4.0E-08 55 1.09 481
M252Y/K288W/V308P/N434Y F696 4.4E-08 56.4 0.93 486
M252Y/K288Y/V308P/N434Y F697 3.1E-08 65.1 0.56 572
S239K/M252Y/V308P/N325G/N434Y F698 2.2E-08 62.8 0.69 492
M252Y/N286E/T307Q/Q311A/N434Y F699 2.3E-08 68.3 0.74 551
S239K/M252Y/N286E/T307Q/Q311A/N434Y F705 7.1E-09 53.7 0.71 530
M252Y/N286E/V308P/M428I/N434Y F706 1.8E-08 62 0.86 535
M252Y/N286E/T307Q/Q311A/M428I/N434Y F707 5.9E-09 54.5 0.73 447
M252Y/N286E/T307Q/V308P/Q311A/N434Y F708 4.1E-09 53.6 0.43 490
M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y F709 2.0E-08 68.3 0.77
594 S239K/M252Y/N286E/T307Q/Q311A/M428I/N434Y F710 1.5E-08 48.6
1.68 614 P238D/M252Y/N286E/T307Q/Q311A/M428I/N434Y F711 6.5E-08
70.7 0.79 489 S239K/M252Y/T307Q/Q311A/N434Y F712 6.0E-08 55.2 0.89
508 P238D/M252Y/T307Q/Q311A/N434Y F713 2.0E-08 52.7 0.88 571
P238D/M252Y/N286E/T307Q/Q311A/N434Y F714 2.3E-07 57.9 0.70 437
P238D/M252Y/N325S/N434Y F715 2.3E-07 57.6 2.42 451
P238D/M252Y/N325M/N434Y F718 2.8E-07 56.3 0.52 348
P238D/M252Y/Q295M/N434Y F719 7.4E-08 57.3 0.76 411
P238D/M252Y/N325G/N434Y F720 2.4E-08 56.4 0.66 385
M252Y/T307Q/V308P/Q311A/N434Y F721 1.5E-08 55.3 0.77 428
M252Y/T307Q/V308P/Q311A/M428I/N434Y F722 2.7E-07 53.7 0.43 322
P238D/M252Y/A327G/N434Y F723 2.8E-07 45.3 7.75 347
P238D/M252Y/L328D/N434Y F724 2.5E-07 43 24.31 355
P238D/M252Y/L328E/N434Y F725 4.2E-08 61.5 0.56 485
L235K/G237R/S239K/M252Y/V308P/N434Y F729 9.2E-07 68.3 0.61 365
T307A/Q311A/N434Y F730 6.0E-07 69.1 0.52 299 T307Q/Q311A/N434Y F731
8.5E-07 67.9 0.63 316 T307A/Q311H/N434Y F732 6.8E-07 69 0.66 227
T307Q/Q311H/N434Y F733 3.2E-07 48.8 2.59 276 M252Y/L328E/N434Y F734
3.1E-07 46.8 9.34 340 G236D/M252Y/L328E/N434Y F736 3.1E-07 52.2
2.27 298 M252Y/S267M/L328E/N434Y F737 3.1E-07 48.6 2.82 298
M252Y/S267L/L328E/N434Y F738 3.5E-07 59.8 0.91 468
P238D/M252Y/T307P/N434Y F739 2.2E-07 68.1 0.77 430
M252Y/T307P/Q311A/N434Y F740 2.9E-07 67.7 0.76 360
M252Y/T307P/Q311H/N434Y F741 3.1E-07 52.9 1.14 322
P238D/T250A/M252Y/N434Y F744 9.9E-07 58.6 1.09 357
P238D/T250F/M252Y/N434Y F747 2.8E-07 59.4 0.93 357
P238D/T250I/M252Y/N434Y F749 5.1E-07 58.6 1.13 357
P238D/T250L/M252Y/N434Y F750 3.0E-07 53 1.34 357
P238D/T250M/M252Y/N434Y F753 1.8E-07 45 6.13 345
P238D/T250Q/M252Y/N434Y F755 3.5E-07 47.8 1.24 322
P238D/T250S/M252Y/N434Y F756 3.7E-07 59.9 0.81 357
P238D/T250V/M252Y/N434Y F757 1.2E-06 55.3 1.29 357
P238D/T250W/M252Y/N434Y F758 1.4E-06 60 0.77 357
P238D/T250Y/M252Y/N434Y F761 1.1E-06 59.7 0.56 191 P238D/N434Y F762
3.6E-08 68.8 0.79 561 L235K/S239K/M252Y/N286E/T307Q/Q311A/N434Y
F763 3.5E-08 69.2 0.76 557
L235R/S239K/M252Y/N286E/T307Q/Q311A/N434Y F764 6.3E-07 60 0.48 377
P238D/T307Q/Q311A/N434Y F765 8.5E-08 56.8 1.06 407
P238D/M252Y/T307Q/L309E/Q311A/N434Y F766 6.0E-07 69.2 0.61 286
T307A/L309E/Q311A/N434Y F767 4.3E-07 69.9 0.63 198
T307Q/L309E/Q311A/N434Y F768 6.4E-07 68.8 0.65 286
T307A/L309E/Q311H/N434Y F769 4.6E-07 69.5 0.77 190
T307Q/L309E/Q311H/N434Y F770 3.0E-07 61.8 0.84 226
M252Y/T256A/N434Y F771 4.0E-07 60 0.57 275 M252Y/E272A/N434Y F772
3.8E-07 62.7 0.57 293 M252Y/K274A/N434Y F773 3.9E-07 62 0.50 285
M252Y/V282A/N434Y F774 4.0E-07 61.5 0.64 314 M252Y/N286A/N434Y F775
6.2E-07 56.1 9.28 243 M252Y/K338A/N434Y F776 3.9E-07 61.9 0.64 273
M252Y/K340A/N434Y F777 3.9E-07 64.2 0.59 278 M252Y/E345A/N434Y F779
3.9E-07 64.2 0.56 273 M252Y/N361A/N434Y F780 3.9E-07 64.3 0.65 256
M252Y/Q362A/N434Y F781 3.7E-07 64.2 0.58 307 M252Y/S375A/N434Y F782
3.5E-07 63.9 0.93 243 M252Y/Y391A/N434Y F783 4.0E-07 64.2 0.50 514
M252Y/D413A/N434Y F784 5.0E-07 65.6 0.70 367 M252Y/L309A/N434Y F785
7.4E-07 64.7 0.70 359 M252Y/L309H/N434Y F786 2.8E-08 62.6 0.61 463
M252Y/S254T/N286E/T307Q/Q311A/N434Y F787 8.8E-08 66.1 0.72 300
M252Y/S254T/T307Q/L309E/Q311A/N434Y F788 4.1E-07 65.5 0.53 251
M252Y/N315A/N434Y F789 1.5E-07 64.3 0.67 243 M252Y/N315D/N434Y F790
2.7E-07 63.9 0.97 265 M252Y/N315E/N434Y F791 4.4E-07 60.6 1.27 243
M252Y/N315F/N434Y F792 4.4E-07 65 0.59 243 M252Y/N315G/N434Y F793
3.3E-07 61.4 0.75 251 M252Y/N315I/N434Y F794 4.1E-07 62.8 0.64 243
M252Y/N315K/N434Y F795 3.1E-07 62.8 0.85 265 M252Y/N315L/N434Y F796
3.4E-07 64.4 0.85 251 M252Y/N315M/N434Y F798 3.5E-07 64.3 0.72 273
M252Y/N315Q/N434Y F799 4.1E-07 62.7 1.36 243 M252Y/N315R/N434Y F800
3.8E-07 65.8 0.61 243 M252Y/N315S/N434Y F802 3.3E-07 61.5 0.67 273
M252Y/N315V/N434Y F803 3.6E-07 59.6 0.86 251 M252Y/N315W/N434Y F804
4.0E-07 60.5 1.55 251 M252Y/N315Y/N434Y F805 3.0E-07 65 0.70 397
M252Y/N325A/N434Y F806 3.1E-07 64.3 0.70 262 M252Y/N384A/N434Y F807
3.2E-07 64.2 0.71 243 M252Y/N389A/N434Y F808 3.2E-07 64.1 0.69 274
M252Y/N389A/N390A/N434Y F809 2.2E-07 63.3 0.56 202
M252Y/S254T/T256S/N434Y F810 2.2E-07 65.2 0.68 360
M252Y/A378V/N434Y F811 4.9E-07 59.4 1.03 301 M252Y/E380S/N434Y F812
2.7E-07 62.8 0.69 267 M252Y/E382V/N434Y F813 2.8E-07 64.3 0.77 284
M252Y/S424E/N434Y F814 1.2E-07 63.3 0.90 188 M252Y/N434Y/Y436I F815
5.5E-07 62.4 0.65 218 M252Y/N434Y/T437R F816 3.6E-07 63.1 0.73 503
P238D/T250V/M252Y/T307P/N434Y F817 9.8E-08 60.6 0.78 543
P238D/T250V/M252Y/T307Q/Q311A/N434Y F819 1.4E-07 49.1 0.74 384
P238D/M252Y/N286E/N434Y F820 3.4E-07 70.4 0.56 312
L235K/S239K/M252Y/N434Y F821 3.1E-07 71 0.67 309
L235R/S239K/M252Y/N434Y F823 1.1E-06 44.2 10.39 367
P238D/T250Y/M252Y/W313F/N434Y F828 2.5E-06 60.8 0.80 361
P238D/T250V/M252Y/I253V/N434Y F831 1.6E-06 59.1 0.70 333
P238D/T250V/M252Y/R255A/N434Y F832 2.6E-06 52.6 1.46 290
P238D/T250V/M252Y/R255D/N434Y F833 8.0E-07 56.6 0.82 343
P238D/T250V/M252Y/R255E/N434Y F834 8.1E-07 55 0.96 386
P238D/T250V/M252Y/R255F/N434Y F836 5.0E-07 58.9 0.66 317
P238D/T250V/M252Y/R255H/N434Y F837 5.6E-07 49.1 2.06 365
P238D/T250V/M252Y/R255I/N434Y F838 4.3E-07 56.2 1.02 351
P238D/T250V/M252Y/R255K/N434Y F839 3.4E-07 58.1 0.76 376
P238D/T250V/M252Y/R255L/N434Y F840 4.2E-07 56.6 0.77 379
P238D/T250V/M252Y/R255M/N434Y F841 1.1E-06 59.6 0.70 330
P238D/T250V/M252Y/R255N/N434Y F843 6.6E-07 57.2 0.72 343
P238D/T250V/M252Y/R255Q/N434Y F844 1.3E-06 58.3 0.69 347
P238D/T250V/M252Y/R255S/N434Y F847 3.4E-07 47.1 3.82 355
P238D/T250V/M252Y/R255W/N434Y F848 8.3E-07 55.7 0.87 368
P238D/T250V/M252Y/R255Y/N434Y F849 3.3E-07 61.1 0.84 331
M252Y/D280A/N434Y F850 2.9E-07 62.2 0.64 310 M252Y/D280E/N434Y F852
3.3E-07 61.8 0.61 285 M252Y/D280G/N434Y F853 3.2E-07 58.4 2.55 302
M252Y/D280H/N434Y F855 3.2E-07 52.9 6.55 366 M252Y/D280K/N434Y F858
3.2E-07 62.6 0.60 357 M252Y/D280N/N434Y F860 3.3E-07 61.1 0.82 365
M252Y/D280Q/N434Y F861 3.2E-07 49.2 9.26 363 M252Y/D280R/N434Y F862
3.0E-07 61 0.72 310 M252Y/D280S/N434Y F863 2.7E-07 55.3 16.98 326
M252Y/D280T/N434Y F867 2.8E-07 64.4 0.75 262
M252Y/N384A/N389A/N434Y F870 7.3E-08 69.9 0.93 499
L235K/S239K/M252Y/T307Q/Q311A/N434Y F871 7.1E-08 70.7 0.92 495
L235R/S239K/M252Y/T307Q/Q311A/N434Y F872 1.3E-07 68.8 0.85 374
L235K/S239K/M252Y/N286E/N434Y F873 1.2E-07 69 0.92 371
L235R/S239K/M252Y/N286E/N434Y F875 4.8E-07 63.7 0.88 165
M252Y/N434Y/Y436A F877 8.3E-07 65.2 0.99 147 M252Y/N434Y/Y436E F878
1.9E-07 65 0.68 210 M252Y/N434Y/Y436F F879 9.2E-07 64.2 0.89 172
M252Y/N434Y/Y436G F880 3.9E-07 65 0.81 170 M252Y/N434Y/Y436H F881
3.1E-07 62.7 0.93 183 M252Y/N434Y/Y436K F882 1.3E-07 64 1.04 188
M252Y/N434Y/Y436L F883 2.1E-07 64.5 0.83 222 M252Y/N434Y/Y436M F884
4.0E-07 64.1 0.84 177 M252Y/N434Y/Y436N F888 4.8E-07 63.9 0.83 163
M252Y/N434Y/Y436S F889 2.2E-07 63.4 0.84 171 M252Y/N434Y/Y436T F890
1.1E-07 63.4 0.73 200 M252Y/N434Y/Y436V F891 1.7E-07 62.9 2.54 208
M252Y/N434Y/Y436W F892 7.1E-08 63.9 1.83 159
M252Y/S254T/N434Y/Y436I F893 9.8E-08 70.2 0.73 257
L235K/S239K/M252Y/N434Y/Y436I F894 9.2E-08 70.6 0.87 253
L235R/S239K/M252Y/N434Y/Y436I F895 2.1E-08 68.7 0.76 573
L235K/S239K/M252Y/N286E/T307Q/Q311A/N315E/N434Y F896 2.0E-08 68.7
0.65 569 L235R/S239K/M252Y/N286E/T307Q/Q311A/N315E/N434Y F897
9.7E-08 64.6 0.87 262 M252Y/N315D/N384A/N389A/N434Y F898 1.7E-07
64.1 0.92 283 M252Y/N315E/N384A/N389A/N434Y F899 1.1E-07 58.4 0.72
243 M252Y/N315D/G316A/N434Y F900 1.7E-07 52.3 2.50 243
M252Y/N315D/G316D/N434Y F901 1.3E-07 55.6 0.67 243
M252Y/N315D/G316E/N434Y F902 2.2E-07 57.2 0.90 266
M252Y/N315D/G316F/N434Y F903 2.3E-07 58.6 0.84 243
M252Y/N315D/G316H/N434Y F904 1.0E-07 48.6 3.34 266
M252Y/N315D/G316I/N434Y F905 1.3E-07 54.7 0.70 243
M252Y/N315D/G316K/N434Y F906 1.5E-07 54 0.59 266
M252Y/N315D/G316L/N434Y F907 1.3E-07 55.8 0.62 266
M252Y/N315D/G316M/N434Y F908 1.5E-07 58.1 0.87 243
M252Y/N315D/G316N/N434Y F910 1.4E-07 55.8 0.68 243
M252Y/N315D/G316Q/N434Y F911 1.3E-07 56.3 0.71 243
M252Y/N315D/G316R/N434Y F912 1.2E-07 59.1 0.57 243
M252Y/N315D/G316S/N434Y F913 1.1E-07 53.6 0.58 243
M252Y/N315D/G316T/N434Y F914 1.5E-07 48.9 2.04 266
M252Y/N315D/G316V/N434Y F915 2.3E-07 54.8 0.61 243
M252Y/N315D/G316W/N434Y F917 2.5E-07 63.4 0.64 314
M252Y/N286S/N434Y F918 2.8E-07 61.8 0.72 329
M252Y/D280E/N384A/N389A/N434Y F919 3.3E-07 61.7 0.67 303
M252Y/D280G/N384A/N389A/N434Y F920 2.5E-07 63.3 0.72 332
M252Y/N286S/N384A/N389A/N434Y F921 1.2E-07 62.3 0.74 324
M252Y/N286E/N384A/N389A/N434Y F922 5.9E-08 69 0.88 319
L235K/S239K/M252Y/N286E/N434Y/Y436I F923 6.0E-08 68.9 0.87 316
L235R/S239K/M252Y/N286E/N434Y/Y436I F924 3.4E-08 70.5 0.78 444
L235K/S239K/M252Y/T307Q/Q311A/N434Y/Y436I F925 3.2E-08 70.8 0.75
440 L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436I
F926 1.1E-07 70.6 0.87 228 L235K/S239K/M252Y/S254T/N434Y/Y436I F927
1.0E-07 70.8 0.80 224 L235R/S239K/M252Y/S254T/N434Y/Y436I F928
2.9E-08 63.9 0.76 375 M252Y/T307Q/Q311A/N434Y/Y436I F929 2.9E-08
64.2 0.87 346 M252Y/S254T/T307Q/Q311A/N434Y/Y436I F930 1.4E-07 58.6
0.78 419 P238D/T250V/M252Y/N286E/N434Y F931 1.2E-07 60.1 1.00 301
P238D/T250V/M252Y/N434Y/Y436I F932 3.2E-07 69.7 0.60 278
T250V/M252Y/N434Y F933 3.0E-07 63.2 0.67 323
L234R/P238D/T250V/M252Y/N434Y F934 3.1E-07 63.2 0.70 361
G236K/P238D/T250V/M252Y/N434Y F935 3.2E-07 64.2 0.61 354
G237K/P238D/T250V/M252Y/N434Y F936 3.2E-07 64.2 0.73 382
G237R/P238D/T250V/M252Y/N434Y F937 3.1E-07 66.2 0.65 320
P238D/S239K/T250V/M252Y/N434Y F938 1.6E-07 69.9 0.85 269
L235K/S239K/M252Y/N434Y/Y436V F939 1.5E-07 70 0.83 265
L235R/S239K/M252Y/N434Y/Y436V F940 1.5E-07 59.9 1.04 313
P238D/T250V/M252Y/N434Y/Y436V F941 1.2E-08 61.4 1.17 449
M252Y/N286E/T307Q/Q311A/N434Y/Y436V F942 4.2E-08 70.3 0.98 455
L235K/S239K/M252Y/T307Q/Q311A/N434Y/Y436V F943 4.0E-08 70.4 0.77
452 L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436V F944 1.7E-07 69.2
1.06 235 T250V/M252Y/N434Y/Y436V F945 1.7E-08 61 0.95 416
T250V/M252Y/V308P/N434Y/Y436V F946 4.3E-08 69 1.25 421
T250V/M252Y/T307Q/Q311A/N434Y/Y436V F947 1.1E-08 60.5 1.18 376
T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F954 5.3E-07 62.5 3.12
217 M252Y/N434Y/H435K/Y436V F957 7.7E-07 62.4 3.82 186
M252Y/N434Y/H435N/Y436V F960 8.0E-07 62.3 1.36 191
M252Y/N434Y/H435R/Y436V F966 3.1E-07 63.1 1.06 246
M252Y/S254A/N434Y F970 2.5E-06 64.8 0.90 214 M252Y/S254G/N434Y F971
2.6E-06 64.7 0.75 230 M252Y/S254H/N434Y F972 2.6E-07 58.7 2.25 239
M252Y/S254I/N434Y F978 1.3E-06 63.4 0.82 235 M252Y/S254Q/N434Y F980
1.8E-07 59.7 0.81 261 M252Y/S254V/N434Y F987 4.0E-08 59.5 0.87 500
P238D/T250V/M252Y/T307Q/Q311A/N434Y/Y436V F988 6.9E-08 56.8 1.08
375 P238D/T250V/M252Y/N286E/N434Y/Y436V F989 1.4E-08 61.6 0.82 446
L235R/S239K/M252Y/V308P/N434Y/Y436V F990 9.4E-09 61 0.88 406
L235R/S239K/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F991 1.3E-08 68.4
1.24 514 L235R/S239K/M252Y/N286E/T307Q/Q311A/N434Y/Y436V F992
5.1E-08 66.7 1.34 495
L235R/S239K/M252Y/T307Q/Q311A/M428I/N434Y/Y436V F993 3.8E-08 63.4
0.93 387 M252Y/T307Q/Q311A/N434Y/Y436V F994 2.8E-07 67 0.67 333
M252Y/N325G/N434Y F995 2.9E-07 59.9 0.71 279
L235R/P238D/S239K/M252Y/N434Y F996 1.3E-07 58.6 1.04 235
L235R/P238D/S239K/M252Y/N434Y/Y436V F997 3.8E-07 69.1 2.40 300
K248I/T250V/M252Y/N434Y/Y436V F998 8.5E-07 66.2 2.51 262
K248Y/T250V/M252Y/N434Y/Y436V F999 2.1E-07 67.4 1.13 273
T250V/M252Y/E258H/N434Y/Y436V F1008 1.7E-07 74.5 1.18 300
L235R/S239K/T250V/M252Y/N434Y/Y436V F1009 1.2E-08 66.6 0.37 441
L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1010 1.9E-07
70.6 0.29 513 L235R/S239K/M252Y/T307A/Q311H/N434Y F1011 4.5E-08
62.2 0.43 459 T250V/M252Y/V308P/N434Y F1012 4.7E-08 67.9 0.31 524
L235R/S239K/T250V/M252Y/V308P/N434Y F1013 3.0E-08 61.8 0.44 419
T250V/M252Y/T307Q/V308P/Q311A/N434Y F1014 3.2E-08 67.2 0.40 485
L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y F1015 2.2E-08 63
0.34 450 L235R/S239K/M252Y/T307Q/V308P/Q311A/N434Y F1016 3.8E-09
58.5 0.61 438 T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F1017
4.2E-09 65.1 0.56 504
L235R/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F1018
3.2E-09 60 0.52 469
L235R/S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F1019 3.4E-07
62.4 0.45 446 P238D/T250V/M252Y/N325G/N434Y F1020 8.5E-08 63 0.54
633 P238D/T250V/M252Y/T307Q/Q311A/N325G/N434Y F1021 3.3E-07 61.9
0.47 510 P238D/T250V/M252Y/N325A/N434Y F1026 8.4E-08 63.3 0.47 404
M252Y/T307A/Q311H/N434Y/Y436V F1027 8.6E-08 70.1 0.39 469
L235R/S239K/M252Y/T307A/Q311H/N434Y/Y436V F1032 4.3E-08 61.7 0.67
589 P238D/T250V/M252Y/T307Q/Q311A/N325G/N434Y/Y436V F1033 1.0E-06
58.1 1.89 169 P238D/N434W F1034 1.5E-08 61.1 0.83 450
L235K/S239K/M252Y/V308P/N434Y/Y436V F1035 1.0E-08 60.1 1.69 410
L235K/S239K/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1036 1.4E-08 68.9
0.87 518 L235K/S239K/M252Y/N286E/T307Q/Q311A/N434Y/Y436V F1037
6.1E-08 67.7 1.44 498
L235K/S239K/M252Y/T307Q/Q311A/M428I/N434Y/Y436V F1038 2.8E-07 60.3
0.78 264 L235K/P238D/S239K/M252Y/N434Y F1039 1.3E-07 58.9 0.81 220
L235K/P238D/S239K/M252Y/N434Y/Y436V F1040 2.0E-07 74.4 1.08 304
L235K/S239K/T250V/M252Y/N434Y/Y436V F1041 1.4E-08 63.5 0.84 445
L235K/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1042 2.0E-07
70.3 0.82 516 L235K/S239K/M252Y/T307A/Q311H/N434Y F1043 5.2E-08
64.8 0.64 528 L235K/S239K/T250V/M252Y/V308P/N434Y F1044 3.5E-08 64
0.53 488 L235K/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y F1045
2.5E-08 62.8 0.59 453 L235K/S239K/M252Y/T307Q/V308P/Q311A/N434Y
F1046 4.5E-09 64.5 0.96 507
L235K/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F1047
3.4E-09 60 0.96 472
L235K/S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F1048 9.9E-08
69.9 1.16 473 L235K/S239K/M252Y/T307A/Q311H/N434Y/Y436V F1050
3.5E-09 58.2 0.90 481
T250V/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436V F1051 3.9E-09
65 0.72 547
L235R/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436V
F1052 3.2E-09 59.6 1.08 512
L235R/S239K/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436V F1058
1.3E-07 63.7 0.74 214 M252Y/Q386E/N434Y/Y436V F1059 1.4E-07 63.1
0.63 214 M252Y/Q386R/N434Y/Y436V F1060 1.4E-07 63.3 0.62 214
M252Y/Q386S/N434Y/Y436V F1061 1.2E-07 64.7 0.60 200
M252Y/P387E/N434Y/Y436V F1062 1.2E-07 61 0.58 208
M252Y/P387R/N434Y/Y436V F1063 1.4E-07 63 0.58 214
