U.S. patent application number 13/595139 was filed with the patent office on 2013-01-10 for antigen-binding molecule capable of binding to two or more antigen molecules repeatedly.
This patent application is currently assigned to CHUGAI SEIYAKU KABUSHIKI KAISHA. Invention is credited to Tomoyuki Igawa, Shinya Ishii, Atsuhiko Maeda, Takashi Nakai.
Application Number | 20130011866 13/595139 |
Document ID | / |
Family ID | 41161956 |
Filed Date | 2013-01-10 |
United States Patent
Application |
20130011866 |
Kind Code |
A1 |
Igawa; Tomoyuki ; et
al. |
January 10, 2013 |
ANTIGEN-BINDING MOLECULE CAPABLE OF BINDING TO TWO OR MORE ANTIGEN
MOLECULES REPEATEDLY
Abstract
The present inventors discovered that antibodies having weaker
antigen-binding activity at the early endosomal pH in comparison
with that at the pH of plasma are capable of binding to multiple
antigen molecules with a single antibody molecule, have long
half-lives in plasma, and have improved durations of time in which
they can bind to antigen.
Inventors: |
Igawa; Tomoyuki; (Shizuoka,
JP) ; Ishii; Shinya; (Shizuoka, JP) ; Maeda;
Atsuhiko; (Shizuoka, JP) ; Nakai; Takashi;
(Shizuoka, JP) |
Assignee: |
CHUGAI SEIYAKU KABUSHIKI
KAISHA
Tokyo
JP
|
Family ID: |
41161956 |
Appl. No.: |
13/595139 |
Filed: |
August 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12936587 |
Jan 3, 2011 |
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PCT/JP2009/057309 |
Apr 10, 2009 |
|
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13595139 |
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Current U.S.
Class: |
435/7.92 ;
436/501 |
Current CPC
Class: |
C07K 2317/565 20130101;
C12N 15/1037 20130101; A61P 37/00 20180101; C07K 2317/77 20130101;
C07K 2317/92 20130101; C07K 2317/24 20130101; C07K 2317/56
20130101; C07K 16/248 20130101; C12N 2760/16111 20130101; A61K
2039/545 20130101; A61P 43/00 20180101; C12N 2760/16134 20130101;
C12N 2770/20071 20130101; C07K 16/2866 20130101; A61K 39/12
20130101; A61K 2039/505 20130101; C07K 16/244 20130101; G01N
33/6854 20130101; C07K 2317/76 20130101; A61K 2039/544 20130101;
A61K 2039/552 20130101; C07K 2317/622 20130101; A61K 39/145
20130101; G01N 2500/00 20130101 |
Class at
Publication: |
435/7.92 ;
436/501 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 11, 2008 |
JP |
2008-104147 |
Sep 26, 2008 |
JP |
2008-247713 |
Mar 19, 2009 |
JP |
2009-068744 |
Claims
1.-27. (canceled)
28. A method of screening for an antibody, which comprises the
steps of: (a) determining the antigen-binding activity of an
antibody at pH 6.7 to pH 10.0; (b) determining the antigen-binding
activity of the antibody at pH 4.0 to pH 6.5; and (c) selecting an
antibody whose antigen-binding activity at pH 6.7 to pH 10.0 is
greater than the antigen-binding activity at pH 4.0 to pH 6.5,
wherein the antibody is used as a pharmaceutical composition and is
any one of the following (i) to (vi): (i) an antibody that is
superior in retention in plasma; (ii) an antibody that is capable
of binding to an antigen two or more times when measured in a
nonhuman animal comprising a cell expressing FcRn; (iii) an
antibody that is capable of binding to more antigens as compared to
the number of its antigen-binding sites when measured in a nonhuman
animal comprising a cell expressing FcRn; (iv) an antibody that
dissociates an extracellularly-bound antigen within a cell; (v) an
antibody that is bound to an antigen and internalized into a cell,
and released to the outside of the cell in an antigen-free form; or
(vi) an antibody that has increased ability to eliminate an antigen
in plasma.
29. The screening method of claim 28, which comprises the step of
selecting an antibody whose antigen-binding activity at pH 6.7 to
pH 10.0 is twice or higher that of the antigen-binding activity at
pH 4.0 to pH 6.5.
30. A method of screening for an antibody, which comprises the
steps of: (a) binding an antibody to an antigen under a condition
of pH 6.7 to pH 10.0; (b) placing the antibody bound to the antigen
of (a) under a condition of pH 4.0 to pH 6.5; and (c) obtaining an
antibody that has dissociated under the condition of pH 4.0 to pH
6.5, wherein the antibody is used as a pharmaceutical composition
and is any one of the following (i) to (vi): (i) an antibody that
is superior in retention in plasma; (ii) an antibody that is
capable of binding to an antigen two or more times when measured in
a nonhuman animal comprising a cell expressing FcRn; (iii) an
antibody that is capable of binding to more antigens as compared to
the number of its antigen-binding sites when measured in a nonhuman
animal comprising a cell expressing FcRn; (iv) an antibody that
dissociates an extracellularly-bound antigen within a cell; (v) an
antibody that is bound to an antigen and internalized into a cell,
and released to the outside of the cell in an antigen-free form; or
(vi) an antibody that has increased ability to eliminate an antigen
in plasma.
31. A method of screening for an antibody whose binding activity at
a first pH is greater than that at a second pH, which comprises the
steps of: (a) binding an antibody to an antigen-immobilized column
under the condition of a first pH; (b) eluting the antibody bound
to the column at the first pH from the column under the condition
of a second pH; and (c) collecting the eluted antibody, wherein the
first pH is 6.7 to 10.0 and the second pH is 4.0 to 6.5, and
wherein the antibody is used as a pharmaceutical composition and is
any one of the following (i) to (vi): (i) an antibody that is
superior in retention in plasma; (ii) an antibody that is capable
of binding to an antigen two or more times when measured in a
nonhuman animal comprising a cell expressing FcRn; (iii) an
antibody that is capable of binding to more antigens as compared to
the number of its antigen-binding sites when measured in a nonhuman
animal comprising a cell expressing FcRn; (iv) an antibody that
dissociates an extracellularly-bound antigen within a cell; (v) an
antibody that is bound to an antigen and internalized into a cell,
and released to the outside of the cell in an antigen-free form; or
(vi) an antibody that has increased ability to eliminate an antigen
in plasma.
32. A method of screening for an antibody whose binding activity at
a first pH is greater than that at a second pH, which comprises the
steps of: (a) binding an antibody library to an antigen-immobilized
column under the condition of a first pH; (b) eluting the antibody
from the column under the condition of a second pH; (c) amplifying
the gene encoding the eluted antibody; and (d) obtaining the eluted
antibody, wherein the first pH is 6.7 to 10.0 and the second pH is
4.0 to 6.5, and wherein the antibody is used as a pharmaceutical
composition and is any one of the following (i) to (vi): (i) an
antibody that is superior in retention in plasma; (ii) an antibody
that is capable of binding to an antigen two or more times when
measured in a nonhuman animal comprising a cell expressing FcRn;
(iii) an antibody that is capable of binding to more antigens as
compared to the number of its antigen-binding sites when measured
in a nonhuman animal comprising a cell expressing FcRn; (iv) an
antibody that dissociates an extracellularly-bound antigen within a
cell; (v) an antibody that is bound to an antigen and internalized
into a cell, and released to the outside of the cell in an
antigen-free form; or (vi) an antibody that has increased ability
to eliminate an antigen in plasma.
33. (canceled)
34. The screening method of claim 28, wherein at least one or more
amino acids of the antibody has been substituted with histidine, or
at least one histidine has been inserted into the antibody.