M252Y/P387S/N434Y/Y436V F1064 1.3E-07 63 0.73 200
M252Y/V422E/N434Y/Y436V F1065 1.4E-07 62.9 0.54 242
M252Y/V422R/N434Y/Y436V F1066 1.4E-07 62.9 0.75 223
M252Y/V422S/N434Y/Y436V F1067 1.3E-07 63.5 0.73 241
M252Y/S424E/N434Y/Y436V F1068 1.7E-07 62.7 0.47 250
M252Y/S424R/N434Y/Y436V F1069 1.4E-07 62.9 0.87 186
M252Y/N434Y/Y436V/Q438E F1070 1.7E-07 62.9 0.62 209
M252Y/N434Y/Y436V/Q438R F1071 1.2E-07 63.1 0.57 235
M252Y/N434Y/Y436V/Q438S F1072 1.3E-07 63.3 0.75 133
M252Y/N434Y/Y436V/S440E F1073 1.3E-07 63 0.56 166
M252Y/N434Y/Y436V/S440R F1074 1.3E-07 57.7 0.67 200
S239D/M252Y/N434Y/Y436V F1075 1.4E-07 58.8 0.74 237
M252Y/K326D/L328Y/N434Y/Y436V F1076 1.3E-07 52.8 0.55 237
S239D/M252Y/K326D/L328Y/N434Y/Y436V F1077 2.0E-06 58 2.77 271
K248N/M252Y/N434Y F1078 4.7E-07 57.3 1.26 324
M252Y/E380N/E382S/N434Y F1079 3.4E-07 62.2 0.75 270
M252Y/E382N/N384S/N434Y F1080 3.2E-07 64 0.66 286 M252Y/S424N/N434Y
F1081 6.2E-07 63.6 0.93 169 M252Y/N434Y/Y436N/Q438T F1082 2.8E-07
64.5 8.15 261 M252Y/N434Y/Q438N F1083 3.5E-07 64 0.81 188
M252Y/N434Y/S440N F1094 2.6E-07 63.8 0.61 230 M252Y/N434Y/S442N
F1095 2.9E-07 64.3 0.64 265 M252Y/S383N/G385S/N434Y F1096 2.7E-07
64.3 0.66 257 M252Y/Q386T/N434Y F1097 2.8E-07 64.2 0.69 279
M252Y/G385N/P387S/N434Y F1098 2.6E-07 58.9 0.63 243
S239D/M252Y/N434Y F1099 2.6E-07 60 0.48 280 M252Y/K326D/L328Y/N434Y
F1100 2.4E-07 54.5 0.61 280 S239D/M252Y/K326D/L328Y/N434Y F1101
6.6E-08 59.4 0.47 430 S239D/M252Y/T307Q/Q311A/N434Y F1102 6.5E-08
60.2 0.58 467 M252Y/T307Q/Q311A/K326D/L328Y/N434Y F1103 6.1E-08
54.8 0.83 467 S239D/M252Y/T307Q/Q311A/K326D/L328Y/N434Y F1104
1.8E-07 62.9 0.61 242 M252Y/V422E/S424R/N434Y/Y436V F1105 1.5E-07
62.6 0.59 258 M252Y/V422S/S424R/N434Y/Y436V F1106 1.4E-07 63.1 0.60
158 M252Y/N434Y/Y436V/Q438R/S440E F1107 1.2E-07 63.2 0.75 208
M252Y/V422D/N434Y/Y436V F1108 1.3E-07 63.2 0.59 234
M252Y/V422K/N434Y/Y436V F1109 1.3E-07 63.4 0.58 200
M252Y/V422T/N434Y/Y436V F1110 1.3E-07 63.4 0.61 208
M252Y/V422Q/N434Y/Y436V F1111 1.6E-07 63.1 0.65 242
M252Y/S424K/N434Y/Y436V F1112 1.2E-07 63.1 0.57 209
M252Y/N434Y/Y436V/Q438K F1113 1.2E-07 63.6 0.68 133
M252Y/N434Y/Y436V/S440D F1114 1.3E-07 63.5 0.57 145
M252Y/N434Y/Y436V/S440Q F1115 1.3E-07 64 0.75 243
M252Y/S424N/N434Y/Y436V F1116 7.4E-08 64.4 0.58 473
M252Y/T307Q/Q311A/S424N/N434Y F1117 4.9E-08 69.3 0.71 465
T250V/M252Y/T307Q/Q311A/S424N/N434Y/Y436V F1118 1.3E-08 61 0.72 419
T250V/M252Y/T307Q/V308P/Q311A/S424N/N434Y/Y436V F1119 1.0E-08 60.5
0.72 376 T250V/M252Y/T307Q/V308P/Q311A/V422E/N434Y/Y436V F1120
1.0E-08 59.7 0.61 426
T250V/M252Y/T307Q/V308P/Q311A/S424R/N434Y/Y436V F1121 1.0E-08 59.9
0.54 418 T250V/M252Y/T307Q/V308P/Q311A/V422E/S424R/N434Y/Y436V
F1122 1.4E-08 59.6 0.41 385
T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R F1123 9.5E-09 60.8
0.61 309 T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/S440E F1124
1.2E-08 59.9 0.61 334
T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R/S440E F1125 5.2E-08
64.3 0.59 346 M252Y/T307Q/N434Y/Y436V F1126 9.0E-08 64.3 0.58 412
M252Y/T307A/N434Y/Y436V F1127 7.9E-08 62.8 0.53 338
M252Y/Q311A/N434Y/Y436V F1128 1.2E-07 62.3 0.41 274
M252Y/Q311H/N434Y/Y436V F1129 4.5E-08 63.2 0.51 315
M252Y/T307Q/Q311H/N434Y/Y436V F1130 5.5E-08 63.7 0.54 453
M252Y/T307A/Q311A/N434Y/Y436V F1131 1.3E-07 69.5 0.81 265
L235R/S239K/M252Y/V422E/N434Y/Y436V F1132 1.4E-07 69.1 0.87 288
L235R/S239K/M252Y/V422S/N434Y/Y436V F1133 1.6E-07 69.5 0.71 316
L235R/S239K/M252Y/S424R/N434Y/Y436V F1134 1.7E-07 70.4 0.58 274
L235R/S239K/M252Y/N434Y/Y436V/Q438R F1135 1.3E-07 69.8 0.72 198
L235R/S239K/M252Y/N434Y/Y436V/S440E F1136 1.6E-07 68.5 0.74 308
L235R/S239K/M252Y/V422E/S424R/N434Y/Y436V F1137 1.6E-07 69.4 0.82
323 L235R/S239K/M252Y/V422S/S424R/N434Y/Y436V F1138 1.7E-07 70.5
0.67 223 L235R/S239K/M252Y/N434Y/Y436V/Q438R/S440E F1139 1.5E-07
71.2 0.58 308 L235R/S239K/M252Y/S424N/N434Y/Y436V F1140 1.6E-07
62.6 0.89 201 M252Y/V422E/S424R/N434Y/Y436V/Q438R/S440E F1141
1.8E-07 62.7 0.94 216 M252Y/V422S/S424R/N434Y/Y436V/Q438R/S440E
F1142 1.9E-07 70 0.80 266
L235R/S239K/M252Y/V422E/S424R/N434Y/Y436V/Q438R/S440E F1143 2.0E-07
70.2 0.90 281 L235R/S239K/M252Y/V422S/S424R/N434Y/Y436V/Q438R/S440E
F1144 1.4E-08 66.7 0.81 400
L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R/S440E
F1145 5.2E-08 68.9 0.89 380
T250V/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E F1146 6.2E-08 75.6
0.85 445
L235R/S239K/T250V/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E F1147
7.2E-08 64.1 0.68 367 M252Y/T307Q/Q311A/N434Y/Q438R/S440E F1148
7.6E-08 70.9 0.69 432
L235R/S239K/M252Y/T307Q/Q311A/N434Y/Q438R/S440E F1151 2.5E-07 71.3
0.52 352 L235R/S239K/M252Y/S424N/N434Y F1152 7.4E-08 71.3 0.56 538
L235R/S239K/M252Y/T307Q/Q311A/S424N/N434Y F1153 4.8E-08 76.7 0.90
530 L235R/S239K/T250V/M252Y/T307Q/Q311A/S424N/N434Y/Y436V F1154
1.3E-08 67.6 0.68 484
L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/S424N/N434Y/Y436V F1157
2.1E-07 64.2 0.13 180 M252Y/N434Y/Q438R/S440E F1158 2.4E-07 70.9
0.67 245 L235R/S239K/M252Y/N434Y/Q438R/S440E F1159 4.8E-07 67.9
0.54 134 S424N/N434W F1160 2.9E-07 54 2.75 370 V308F/S424N/N434Y
F1161 1.1E-06 67.3 0.77 157 I332V/S424N/N434Y F1162 3.4E-07 58.1
0.93 313 P238D/T250Y/M252Y/N434Y/Y436V F1163 1.5E-07 60.2 0.84 543
P238D/T250Y/M252Y/T307Q/Q311A/N434Y F1164 7.0E-08 58.2 0.91 500
P238D/T250Y/M252Y/T307Q/Q311A/N434Y/Y436V F1165 1.6E-08 46.6 1.26
454 P238D/T250Y/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1174 4.9E-07
60.3 1.38 145 P257I/N434H F1176 2.0E-06 57 1.10 215 V308F F1178
8.7E-07 60.1 1.09 353 V259I/V308F/M428L F1183 1.3E-06 63.9 0.61 203
E380A/M428L/N434S F1184 1.0E-06 70.1 0.40 329 T307A/M428L/N434S
F1185 9.2E-07 64.6 0.55 415 T307A/E380A/M428L/N434S F1188 1.7E-06
64.3 0.56 322 T307A/E380A/N434H F1189 1.6E-07 63 0.52 161
M252Y/H433D/N434Y/Y436V/Q438R/S440E F1190 2.4E-07 63.7 0.58 161
M252Y/H433E/N434Y/Y436V/Q438R/S440E F1191 2.1E-07 63 0.70 147
M252Y/N434Y/Y436V/T437A/Q438R/S440E F1192 1.3E-07 62.3 0.82 133
M252Y/N434Y/Y436V/T437G/Q438R/S440E F1194 1.6E-07 60.3 0.84 168
M252Y/N434Y/Y436V/Q438R/K439D/S440E F1195 1.8E-07 60.5 2.91 156
M252Y/N434Y/Y436V/Q438R/S440E/L441A F1196 1.5E-07 60.4 0.90 161
M252Y/N434Y/Y436V/Q438R/S440E/L441E F1197 9.5E-08 63.2 0.48 129
M252Y/S254T/N434Y/Y436V/Q438R/S440E F1198 7.8E-08 60.8 0.68 67
M252Y/T256E/N434Y/Y436V/Q438R/S440E F1199 6.2E-08 61.2 0.74 67
M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F1200 1.3E-07 68.8 0.68
164 T250V/M252Y/S254T/N434Y/Y436V/Q438R/S440E F1201 1.1E-07 66.3
0.97 101 T250V/M252Y/T256E/N434Y/Y436V/Q438R/S440E F1202 8.8E-08 66
0.77 101 T250V/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F1203
1.5E-07 62.4 0.60 90 M252Y/T256Q/N434Y/Y436V/Q438R/S440E F1204
1.2E-07 62.6 0.35 64 M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E
F1205 2.0E-07 68.4 0.87 125
T250V/M252Y/T256Q/N434Y/Y436V/Q438R/S440E F1206 1.7E-07 68.5 0.71
99 T250V/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E F1207 1.1E-06
59.6 0.70 116 I332E/M428L/N434S F1208 5.7E-07 57.5 2.22 114
L251A/M252Y/N434Y/Y436V F1211 1.2E-06 55.8 3.02 89
L251H/M252Y/N434Y/Y436V F1216 1.2E-06 55.8 3.33 91
L251S/M252Y/N434Y/Y436V F1217 1.1E-06 55.5 2.65 116
L251T/M252Y/N434Y/Y436V F1218 2.5E-07 57.3 1.11 200
L251V/M252Y/N434Y/Y436V F1229 2.8E-06 62.9 0.76 204
M252Y/I253V/N434Y/Y436V F1230 1.1E-07 63.1 0.68 145
M252Y/N434Y/Y436V/Q438R/S440D F1231 9.7E-08 63 0.87 145
M252Y/N434Y/Y436V/Q438K/S440E F1232 9.8E-08 63.2 0.86 145
M252Y/N434Y/Y436V/Q438K/S440D F1243 1.3E-07 70.6 0.73 194
L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440E F1244 1.0E-07 69.2
0.85 132 L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438R/S440E F1245
8.2E-08 69.3 0.97 132
L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F1246 1.7E-07
75.1 0.74 229 L235R/S239K/T250V/M252Y/S254T/N434Y/Y436V/Q438R/S440E
F1247 1.5E-07 72.9 1.20 167
L235R/S239K/T250V/M252Y/T256E/N434Y/Y436V/Q438R/S440E
F1248 1.2E-07 73 0.94 167
L235R/S239K/T250V/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F1249
2.1E-07 70.5 0.78 155
L235R/S239K/M252Y/T256Q/N434Y/Y436V/Q438R/S440E F1250 1.7E-07 70.7
0.72 129 L235R/S239K/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E
F1251 2.8E-07 74.6 1.13 190
L235R/S239K/T250V/M252Y/T256Q/N434Y/Y436V/Q438R/S440E F1252 2.3E-07
75 0.90 164
L235R/S239K/T250V/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E F1253
1.1E-07 70.9 0.87 436
L235R/S239K/M252Y/T307A/N434Y/Y436V/Q438R/S440E F1254 6.4E-08 71.6
0.67 369 L235R/S239K/M252Y/T307Q/N434Y/Y436V/Q438R/S440E F1255
1.1E-07 69.9 0.74 362
L235R/S239K/M252Y/Q311A/N434Y/Y436V/Q438R/S440E F1256 1.6E-07 69.6
0.74 298 L235R/S239K/M252Y/Q311H/N434Y/Y436V/Q438R/S440E F1257
7.8E-08 70.5 0.83 477
L235R/S239K/M252Y/T307A/Q311A/N434Y/Y436V/Q438R/S440E F1258 1.1E-07
70.1 0.60 428 L235R/S239K/M252Y/T307A/Q311H/N434Y/Y436V/Q438R/S440E
F1259 4.5E-08 71 0.79 410
L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E F1260 6.5E-08
70.7 0.49 338 L235R/S239K/M252Y/T307Q/Q311H/N434Y/Y436V/Q438R/S440E
F1261 1.4E-07 70.3 0.69 210
L235R/S239K/M252Y/N434Y/Y436V/Q438R/S440D F1262 1.3E-07 70.1 0.70
210 L235R/S239K/M252Y/N434Y/Y436V/Q438K/S440E F1263 1.2E-07 70.3
0.67 210 L235R/S239K/M252Y/N434Y/Y436V/Q438K/S440D F1264 1.3E-07
68.6 0.67 206 L235R/S239K/M252Y/T256A/N434Y/Y436V/Q438R/S440E F1265
1.6E-07 65.8 0.72 182
L235R/S239K/M252Y/T256G/N434Y/Y436V/Q438R/S440E F1266 1.0E-07 69.7
0.70 210 L235R/S239K/M252Y/T256N/N434Y/Y436V/Q438R/S440E F1267
1.5E-07 69 0.73 226 L235R/S239K/M252Y/S254A/N434Y/Y436V/Q438R/S440E
F1268 2.0E-07 70.3 0.70 226
L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438R/S440E F1269 1.7E-07 70.5
0.80 212 L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438K/S440D F1270
1.2E-07 69 0.66 212 L235R/S239K/M252Y/S254A/N434Y/Y436V/Q438K/S440D
F1271 2.0E-07 69 0.69 228
L235R/S239K/M252Y/S254A/H433D/N434Y/Y436V/Q438R/S440E F1272 1.7E-07
69.2 0.86 215 L235R/S239K/M252Y/S254A/H433D/N434Y/Y436V/Q438K/S440D
F1273 1.5E-07 70.4 0.79 142
L235R/S239K/M252Y/T256Q/N434Y/Y436V/Q438K/S440D F1274 2.5E-07 70.4
0.70 158 L235R/S239K/M252Y/T256Q/H433D/N434Y/Y436V/Q438R/S440E
F1275 2.1E-07 70.7 0.78 144
L235R/S239K/M252Y/T256Q/H433D/N434Y/Y436V/Q438K/S440D F1276 1.0E-07
68.7 0.77 192 L235R/S239K/M252Y/T256A/N434Y/Y436V/Q438K/S440D F1277
1.7E-07 68.8 0.69 208
L235R/S239K/M252Y/T256A/H433D/N434Y/Y436V/Q438R/S440E F1278 1.4E-07
68.9 0.97 195 L235R/S239K/M252Y/T256A/H433D/N434Y/Y436V/Q438K/S440D
F1279 1.2E-07 66.2 0.79 169
L235R/S239K/M252Y/T256G/N434Y/Y436V/Q438K/S440D F1280 2.1E-07 66.1
1.03 185 L235R/S239K/M252Y/T256G/H433D/N434Y/Y436V/Q438R/S440E
F1281 1.7E-07 66.4 0.99 171
L235R/S239K/M252Y/T256G/H433D/N434Y/Y436V/Q438K/S440D F1282 7.7E-08
69.8 0.77 196 L235R/S239K/M252Y/T256N/N434Y/Y436V/Q438K/S440D F1283
1.3E-07 69.8 0.81 212
L235R/S239K/M252Y/T256N/H433D/N434Y/Y436V/Q438R/S440E F1284 1.1E-07
70 1.02 198 L235R/S239K/M252Y/T256N/H433D/N434Y/Y436V/Q438K/S440D
F1285 9.4E-08 70.4 0.72 181
L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440D F1286 1.6E-07 70.4
0.86 197 L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438R/S440E
F1287 1.5E-07 71.2 0.63 183
L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438K/S440D F1288 7.9E-08
68.8 0.92 118 L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440D F1289
1.3E-07 68.8 0.76 134
L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438R/S440E F1290 1.1E-07
69.2 0.91 121 L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438K/S440D
F1291 1.5E-07 70.4 0.62 275 L235R/S239K/M252Y/H433D/N434Y/Y436V
F1292 4.2E-07 71.1 0.66 95
L235R/S239K/H433D/N434W/Y436V/Q438R/S440E F1293 1.6E-07 68.6 0.73
153 L235R/S239K/M252Y/T256E/N434Y/Q438R/S440E F1294 2.0E-07 68.9
0.90 106 L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438R/S440E F1295
9.8E-08 68.7 0.81 128
L235R/S239K/M252Y/T256E/N434Y/Y436F/Q438R/S440E F1296 2.3E-07 68.8
0.76 152 L235R/S239K/M252Y/T256E/H433D/N434Y/Q438R/S440E F1297
2.5E-07 69.1 0.81 107
L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438R/S440E F1298 1.5E-07
69.1 0.82 106 L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438R/S440E
F1299 1.5E-07 68.7 0.72 140
L235R/S239K/M252Y/T256E/N434Y/Q438K/S440D F1300 1.6E-07 69 0.86 118
L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438K/S440D F1301 8.3E-08 69
0.85 140 L235R/S239K/M252Y/T256E/N434Y/Y436F/Q438K/S440D F1302
2.2E-07 69.2 0.75 138
L235R/S239K/M252Y/T256E/H433D/N434Y/Q438K/S440D F1303 2.1E-07 69.1
0.81 119 L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438K/S440D
F1304 1.2E-07 69.4 0.75 118
L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438K/S440D F1305 2.0E-07
70.8 0.77 212 L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438R/S440D F1306
1.9E-07 70.5 0.70 238
L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438K/S440E F1307 1.4E-07 69.5
0.68 337 L235R/S239K/M252Y/V422A/S424A/N434Y/Y436V F1308 2.1E-07
68.5 0.56 345 L235R/S239K/M252Y/V422L/S424L/N434Y/Y436V F1309
1.3E-07 70.2 0.83 284 L235R/S239K/M252Y/N434Y/Y436V/Q438A/S440A
F1310 2.3E-07 68.2 1.27 325
L235R/S239K/M252Y/N434Y/Y436V/Q438L/S440L F1311 1.7E-07 70.6 0.59
347 L235R/S239K/M252Y/V422A/S424A/H433D/N434Y/Y436V F1312 1.8E-07
70.4 0.55 355 L235R/S239K/M252Y/V422L/S424L/H433D/N434Y/Y436V F1313
1.8E-07 70.5 0.75 300
L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438A/S440A F1314 2.3E-07 69.4
1.15 302 L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438L/S440L F1315
1.5E-07 67.5 0.67 258 G237K/S239K/M252Y/N434Y/Y436V F1316 1.5E-07
67.7 0.70 260 G237R/S239K/M252Y/N434Y/Y436V F1317 1.4E-07 65.8 0.59
340 S239K/M252Y/P329K/N434Y/Y436V F1318 1.4E-07 66.2 0.57 317
S239K/M252Y/P329R/N434Y/Y436V F1319 2.7E-07 60.2 0.54 310
M252Y/L328Y/N434Y F1320 1.2E-07 70.5 0.71 181
L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440D F1321 1.0E-07 70.4
0.73 181 L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440E F1322
1.6E-07 70.7 0.77 183
L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438R/S440D F1323 1.5E-07
70.8 0.88 209 L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438K/S440E
F1324 1.3E-07 63.8 0.52 223 L234A/L235A/M252Y/N434Y/Y436V F1325
2.1E-07 55.1 1.87 249 L234A/L235A/M252Y/N297A/N434Y/Y436V F1326
1.1E-08 60.4 0.62 399
L234A/L235A/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1327 1.4E-08
48.2 0.91 425
L234A/L235A/T250V/M252Y/N297A/T307Q/V308P/Q311A/N434Y/Y436V F1328
1.5E-07 71.2 0.68 258
L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438R/S440E F1329 1.3E-07 71.2
0.74 229 L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440E
F1330 1.0E-07 69.8 0.80 167
L235R/G236R/S239K/M252Y/T256E/N434Y/Y436V/Q438R/S440E F1331 7.7E-08
70 0.90 167
L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F1333
1.2E-07 70.4 0.60 300 L235R/G236R/S239K/M252Y/N434Y/Y436V F1334
1.0E-07 71.1 0.59 245
L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438K/S440D F1335 8.8E-08 71.3
0.54 216 L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440D
F1336 7.2E-08 69.8 0.84 153
L235R/G236R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440D F1337 7.4E-08
68.9 0.69 118 L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440E F1338
1.0E-07 69.2 0.84 146
L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438K/S440E F1339 2.5E-07
69.5 0.59 144
L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436T/Q438K/S440E F1340
5.6E-08 69.1 0.76 118
L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438K/S440E F1341 3.2E-07
70.7 0.44 181 L235R/S239K/M252Y/S254T/N434Y/Y436T/Q438K/S440E F1342
2.5E-07 69.2 0.68 118
L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438K/S440E F1343 2.0E-07 69.1
0.56 118 L235R/S239K/M252Y/S254T/T256E/N434Y/Y436T/Q438K/S440E
F1344 4.0E-07 70.5 0.53 210
L235R/S239K/M252Y/N434Y/Y436T/Q438K/S440E F1345 1.0E-07 71.2 0.77
245 L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438K/S440E F1346 8.6E-08
71.3 0.70 216 L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440E
F1347 7.1E-08 69.9 0.95 153
L235R/G236R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440E F1348 5.5E-08
70.1 0.72 153
L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438K/S440E F1349
3.4E-07 70.2 0.67 198 L235R/S239K/M252Y/N434Y/Y436T/Q438R/S440E
F1350 1.2E-07 70.6 0.70 232
L235R/S239K/M252Y/N434Y/Y436F/Q438K/S440E F1351 1.6E-07 70.4 0.68
220 L235R/S239K/M252Y/N434Y/Y436F/Q438R/S440E F1352 3.9E-07 70.5
0.75 236 L235R/S239K/M252Y/H433D/N434Y/Y436T/Q438K/S440E F1353
4.3E-07 70.4 0.73 199
L235R/S239K/M252Y/H433D/N434Y/Y436T/Q438R/S440E F1354 2.3E-07 70.7
0.66 210 L235R/S239K/M252Y/H433D/N434Y/Y436F/Q438K/S440E F1355
2.5E-07 70.8 0.64 198
L235R/S239K/M252Y/H433D/N434Y/Y436F/Q438R/S440E F1356 1.6E-07 58.9
0.62 412 G236R/M252Y/L328R/N434Y/Y436V F1357 2.8E-07 70.5 0.61 169
L235R/S239K/M252Y/S254T/N434Y/Y436T/Q438R/S440E F1358 9.1E-08 71
0.64 203 L235R/S239K/M252Y/S254T/N434Y/Y436F/Q438K/S440E F1359
1.3E-07 71.1 0.67 191
L235R/S239K/M252Y/S254T/N434Y/Y436F/Q438R/S440E F1360 3.1E-07 70.6
0.57 207 L235R/S239K/M252Y/S254T/H433D/N434Y/Y436T/Q438K/S440E
F1361 3.5E-07 70.5 0.63 170
L235R/S239K/M252Y/S254T/H433D/N434Y/Y436T/Q438R/S440E F1362 1.4E-07
71 0.67 181 L235R/S239K/M252Y/S254T/H433D/N434Y/Y436F/Q438K/S440E
F1363 1.9E-07 71 0.48 169
L235R/S239K/M252Y/S254T/H433D/N434Y/Y436F/Q438R/S440E F1364 7.5E-08
69 0.84 140 L235R/S239K/M252Y/T256E/N434Y/Y436F/Q438K/S440E F1365
3.1E-07 69.1 0.78 144
L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438K/S440E F1366 1.2E-07
69.3 0.71 118 L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438K/S440E
F1367 1.8E-07 69.2 0.68 106
L235R/S239K/M252Y/S254T/T256E/N434Y/Y436T/Q438R/S440E F1368 5.5E-08
69 0.89 140 L235R/S239K/M252Y/S254T/T256E/N434Y/Y436F/Q438K/S440E
F1369 7.6E-08 69.2 0.84 128
L235R/S239K/M252Y/S254T/T256E/N434Y/Y436F/Q438R/S440E F1370 9.1E-08
69.5 0.77 146
L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438K/S440E F1371
1.1E-07 69.1 0.51 134
L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438R/S440E F1372
2.3E-07 69.4 0.70 107
L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436T/Q438R/S440E F1373
8.7E-08 69.5 0.77 118
L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436F/Q438K/S440E F1374
1.2E-07 69.3 0.54 106
L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436F/Q438R/S440E F1375
1.0E-07 69.9 0.47 236 L235R/S239K/M252Y/S254T/N434Y/Y436V F1376
9.1E-08 68.7 0.70 173 L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V
F1377 8.3E-08 68.4 0.73 173 L235R/S239K/M252Y/T256E/N434Y/Y436V
F1378 3.6E-07 70.6 0.65 236 L235R/S239K/M252Y/N434Y/Y436T F1379
2.8E-07 71.1 0.68 275 L235R/S239K/M252Y/N434Y/Y436F F1410 1.9E-06
61.1 0.59 183 V308P/I332V F1411 1.7E-07 60.2 0.55 299
V308P/I332V/M428L/N434S F1413 3.7E-08 68.3 2.19 332
L235R/S239K/M252Y/S254T/T256E/T307Q/Q311A/H433D/N434Y/Y436V/Q438K/S440E
F1414 5.6E-08 69.7 0.76 292
L235R/S239K/M252Y/S254T/T256E/T307Q/H433D/N434Y/Y436V/Q438K/S440E
F1415 5.9E-08 68.4 2.01 284
L235R/S239K/M252Y/S254T/T256E/Q311A/H433D/N434Y/Y436V/Q438K/S440E
F1416 1.3E-08 60.1 1.20 327
L235R/S239K/M252Y/S254T/T256E/V308P/H433D/N434Y/Y436V/Q438K/S440E
F1417 5.9E-08 68.6 1.72 121
L235R/S239K/M252Y/S254T/T256E/H433D/N434W/Y436V/Q438K/S440E F1418
7.5E-08 68.4 1.25 134
L235R/S239K/M252Y/S254T/T256E/H433D/N434W/Y436V/Q438R/S440E F1419
1.5E-07 69.5 0.71 226
L235R/S239K/M252Y/H433D/N434W/Y436V/Q438R/S440E F1420 1.3E-07 69.5
0.70 212 L235R/S239K/M252Y/H433D/N434W/Y436V/Q438K/S440E F1421
3.2E-08 58.9 2.59 348 V308P/M428L/N434W F1422 1.9E-08 59.8 1.27 315
L235R/S239K/M252Y/T256E/V308P/H433D/N434Y/Y436V/Q438R/S440E F1423
1.6E-08 45.4 4.03 157
L235R/S239K/M252Y/T256E/V302D/V308P/H433D/N434Y/Y436V/Q438R/S440E
F1424 1.6E-08 49 0.90 159
L235R/S239K/M252Y/T256E/V302E/V308P/H433D/N434Y/Y436V/Q438R/S440E
F1425 1.9E-08 49.4 2.04 185
L235R/S239K/M252Y/T256E/V303D/V308P/H433D/N434Y/Y436V/Q438R/S440E
F1426 1.8E-08 59.7 1.53 212
L235R/S239K/M252Y/T256E/V303E/V308P/H433D/N434Y/Y436V/Q438R/S440E
F1428 1.5E-08 45 8.91 243
L235R/S239K/M252Y/T256E/S304E/V308P/H433D/N434Y/Y436V/Q438R/S440E
F1430 3.1E-08 48.6 1.97 156
L235R/S239K/M252Y/T256E/V305E/V308P/H433D/N434Y/Y436V/Q438R/S440E
F1433 4.5E-08 46.2 9.04 181
L235R/S239K/M252Y/T256E/T307D/V308P/H433D/N434Y/Y436V/Q438R/S440E
F1434 3.6E-08 49.5 2.54 206
L235R/S239K/M252Y/T256E/T307E/V308P/H433D/N434Y/Y436V/Q438R/S440E
[0785] The variants (IgG1-F600 to IgG-F1434) each comprising a
heavy chain prepared as described above and L(WT)-CK (SEQ ID NO: 2)
were expressed and purified by the method known to those skilled in
the art described in Reference Example 2 of WO2011/122011.
1-2. Evaluation of FcRn Binding Affinity of Fc Variants Using
Biacore
[0786] hFcRn binding affinity of new Fc variants prepared in
Example 1 (F600-F1434) and previous Fc variants prepared in Example
1 of WO2011/122011 (F1-F599) was evaluated using Biacore T100 (GE
Healthcare). For this purpose, human FcRn was prepared as described
in Reference Example A2. An appropriate amount of protein L
(ACTIGEN) was immobilized onto Sensor chip CM4 (GE Healthcare) by
the amino coupling method, and the chip was allowed to capture an
antibody of interest. Then, diluted FcRn solutions and running
buffer (as a reference solution) were injected to allow human FcRn
to interact with the antibody captured on the sensor chip. The
running buffer used comprised 50 mmol/l sodium phosphate, 150
mmol/l NaCl, and 0.05% (w/v) Tween20 (pH 7.0). FcRn was diluted
using each buffer. The chip was regenerated using 10 mmol/l
glycine-HCl (pH 1.5). Assays were carried out exclusively at 25
degrees C. The association rate constant ka (1/Ms) and dissociation
rate constant ka (1/s), both of which are kinetic parameters, were
calculated based on the sensorgrams obtained in the assays, and KD
(M) of each antibody for human FcRn was determined from these
values. Each parameter was calculated using Biacore T100 Evaluation
Software (GE Healthcare). The binding affinity of all Fc variants
is shown in Table 16.
1-3. Evaluation of Stability of Fc Variants Using Differential
Scanning Fluorimetry (DSF)
[0787] Stability of new Fc variants prepared in Example 1
(F600-F1434) and previous Fc variants prepared in Example 1 of
WO2011/122011 (F1-F599) was evaluated using differential scanning
fluorimetry (DSF). This method consists of measuring the
fluorescence intensity of a polarity sensitive probe at gradually
increasing temperatures, and obtaining the transition temperature
of exposure of the hydrophobic regions of proteins. It is already
reported that the transition temperatures acquired using DSF are in
a good correlation with the melting temperatures acquired using
differential scanning calorimetry (Journal of Pharmaceutical
Science 2010; 4: 1707-1720). The SYPRO orange dye (Molecular
Probes) was diluted into PBS (Sigma), and added to the protein
solutions. Each sample was used with 20 microliter of the dyed
solution. The fluorescence emission was collected at 555 nm with a
fixed excitation wavelength at 470 nm. During the DSF experiment,
the temperature was increased from 30 to 99 degrees C. and at 0.4
degrees C. increments with an equilibration time of 6 seconds at
each temperature prior to measurement. The data were analyzed using
Rotor-Gene Q Series Software (QIAGEN). The temperature of the
fluorescence transition is defined as the melting temperature (Tm).
Tm values of the Fc variants F1-F1434 are shown in Table 16.
1-4. Evaluation of Purity of Fc Variants Using Size Exclusion
Chromatography (SEC)
[0788] High molecular weight species percentage (HMW (%)) of the
new Fc variants prepared in Example 1 (F600-F1434) and previous Fc
variants prepared in Example 1 of WO2011/122011 (F1-F599) was
evaluated using size exclusion chromatography (SEC). SEC was
performed in ACQUITY UPLC H-Class system (waters). The antibodies
were injected onto a BEH200 SEC column (1.7 micrometer,
4.6.times.150 mm, waters). The mobile phase was 0.05 M sodium
phosphate, 0.3 M sodium chloride (pH7.0, Isekyu), running
isocratically at a flow rate of 0.3 mL/min. Eluted protein was
detected by UV absorbance at 215 nm. The data were analyzed using
Empower2 (waters). Peaks eluting earlier than the antibody monomer
peak were recorded in the HMW components percentile. The HMW (%) of
all Fc variants (F1-F1434) are shown in Table 16.
1-5. Evaluation of Immunogenicity Risk of Fc Variants Using in
Silico Immunogenicity Prediction Tool Epibase
[0789] Clinical utility and efficacy of the therapeutic antibodies
can be limited by the production of anti-drug antibodies (ADAs),
since ADA can influence their efficacy and pharmacokinetics and
sometimes lead to serious side effects. Although many factors
influence the immunogenicity of therapeutic antibodies, a number of
reports describe the importance of effector T-cell epitopes present
in the therapeutic protein.
[0790] In silico tools to predict T-cell epitopes, such as Epibase
(Lonza), iTope/TCED (Antitope) and EpiMatrix (EpiVax) have been
developed. By using these in silico tools, the presence of T-cell
epitope in each amino acid sequence can be predicted (Expert Opin
Biol Ther. 2007 March; 7(3):405-18.), allowing the evaluation of
potential immunogenicity of the Fc variants. Epibase Light (Lonza)
was used to evaluate the potential immunogenicity of the Fc
variants.
[0791] Epibase Light (Lonza) is an in silico tool to calculate the
binding affinity of 9-mer peptide to major DRB1 alleles using
FASTER algorism (Expert Opin Biol Ther. 2007 March; 7(3):405-18.).
Epibase Light (Lonza) identifies T-cell epitopes with strong
binding and medium binding to MHC class II. In silico
immunogenicity score for each Fc variants was calculated using the
following formula incorporated in Epibase Light (Lonza) system.
Immunogenicity score=Sum ((each DRB1 allotype population
frequency).times.(number of critical epitopes)).
[0792] For DRB1 allotype population frequency used in the formula,
following DRB1 allotype population frequency based on Caucasian
population was used.
[0793] DRB1*0701 (25.3%), DRB1*1501 (23.1%), DRB1*0301 (21.7%),
DRB1*0101 (15.3%), DRB1*0401 (13.8%), DRB1*1101 (11.8%), DRB1*1302
(8.0%), DRB1*1401 (4.9%), DRB1*0403 (2.3%), DRB1*0901 (1.8%)
[0794] The total number of any strong and medium binding epitopes
identified in constant region (CH1-hinge-CH2-CH3) of the variants
by FASTER algorism was used as number of critical epitopes in the
formula. Filtered epitopes are those with human antibody germline
sequence or junction regions between variable region and constant
region, and only non-filtered epitopes are considered (counted as
critical epitope) in the immunogenicity score calculation.
[0795] Immunogenicity score of amino acid sequence of new Fc
variants described in Example 1 (F600-F1434) and previous Fc
variants described in Example 1 of WO2011/122011 (F1-F599) was
calculated using above described Epibase Light (Lonza) system.
Immunogenicity score of all Fc variants (F1-F1434) are shown in
Table 16.
[Example 2] Identification FcRn Binding Improved Fc Variants with
High Stability, Low High Molecular Weight Species and Low
Immunogenicity Risk
[0796] 2-1. Analysis of Previous and New Fc Variants by Plotting
Tm, HMW (%) and Immunogenicity Score Against hFcRn Binding
Affinity
[0797] hFcRn binding affinity and Tm of previous Fc variants
(F1-F599) described in Example 1 of WO2011/122011 and new Fc
variants (F600-F1052) generated and evaluated in Example 1 were
plotted and are shown in FIG. 2. hFcRn binding affinity and HMW (%)
of previous and new Fc variants were plotted and are shown in FIG.
3. hFcRn binding affinity and immunogenicity score of Fc variants
F1-F599 and new Fc variants (F600-F1052) were plotted and are shown
in FIG. 4.
[0798] The new Fc variants (F600-F1052) and previous Fc variants
(F1-F599) variants having Ser239Lys or Asp270Phe mutation were
deleted from the plots. Since Ser239Lys and Asp270Phe mutation
improved the stability (Tm) while it did not improve FcRn binding
affinity and reduced the binding affinity to all human Fc gamma
receptors, in the following detailed analysis of Group 1-4,
stability of Fc variants should be compared within the variants
that do not have Ser239Lys nor Asp270Phe mutation.
[0799] In addition, new Fc variants (F600-F1052) and previous Fc
variants (F1-F599) variants having Pro257Xxx (Xxx is Ala or Val or
Ile or Leu or Thr) or Met252Trp mutation were deleted from the
plots although these variants improve FcRn binding affinity.
Pro257Xxx and Met252Trp mutation did not exhibit significant
reduction in Tm suggesting that variants with Pro257Xxx and
Met252Trp mutation have high stability. Nevertheless, these
variants having Pro257Xxx or Met252Trp mutations showed significant
aggregation and precipitation during an accelerated stability study
or when stored refrigerated. Due to their detrimental stability, Fc
variants with Pro257Xxx and Met252Trp mutation are not acceptable
for pharmaceutical development and therefore, in the following
detailed analysis of Group 1-4, such Fc variants should be deleted
from the plots.
2-2. Detailed Analysis of Group 1 (Binding Affinity to hFcRn
Stronger than 15 nM)
[0800] New Fc variants (F600-F1052) generated and evaluated in
Example 1, and previous Fc variants (F1-F599) described in Example
1 of WO2011/122011, with binding affinity to hFcRn stronger than 15
nM (described as Group 1 hereafter), were analyzed in detail by
plotting hFcRn binding affinity in X-axis and Tm, HMW (%) and
immunogenicity score in Y-axis.