35.-50. (canceled)
51. The screening method of claim 29, wherein at least one or more
amino acids of the antibody has been substituted with histidine, or
at least one histidine has been inserted into the antibody.
52. The screening method of claim 30, wherein at least one or more
amino acids of the antibody has been substituted with histidine, or
at least one histidine has been inserted into the antibody.
53. The screening method of claim 31, wherein at least one or more
amino acids of the antibody has been substituted with histidine, or
at least one histidine has been inserted into the antibody.
54. The screening method of claim 32, wherein at least one or more
amino acids of the antibody has been substituted with histidine, or
at least one histidine has been inserted into the antibody.
Description
TECHNICAL FIELD
[0001] The present invention relates to methods for improving the
pharmacokinetics of antigen-binding molecules and methods for
increasing the number of times of antigen-binding of
antigen-binding molecules, as well as antigen-binding molecules
having improved pharmacokinetics, antigen-binding molecules having
increased number of times of antigen-binding, and methods for
producing such molecules.
BACKGROUND ART
[0002] Antibodies are drawing attention as pharmaceuticals as they
are highly stable in plasma and have few adverse effects. At
present, a number of IgG-type antibody pharmaceuticals are
available on the market and many more antibody pharmaceuticals are
currently under development (Non-Patent Documents 1 and 2).
Meanwhile, various technologies applicable to second-generation
antibody pharmaceuticals have been developed, including those that
enhance effector function, antigen-binding ability,
pharmacokinetics, and stability, and those that reduce the risk of
immunogenicity (Non-Patent Document 3). In general, the requisite
dose of an antibody pharmaceutical is very high. This, in turn, has
led to problems, such as high production cost, as well as the
difficulty in producing subcutaneous formulations. In theory, the
dose of an antibody pharmaceutical may be reduced by improving
antibody pharmacokinetics or improving the affinity between
antibodies and antigens.
[0003] The literature has reported methods for improving antibody
pharmacokinetics using artificial substitution of amino acids in
constant regions (Non-Patent Documents 4 and 5). Similarly,
affinity maturation has been reported as a technology for enhancing
antigen-binding ability or antigen-neutralizing activity
(Non-Patent Document 6). This technology enables enhancement of
antigen-binding activity by introduction of amino acid mutations
into the CDR region of a variable region or such. The enhancement
of antigen-binding ability enables improvement of in vitro
biological activity or reduction of dosage, and further enables
improvement of in vivo efficacy (Non-Patent Document 7).
[0004] The antigen-neutralizing capacity of a single antibody
molecule depends on its affinity. By increasing the affinity, an
antigen can be neutralized by smaller amount of an antibody.
Various methods can be used to enhance the antibody affinity.
Furthermore, if the affinity could be made infinite by covalently
binding the antibody to the antigen, a single antibody molecule
could neutralize one antigen molecule (a divalent antibody can
neutralize two antigen molecules). However, the stoichiometric
neutralization of one antibody against one antigen (one divalent
antibody against two antigens) is the limit of pre-existing
methods, and thus it is impossible to completely neutralize antigen
with the smaller amount of antibody than the amount of antigen. In
other words, the affinity enhancing effect has a limit (Non-Patent
Document 9). To prolong the neutralization effect of a neutralizing
antibody for a certain period, the antibody must be administered at
a dose higher than the amount of antigen produced in the body
during the same period. With the improvement of antibody
pharmacokinetics or affinity maturation technology alone described
above, there is thus a limitation in the reduction of the required
antibody dose.
[0005] Accordingly, in order to sustain antibody's
antigen-neutralizing effect for a target period with smaller amount
of the antibody than the amount of antigen, a single antibody must
neutralize multiple antigens. Methods for neutralizing multiple
antigens with a single antibody include antigen inactivation using
catalytic antibodies, which are antibodies conferred with a
catalytic function. When the antigen is a protein, it can be
inactivated by hydrolyzing its peptide bonds. An antibody can
repeatedly neutralize antigens by catalyzing such hydrolysis
(Non-Patent Document 8). There are many previous reports published
on catalytic antibodies and technologies for producing them.