[0801] Detail analysis of Group 1 by plotting hFcRn binding
affinity (KD stronger than 15 nM) in X-axis and Tm, HMW (%) and
immunogenicity score in Y-axis are shown respectively in FIGS. 5, 6
and 7.
[0802] As for developability criteria of Fc variants in Group 1, Tm
criteria was set as higher than 57.5 degrees C., HMW (%) criteria
was set as lower than 2%, and immunogenicity score was set as lower
than 500.
[0803] Fc variants in Group 1 which satisfies all the
developability criteria (Tm higher than 57.5 degrees C., HMW (%)
lower than 2%, and immunogenicity score lower than 500) are shown
in Table 17.
TABLE-US-00017 TABLE 17 Immuno- Variant hFcRn Tm HMW genicity name
KD (M) (.degree. C.) (%) score Mutation F941 1.2E-08 61.4 1.2 449
M252Y/N286E/T307Q/ Q311A/N434Y/Y436V F947 1.1E-08 60.5 1.2 376
T250V/M252Y/T307Q/ V308P/Q311A/N434Y/ Y436V F1016 3.8E-09 58.5 0.6
438 T250V/M252Y/N286E/ T307Q/V308P/Q311A/ N434Y/Y436V F1050 3.5E-09
58.2 0.9 481 T250V/M252Y/N286E/ T307Q/V308P/Q311A/
M428I/N434Y/Y436V
[0804] None of the previous Fc variants (F1-F599) had an affinity
stronger than 15 nM, whereas the several new Fc variants generated
in EXAMPLE 1 were stronger than 15 nM and met all the
developability criteria. Such Group 1 new Fc variants described in
Table 17 are extremely valuable for Fc domain to enable very rapid
and extensive antigen elimination from plasma especially when used
in combination with pH-dependent antigen-binding domain.
2-3. Detailed Analysis of Group 2 (Binding Affinity to hFcRn
Between 15 nM and 50 nM)
[0805] New Fc variants (F600-F1052) generated and evaluated in
Example 1, and previous Fc variants (F1-F599) described in Example
1 of WO2011/122011, with binding affinity to hFcRn between 15 nM
and 50 nM (hereafter called "Group 2"), were analyzed in detail by
plotting hFcRn binding affinity on the X-axis and Tm, HMW (%) and
immunogenicity score on Y-axis.
[0806] Detailed analysis of Group 2 by plotting hFcRn binding
affinity (KD between 15 nM and 50 nM) on the x-axis, and Tm, HMW
(%) or immunogenicity score on the Y-axis are shown in FIGS. 8, 9
and 10, respectively.
[0807] As for developability criteria of Fc variants in Group 2, Tm
criteria was set as higher than 60 degrees C., HMW (%) criteria was
set as lower than 2%, and immunogenicity score was set as lower
than 500.
[0808] Fc variants in Group 2 which satisfies all the
developability criteria (Tm higher than 60 degrees C., HMW (%)
lower than 2%, and immunogenicity score lower than 500) are shown
in Table 18.
TABLE-US-00018 TABLE 18 Immuno- Variant hFcRn Tm HMW genicity name
KD (M) (.degree. C.) (%) score Mutation F928 2.9E-08 63.9 0.8 375
M252Y/T307Q/Q311A/ N434Y/Y436I F929 2.9E-08 64.2 0.9 346
M252Y/S254T/T307Q/ Q311A/N434Y/Y436I F945 1.7E-08 61 1 416
T250V/M252Y/V308P/ N434Y/Y436V F946 4.3E-08 69 1.3 421
T250V/M252Y/T307Q/ Q311A/N434Y/Y436V F993 3.8E-08 63.4 0.9 387
M252Y/T307Q/Q311A/ N434Y/Y436V F1011 4.5E-08 62.2 0.4 459
T250V/M252Y/V308P/ N434Y
[0809] None of the previous Fc variants (F1-F599) satisfied all the
developability criteria, but several of the new Fc variants
generated in Example 1 met all. Such Fc variants of Group 2 which
meet the developability criteria are extremely valuable to enable
rapid and extensive antigen elimination from plasma especially when
used in combination with pH-dependent antigen-binding domain.
2-4. Detailed Analysis of Group 3 (Binding Affinity to hFcRn
Between 50 nM and 150 nM)
[0810] New Fc variants (F600-F1052) generated and evaluated in
Example 1, and previous Fc variants (F1-F599) described in Example
1 of WO2011/122011, with binding affinity to hFcRn between 50 nM
and 150 nM (called hereinafter "Group 3"), were analyzed in detail
by plotting hFcRn binding affinity on the X-axis and Tm, HMW (%)
and immunogenicity score on the Y-axis.
[0811] Detail analysis of Group 3 by plotting hFcRn binding
affinity (KD between 50 nM and 150 nM) in X-axis, and Tm, HMW (%)
or immunogenicity score on Y-axis are shown in FIGS. 11, 12 and 13,
respectively.
[0812] As for developability criteria of Fc variants in Group 3, Tm
criteria was set as higher than 63.0 degrees C., HMW (%) criteria
was set as lower than 2%, and immunogenicity score was set as lower
than 250.
[0813] Fc variants in Group 3 which satisfies all the
developability criteria (Tm higher than 63.0 degrees C., HMW (%)
lower than 2%, and immunogenicity score lower than 250) are shown
in Table 19.
TABLE-US-00019 TABLE 19 Immuno- Variant hFcRn Tm HMW genicity name
KD (M) (.degree. C.) (%) score Mutation F789 1.5E-07 64.3 0.7 243
M252Y/N315D/N434Y F814 1.2E-07 63.3 0.9 188 M252Y/N434Y/Y436I F882
1.3E-07 64 1 188 M252Y/N434Y/Y436L F890 1.1E-07 63.4 0.7 200
M252Y/N434Y/Y436V F892 7.1E-08 63.9 1.8 159 M252Y/S254T/N434Y/
Y436I
[0814] None of the previous Fc variants (F1-F599) satisfied all the
developability criteria, whereas the several new Fc variants
generated in Example 1 met all. Such new Fc variants of Group 3
which meet all developability criteria are extremely valuable to
enable moderate and sustained antigen elimination from plasma
especially used in combination with pH-dependent antigen-binding
domain.
2-5. Detailed Analysis of Group 4 (Binding Affinity to hFcRn
Between 150 nM and 700 nM)
[0815] New Fc variants (F600-F1052) generated and evaluated in
Example 1, and previous Fc variants (F1-F599) described in Example
1 of WO2011/122011, with binding affinity to hFcRn between 150 nM
and 700 nM (called hereinafter "Group 4"), were analyzed in detail
by plotting hFcRn binding affinity on X-axis and Tm, HMW (%) and
immunogenicity score on Y-axis.
[0816] Detail analysis of Group 4 by plotting hFcRn binding
affinity (KD between 150 nM and 700 nM) on X-axis, and Tm, HMW (%)
or immunogenicity score on Y-axis are shown in FIGS. 14, 15 and 16,
respectively.
[0817] As for developability criteria of Fc variants in Group 4, Tm
criteria was set as higher than 66.5 degrees C., HMW (%) criteria
was set as lower than 2%, and immunogenicity score was set as lower
than 250.
[0818] Fc variants in Group 4 which satisfies all the
developability criteria (Tm higher than 66.5 degrees C., HMW (%)
lower than 2%, and immunogenicity score lower than 250) are shown
in Table 20.
TABLE-US-00020 TABLE 20 Immuno- Variant hFcRn Tm HMW genicity name
KD (M) (.degree. C.) (%) score Mutation F732 6.8E-07 69 0.7 227
T307Q/Q311H/N434Y F767 4.3E-07 69.9 0.6 198 T307Q/L309E/Q311A/
N434Y F769 4.6E-07 69.5 0.8 190 T307Q/L309E/Q311H/ N434Y F944
1.7E-07 69.2 1.1 235 T250V/M252Y/N434Y/ Y436V
[0819] None of the previous Fc variants (F1-F599) satisfied all the
developability criteria, whereas the several new Fc variants
generated in Example 1 met them all. Such new Fc variants of Group
4 which meet all developability criteria are extremely valuable to
enable moderate and sustained antigen elimination from plasma
especially used in combination with pH-dependent antigen-binding
domain.
[0820] In summary, new Fc variants described in Table 17 to 20 have
high Tm, low HMW (%), and low immunogenicity score which are
suitable for pharmaceutical development of antigen-binding molecule
capable of removing antigen from the plasma.
[Example 3] In Vivo Antigen Elimination Study of New Fc Variants in
Human IL-6 Receptor Steady-State Infusion Model Using Human FcRn
Transgenic
3-1. Preparation of Antibodies for In Vivo Study
[0821] pH-dependent anti-human IL6 receptor IgG1 antibody, Fv4-IgG1
comprising VH3-IgG1 (SEQ ID NO: 1) and VL3-CK (SEQ ID NO: 3),
previous Fc variant Fv4-F11 comprising VH3-F11 (SEQ ID NO: 4) and
VL3-CK (SEQ ID NO: 3), new Fc variants, Fv4-F652 comprising
VH3-F652 (SEQ ID NO: 5) and VL3-CK (SEQ ID NO: 3), and Fv4-F890
comprising VH3-F890 (SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 3), and
Fv4-F946 comprising VH3-F946 (SEQ ID NO: 7) and VL3-CK (SEQ ID NO:
3) were expressed and purified by the method known to those skilled
in the art described in Reference Example 2 of WO2011/122011.
[0822] In vivo antigen elimination study of Fv4-IgG1, Fv4-F11,
Fv4-F652, Fv4-F890 and Fv4-F946 were performed in human IL-6
receptor steady-state infusion model using human FcRn
transgenic.
3-2. In Vivo Study of Antibodies by Steady-State Infusion Model
Using Human FcRn Transgenic Mouse Line 32
[0823] An in vivo test was conducted by steady-state infusion model
using human FcRn transgenic mouse line 32. An infusion pump
(MINI-OSMOTIC PUMP MODEL 2004; alzet) containing soluble human IL-6
receptor was implanted under the skin on the back of human FcRn
transgenic mouse line 32 (B6.mFcRn-/-.hFcRn Tg line 32+/+ mouse
(B6.mFcRn-/- hFCRN Tg32 B6.Cg-Fcgrt<tm1Dcr>Tg(FCGRT)32Dcr),
Jackson Laboratories; Methods Mol Biol. (2010) 602: 93-104) to
prepare model animals in which the plasma concentration of soluble
human IL-6 receptor was kept constant. Anti-human IL-6 receptor
antibodies were administered to the model animals to assess the in
vivo dynamics after administration of soluble human IL-6 receptor.
Monoclonal anti-mouse CD4 antibody (in house) was administered at
20 mg/kg before implanting infusion pump, and 7 and 17 days after
antibody administration into the caudal vein to suppress the
production of neutralizing antibody against soluble human IL-6
receptor. Then, an infusion pump containing 92.8 microgram/ml
soluble human IL-6 receptor was implanted under the skin on the
back of the mice. Three days after implantation of an infusion
pump, anti-human IL-6 receptor antibodies were administered once
into the caudal vein. In study 1, Fv4-IgG1, Fv4-F652, Fv4-F890 and
Fv4-F946 were administered at as dosage of 1 mg/kg together with
approximately 1 g/kg Sanglopor (CSL Behring), and in study 2,
Fv4-IgG1, Fv4-F11 and Fv4-F652 were administered at 1 mg/kg. In
both studies, no antibody was administered to the control group (no
antibody injection). Blood was collected at appropriate time points
after the administration of the anti-human IL-6 receptor antibody.
The collected blood was immediately centrifuged at 15,000 rpm and 4
degrees C. for 15 minutes to separate plasma. The separated plasma
was stored in a refrigerator at -20 degrees C. or below before the
assay.
3-3. Measurement of Anti-Human IL-6 Receptor Antibody Plasma
Concentration by ELISA
[0824] The concentration of anti-human IL-6 receptor antibody in
mouse plasma was measured by ELISA. Anti-human IgG (gamma-chain
specific) F(ab')2 antibody fragment (Sigma) was dispensed onto a
Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and allowed to
stand overnight at 4 degrees C. to prepare anti-human
IgG-immobilized plates. Calibration curve samples having plasma
concentrations of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, and 0.0125
microgram/ml, and mouse plasma samples diluted 100-fold or more
were prepared. 200 microliter of 20 ng/ml hsIL-6R were added to 100
microliter of the calibration curve samples and plasma samples, and
then the samples were allowed to stand for one hour at room
temperature. Subsequently, the samples were dispensed onto the
anti-human IgG-immobilized plates, and allowed to stand for one
hour at room temperature. Then, Biotinylated Anti-Human IL-6R
Antibody (R&D) was added to react for one hour at room
temperature. Subsequently, Streptavidin-PolyHRP80 (Stereospecific
Detection Technologies) was added to react for one hour at room
temperature, and chromogenic reaction was carried out using TMP One
Component HRP Microwell Substrate (BioFX Laboratories) as a
substrate. After stopping the reaction with 1 N sulfuric acid
(Showa Chemical), the absorbance at 450 nm was measured by a
microplate reader. The concentration in mouse plasma was calculated
from the absorbance of the calibration curve using the analytical
software SOFTmax PRO (Molecular Devices).
3-4. Measurement of hsIL-6R Plasma Concentration by
Electrochemiluminescence Assay
[0825] The concentration of hsIL-6R in mouse plasma was measured by
electrochemiluminescence. hsIL-6R calibration curve samples
adjusted to concentrations of 2,000, 1,000, 500, 250, 125, 62.5,
and 31.25 pg/ml, and mouse plasma samples diluted 50-fold or more
were prepared. The samples were mixed with a solution of Monoclonal
Anti-human IL-6R Antibody (R&D) ruthenium-labeled with
Sulfo-Tag NHS Ester (Meso Scale Discovery), Biotinylated Anti-human
IL-6R Antibody (R&D, Systems Inc., USA), and tocilizumab
(Chugai Pharmaceutical Co., Ltd.)), and then allowed to react
overnight at 37 degrees C. The final concentration of tocilizumab
as an anti-human IL-6 receptor antibody was 333 microgram/ml, which
is in excess of the concentration of anti-human IL-6 receptor
antibody contained in the samples, for the purpose of binding
nearly all of the hsIL-6R molecules in the samples to tocilizumab.
Subsequently, the samples were dispensed into an MA400 PR
Streptavidin Plate (Meso Scale Discovery), and allowed to react for
one hour at room temperature, and washing was performed.
Immediately after Read Buffer T (.times.4) (Meso Scale Discovery)
was dispensed, the measurement was performed by the Sector PR 400
Reader (Meso Scale Discovery). The hsIL-6R concentration was
calculated based on the response of the calibration curve using the
analytical software SOFTmax PRO (Molecular Devices).
3-5. Result of Study 1; In Vivo Antigen Elimination Effect of New
Fc Variants
[0826] FIG. 17 shows plasma hsIL-6R concentration time profile and
FIG. 18 shows plasma antibody concentration time profile after
injection of Fv4-IgG1, Fv4-F652, Fv4-F890 and Fv4-F946. Compared to
Fv4-IgG1 and control (no antibody injection), Fv4-F652, Fv4-F890
and Fv4-F946 having new Fc variants with improved binding to FcRn
at neutral pH exhibited significant reduction of plasma hsIL-6R
concentration demonstrating in vivo antigen elimination effect of
pH-dependent antigen binding antibody with improved binding to FcRn
at neutral pH. Despite that Fv4-F652 and Fv4-F890 demonstrated
30-fold and 10-fold antigen elimination effect at day 7 compared to
Fv4-IgG1, respectively, plasma antibody concentration time profile
of Fv4-F652 and Fv4-F890 were comparable to Fv4-IgG1.
[0827] Therefore, this study demonstrated that Fv4-F652 and
Fv4-F890 were able to selectively eliminate soluble antigen from
plasma while maintaining antibody pharmacokinetics comparable to
that of Fv4-IgG1. Fv4-F890 belongs to Group 3, and this study
demonstrated that Fc variants in Group 3 can reduce then plasma
antigen concentration by approximately 10-fold while maintaining
the antibody pharmacokinetic comparable to IgG1. This means that
applying Group 3 Fc variant to pH-dependent antigen binding IgG1
antibody can lower the antibody dosage by 10-fold. Such reduction
in antibody dosage by Group 3 Fc variant is especially meaningful
when antibody dosage needs to be reduced, and simultaneously
requires infrequent dosing.
[0828] On the other hand, Fv4-F946 demonstrated 100-fold reduction
of plasma hsIL-6R concentration compared to Fv4-IgG1, and antibody
clearance of Fv4-F946 was larger than Fv4-IgG1. Fv4-F946 belongs to
Group 2, and this study demonstrated that Fc variants in Group 2
can reduce the plasma antigen concentration by approximately
100-fold although the antibody clearance is larger than IgG1. This
means that applying Group 2 Fc variant to pH-dependent antigen
binding IgG1 antibody can reduce the total plasma antigen
concentration by approximately 100-fold. In case the target plasma
antigen concentration is too high to neutralize by realistic
antibody dosage (i.e 100 mg/kg), 100-fold reduction of total
antigen concentration regardless of the increase of antigen
clearance by Group 2 Fc variant means that target antigen can be
neutralized by less than 10 mg/kg, which is a realistic antibody
dosage.
[0829] hFcRn binding affinity of Fv4-F652 and Fv4-F890 were
measured in multiplicates, and affinity against hFcRn was 2.4E-07 M
(n=7) for F652, and 1.1E-07 M (n=12) for F890. Previous studies
described in Example 1 of WO2011/122011 revealed that the extent of
antigen elimination and antibody clearance correlated with binding
affinity to FcRn at neutral pH. As shown in FIG. 17, Fv4-F652
exhibited larger extent of antigen elimination compared to Fv4-F890
although antibody pharmacokinetics was comparable to Fv4-F890.
Therefore, it was suggested that specific mutation in F652
contributed to enhanced antigen sweeping effect.
[0830] In order to identify which residue contributed to the
enhanced antigen sweeping effect of F652, study 2 was performed
using F11 (Met252Tyr, Asn434Tyr double mutant) and F652 (Pro238Asp,
Met252Tyr, Asn434Tyr triple mutant). Affinity against hFcRn was
3.1E-07 M (n=12) for F11, which was comparable to the affinity
measured for F652.
3-6. Result of Study 2; In Vivo Antigen Elimination Effect of
Pro238Asp Mutation
[0831] FIG. 19 shows plasma hsIL-6R concentration time profile and
FIG. 20 shows plasma antibody concentration time profile after
injection of Fv4-IgG1, Fv4-F11 and Fv4-F652. Although Fv4-F11
exhibited reduction of plasma hsIL-6R concentration, Fv4-F652
exhibited larger reduction of plasma hsIL-6R concentration. Fv4-F11
and Fv4-F652 exhibited comparable plasma antibody concentration
time profile.
[0832] Therefore, this study demonstrated that Pro238Asp mutation
were able to enhance antigen elimination plasma while maintaining
antibody pharmacokinetics comparable to Fv4-IgG1. Therefore,
Pro238Asp mutation is extremely valuable for enhancing antigen
elimination by pH-dependent antigen binding antibody.
[Example 4] Elimination of Rheumatoid Factor Binding to FcRn
Binding Improved Fc Variants by Site-Directed Mutagenesis
[0833] Clinical utility and efficacy of the therapeutic antibodies
can be limited by the production of anti-drug antibodies (ADAs),
since ADA can influence their efficacy and pharmacokinetics and
sometimes lead to serious side effects. Many factors influence the
immunogenicity of therapeutic antibodies, and the presence of
effector T-cell epitopes is one of the factors. In addition,
presence of pre-existing antibodies against therapeutic antibody
can also be problematic from the point of ADA. Especially in case
of therapeutic antibody for patients with autoimmune disease such
as rheumatoid arthritis, rheumatoid factor, an autoantibody against
human IgG, could be an issue of pre-existing antibody. Recently, it
was reported that humanized anti-CD4 IgG1 antibody with Asn434His
mutation elicited significant rheumatoid factor binding (Clin
Pharmacol Ther. 2011 February; 89(2):283-90). Detail study have
confirmed that Asn434His mutation in the human IgG1 increased the
binding of rheumatoid factor to the Fc region of the antibody
compared to the parent human IgG1.
[0834] Rheumatoid factor is a polyclonal autoantibody against human
IgG, and their epitope in human IgG varies among the clone, but
their epitope seems to be located in the CH2/CH3 interface region
as well as CH3 domain which could overlap with the FcRn binding
epitope. Therefore, mutations to increase the binding affinity to
FcRn might also increase the binding affinity to specific clone of
rheumatoid factor.
[0835] Previous studies have demonstrated that Fc-engineering to
increase the binding affinity to FcRn at acidic pH improved the
endosomal recycling efficiency and prolonged the pharmacokinetics
of the antibody. For example, M252Y/S254T/T256E (YTE) variant (J
Biol Chem 2006 281:23514-23524.), M428L/N434S (LS) variant (Nat
Biotechnol, 2010 28:157-159.) and N434H variant (Clinical
Pharmacology & Therapeutics (2011) 89(2):283-290.) showed
improvement in half-life relative to native IgG1.