However, there have been no reports of catalytic antibodies having
sufficient catalytic activity as a pharmaceutical agent.
Specifically, in an antibody in vivo study for a certain antigen,
there has been no publication of catalytic antibodies which can
produce a comparable or stronger effect even at low doses or
produce a more prolonged effect even at a same dose as compared to
an ordinary non-catalytic neutralizing antibody.
[0006] As described above, there have been no reports of antibodies
that can produce a more superior in vivo effect than ordinary
neutralizing antibodies through a single antibody neutralizing
multiple antigen molecules. Thus, from the viewpoint of dose
reduction and prolongation of the durability, there is a need for
new technologies that permit the production of novel antibody
molecules having a stronger in vivo effect than ordinary
neutralizing antibodies by individually neutralizing multiple
antigen molecules.
[0007] Prior art documents related to the present invention are
shown below:
PRIOR ART DOCUMENTS
Non-Patent Documents
[0008] Non-Patent Document 1: Monoclonal antibody successes in the
clinic. Janice M Reichert, Clark J Rosensweig, Laura B Faden &
Matthew C Dewitz, Nature Biotechnology 23, 1073-1078 (2005) [0009]
Non-Patent Document 2: Pavlou A K, Belsey M J. The therapeutic
antibodies market to 2008. Eur J Pharm Biopharm. 2005 April;
59(3):389-96 [0010] Non-Patent Document 3: Kim S J, Park Y, Hong H
J. Antibody engineering for the development of therapeutic
antibodies. Mol Cells. 2005 Aug. 31; 20(1):17-29. Review [0011]
Non-Patent Document 4: Hinton P R, Xiong J M, Johlfs M G, Tang M T,
Keller S, Tsurushita N. An engineered human IgG1 antibody with
longer serum half-life. J Immunol. 2006 Jan. 1; 176(1):346-56
[0012] Non-Patent Document 5: Ghetie V, Popov S, Borvak J, Radu C,
Matesoi D, Medesan C, Ober R J, Ward E S. Increasing the serum
persistence of an IgG fragment by random mutagenesis. Nat
Biotechnol. 1997 July; 15(7):637-40 [0013] Non-Patent Document 6:
Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt R R,
Takeuchi T, Lerner R A, Crea R. A general method for greatly
improving the affinity of antibodies by using combinatorial
libraries. Proc Natl Acad Sci USA. 2005 Jun. 14; 102(24):8466-71.
Epub 2005 Jun. 6 [0014] Non-Patent Document 7: Wu H, Pfarr D S,
Johnson S, Brewah Y A, Woods R M, Patel N K, White W I, Young J F,
Kiener P A. Development of Motavizumab, an Ultra-potent Antibody
for the Prevention of Respiratory Syncytial Virus Infection in the
Upper and Lower Respiratory Tract. J Mol Biol. 2007, 368, 652-665
[0015] Non-Patent Document 8: Hanson C V, Nishiyama Y, Paul S.
Catalytic antibodies and their applications. Curr Opin Biotechnol.
2005 December; 16(6):631-6 [0016] Non-Patent Document 9:
Rathanaswami P, Roalstad S, Roskos L, Su Q J, Lackie S, Babcook J.
Demonstration of an in vivo generated sub-picomolar affinity fully
human monoclonal antibody to interleukin-8 Biochem Biophys Res
Commun. 2005 Sep. 9; 334(4):1004-13
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0017] The above noted circumstances led to the discoveries of the
present invention. Accordingly, an objective of the present
invention is to provide methods for binding antigen-binding
molecules to the antigens multiple times and methods for improving
the pharmacokinetics of antigen-binding molecules, as well as
antigen-binding molecules that are capable of binding to the
antigens multiple times, antigen-binding molecules having improved
pharmacokinetics, pharmaceutical compositions containing such
antigen-binding molecules, and methods for producing such molecules
and compositions.
Means for Solving the Problems
[0018] Dedicated studies on methods for binding polypeptides having
antigen-binding ability, such as antigen-binding molecules, to the
antigens multiple times, and methods for improving the half-lives
of such molecules in plasma (blood) (improving their
pharmacokinetics) were conducted herein. As a result, it was
discovered that if the antigen-binding activity of an
antigen-binding molecule at the early endosomal pH is lower than
its antigen-binding activity at the pH of plasma (blood), it would
be able to bind to antigens multiple times and have a longer
half-life in plasma.
[0019] Accordingly, the present invention relates to methods for
binding antigen-binding molecules to antigens multiple times,
methods for improving the pharmacokinetics of antigen-binding
molecules, and methods for producing antigen-binding molecules with
improved pharmacokinetics; the present invention also relates to
antigen-binding molecules that are capable of binding to antigens
multiple times and antigen-binding molecules with improved
pharmacokinetics. More specifically, the present invention
provides:
[1] an antigen-binding molecule having a KD(pH5.8)/KD(pH7.4) value,
defined as the ratio of KD for the antigen at pH 5.8 and KD for the
antigen at pH 7.4, of 2 or higher; [2] the antigen-binding molecule
of [1], wherein the KD(pH5.8)/KD(pH7.4) value is 10 or higher; [3]
the antigen-binding molecule of [1], wherein the
KD(pH5.8)/KD(pH7.4) value is 40 or higher; [4] the antigen-binding
molecule of any one of [1] to [3], wherein at least one amino acid
of the antigen-binding molecule has been substituted with
histidine, or at least one histidine has been inserted into the
antigen-binding molecule; [5] the antigen-binding molecule of any
one of [1] to [4], wherein the antigen-binding molecule has an
antagonistic activity; [6]. the antigen-binding molecule of any one
of [1] to [5], wherein the antigen-binding molecule binds to a
membrane antigen or a soluble antigen; [7] the antigen-binding
molecule of any one of [1] to [6], wherein the antigen-binding
molecule is an antibody; [8] a pharmaceutical composition
comprising the antigen-binding molecule of any one of [1] to [7];
[9] a method for improving the pharmacokinetics of an
antigen-binding molecule by impairing the antigen-binding activity
of the antigen-binding molecule at pH 5.8 as compared to that at pH
7.4; [10] a method for increasing the number of times of
antigen-binding for an antigen-binding molecule by impairing the
antigen-binding activity of the antigen-binding molecule at pH 5.8
as compared to that at pH 7.4; [11] a method for increasing the
number of antigens that can be bound by an antigen-binding molecule
by impairing the antigen-binding activity of the antigen-binding
molecule at pH 5.8 as compared to that at pH 7.4; [12] a method for
dissociating within a cell an antigen from an extracellularly-bound
antigen-binding molecule by impairing the antigen-binding activity
of the antigen-binding molecule at pH 5.8 as compared to that at pH
7.4; [13] a method for releasing an antigen-binding molecule, which
has been bound to an antigen and internalized into a cell, in an
antigen-free form to the outside of the cell by impairing the
antigen-binding activity of the antigen-binding molecule at pH 5.8
as compared to that at pH 7.4; [14] a method for increasing the
ability of an antigen-binding molecule to eliminate an antigen in
plasma by impairing the antigen-binding activity of the
antigen-binding molecule at pH 5.8 as compared to that at pH 7.4;
[15] the method of any one of [9] to [14], wherein the
KD(pH5.8)/KD(pH7.4) value, defined as the ratio of KD for the
antigen at pH 5.8 and KD for the antigen at pH 7.4, is 2 or higher;
[16] the method of any one of [9] to [14], wherein the
KD(pH5.8)/KD(pH7.4) value is 10 or higher; [17] the method of any
one of [9] to [14], wherein the KD(pH5.8)/KD(pH7.4) value is 40 or
higher; [18] a method for improving the pharmacokinetics of an
antigen-binding molecule by substituting at least one amino acid of