[0836] To achieve antigen elimination from plasma, Fc regions of
the antigen-binding molecule (antibody) which interacts with FcRn
(Nat Rev Immunol. 2007 September; 7(9):715-25) was engineered to
have improved binding affinity to FcRn at neutral pH, such
engineered Fc variants include F11 variant, F68 variant, F890
variant and F947 variant. The mechanism of antigen elimination from
plasma by pH-dependent antigen binding antibody with improved
binding affinity to FcRn at neutral pH in comparison to the
conventional antibody is shown in FIG. 1.
[0837] Such Fc variant with improved FcRn-binding (either at pH 6.0
and/or neutral pH) could exhibit increased binding to rheumatoid
factor as in the case of previously reported Asn434His mutation.
Therefore, we tested whether these FcRn binding improved Fc
variants would exhibit increased binding to rheumatoid factor.
Variant antibodies used in the following study were Fv4-hIgG1,
Fv4-YTE, Fv4-LS, Fv4-N434H, Fv4-F11, Fv4-F68, Fv4-890 and
Fv4-F947.
4-1. Rheumatoid Factor Binding Study of FcRn Binding Improved Fc
Variants
[0838] Binding assay against rheumatoid factor was performed by
Electrochemiluminescence (ECL) at pH7.4. The assays were performed
with the serum of 15 or 30 individual RA patients (Proteogenex).
50-fold diluted serum samples, Biotin labeled test antibody (1
microgram/mL) and SULFO-TAG NHS Ester (Meso Scale Discovery)
labeled test antibody (1 microgram/mL) were mixed and incubated for
3 hr at room temperature. Then, the mixtures were added to
Streptavidin coated MULTI-ARRAY 96 well plates (Meso Scale
Discovery), and the plates were incubated for 2 hr at room
temperature and washed. After Read Buffer T (.times.4) (Meso Scale
Discovery) was added to each well, plates were immediately set on
the SECTOR imager 2400 Reader (Meso Scale Discovery) and the
chemiluminescence was measured.
[0839] Results of this study are shown in FIGS. 21 and 22. FIGS. 21
and 22 are the ECL response of the serum from 15 or 30 individual
RA patients. Fv4-hIgG1 with native human IgG1 (FIGS. 21-1 and 22-1)
showed only weak rheumatoid factor binding, whereas all the FcRn
binding improved Fc variants (Fv4-YTE (FIG. 21-2), Fv4-LS (FIG.
21-3), Fv4-N434H (FIG. 22-2), Fv4-F11 (FIG. 22-3), Fv4-F68 (FIG.
22-4), Fv4-890 (FIG. 22-5) and Fv4-F947 (FIG. 22-6)) significantly
enhanced the rheumatoid factor binding in more than the two donors.
This study clearly demonstrates that immunogenicity related to the
pre-existing rheumatoid factor can be an issue when considering the
clinical development of the therapeutic antibody with improved
binding affinity to FcRn for autoimmune disease such as rheumatoid
arthritis. FIG. 23 shows the mean, geomean and median of the ECL
response of the serum of the above mentioned antibody variants with
the blood of fifteen RA patients.
[0840] Therefore, in the next study, we have generated panels of
variants that could potentially reduce the polyclonal rheumatoid
factor binding while maintaining FcRn binding capability.
4-2. Reduction of Rheumatoid Factor Binding of FcRn Binding
Improved Fc Variants by Introducing Mutations in the Fc Region
[0841] In order to generate the variants with reduced polyclonal
rheumatoid factor binding while maintaining FcRn binding
capability, mutations were rationally introduced to the surface
residues near the CH2/CH3 interface which was assumed not to
interfere with human FcRn/human IgG interaction.
[0842] Fv4-F890 was selected as parent Fc variant, and single
mutation and combined Fc variants of single mutation were
introduced into Fv4-F890. The novel Fc variants F1058 to F1073,
F1107 to F1114, F1104 to F1106, and F1230 to F1232 described in
Table 21 were generated. In addition, Fv4-F947 was selected as
parent Fc variant and same single and combined mutations were
introduced. The novel Fc variants F1119-F1124 described in Table 21
were generated. First the variants were evaluated for their binding
affinity to human FcRn at pH7.0. Results are also described in
Table 21. Compared to either parent Fv4-F890 or Fv4-F947, these
variants did not show significant reduction in binding affinity
against human FcRn, demonstrating that these mutations did not
affect human FcRn binding.
TABLE-US-00021 TABLE 21 hFCRn Variant KD (M) name at pH 7.0
Mutations F890 1.07E-07 M252Y/N434Y/Y436V F1058 1.30E-07
M252Y/Q386E/N434Y/Y436V F1059 1.40E-07 M252Y/Q386R/N434Y/Y436V
F1060 1.40E-07 M252Y/Q386S/N434Y/Y436V F1061 1.20E-07
M252Y/P387E/N434Y/Y436V F1062 1.20E-07 M252Y/P387R/N434Y/Y436V
F1063 1.40E-07 M252Y/P387S/N434Y/Y436V F1064 1.30E-07
M252Y/V422E/N434Y/Y436V F1065 1.40E-07 M252Y/V422R/N434Y/Y436V
F1066 1.40E-07 M252Y/V422S/N434Y/Y436V F1067 1.30E-07
M252Y/S424E/N434Y/Y436V F1068 1.70E-07 M252Y/S424R/N434Y/Y436V
F1069 1.40E-07 M252Y/N434Y/Y436V/Q438E F1070 1.70E-07
M252Y/N434Y/Y436V/Q438R F1071 1.20E-07 M252Y/N434Y/Y436V/Q438S
F1072 1.30E-07 M252Y/N434Y/Y436V/S440E F1073 1.34E-07
M252Y/N434Y/Y436V/S440R F1107 1.20E-07 M252Y/V422D/N434Y/Y436V
F1108 1.30E-07 M252Y/V422K/N434Y/Y436V F1109 1.30E-07
M252Y/V422T/N434Y/Y436V F1110 1.30E-07 M252Y/V422Q/N434Y/Y436V
F1111 1.60E-07 M252Y/S424K/N434Y/Y436V F1112 1.20E-07
M252Y/N434Y/Y436V/Q438K F1113 1.20E-07 M252Y/N434Y/Y436V/S440D
F1114 1.30E-07 M252Y/N434Y/Y436V/S440Q F1104 1.80E-07
M252Y/V422E/S424R/N434Y/Y436V F1105 1.50E-07
M252Y/V422S/S424R/N434Y/Y436V F1106 1.40E-07
M252Y/N434Y/Y436V/Q438R/S440E F1230 1.12E-07
M252Y/N434Y/Y436V/Q438R/S440D F1231 9.73E-08
M252Y/N434Y/Y436V/Q438K/S440E F1232 9.79E-08
M252Y/N434Y/Y436V/Q438K/S440D F947 1.11E-08
T250V/M252Y/T307Q/V308P/Q311A/N434Y/ Y436V F1119 1.00E-08
T250V/M252Y/T307Q/V308P/Q311A/V422E/ N434Y/Y436V F1120 1.00E-08
T250V/M252Y/T307Q/V308P/Q311A/S424R/ N434Y/Y436V F1121 1.00E-08
T250V/M252Y/T307Q/V308P/Q311A/V422E/ S424R/N434Y/Y436V F1122
1.40E-08 T250V/M252Y/T307Q/V308P/Q311A/N434Y/ Y436V/Q438R F1123
9.50E-09 T250V/M252Y/T307Q/V308P/Q311A/N434Y/ Y436V/S440E F1124
1.20E-08 T250V/M252Y/T307Q/V308P/Q311A/N434Y/ Y436V/Q438R/S440E
[0843] Then we performed rheumatoid factor binding study at pH 7
for the variants in Table 21. Results of this study are shown in
FIGS. 24 to 29. These figures show the ECL response of the serum
from fifteen individual RA patients for the following variants of
the antibody: Fv4-IgG1, Fv4-F890, Fv4-F1058 to Fv4-1073 (FIG. 24),
Fv4-F1104 to Fv4-F1106 (FIG. 26), Fv4-F1107 to Fv4-F1114 (FIG. 27),
Fv4-F1230 to Fv4-F1232 (FIG. 28), Fv4-947 and Fv4-F1119 to
Fv4-F1124 (FIG. 29). FIGS. 25-1, 25-2 and 25-3 are the mean,
geomean and median of the ECL response of the serum from fifteen RA
patients for the variants Fv4-IgG1, Fv4-F890, and Fv4-F1058 to
Fv4-1073. Surprisingly, compared to F890 which exhibited strong
rheumatoid factor binding, novel Fc variants with single mutation
to F890, such as F1062, F1064-F1072 and F1107-F1114 exhibited
significant reduction in rheumatoid factor binding. Especially,
F1062, F1064, F1068, F1070, F1072, F1107 to F1109 and F1111-F1113
exhibited comparable rheumatoid factor binding as native IgG1
demonstrating that the increased immunogenicity risk of F890
variant was completely eliminated by introducing additional single
mutation to reduce rheumatoid factor binding without affecting
human FcRn binding. Since rheumatoid factor in patients is a
polyclonal antibody binding to multiple epitopes in the Fc region,
it was surprising that a single mutation significantly eliminated
the binding of rheumatoid factor to the Fc region.
[0844] Furthermore, compared to single mutated Fc F1070 (Q438R) or
F1072 (S440E), double mutated Fc F1106 (Q438R/S440E) showed
significant reduction in rheumatoid factor binding. Likewise,
double mutated Fc F1230 (Q438R/S440D), F1231 (Q438K/S440E) and
F1232 (Q438K/S440D) also showed additional reduction in rheumatoid
factor binding by combination of mutations. Meanwhile, F1104
(V422E/S424R) or F1105 (V422S/S424R) did not show any combination
effect.
[0845] In addition, with Fv4-F939 selected as parent Fc variant,
other mutations for increasing FcRn binding (S254T or T256E) and
for reducing rheumatoid factor binding (H433D) were evaluated.
Novel Fc variants (F1291, F1268, F1269, F1243, F1245, F1321, F1340
and F1323) described in Table 22 were generated. First, the
variants were evaluated for their binding affinity to human FcRn at
pH7.0. Results are also described in Table 22.
TABLE-US-00022 TABLE 22 hFcRn Variant KD (M) name at pH 7.0
Mutations F939 1.5E-07 L235R/S239K/M252Y/N434Y/Y436V F1291 1.5E-07
L235R/S239K/M252Y/H433D/N434Y/Y436V F1268 2.0E-07
L235R/S239K/M252Y/H433D/N434Y/Y436V/ Q438R/S440E F1269 1.7E-07
L235R/S239K/M252Y/H433D/N434Y/Y436V/ Q438K/S440D F1243 1.3E-07
L235R/S239K/M252Y/S254T/N434Y/Y436V/ Q438R/S440E F1245 8.2E-08
L235R/S239K/M252Y/S254T/T256E/N434Y/ Y436V/Q438R/S440E F1321
1.0E-07 L235R/S239K/M252Y/S254T/N434Y/Y436V/ Q438K/S440E F1340
5.6E-08 L235R/S239K/M252Y/S254T/T256E/N434Y/ Y436V/Q438K/S440E
F1323 1.5E-07 L235R/S239K/M252Y/S254T/H433D/N434Y/
Y436V/Q438K/S440E
[0846] Then we performed rheumatoid factor binding study for these
variants as described above. Results of this study are shown in
FIG. 30. Surprisingly, compared to F939, F1291 (single H433D
mutation to F939) exhibited significant reduction in rheumatoid
factor binding in some donors. Similarly, compared to F1321, F1323
(single H433D mutation to F1321) exhibited significant reduction in
rheumatoid factor binding in some donors. Furthermore, Q438R/S440E,
Q438K/S440D and Q438K/S440E mutations showed significant reduction
in rheumatoid factor binding with variants having other mutations
for increasing FcRn binding (S254T or T256E).
4-3. Reduction of Rheumatoid Factor Binding of FcRn Binding
Improved Fc Variants by Introducing Additional N-Glycosylation in
the Fc Region
[0847] Introduction of additional N-glycosylation near the
rheumatoid factor binding epitope may also abrogate rheumatoid
factor binding, due to steric hindrance with bulky N-glycosylation.
Mutation can be selected from the point so that the mutation
introduces N-glycosylation sequence (Asn-Xxx-Ser/Thr) while
maintaining FcRn binding. In order to introduce additional
N-glycosylation sequence into Fc region, single or double
mutation(s) were introduced into Fv4-F11. Novel Fc variants
(F1077-F1083, F1094-F1097) described in Table 23 were generated.
The variants were evaluated for their binding affinity to human
FcRn at pH7.0 and the presence of additional glycosylation by
SDS-Page. Results are described in Table 23. F1077 (K248N), F1080
(S424N), F1081 (Y436N/Q438T) and F1082 (Q438N) were found to have
additional glycosylation, and especially F1080 (S424N) maintained
binding affinity against human FcRn.
TABLE-US-00023 TABLE 23 hFcRn Variant KD (M) name at pH 7.0
Glycosylation Mutations F11 3.3E-07 M252Y/N434Y F1077 2.0E-06 ++
K248N/M252Y/N434Y F1078 4.7E-07 - M252Y/E380N/E382S/N434Y F1079
3.4E-07 - M252Y/E382N/N384S/N434Y F1080 3.2E-07 ++
M252Y/S424N/N434Y F1081 6.2E-07 ++ M252Y/N434Y/Y436N/Q438T F1082
2.8E-07 + M252Y/N434Y/Q438N F1083 3.5E-07 - M252Y/N434Y/S440N F1094
2.6E-07 - M252Y/N434Y/S442N F1095 2.9E-07 - M252Y/S383N/G385S/N434Y
F1096 2.7E-07 - M252Y/Q386T/N434Y F1097 2.8E-07 -
M252Y/G385N/P387S/N434Y
[0848] Therefore, in the next study, S424N mutation was introduced
into Fv4-F890, Fv4-F1115 described in Table 24 was generated and
evaluated for their binding affinity to human FcRn at pH7.0.
Results are also described in Table 24.
TABLE-US-00024 TABLE 24 Variant hFcRn KD name (M) at pH 7.0
Mutations F890 1.1E-07 M252Y/N434Y/Y436V F1115 1.3E-07
M252Y/S424N/N434Y/Y436V
[0849] Then we performed rheumatoid factor binding study for these
variants as described above. Result of this study is shown in FIG.
31. Surprisingly, single S424N mutant, Fv4-F1115, exhibited
significant reduction in rheumatoid factor binding. This result
suggests that the introduction of additional N-glycosylation is
effective approach for abrogating rheumatoid factor binding.
4-4. Reduction of Rheumatoid Factor Binding of YTE, N434H and LS
Variant
[0850] In order to reduce rheumatoid factor binding of Fv4-YTE,
Fv4-N434H and Fv4-LS variants, which improves FcRn binding at
acidic pH and prolongs antibody pharmacokinetics, Q438R/S440E
mutations or 5424N mutation were introduced into these variants.
Novel Fc variants (F1166, F1167, F1172, F1173, F1170 and F1171)
described in Table 25 were generated. First the variants were
evaluated for their binding affinity to human FcRn at pH6.0.
Results are also described in Table 25.
TABLE-US-00025 TABLE 25 Variant hFcRn KD name (M) at pH 6.0
Mutations IgG1 2.4E-06 none YTE 2.1E-07 M252Y/S254T/T256E F1166
2.1E-07 M252Y/S254T/T256E/Q438R/S440E F1167 2.5E-07
M252Y/S254T/T256E/S424N LS 1.6E-07 M428L/N434S F1170 1.5E-07
M428L/N434S/Q438R/S440E F1171 2.4E-07 S424N/M428L/N434S N434H
4.3E-07 N434H F1172 4.0E-07 N434H/Q438R/S440E F1173 5.3E-07
S424N/N434H
[0851] Then we performed rheumatoid factor binding study for these
variants (Fv4-F1166, F1167, F1172, F1173, F1170 and F1171) as
described above. Result of this study is shown in FIG. 32. Compared
to YTE which exhibited strong rheumatoid factor binding in two
donors (90216S and 90214S), F1166 (Q438R/S440E) and F1167 (S424N)
exhibited significant reduction in rheumatoid factor binding.
Furthermore, F1173 and F1171 show that S424N mutation could also
abrogate rheumatoid factor binding of N434H and LS variant.
However, Q438R/S440E mutations could not abrogate rheumatoid factor
binding of N434H and LS variant completely, rheumatoid factor
binding was observed in one or two donors.
4-5. Alternative Mutations for Reduction of Rheumatoid Factor
Binding of LS Variant
[0852] Novel single mutations were introduced into Fv4-LS, Fc
variants (Fv4-F1380 to Fv4-F1392) described in Table 26 were
generated.
TABLE-US-00026 TABLE 26 Variant hFcRn KD name (M) at pH 6.0
Mutations F22 7.1E-08 M428L/N434S (=LS) F1380 7.3E-08
S426D/M428L/N434S F1381 8.6E-08 S426E/M428L/N434S F1382 1.3E-07
S426K/M428L/N434S F1383 1.6E-07 S426R/M428L/N434S F1384 8.6E-08
S426A/M428L/N434S F1385 7.7E-08 S426Q/M428L/N434S F1386 1.6E-07
S426Y/M428L/N434S F1387 1.5E-07 M428L/N434S/Y436M F1388 8.0E-08
M428L/N434S/Y436F F1389 6.8E-08 M428L/N434S/Y436T F1390 4.0E-07
M428L/N434S/Y436H F1391 4.2E-07 M428L/N434S/Y436N F1392 2.7E-07
M428L/N434S/Y436K
[0853] Then we performed rheumatoid factor binding study for the
variants which maintains FcRn binding at pH6.0 (Fv4-F1380,
F1384-F1386, F1388 and F1389). Result of this study is shown in
FIG. 33. These variants exhibited significant reduction in
rheumatoid factor binding in some donors. Especially, Fv4-F1389
exhibited comparable rheumatoid factor binding as native IgG1.
[0854] Therefore, mutation such as Pro387Arg, Val422Glu, Val422Arg,
Val422Ser, Val422Asp, Val422Lys, Val422Thr, Val422Gln, Ser424Glu,
Ser424Arg, Ser424Lys, Ser424Asn, Ser426Asp, Ser426Ala, Ser426Gln,
Ser426Tyr, His433Asp, Tyr436Thr, Gln438Glu, Gln438Arg, Gln438Ser,
Gln438Lys, Ser440Glu, Ser440Asp, Ser440Gln (positions are given in
EU numbering) are extremely useful for reducing the immunogenicity
of antigen-binding molecule containing FcRn binding increased Fc
region (for example F1-F1434) such as pH-dependent antigen binding
antibody with improved binding affinity to FcRn at neutral pH which
is capable of eliminating antigen from plasma and antibody with
improved binding affinity to FcRn at acidic pH which is capable of
improving antibody pharmacokinetics.
[0855] Mutation sites other than EU387, EU422, EU424, EU426, EU433,
EU436, EU438 and EU440 for reducing the binding of rheumatoid
factor without affecting human FcRn binding could be selected from
248-257, 305-314, 342-352, 380-386, 388, 414-421, 423, 425-437,
439, and 441-444 in EU numbering.
[Example 5] Reduction of Rheumatoid Factor Binding of Novel Fc
Variants with Improved Binding to Human FcRn at Neutral pH
[0856] Novel Fc variants (F939, F1378, F1379, F1262, F1138, F1344,
F1349, F1350, F1351, F1261, F1263, F1305, F1306, F1268, F1269,
F1413, F1416, F1419, F1420, F1370, F1371, F1599, F1600, F1566,
F1448, F1601-F1603, F1531, F1604, F1605, F1586, F1592, F1610-F1615,
F1567, F1572, F1576, F1578, F1579, F1641-F1655, F1329, F1331)
described in Table 27 were generated. First the variants were
evaluated for their binding affinity to human FcRn at pH7.0.
Results are also described in Table 27.
TABLE-US-00027 TABLE 27 hFcRn Variant KD (M) name at pH 7.0
Mutations F939 1.5E-07 L235R/S239K/M252Y/N434Y/Y436V F1378 3.6E-07
L235R/S239K/M252Y/N434Y/Y436T F1379 2.8E-07
L235R/S239K/M252Y/N434Y/Y436F F1262 1.3E-07
L235R/S239K/M252Y/N434Y/Y436V/Q438K/ S440E F1138 1.7E-07
L235R/S239K/M252Y/N434Y/Y436V/Q438R/ S440E F1344 4.0E-07
L235R/S239K/M252Y/N434Y/Y436T/Q438K/S440E F1349 3.4E-07
L235R/S239K/M252Y/N434Y/Y436T/Q438R/ S440E F1350 1.2E-07
L235R/S239K/M252Y/N434Y/Y436F/Q438K/S440E F1351 1.6E-07
L235R/S239K/M252Y/N434Y/Y436F/Q438R/S440E F1261 1.4E-07
L235R/S239K/M252Y/N434Y/Y436V/Q438R/ S440D F1263 1.2E-07
L235R/S239K/M252Y/N434Y/Y436V/Q438K/ S440D F1305 2.0E-07
L235R/S239K/M252Y/H433D/N434Y/Y436V/ Q438R/S440D F1306 1.9E-07
L235R/S239K/M252Y/H433D/N434Y/Y436V/ Q438K/S440E F1268 2.0E-07
L235R/S239K/M252Y/H433D/N434Y/Y436V/ Q438R/S440E F1269 1.7E-07
L235R/S239K/M252Y/H433D/N434Y/Y436V/ Q438K/S440D F1413 3.7E-08
L235R/S239K/M252Y/S254T/T256E/T307Q/Q311A/
H433D/N434Y/Y436V/Q438K/S440E F1416 1.3E-08
L235R/S239K/M252Y/S254T/T256E/V308P/H433D/ N434Y/Y436V/Q438K/S440E
F1419 1.5E-07 L235R/S239K/M252Y/H433D/N434W/Y436V/ Q438R/S440E
F1420 1.3E-07 L235R/S239K/M252Y/H433D/N434W/Y436V/ Q438K/S440E
F1370 9.1E-08 L235R/S239K/M252Y/S254T/T256E/H433D/
N434Y/Y436V/Q438K/S440E F1371 1.1E-07
L235R/S239K/M252Y/S254T/T256E/H433D/ N434Y/Y436V/Q438R/S440E F1599
7.4E-08 L235R/S239K/M252Y/S254T/T256E/H433D/
N434Y/Y436V/Q438K/S440D F1600 8.5E-08
L235R/S239K/M252Y/S254T/T256E/H433D/ N434Y/Y436V/Q438R/S440D F1566
4.0E-08 L235R/S239K/M252Y/S254T/T256E/N286E/
H433D/N434Y/Y436V/Q438K/S440E F1448 4.9E-08
L235R/S239K/M252Y/S254T/T256E/N286E/ H433D/N434Y/Y436V/Q438R/S440E
F1601 3.5E-08 L235R/S239K/M252Y/S254T/T256E/N286E/
H433D/N434Y/Y436V/Q438K/S440D F1602 3.6E-08
L235R/S239K/M252Y/S254T/T256E/N286E/ H433D/N434Y/Y436V/Q438R/S440D
F1603 5.9E-08 L235R/S239K/M252Y/S254T/R255L/T256E/H433D/
N434Y/Y436V/Q438K/S440E F1531 7.6E-08
L235R/S239K/M252Y/S254T/R255L/T256E/H433D/ N434Y/Y436V/Q438R/S440E
F1604 6.0E-08 L235R/S239K/M252Y/S254T/R255L/T256E/H433D/
N434Y/Y436V/Q438K/S440D F1605 6.1E-08
L235R/S239K/M252Y/S254T/R255L/T256E/H433D/ N434Y/Y436V/Q438R/S440D
F1586 5.5E-08 L235R/S239K/M252Y/S254T/R255L/T256E/E258D/
H433D/N434Y/Y436V/Q438R/S440E F1592 5.5E-08
L235R/S239K/M252Y/S254T/R255L/T256E/E258I/
H433D/N434Y/Y436V/Q438R/S440E F1610 4.8E-08
L235R/S239K/M252Y/S254T/R255L/T256E/E258D/
H433D/N434Y/Y436V/Q438K/S440E F1611 5.2E-08
L235R/S239K/M252Y/S254T/R255L/T256E/E258I/
H433D/N434Y/Y436V/Q438K/S440E F1612 4.9E-08
L235R/S239K/M252Y/S254T/R255L/T256E/E258D/
H433D/N434Y/Y436V/Q438K/S440D F1613 5.2E-08
L235R/S239K/M252Y/S254T/R255L/T256E/E258I/
H433D/N434Y/Y436V/Q438K/S440D F1614 5.1E-08
L235R/S239K/M252Y/S254T/R255L/T256E/E258D/
H433D/N434Y/Y436V/Q438R/S440D F1615 6.0E-08
L235R/S239K/M252Y/S254T/R255L/T256E/E258I/
H433D/N434Y/Y436V/Q438R/S440D F1567 4.8E-08
L235R/S239K/M252Y/S254T/T256E/H433A/ N434Y/Y436V/Q438R/S440E F1572
4.1E-08 L235R/S239K/M252Y/S254T/T256E/H433K/
N434Y/Y436V/Q438R/S440E F1576 4.3E-08
L235R/S239K/M252Y/S254T/T256E/H433P/N434Y/ Y436V/Q438R/S440E F1578
4.6E-08 L235R/S239K/M252Y/S254T/T256E/H433R/
N434Y/Y436V/Q438R/S440E F1579 5.4E-08
L235R/S239K/M252Y/S254T/T256E/H433S/N434Y/ Y436V/Q438R/S440E F1641
4.1E-08 L235R/S239K/M252Y/S254T/T256E/H433A/
N434Y/Y436V/Q438K/S440E F1642 4.1E-08
L235R/S239K/M252Y/S254T/T256E/H433A/ N434Y/Y436V/Q438R/S440D F1643
3.9E-08 L235R/S239K/M252Y/S254T/T256E/H433A/
N434Y/Y436V/Q438K/S440D F1644 3.5E-08
L235R/S239K/M252Y/S254T/T256E/H433K/ N434Y/Y436V/Q438K/S440E F1645
3.6E-08 L235R/S239K/M252Y/S254T/T256F/H433K/
N434Y/Y436V/Q438R/S440D F1646 3.5E-08
L235R/S239K/M252Y/S254T/T256E/H433K/ N434Y/Y436V/Q438K/S440D F1647
3.8E-08 L235R/S239K/M252Y/S254T/T256E/H433P/
N434Y/Y436V/Q438K/S440E F1648 3.8E-08
L235R/S239K/M252Y/S254T/T256E/H433P/ N434Y/Y436V/Q438R/S440D F1649
3.7E-08 L235R/S239K/M252Y/S254T/T256E/H433P/N434Y/
Y436V/Q438K/S440D F1650 4.0E-08
L235R/S239K/M252Y/S254T/T256E/H433R/ N434Y/Y436V/Q438K/S440E F1651
4.4E-08 L235R/S239K/M252Y/S254T/T256E/H433R/
N434Y/Y436V/Q438R/S440D F1652 4.0E-08
L235R/S239K/M252Y/S254T/T256E/H433R/ N434Y/Y436V/Q438K/S440D F1653
4.5E-08 L235R/S239K/M252Y/S254T/T256E/H433S/N434Y/
Y436V/Q438K/S440E F1654 4.5E-08
L235R/S239K/M252Y/S254T/T256E/H433S/N434Y/ Y436V/Q438R/S440D F1655
4.4E-08 L235R/S239K/M252Y/S254T/T256E/H433S/N434Y/
Y436V/Q438K/S440D F1329 1.3E-07
L235R/G236R/S239K/M252Y/S254T/N434Y/ Y436V/Q438R/S440E F1331
7.7E-08 L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/
Y436V/Q438R/S440E
[0857] Then we performed rheumatoid factor binding study at pH 7.4
for the variants in Table 27. Results of this study are shown in
FIGS. 34 to 94.
[0858] Double mutations for decreasing rheumatoid factor binding
(Q438R/S440E, Q438R/S440D, Q438K/S440E and Q438K/S440D) showed
significant reduction in rheumatoid factor binding to other
mutations for increasing FcRn binding at neutral pH.
5-1. Reduction of Rheumatoid Factor Binding of Novel Fc Variants
with Improved Binding to Human FcRn at Acidic pH
[0859] Novel Fc variants (F1718-F1721, F1671, F1670, F1711-F1713,
F1722-F1725, F1675, F1714-F1717, F1683, F1756-F1759, F1681,
F1749-F1751, F1760-F1763, F1752-F1755, F1685) described in Table 28
were generated. First the variants were evaluated for their binding
affinity to human FcRn at pH6.0. Results are also described in
Table 28.
TABLE-US-00028 TABLE 28 hFcRn Variant KD (M) name at pH 6.0
Mutations F1718 6.1E-08 N434Y/Y436V/Q438R/S440E F1719 5.2E-08
N434Y/Y436V/Q438R/S440D F1720 4.5E-08 N434Y/Y436V/Q438K/S440E F1721
5.5E-08 N434Y/Y436V/Q438K/S440D F1671 9.5E-08
L235R/S239K/N434Y/Y436V F1670 6.4E-08
L235R/S239K/N434Y/Y436V/Q438R/S440E F1711 5.5E-08
L235R/S239K/N434Y/Y436V/Q438R/S440D F1712 5.5E-08
L235R/S239K/N434Y/Y436V/Q438K/S440E F1713 5.1E-08
L235R/S239K/N434Y/Y436V/Q438K/S440D F1722 1.0E-07
H433D/N434Y/Y436V/Q438R/S440E F1723 9.6E-08
H433D/N434Y/Y436V/Q438R/S440D F1724 9.9E-08
H433D/N434Y/Y436V/Q438K/S440E F1725 9.3E-08
H433D/N434Y/Y436V/Q438K/S440D F1675 9.7E-08
L235R/S239K/H433D/N434Y/Y436V F1714 1.1E-07
L235R/S239K/H433D/N434Y/Y436V/Q438R/S440E F1715 1.1E-07
L235R/S239K/H433D/N434Y/Y436V/Q438R/S440D F1716 9.6E-08
L235R/S239K/H433D/N434Y/Y436V/Q438K/S440E F1717 9.5E-08
L235R/S239K/H433D/N434Y/Y436V/Q438K/S440D F1683 5.7E-08
L235R/S239K/N434Y/Y436F/Q438R/S440E F1756 8.0E-08
N434Y/Y436T/Q438R/S440E F1757 7.2E-08 N434Y/Y436T/Q438R/S440D F1758
7.0E-08 N434Y/Y436T/Q438K/S440E F1759 6.3E-08
N434Y/Y436T/Q438K/S440D F1681 8.4E-08
L235R/S239K/N434Y/Y436T/Q438R/S440E F1749 8.7E-08
L235R/S239K/N434Y/Y436T/Q438R/S440D F1750 7.0E-08
L235R/S239K/N434Y/Y436T/Q438K/S440E F1751 6.9E-08
L235R/S239K/N434Y/Y436T/Q438K/S440D F1760 1.2E-07
H433D/N434Y/Y436T/Q438R/S440E F1761 1.3E-07
H433D/N434Y/Y436T/Q438R/S440D F1762 1.1E-07
H433D/N434Y/Y436T/Q438K/S440E F1763 1.1E-07
H433D/N434Y/Y436T/Q438K/S440D F1752 1.5E-07
L235R/S239K/H433D/N434Y/Y436T/Q438R/S440E F1753 1.3E-07
L235R/S239K/H433D/N434Y/Y436T/Q438R/S440D F1754 1.2E-07
L235R/S239K/H433D/N434Y/Y436T/Q438K/S440E F1755 1.3E-07
L235R/S239K/H433D/N434Y/Y436T/Q438K/S440D F1685 8.7E-08
L235R/S239K/N434Y/Q438R/S440E
[0860] Then we performed rheumatoid factor binding study at pH 7.4
for the variants in Table 28. Results of this study are shown in
FIGS. 95 to 130.
[0861] Double mutations for decreasing rheumatoid factor binding
(Q438R/S440E, Q438R/S440D, Q438K/S440E and Q438K/S440D) showed
significant reduction in rheumatoid factor binding to other
mutations for increasing FcRn binding at acidic pH.
[Example 6] In Vivo Antigen Elimination Study of Novel Fc Variants
in Human IL-6 Receptor Steady-State Infusion Model Using Human FcRn
Transgenic Mice
6-1. Preparation of Antibodies for In Vivo Study
[0862] pH-dependent anti-human IL6 receptor IgG1 antibody, Fv4-IgG1
comprising VH3-IgG1 (SEQ ID NO: 1) and VL3-CK (SEQ ID NO: 3), new
Fc variants, Fv4-F1243 comprising VH3-F1243 (SEQ ID NO: 8) and
VL3-CK (SEQ ID NO: 3), and Fv4-F1245 comprising VH3-F1245 (SEQ ID
NO: 9) and VL3-CK (SEQ ID NO: 3) were expressed and purified by the
method known to those skilled in the art described in Example 2 of
WO2011/122011.
[0863] As described in Example 4, Fv4-F1243 and Fv4-F1245 have
novel Fc region with improved binding affinity to human FcRn at
neutral pH, but significantly reduced binding to rheumatoid factor.
In order to evaluate antigen elimination effect of these variants,
an in vivo study of Fv4-IgG1, Fv4-F1243 and Fv4-F1245 was performed
in a human IL-6 receptor steady-state infusion model using human
FcRn transgenic mice.
6-2. In Vivo Study of Antibodies by Steady-State Infusion Model
Using Human FcRn Transgenic Mouse Line 32
[0864] An in vivo test was conducted by steady-state infusion model
using human FcRn transgenic mouse line 32 by the same methods
described in Example 13 of WO2011/122011.
6-3. Result of Study; In Vivo Antigen Elimination Effect of New Fc
Variants
[0865] FIG. 131 shows plasma hsIL-6R concentration time profile and
FIG. 132 shows plasma antibody concentration time profile after
injection of Fv4-IgG1, Fv4-F1243 and Fv4-F1245. Compared to
Fv4-IgG1 and control (no antibody injection), Fv4-F1243 and
Fv4-F1245 having novel Fc variants with improved binding to FcRn at
neutral pH exhibited significant reduction of plasma hsIL-6R
concentration demonstrating in vivo antigen elimination of
pH-dependent antigen binding antibody with improved binding to FcRn
at neutral pH. Fv4-F1243 and Fv4-F1245 demonstrated 10-fold antigen
elimination effect at day 21 or day 7 compared to Fv4-IgG1,
respectively, whereby the plasma antibody concentration time
profile of Fv4-F1243 and Fv4-F1245 was comparable to Fv4-IgG1.
[Example 7] In Vivo PK Study of Novel Fc Variants Using Human FcRn
Transgenic Mice
7-1. Preparation of Antibodies for In Vivo Study
[0866] pH-dependent anti-human IL6 receptor IgG1 antibody, Fv4-IgG1
comprising VH3-IgG1 (SEQ ID NO: 1) and VL3-CK (SEQ ID NO: 3), a new
Fc variant, Fv4-F1389 comprising VH3-F1389 (SEQ ID NO: 10) and
VL3-CK (SEQ ID NO: 3), were expressed and purified by the method
known to those skilled in the art described in Reference Example 2
of WO2011/122011.
[0867] As described in Example 4 and 5, Fv4-F1389 has a novel Fc
region with improved binding affinity to human FcRn at acidic pH,
but significantly reduced binding to rheumatoid factor. In order to
evaluate the pharmacokinetics of this variant, an in vivo study of
Fv4-IgG1 and Fv4-F1389 was performed using human FcRn transgenic
mice.
7-2. In Vivo Study of Antibodies by Using Human FcRn Transgenic
Mouse Line 32
[0868] An in vivo test was conducted using human FcRn transgenic
mouse line 32 by the same methods described in Example 13 of
WO2011/122011.
7-3. Result of In Vivo PK Study of New Fc Variants
[0869] FIG. 133 shows plasma antibody concentration time profile
after injection of Fv4-IgG1 and Fv4-F1389. Compared to Fv4-IgG1,
Fv4-F1389 having novel Fc variants with improved binding to FcRn at
acidic pH and reduced binding to rheumatoid factor exhibited
improved pharmacokinetics. Novel Fc variants described in Table 28
have increased binding affinity to FcRn at pH6.0 to a same level as
F1389. Therefore, these variants are also expected to exhibit
improved pharmacokinetics using human FcRn transgenic mouse line 32
while having reduced binding to rheumatoid factor.
[Example 8] Preparation of Antibodies that Bind to Human IgA in a
Calcium-Dependent Manner
[0870] 8-1. Preparation of Human IgA (hIgA)
[0871] Human IgA (hereinafter also abbreviated as "hIgA") was
prepared as an antigen by using the following recombinant
techniques. hIgA (the variable region is derived from an anti-human
IL-6 Receptor antibody) was expressed by culturing host cells
carrying recombinant vectors inserted with H (WT)-IgA1 (SEQ ID NO:
12) and L (WT) (SEQ ID NO: 13) and purified by a method known to
those skilled in the art using ion-exchange chromatography and gel
filtration chromatography.
8-2. Expression and Purification of Antibodies that Bind to
hIgA
[0872] GA2-IgG1 (heavy chain SEQ ID NO: 14; light chain SEQ ID NO:
15) is an antibody that bind to hIgA. The DNA sequences encoding
heavy chain of GA2-IgG1 (SEQ ID NO: 14) and light chain of GA2-IgG1
(SEQ ID NO: 15) were inserted into animal cell expression plasmids
by a method known to those skilled in the art. The antibody was
expressed by the method described below. Cells of human fetal
kidney cell-derived line FreeStyle 293-F (Invitrogen) were
suspended in FreeStyle 293 Expression Medium (Invitrogen). The cell
suspension was seeded into a 6-well plate (3 mL/well) at a cell
density of 1.33.times.10.sup.6 cells/ml. Then, the constructed
plasmids were introduced into the cells by a lipofection method.
The cells were cultured for four days in a CO.sub.2 incubator (37
degrees C., 8% CO.sub.2, 90 rpm). The antibodies were purified from
the isolated culture supernatants 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
purified antibody solutions was measured using a spectrophotometer.
The antibody concentrations were determined from the measured
values using the absorption coefficient calculated by the PACE
method (Protein Science (1995) 4, 2411-2423).
8-3. Assessment of Obtained Antibodies for Calcium-Dependent
hIgA-Binding Activity
[0873] The antibodies isolated as described in 8-2 were assessed
for their hIgA-binding activity (dissociation constant KD (M))
using Biacore T200 (GE Healthcare). Running buffers used in the
measurement were 0.05% tween20/20 mmol/L ACES/150 mmol/L NaCl (pH
7.4 or 5.8) containing 3 microM or 1.2 mM CaCl.sub.2.
[0874] The antibody was allowed to bind to Sensor chip CM5 (GE
Healthcare) immobilized with a suitable amount of recombinant
Protein A/G (Thermo Scientific) by the amino coupling method. Then,
an appropriate concentration of hIgA (described in 8-1) was
injected as an analyte to allow interaction with the antibody on
the sensor chip. The measurement was carried out at 37 degrees C.
After the measurement, 10 mmol/L glycine-HCl (pH 1.5) was injected
to regenerate the sensor chip. The dissociation constant KD (M) was
calculated from the measurement result by curve fitting analysis
and equilibrium parameter analysis using Biacore T200 Evaluation
Software (GE Healthcare). The result and obtained sensorgrams are
shown in Table 29 and FIG. 134, respectively. It was revealed that
GA2-IgG1 bound strongly to hIgA at a Ca.sup.2+ concentration of 1.2
mM whereas the antibody bound weakly to hIgA at a Ca.sup.2+
concentration of 3 microM. Furthermore, at a Ca.sup.2+
concentration of 1.2 mM, GA2-IgG1 was shown to bind to human IgA
strongly at pH 7.4 but weakly at pH 5.8. More specifically,
GA2-IgG1 was revealed to bind to human IgA in a pH- and
calcium-dependent manner.
TABLE-US-00029 TABLE 29 Antibody name Conditions Fit ka kd KD [M]
GA2-IgG1 pH 7.4, 1:1 binding 4.0E+05 1.6E-02 3.9E-08 1.2 mM Ca
model pH 7.4, Steady State -- -- 6.7E-06 3 microM Ca Affinity pH
5.8, Steady State -- -- 4.0E-06 1.2 mM Ca Affinity pH 5.8, Steady
State -- -- 5.0E-06 3 microM Ca Affinity
[Example 9] Preparation of Antibodies with Modified Fc Region that
Bind to hIgA in a Calcium-Dependent Manner
[0875] Next, to evaluate the effect of FcRn binding on antigen
(hIgA) elimination from plasma, GA2-F760 (heavy chain SEQ ID NO:
16; light chain SEQ ID NO: 15) was constructed by introducing amino
acid substitutions L235R and S239K into GA2-IgG1 to eliminate
binding to FcgammaR. Furthermore GA2-F1331 (heavy chain SEQ ID NO:
17; light chain SEQ ID NO: 15) was constructed by introducing amino
acid substitution G236R, M252Y, S254T, T256E, N434Y, Y436V, Q438R
and S440E into GA2-F760, which binds to FcRn stronger than GA2-F760
at pH 7.4. The modified antibodies were expressed by the method
described above using animal expression plasmids inserted with DNA
sequences encoding GA2-F1331 (heavy chain SEQ ID NO: 17; light
chain SEQ ID NO: 15) and GA2-F760 (heavy chain SEQ ID NO: 16; light
chain SEQ ID NO: 15) by a method known to those skilled in the art.
The antibody concentrations were determined after purification.
GA2-F760 was assessed for its binding to various mouse FcgammaR
(mFcgammaRI, mFcgammaRII, mFcgammaRIII, and mFcgammaRIV). The
result showed that GA2-F760 did not bind to any of the
receptors.
[Example 10] Assessment of the Effect of Ca-Dependent hIgA-Binding
Antibodies on Plasma Retention of an Antigen Using Human FcRn
Transgenic Mice
10-1. In Vivo Test Using Human FcRn Transgenic Mice
[0876] Pharmacokinetics of hIgA and anti-hIgA antibody was assessed
after administration of hIgA (human IgA; prepared as described in
Example 8) alone or in combination with an anti-hIgA antibody to
human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32+/+mouse,
Jackson Laboratories; Methods Mol Biol. (2010) 602: 93-104). A
mixture of hIgA and anti-hIgA antibody was administered once at a
dose of 10 mL/kg via the caudal vein. GA2-F760 and GA2-F1331
described above were the anti-hIgA antibodies that were used.
[0877] In every mixture, the hIgA concentration was 80 microg/mL
and the anti-hIgA antibody concentration was 2.69 mg/mL. Under the
conditions described above, the majority of hIgA is predicted to
bind to the antibody since the anti-hIgA antibody is present in
sufficient excess over hIgA. Blood was collected from the mice
fifteen minutes, one hour, two hours, seven hours, one day, three
days, seven days and fourteen days after administration. The
collected blood was immediately centrifuged for 15 minutes at
12,000 rpm and 4 degrees C. to obtain the plasma. The separated
plasma was stored in a freezer at -20 degrees C. or below until
measurement.
10-2. Determination of Plasma Anti-hIgA Antibody Concentration in
Human FcRn Transgenic Mice by ELISA
[0878] The anti-hIgA antibody concentrations in mouse plasma were
determined by ELISA. First, Anti-Human IgG-immobilized plates were
prepared by aliquoting Anti-Human IgG (gamma-chain specific)
F(ab')2 Fragment Antibody (SIGMA) to each well of Nunc-Immuno
Plate, MaxiSorp (Nalge nunc International) and allowing the plates
to stand at 4 degrees C. overnight. Anti-hIgA antibody standard
curve samples prepared as standard solutions at plasma
concentrations of 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.01563, and
0.007813 microg/mL and assay samples prepared by diluting mouse
plasma samples 100-fold or more were aliquoted into the Anti-Human
IgG-immobilized plates, and then the plates were incubated at 25
degrees C. for one hour. Next, Goat Anti-Human IgG (gamma-chain
specific) Biotin (BIOT) Conjugate (Southern Biotechnology
Associates Inc.) was aliquoted into each well of the plates, and
then the plates were incubated at 25 degrees C. for one hour. Then,
Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was
added to each well of the plates, after which the plates were
incubated at 25 degrees C. for one hour. The chromogenic reaction
using TMB One Component HRP Microwell Substrate (BioFX
Laboratories) as a substrate was terminated with 1N sulfuric acid
(Showa Chemical), and then the reaction mixture in each well was
measured using a microplate reader to measure the absorbance at 450
nm. The anti-hIgA antibody concentration in mouse plasma was
calculated from the absorbance of the standard curve using analysis
software SOFTmax PRO (Molecular Devices). The time course of plasma
antibody concentrations of GA2-F1331, and GA2-F760 in human FcRn
transgenic mice, which were determined by the method described
above, is shown in FIG. 135.
10-3. Determination of hIgA Concentration in Plasma by ELISA
[0879] hIgA concentrations in mouse plasma were measured by ELISA.
First, Anti-Human IgA-immobilized plates were prepared by
aliquoting Goat anti-Human IgA Antibody (BETHYL) into each well of
Nunc-Immuno Plate, MaxiSorp (Nalge nunc International) and allowing
the plates to stand at 4 degrees C. overnight. hIgA standard curve
samples were prepared as standard solutions at plasma
concentrations of 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, and 0.00625
microg/mL and assay samples were prepared by diluting mouse plasma
samples 100-fold or more. Each sample (100 microL) was mixed with
200 microL of 500 ng/mL hsIL-6R at room temperature for one hour,
and then it was aliquoted at 100 microL/well into the Anti-Human
IgA-immobilized plates. The resulting plates were allowed to stand
at room temperature for one hour. Next, after adding Biotinylated
Anti-human IL-6R Antibody (R&D) into each well of the plates,
the plates were incubated at room temperature for one hour. Then,
after aliquoting Streptavidin-PolyHRP80 (Stereospecific Detection
Technologies) into each well of the plates, the plates were
incubated at room temperature for one hour. The chromogenic
reaction using as a substrate TMB One Component HRP Microwell
Substrate (BioFX Laboratories) was terminated with 1N sulfuric acid
(Showa Chemical), and then the reaction mixture in each well was
measured using a microplate reader to measure the absorbance at 450
nm. The concentration in mouse plasma was calculated from the
absorbance of the standard curve using analysis software SOFTmax
PRO (Molecular Devices). The time course of plasma hIgA
concentrations in human FcRn transgenic mice after intravenous
administration, as determined by the method described above, is
shown in FIG. 136.
[0880] The result showed that the elimination of hIgA was markedly
accelerated when hIgA was administered in combination with
GA2-F1331, an antibody that exhibits strong human FcRn binding, as
compared to when hIgA was administered in combination with
GA2-F760, which has very weak affinity to human FcRn.
[Example 11] Preparation of pH-Dependent Anti-IgE Antibody
11-1. Preparation of Anti-Human IgE Antibody
[0881] To prepare pH-dependent anti-human IgE antibodies, human IgE
(heavy chain SEQ ID NO: 18; light chain SEQ ID NO: 19) (the
variable region is derived from an anti-human glypican3 antibody)
as an antigen was expressed using FreeStyle293 (Life Technologies).
Human IgE was prepared by purifying the expressed human IgE using a
conventional chromatographic method known to those skilled in the
art.
[0882] An antibody that binds to human IgE in a pH-dependent manner
was selected from a number of obtained antibodies. The selected
anti-human IgE antibody was expressed using human IgG1 heavy chain
constant region and human light chain constant region, and then
purified. The produced antibody was named clone 278 (heavy chain
SEQ ID NO: 20; light chain SEQ ID NO: 21).
11-2. Assessment of Anti-Human IgE Antibodies for their Binding
Activity and pH-Dependent Binding Activity
[0883] Antibodies capable of dissociating from antigens within the
endosome can be created not only by designing them so as to bind to
antigens in a pH-dependent manner, but also by designing them so as
to bind to antigens in a Ca-dependent manner. Thus, clone 278 and
the control Xolair (omalizumab; Novartis) whose IgE-binding
activity does not depend on pH/Ca were assessed for their pH
dependency and pH/Ca dependency of the human IgE (hIgE)-binding
activity.
[0884] More specifically, the hIgE-binding activities (dissociation
constant KD (M)) of clone 278 and Xolair were assessed using
Biacore T200 (GE Healthcare). Running buffers used in the assay
were:
[0885] 1.2 mmol/l CaCl.sub.2/0.05% tween20, 20 mmol/l ACES, 150
mmol/l NaCl, pH 7.4;
[0886] 1.2 mmol/l CaCl.sub.2/0.05% tween20, 20 mmol/l ACES, 150
mmol/l NaCl, pH 5.8; and
[0887] 3 micromol/l CaCl.sub.2/0.05% tween20, 20 mmol/l ACES, 150
mmol/l NaCl, pH 5.8.
[0888] A chemically-synthesized peptide having a human glypican 3
protein-derived sequence (SEQ ID NO: 22) whose C-terminal Lys is
biotinylated (hereinafter abbreviated as "biotinylated GPC3
peptide") was added in an appropriate amount and immobilized onto
Sensor chip SA (GE Healthcare) based on the affinity between biotin
and streptavidin. Human IgE was immobilized onto the chip by
injecting it at an appropriate concentration so as to be trapped by
the biotinylated GPC3 peptide. As an analyte, clone 278 was
injected at an appropriate concentration and allowed to interact
with the human IgE on the sensor chip. Then, 10 mmol/L glycine-HCl
(pH 1.5) was injected to regenerate the sensor chip. The
interaction was always measured at 37 degrees C. The measurement
result was analyzed by curve fitting using Biacore T200 Evaluation
Software (GE Healthcare) to calculate the association rate constant
ka (1/Ms) and dissociation rate constant kd (1/s). The dissociation
constant KD (M) was calculated from the above-described constants.
Furthermore, the KD ratios in each antibody under the conditions of
[pH 5.8, 1.2 mM Ca] to [pH 7.4, 1.2 mM Ca] were calculated to
assess the pH-dependent binding, while the KD ratios in each
antibody under the conditions of [pH 5.8, 3 microM Ca] to [pH 7.4,
1.2 mM Ca] were calculated to assess the pH/Ca-dependent binding.
The result is shown in Table 30.
TABLE-US-00030 TABLE 30 pH pH/Ca dependency dependency KD (pH 5.8,
KD (pH 5.8, 1.2 mM 3 microM Antibody Ca)/KD (pH Ca)/KD (pH name
Buffer ka kd KD 7.4, 1.2 mM 7.4, 1.2 mM (abbreviation) conditions
(1/Ms) (1/s) (M) Ca) Ca) Clone 278 pH 7.4, 1.5E+06 3.6E-03 2.4E-09
842.5 1636.5 1.2 mM Ca pH 5.8, 1.2E+05 2.3E-01 2.0E-06 1.2 mM Ca pH
5.8, 6.2E+04 2.4E-01 3.9E-06 3 microM Ca Xolair pH 7.4, 2.5E+06
1.1E-02 4.4E-09 2.3 2.9 1.2 mM Ca pH 5.8, 2.4E+06 2.4E-02 9.9E-09
1.2 mM Ca pH 5.8, 1.4E+06 1.7E-02 1.3E-08 3 microM Ca
[Example 12] Preparation of Antibodies with Modified Fc Region that
Bind to Human IgE for In Vivo Testing
[0889] Next, to evaluate the effect of FcRn binding on antigen
(human IgE) elimination from plasma, 278-F760 (heavy chain SEQ ID
NO: 23; light chain SEQ ID NO: 21) was constructed to eliminate
binding to FcgammaR. Furthermore 278-F1331 (heavy chain SEQ ID NO:
24; light chain SEQ ID NO: 21) was constructed by introducing amino
acid substitution G236R, M252Y, S254T, T256E, N434Y, Y436V, Q438R
and S440E into 278-F760, which binds to FcRn stronger than 278-F760
at pH 7.4. The modified antibodies were expressed by the method
described above using animal expression plasmids inserted with DNA
sequences encoding 278-F1331 (heavy chain SEQ ID NO: 24; light
chain SEQ ID NO: 21) and 278-F760 (heavy chain SEQ ID NO: 23; light
chain SEQ ID NO: 21) by a method known to those skilled in the art.
The antibody concentrations were determined after purification.
[Example 13] In Vivo Assessment of Clone 278
[0890] 13-1. Preparation of Human IgE (hIgE(Asp6)) for In Vivo
Assessment
[0891] hIgE(Asp6) (the variable region is derived from an
anti-human glypican3 antibody), which is a human IgE for in vivo
assessment consisting of a heavy chain (SEQ ID NO: 25) and a light
chain (SEQ ID NO: 19), was produced by the same method as described
in Example 11. hIgE(Asp6) is a modified molecule resulting from
asparagine-to-aspartic acid alteration at the six N-linked
glycosylation sites in human IgE so that the heterogeneity in the
N-linked sugar chain of human IgE is not affected by time-dependent
changes in the plasma concentration of human IgE as an antigen.
13-2. Assessment of Clone 278 for the Effect of Accelerating Human
IgE Elimination Using Human FcRn Transgenic Mice
[0892] Pharmacokinetics of hIgE(Asp6) and anti-human IgE antibody
was assessed after administration of hIgE(Asp6) in combination with
an anti-hIgE antibody (278-F760 and 278-F1331) and Sanglopor (Human
normal Immunoglobulin, CSL Behring) to human FcRn transgenic mice
(B6.mFcRn-/-.hFcRn Tg line 32+/+ mouse, Jackson Laboratories;
Methods Mol Biol. (2010) 602: 93-104). A mixture of hIgE(Asp6),
anti-human IgE antibody and Sanglopor (the concentrations are shown
in Table 31) was administered once at a dose of 10 mL/kg via the
caudal vein. Under the conditions described above, hIgE(Asp6) is
predicted to bind almost completely to the antibody since each
antibody is present sufficiently in excess over hIgE(Asp6). Blood
was collected from the mice five minutes, two hours, seven hours,
one day, two days, four or five days, seven days, fourteen days,
twenty-one days, and twenty-eight days after administration. The
collected blood was immediately centrifuged at 15,000 rpm and 4
degrees C. for 5 minutes to obtain plasma. The separated plasma was
stored in a freezer at -20 degrees C. or below until
measurement.
TABLE-US-00031 TABLE 31 hIgE(Asp6) Anti-hIgE antibody Sanglopor
concentration in concentration in concentration in administered
administered adminstered Anti-hIgE solution solution solution
antibody (microgram/mL) (microgram/mL) (mg/mL) 278-F760 20 100 100
278-F1331 20 100 100
13-3. Determination of Plasma Anti-Human IgE Antibody Concentration
in Human FcRn Transgenic Mice
[0893] Anti-hIgE antibody concentrations in mouse plasma were
determined by electrochemiluminescence (ECL) assay. Standard curve
samples were prepared at plasma concentrations of 32, 16, 8, 4, 2,
1, 0.5, and 0.25 microgram/mL. The standard curve samples and mouse
plasma assay samples were aliquoted into ECL plates immobilized
with hIgE(Asp6). The plates were allowed to stand at 4 degrees C.
overnight. Then, Anti Rabbit Antibody (Goat), SULFO-TAG Labeled
(Meso Scale Discovery) was reacted at room temperature for one
hour. Immediately after Read Buffer T (.times.4) (Meso Scale
Discovery) was dispensed, the measurement was performed by the
Sector Imager 2400 Reader (Meso Scale Discovery). The concentration
in mouse plasma was calculated from the response of the standard
curve using analysis software SOFTmax PRO (Molecular Devices). A
time course of the plasma antibody concentration after intravenous
administration, which was determined by the method described above,
is shown in FIG. 137.
13-4. Determination of Plasma hIgE(Asp6) Concentration in Human
FcRn Transgenic Mice
[0894] hIgE(Asp6) concentrations in mouse plasma were determined by
ELISA. Standard curve samples were prepared at plasma
concentrations of 192, 96, 48, 24, 12, 6, and 3 ng/mL. Xolair
(Novartis) was added at 10 microgram/mL to the standard curve
samples and mouse plasma assay samples to equalize the immune
complex of hIgE(Asp6) and anti-hIgE antibody. After 30 minutes of
incubation at room temperature, the standard curve samples and
mouse plasma assay samples were aliquoted into immunoplates
(MABTECH) immobilized with anti-human IgE antibody or immunoplates
(Nunc F96 MicroWell Plate (Nalge nunc International)) immobilized
with anti-human IgE antibody (clone 107; MABTECH). The plates were
allowed to stand at room temperature for two hours or at 4 degrees
C. overnight. Then, human GPC3 core protein (SEQ ID NO: 26),
anti-GPC3 antibody biotinylated with NHS-PEG4-Biotin (Thermo Fisher
Scientific) (prepared in Chugai pharmaceutical Co., Ltd.), and
Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) were
reacted sequentially for one hour each. The chromogenic reaction
using as a substrate TMB One Component HRP Microwell Substrate
(BioFX Laboratories) was terminated with IN sulfuric acid (Showa
Chemical), and then the concentration in mouse plasma was
determined by a method in which the color development is assessed
by measuring the absorbance at 450 nm using a microplate reader or
a method in which a luminescent reaction is carried out using
SuperSignal.RTM. ELISA Pico Chemiluminescent Substrate (Thermo
Fisher Scientific) as a substrate and the luminescence intensity is
measured with a microplate reader. The concentration in mouse
plasma was calculated from the absorbance or luminescence intensity
of the standard curve using analysis software SOFTmax PRO
(Molecular Devices). The time course of plasma hIgE(Asp6)
concentration after intravenous administration, which was
determined by the method described above, is shown in FIG. 138.
[0895] The result showed that the elimination of human IgE was
significantly accelerated when human IgE was administered in
combination with 278-F1331, which binds to human FcRn much stronger
than 278-F760. Specifically, it was demonstrated that not only in
the case of IL6R and IgA, but also in the case of IgE, a
pH-dependent antigen binding antibody having increased binding
activity to FcRn can accelerate antigen clearance from plasma and
decrease concentration of antigen in plasma.
[Reference Example A1] Preparation of Soluble Human IL-6 Receptor
(hsIL-6R)
[0896] Recombinant human IL-6 receptor as an antigen was prepared
as follows. A cell line constitutively expressing soluble human
IL-6 receptor (hereinafter referred to as hsIL-6R) having the amino
acid sequence of positions 1 to 357 from the N terminus as reported
in J. Immunol. 152: 4958-4968 (1994) was established by a method
known to those skilled in the art. The cells were cultured to
express hsIL-6R. The hsIL-6R was purified from the culture
supernatant by two steps: Blue Sepharose 6 FF column chromatography
and gel filtration chromatography. A fraction eluted as the main
peak in the final stage was used as the final purification
product.
[Reference Example A2] Preparation of Human FcRn
[0897] FcRn is a heterodimer of FcRn alpha chain and
beta2-microglobulin. Oligo-DNA primers were prepared based on the
published human FcRn gene sequence (J Exp Med. 1994 Dec. 1; 180(6):
2377-81). A DNA fragment encoding the whole gene was prepared by
PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech)
as a template and the prepared primers. Using the obtained DNA
fragment as a template, a DNA fragment encoding the extracellular
domain containing the signal region (Met1-Leu290) was amplified by
PCR, and inserted into a mammalian cell expression vector.
Likewise, oligo-DNA primers were prepared based on the published
human beta2-microglobulin gene sequence (Proc. Natl. Acad. Sci.
U.S.A. 99 (26): 16899-16903 (2002)). A DNA fragment encoding the
whole gene was prepared by PCR using human cDNA (Human Placenta
Marathon-Ready cDNA, Clontech) as a template and the prepared
primers. Using the obtained DNA fragment as a template, a DNA
fragment encoding the whole protein containing a signal region
(Met1-Met119) was amplified by PCR and inserted into a mammalian
cell expression vector.
[0898] Soluble human FcRn was expressed by the following procedure.
The plasmids constructed for expressing human FcRn alpha chain (SEQ
ID NO: 27) and beta2-microglobulin (SEQ ID NO: 28) were introduced
into cells of the human embryonic kidney cancer-derived cell line
HEK293H (Invitrogen) by the lipofection method using PEI
(Polyscience). The resulting culture supernatant was collected, and
FcRn was purified using IgG Sepharose 6 Fast Flow (Amersham
Biosciences), followed by further purification using HiTrap Q HP
(GE Healthcare) (J Immunol. 2002 Nov. 1; 169(9): 5171-80).
[Reference Example A3] Preparation of Human IgA (hIgA)
[0899] hIgA comprising H (WT)-IgA1 (SEQ ID NO: 12) and L (WT) (SEQ
ID NO: 13) was expressed and purified by the method known to those
skilled in the art using rProtein L-agarose (ACTIgen) followed by
gel filtration chromatography.
[Reference Example A4] Preparation of Soluble Human Plexin A1
(hsPlexin A1)
[0900] Recombinant soluble human plexin A1 as an antigen
(hereinafter referred to as hsPlexin A1) was prepared as follows.
hsPlexin A1 was constructed by reference to NCBI Reference Sequence
(NP_115618). Specially, hsPlexin A1 was comprised of the amino acid
sequence of positions 27-1243 from the above-mentioned NCBI
Reference FLAG-tag (DYKDDDDK, SEQ ID NO: 29) was connected to its C
terminus. hsPlexin A1 was transiently expressed using FreeStyle293
(Invitrogen) and purified from the culture supernatant by two
steps: anti-FLAG column chromatography and gel filtration
chromatography. A fraction eluted as the main peak in the final
stage was used as the final purification product.
INDUSTRIAL APPLICABILITY
[0901] When a conventional antibody targeting soluble antigen is
administered to a subject, the antigen binds to the antibody and
persists stably in plasma. Since an antigen bound to an antibody
has a significantly longer half-life than an antigen alone, the
antigen concentration increases after the injection of a
conventional antibody to approximately 10 to 1000-folds of total
plasma antigen concentration from the baseline. Such an increase of
the total plasma antigen concentration is not preferable for a
therapeutic antibody, because the antibody concentration (dosage)
has to be 10 to 1000-fold higher than necessary compared to when no
substantial increase in total plasma antigen concentration occurs.
Therefore, an antibody which eliminates the antigen from plasma and
also reduces the total plasma antigen concentration compared to a
conventional antibody is extremely valuable since the required
dosage would be 10 to 1000-fold lower than that required for a
conventional antibody.
[0902] The present inventors conducted dedicated studies on
modified FcRn-binding domains which have an enhanced affinity for
FcRn at neutral pH and antigen-binding molecules comprising said
FcRn-binding domain which have low immunogenicity, high stability
and form only a few aggregates. As a result, it was discovered that
substitutions at specific positions of the FcRn-binding domain
increases the affinity for the FcRn at neutral pH without
substantially increasing the immunogenicity and the ratio of high
molecular weight species, and without substantially decreasing
stability of antigen-binding molecules comprising the FcRn-binding
domain. The antigen-binding molecules comprising the FcRn-binding
domain of the present invention are superior in pharmacokinetics in
facilitating the reduction of the plasma antigen concentration and
meet the developability criteria of low immunogenicity, high
stability and very few aggregates.
[0903] Furthermore, Fc-engineering to increase the binding affinity
to FcRn at neutral or acidic pH can improve the endosomal recycling
efficiency and the pharmacokinetics of the antibody. However,
modifications of the amino acid sequence of an antibody (e.g. amino
acid substitutions and insertions) can also increase the
immunogenicity of the therapeutic antibody which, in turn, can
result in a cytokine storm and/or production of anti-drug
antibodies (ADA).
[0904] The present inventors conducted dedicated studies on
antigen-binding molecules comprising a modified FcRn-binding domain
whose binding activity for a pre-existing anti-drug antibody (ADA)
was increased at neutral pH due to substitutions in the
FcRn-binding domain that increased the affinity for FcRn at neutral
pH or acidic pH. As a result, it was discovered that other
substitutions at specific positions of the FcRn-binding domain
decrease the binding activity for a pre-existing anti-drug antibody
(ADA) at neutral pH while maintaining to a high extent the
increased FcRn-binding activity in the respective pH ranges. The
antigen-binding molecules of the present invention are superior in
pharmacokinetics in facilitating the reduction of the plasma
antigen concentration without increasing the antibody
clearance.
SEQUENCE LISTING
Sequence CWU 1
1
291447PRTArtificial SequenceAn artificially synthesized peptide
sequence 1Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile
Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu
Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr
Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150
155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265
270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 4452214PRTArtificial SequenceAn
artificially synthesized peptide sequence 2Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr
Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 2103214PRTArtificial SequenceAn
artificially synthesized peptide sequence 3Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Ser Val Thr Ile
Thr Cys Gln Ala Ser Thr Asp Ile Ser Ser His 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45Tyr Tyr Gly
Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala65 70 75
80Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 2104447PRTArtificial SequenceAn
artificially synthesized peptide sequence 4Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr
Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30His Ala Trp Ser
Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45Ile Gly Phe
Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60Gln Gly
Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu
Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200
205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Tyr Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala
Leu His Tyr His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
4455447PRTArtificial SequenceAn artificially synthesized peptide
sequence 5Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile
Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu
Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr
Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150
155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Asp225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser 245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265
270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu 420 425 430Ala Leu His Tyr His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 4456447PRTArtificial SequenceAn
artificially synthesized peptide sequence 6Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr
Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30His Ala Trp Ser
Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45Ile Gly Phe
Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60Gln Gly
Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu
Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200
205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Tyr Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala
Leu His Tyr His Val Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
4457447PRTArtificial SequenceAn artificially synthesized peptide
sequence 7Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile
Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu
Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr
Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150
155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Tyr Ile Ser
245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Gln Val Leu His
Ala Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Tyr His Val Thr
Gln Lys Ser Leu Ser Leu Ser Pro 435 440 4458447PRTArtificial
SequenceAn artificially synthesized peptide sequence 8Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu
Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30His
Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40
45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr
Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Arg Gly Gly Pro225 230 235 240Lys Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Tyr Ile Thr 245 250 255Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310
315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425
430Ala Leu His Tyr His Val Thr Arg Lys Glu Leu Ser Leu Ser Pro 435
440 4459447PRTArtificial SequenceAn artificially synthesized
peptide sequence 9Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His
Ser Ile Ser His Asp 20 25 30His Ala Trp Ser Trp Val Arg Gln Pro Pro
Gly Glu Gly Leu Glu Trp 35 40 45Ile Gly Phe Ile Ser Tyr Ser Gly Ile
Thr Asn Tyr Asn Pro Ser Leu 50 55 60Gln Gly Arg Val Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Arg Gly Gly Pro225 230 235 240Lys
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr 245 250
255Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 420 425 430Ala Leu His Tyr His Val Thr Arg
Lys Glu Leu Ser Leu Ser Pro 435 440 44510447PRTArtificial
SequenceAn artificially synthesized peptide sequence 10Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu
Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30His
Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40
45Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
50 55 60Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr
Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310
315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 420 425
430Ala Leu His Ser His Thr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
440 445115PRTArtificial SequenceAn artificially synthesized peptide
sequence 11Gly Gly Gly Gly Ser1 512464PRTArtificial SequenceAn
artificially synthesized peptide sequence 12Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Arg Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30His Ala Trp Ser
Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45Ile Gly Tyr
Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60Lys Ser
Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser65 70 75
80Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln
Gly 100 105 110Ser Leu Val Thr Val Ser Ser Ala Ser Pro Thr Ser Pro
Lys Val Phe 115 120 125Pro Leu Ser Leu Cys Ser Thr Gln Pro Asp Gly
Asn Val Val Ile Ala 130 135 140Cys Leu Val Gln Gly Phe Phe Pro Gln
Glu Pro Leu Ser Val Thr Trp145 150 155 160Ser Glu Ser Gly Gln Gly
Val Thr Ala Arg Asn Phe Pro Pro Ser Gln 165 170 175Asp Ala Ser Gly
Asp Leu Tyr Thr Thr Ser Ser Gln Leu Thr Leu Pro 180 185 190Ala Thr
Gln Cys Leu Ala Gly Lys Ser Val Thr Cys His Val Lys His 195 200
205Tyr Thr Asn Pro Ser Gln Asp Val Thr Val Pro Cys Pro Val Pro Ser
210 215 220Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser
Pro Ser225 230 235 240Cys Cys His Pro Arg Leu Ser Leu His Arg Pro
Ala Leu Glu Asp Leu 245 250 255Leu Leu Gly Ser Glu Ala Asn Leu Thr
Cys Thr Leu Thr Gly Leu Arg 260 265 270Asp Ala Ser Gly Val Thr Phe
Thr Trp Thr Pro Ser Ser Gly Lys Ser 275 280 285Ala Val Gln Gly Pro
Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val 290 295 300Ser Ser Val
Leu Pro Gly Cys Ala Glu Pro Trp Asn His Gly Lys Thr305 310 315
320Phe Thr Cys Thr Ala Ala Tyr Pro Glu Ser Lys Thr Pro Leu Thr Ala
325 330 335Thr Leu Ser Lys Ser Gly Asn Thr Phe Arg Pro Glu Val His
Leu Leu 340 345 350Pro Pro Pro Ser Glu Glu Leu Ala Leu Asn Glu Leu
Val Thr Leu Thr 355 360 365Cys Leu Ala Arg Gly Phe Ser Pro Lys Asp
Val Leu Val Arg Trp Leu 370 375 380Gln Gly Ser Gln Glu Leu Pro Arg
Glu Lys Tyr Leu Thr Trp Ala Ser385 390 395 400Arg Gln Glu Pro Ser
Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile 405 410 415Leu Arg Val
Ala Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys 420 425 430Met
Val Gly His Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile 435 440
445Asp Arg Leu Ala Gly Lys Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
450 455 46013214PRTArtificial SequenceAn artificially synthesized
peptide sequence 13Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Arg Leu His Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr
Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21014451PRTArtificial SequenceAn artificially synthesized peptide
sequence 14Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Pro Arg Trp Glu Thr
Ala Ile Ser Ser Asp Ala Phe Asp Ile 100 105 110Trp Gly Gln Gly Thr
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val
Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170
175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val 195 200 205Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys 210 215 220Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu225 230 235 240Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295
300Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu305 310 315 320Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala 325 330 335Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro 340 345 350Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr385 390 395 400Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410
415Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 435 440 445Leu Ser Pro 45015214PRTArtificial SequenceAn
artificially synthesized peptide sequence 15Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Asp Asp 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Glu Ala
Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Ser Ser Ser Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21016451PRTArtificial SequenceAn
artificially synthesized peptide sequence 16Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile
Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Pro Arg Trp Glu Thr Ala Ile Ser Ser Asp Ala Phe Asp
Ile 100 105 110Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser
Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200
205Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu225 230 235 240Arg Gly Gly Pro Lys Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 245 250 255Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val 260 265 270Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu305 310 315
320Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro 340 345 350Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln 355 360 365Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala 370 375 380Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr385 390 395 400Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440
445Leu Ser Pro 45017451PRTArtificial SequenceAn artificially
synthesized peptide sequence 17Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile
Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr
Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Pro Arg Trp Glu Thr Ala Ile Ser Ser Asp Ala Phe Asp Ile 100 105
110Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu225 230
235 240Arg Arg Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr 245 250 255Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val
Val Asp Val 260 265 270Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val 275 280 285Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser 290 295 300Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu305 310 315 320Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345
350Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
355 360 365Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala 370 375 380Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr385 390 395 400Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu 405 410 415Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430Val Met His Glu Ala
Leu His Tyr His Val Thr Arg Lys Glu Leu Ser 435 440 445Leu Ser Pro
45018543PRTHomo sapiens 18Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Leu Asp Pro Lys Thr
Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Leu
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Phe
Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val
Ser Ser Ala Ser Thr Gln Ser Pro Ser Val Phe Pro Leu Thr Arg 115 120
125Cys Cys Lys Asn Ile Pro Ser Asn Ala Thr Ser Val Thr Leu Gly Cys
130 135 140Leu Ala Thr Gly Tyr Phe Pro Glu Pro Val Met Val Thr Trp
Asp Thr145 150 155 160Gly Ser Leu Asn Gly Thr Thr Met Thr Leu Pro
Ala Thr Thr Leu Thr 165 170 175Leu Ser Gly His Tyr Ala Thr Ile Ser
Leu Leu Thr Val Ser Gly Ala 180 185 190Trp Ala Lys Gln Met Phe Thr
Cys Arg Val Ala His Thr Pro Ser Ser 195 200 205Thr Asp Trp Val Asp
Asn Lys Thr Phe Ser Val Cys Ser Arg Asp Phe 210 215 220Thr Pro Pro
Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly225 230 235
240His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr
245 250 255Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val
Met Asp 260 265 270Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly
Glu Leu Ala Ser 275 280 285Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys
His Trp Leu Ser Asp Arg 290 295 300Thr Tyr Thr Cys Gln Val Thr Tyr
Gln Gly His Thr Phe Glu Asp Ser305 310 315 320Thr Lys Lys Cys Ala
Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu 325 330 335Ser Arg Pro
Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile 340 345 350Thr
Cys Leu Val Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu 355 360
365Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys
370 375 380Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr
Leu Pro385 390 395 400Val Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr
Tyr Gln Cys Arg Val 405 410 415Thr His Pro His Leu Pro Arg Ala Leu
Met Arg Ser Thr Thr Lys Thr 420 425 430Ser Gly Pro Arg Ala Ala Pro
Glu Val Tyr Ala Phe Ala Thr Pro Glu 435 440 445Trp Pro Gly Ser Arg
Asp Lys Arg Thr Leu Ala Cys Leu Ile Gln Asn 450 455 460Phe Met Pro
Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu Val Gln465 470 475
480Leu Pro Asp Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly
485 490 495Ser Gly Phe Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala
Glu Trp 500 505 510Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala Val His
Glu Ala Ala Ser 515 520 525Pro Ser Gln Thr Val Gln Arg Ala Val Ser
Val Asn Pro Gly Lys 530 535 54019219PRTHomo sapiens 19Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn
Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155 160Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185
190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21520443PRTArtificial SequenceAn artificially synthesized peptide
sequence 20Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly
Thr Pro1 5 10 15Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser
Ser Tyr His 20 25 30Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Ile Gly 35 40 45Val Ile Asn Ser Ala Gly Asn Thr Tyr Tyr Ala
Ser Trp Ala Lys Gly 50 55 60Arg Phe Thr Val Ser Lys Thr Ser Thr Thr
Val Asp Leu Asn Leu Thr65 70 75 80Ser Leu Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ala Arg Tyr Val 85 90 95Phe Ser Ser Gly Ser His Asp
Ile Trp Gly Pro Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala145 150
155 160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly 165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys 195 200 205Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro
Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys 260 265 270Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310
315 320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser 340 345 350Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425
430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
44021217PRTArtificial SequenceAn artificially synthesized peptide
sequence 21Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala
Val Gly1 5 10 15Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile
Gly Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
Lys Glu Leu Ile 35 40 45Tyr Gly Thr Ser Thr Leu Glu Ser Gly Val Pro
Ser Arg Phe Ile Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
Ile Ser Gly Val Glu Cys65 70 75 80Ala Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Gly Tyr Ser Glu Asp Asn 85 90 95Ile Asp Asn Ala Phe Gly Gly
Gly Thr Glu Val Val Val Lys Arg Thr 100 105 110Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly145 150
155 160Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr 165 170 175Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His 180 185 190Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val 195 200 205Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 2152232PRTArtificial SequenceAn artificially synthesized
peptide sequence 22Val Asp Asp Ala Pro Gly Asn Ser Gln Gln Ala Thr
Pro Lys Asp Asn1 5 10 15Glu Ile Ser Thr Phe His Asn Leu Gly Asn Val
His Ser Pro Leu Lys 20 25 3023443PRTArtificial SequenceAn
artificially synthesized peptide sequence 23Gln Ser Leu Glu Glu Ser
Gly Gly Arg Leu Val Thr Pro Gly Thr Pro1 5 10 15Leu Thr Leu Thr Cys
Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr His 20 25 30Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45Val Ile Asn
Ser Ala Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60Arg Phe
Thr Val Ser Lys Thr Ser Thr Thr Val Asp Leu Asn Leu Thr65 70 75
80Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Tyr Val
85 90 95Phe Ser Ser Gly Ser His Asp Ile Trp Gly Pro Gly Thr Leu Val
Thr 100 105 110Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro 115 120 125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val 130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala145 150 155 160Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200
205Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220Pro Pro Cys Pro Ala Pro Glu Leu Arg Gly Gly Pro Lys Val
Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315
320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser 340 345 350Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 44024443PRTArtificial
SequenceAn artificially synthesized peptide sequence 24Gln Ser Leu
Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro1 5 10 15Leu Thr
Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr His 20 25 30Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40
45Val Ile Asn Ser Ala Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly
50 55 60Arg Phe Thr Val Ser Lys Thr Ser Thr Thr Val Asp Leu Asn Leu
Thr65 70 75 80Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala
Arg Tyr Val 85 90 95Phe Ser Ser Gly Ser His Asp Ile Trp Gly Pro Gly
Thr Leu Val Thr 100 105 110Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro 115 120 125Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val 130 135 140Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala145 150 155 160Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185
190Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Leu Arg Arg Gly Pro
Lys Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu
Tyr Ile Thr Arg Glu Pro Glu 245 250 255Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys 260 265 270Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310
315 320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser 340 345 350Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Tyr 420 425
430His Val Thr Arg Lys Glu Leu Ser Leu Ser Pro 435
44025543PRTArtificial SequenceAn artificially synthesized peptide
sequence 25Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Asp Tyr 20 25 30Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
Tyr Ser Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Phe Tyr Ser Tyr Thr
Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala Ser
Thr Gln Ser Pro Ser Val Phe Pro Leu Thr Arg 115 120 125Cys Cys Lys
Asn Ile Pro Ser Asp Ala Thr Ser Val Thr Leu Gly Cys 130 135 140Leu
Ala Thr Gly Tyr Phe Pro Glu Pro Val Met Val Thr Trp Asp Thr145 150
155 160Gly Ser Leu Asp Gly Thr Thr Met Thr Leu Pro Ala Thr Thr Leu
Thr 165 170 175Leu Ser Gly His Tyr Ala Thr Ile Ser Leu Leu Thr Val
Ser Gly Ala 180 185 190Trp Ala Lys Gln Met Phe Thr Cys Arg Val Ala
His Thr Pro Ser Ser 195 200 205Thr Asp Trp Val Asp Asp Lys Thr Phe
Ser Val Cys Ser Arg Asp Phe 210 215 220Thr Pro Pro Thr Val Lys Ile
Leu Gln Ser Ser Cys Asp Gly Gly Gly225 230 235 240His Phe Pro Pro
Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr 245 250 255Pro Gly
Thr Ile Asp Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp 260 265
270Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser
275 280 285Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser
Asp Arg 290 295 300Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr
Phe Glu Asp Ser305 310 315 320Thr Lys Lys Cys Ala Asp Ser Asn Pro
Arg Gly Val Ser Ala Tyr Leu 325 330 335Ser Arg Pro Ser Pro Phe Asp
Leu Phe Ile Arg Lys Ser Pro Thr Ile 340 345 350Thr Cys Leu Val Val
Asp Leu Ala Pro Ser Lys Gly Thr Val Asp Leu 355 360 365Thr Trp Ser
Arg Ala Ser Gly Lys Pro Val Asp His Ser Thr Arg Lys 370 375 380Glu
Glu Lys Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu Pro385 390
395 400Val Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg
Val 405 410 415Thr His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr
Thr Lys Thr 420 425 430Ser Gly Pro Arg Ala Ala Pro Glu Val Tyr Ala
Phe Ala Thr Pro Glu 435 440 445Trp Pro Gly Ser Arg Asp Lys Arg Thr
Leu Ala Cys Leu Ile Gln Asn 450 455 460Phe Met Pro Glu Asp Ile Ser
Val Gln Trp Leu His Asn Glu Val Gln465 470 475 480Leu Pro Asp Ala
Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly 485 490 495Ser Gly
Phe Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu Trp 500 505
510Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala Val His Glu Ala Ala Ser
515 520 525Pro Ser Gln Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly
Lys 530 535 54026545PRTHomo sapiens 26Gln Pro Pro Pro Pro Pro Pro
Asp Ala Thr Cys His Gln Val Arg Ser1 5 10 15Phe Phe Gln Arg Leu Gln
Pro Gly Leu Lys Trp Val Pro Glu Thr Pro 20 25 30Val Pro Gly Ser Asp
Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys 35 40 45Cys Ser Arg Lys
Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn 50 55 60Met Glu Gln
Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile65 70 75 80Ile
Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg 85 90
95His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser
100 105 110Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr
Asp Val 115 120 125Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp
Asp Met Val Asn 130 135 140Glu Leu Phe Asp Ser Leu Phe Pro Val Ile
Tyr Thr Gln Leu Met Asn145 150 155 160Pro Gly Leu Pro Asp Ser Ala
Leu Asp Ile Asn Glu Cys Leu Arg Gly 165 170 175Ala Arg Arg Asp Leu
Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met 180 185 190Thr Gln Val
Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala 195 200 205Leu
Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe 210 215
220Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser
Tyr225 230 235 240Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly
Tyr Cys Asn Val 245 250 255Val Met Gln Gly Cys Met Ala Gly Val Val
Glu Ile Asp Lys Tyr Trp 260 265 270Arg Glu Tyr Ile Leu Ser Leu Glu
Glu Leu Val Asn Gly Met Tyr Arg 275 280 285Ile Tyr Asp Met Glu Asn
Val Leu Leu Gly Leu Phe Ser Thr Ile His 290 295 300Asp Ser Ile Gln
Tyr Val Gln Lys Asn Ala Gly Lys Leu Thr Thr Thr305 310 315 320Ile
Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala 325 330
335Tyr Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala
340 345 350His Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu
Leu Ile 355 360 365Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala
Leu Pro Gly Tyr 370 375 380Ile Cys Ser His Ser Pro Val Ala Glu Asn
Asp Thr Leu Cys Trp Asn385 390 395 400Gly Gln Glu Leu Val Glu Arg
Tyr Ser Gln Lys Ala Ala Arg Asn Gly 405 410 415Met Lys Asn Gln Phe
Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu 420 425 430Pro Val Val
Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu 435 440 445Leu
Arg Thr Met Ser Met Pro Lys Gly Arg Val Leu Asp Lys Asn Leu 450 455
460Asp Glu Glu Gly Phe Glu Ala Gly Asp Cys Gly Asp Asp Glu Asp
Glu465 470 475 480Cys Ile Gly Gly Ala Gly Asp Gly Met Ile Lys Val
Lys Asn Gln Leu 485 490 495Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu
Asp Val Asp Asp Ala Pro 500 505 510Gly Asn Ser Gln Gln Ala Thr Pro
Lys Asp Asn Glu Ile Ser Thr Phe 515 520 525His Asn Leu Gly Asn Val
His Ser Pro Leu Lys His His His
His His 530 535 540His54527365PRTHomo sapiens 27Met Gly Val Pro Arg
Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe1 5 10 15Leu Leu Pro Gly
Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr 20 25 30His Leu Thr
Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp 35 40 45Val Ser
Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser Leu 50 55 60Arg
Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val65 70 75
80Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys
85 90 95Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr
Thr 100 105 110Leu Gln Gly Leu Leu Gly Cys Glu Leu Gly Pro Asp Asn
Thr Ser Val 115 120 125Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu
Phe Met Asn Phe Asp 130 135 140Leu Lys Gln Gly Thr Trp Gly Gly Asp
Trp Pro Glu Ala Leu Ala Ile145 150 155 160Ser Gln Arg Trp Gln Gln
Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr 165 170 175Phe Leu Leu Phe
Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg 180 185 190Gly Arg
Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys 195 200
205Ala Arg Pro Ser Ser Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe
210 215 220Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn
Gly Leu225 230 235 240Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro
Asn Ser Asp Gly Ser 245 250 255Phe His Ala Ser Ser Ser Leu Thr Val
Lys Ser Gly Asp Glu His His 260 265 270Tyr Cys Cys Ile Val Gln His
Ala Gly Leu Ala Gln Pro Leu Arg Val 275 280 285Glu Leu Glu Ser Pro
Ala Lys Ser Ser Val Leu Val Val Gly Ile Val 290 295 300Ile Gly Val
Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu Leu305 310 315
320Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu Arg
325 330 335Gly Asp Asp Thr Gly Val Leu Leu Pro Thr Pro Gly Glu Ala
Gln Asp 340 345 350Ala Asp Leu Lys Asp Val Asn Val Ile Pro Ala Thr
Ala 355 360 36528119PRTHomo sapiens 28Met Ser Arg Ser Val Ala Leu
Ala Val Leu Ala Leu Leu Ser Leu Ser1 5 10 15Gly Leu Glu Ala Ile Gln
Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg 20 25 30His Pro Ala Glu Asn
Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser 35 40 45Gly Phe His Pro
Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu 50 55 60Arg Ile Glu
Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp65 70 75 80Ser
Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp 85 90
95Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile
100 105 110Val Lys Trp Asp Arg Asp Met 115298PRTArtificial
SequenceAn artificially synthesized peptide sequence 29Asp Tyr Lys
Asp Asp Asp Asp Lys1 5
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