the antigen-binding molecule with histidine, or inserting at least
one histidine into the antigen-binding molecule; [19] a method for
increasing the number of times of antigen-binding for an
antigen-binding molecule by substituting at least one amino acid of
the antigen-binding molecule with histidine, or inserting at least
one histidine into the antigen-binding molecule; [20] a method for
increasing the number of antigens that can be bound by an
antigen-binding molecule by substituting at least one amino acid of
the antigen-binding molecule with histidine, or inserting at least
one histidine into the antigen-binding molecule; [21] a method for
dissociating within a cell an antigen from an extracellularly-bound
antigen-binding molecule by substituting at least one amino acid of
the antigen-binding molecule with histidine, or inserting at least
one histidine into the antigen-binding molecule; [22] a method for
releasing an antigen-binding molecule, which has been bound to an
antigen and internalized into a cell, in an antigen-free form to
the outside of the cell, by substituting at least one amino acid of
the antigen-binding molecule with histidine, or inserting at least
one histidine into the antigen-binding molecule; [23] a method for
increasing the ability of an antigen-binding molecule to eliminate
an antigen in plasma by substituting at least one amino acid of the
antigen-binding molecule with histidine, or inserting at least one
histidine into the antigen-binding molecule; [24] the method of any
one of [18] to [23], wherein the histidine substitution or
insertion increases the KD(pH5.8)/KD(pH7.4) value, defined as the
ratio of the antigen-binding activity at pH 5.8 and the
antigen-binding activity at pH 7.4, as compared to the
KD(pH5.8)/KD(pH7.4) value before the histidine substitution or
insertion; [25] the method of any one of [9] to [24], wherein the
antigen-binding molecule has an antagonistic activity; [26] the
method of any one of [9] to [25], wherein the antigen-binding
molecule binds to a membrane antigen or a soluble antigen; [27] the
method of any one of [9] to [26], wherein the antigen-binding
molecule is an antibody; [28] a method of screening for an
antigen-binding molecule, which comprises the steps of: (a)
determining the antigen-binding activity of an antigen-binding
molecule at pH 6.7 to pH 10.0; (b) determining the antigen-binding
activity of the antigen-binding molecule at pH 4.0 to pH 6.5; and
(c) selecting an antigen-binding molecule whose antigen-binding
activity at pH 6.7 to pH 10.0 is greater than the antigen-binding
activity at pH 4.0 to pH 6.5; [29] the screening method of [28],
which comprises the step of selecting an antibody whose
antigen-binding activity at pH 6.7 to pH 10.0 is twice or higher
that of the antigen-binding activity at pH 4.0 to pH 6.5; [30] a
method of screening for an antigen-binding molecule, which
comprises the steps of: (a) binding an antigen-binding molecule to
an antigen under a condition of pH 6.7 to pH 10.0; (b) placing the
antigen-binding molecule that bound to the antigen of (a) under a
condition of pH 4.0 to pH 6.5; and (c) obtaining an antigen-binding
molecule that dissociated under the condition of pH 4.0 to pH 6.5;
[31] a method of screening for an antigen-binding molecule whose
binding activity at a first pH is greater than that at a second pH,
which comprises the steps of: (a) binding an antigen-binding
molecule to an antigen-immobilized column under the condition of a
first pH; (b) eluting the antigen-binding molecule that had bound
to the column at the first pH from the column under the condition
of a second pH; and (c) collecting the eluted antigen-binding
molecule; [32] a method of screening for an antigen-binding
molecule whose binding activity at a first pH is greater than that
at a second pH, which comprises the steps of: (a) binding an
antigen-binding molecule library to an antigen-immobilized column
under the condition of a first pH; (b) eluting the antigen-binding
molecule from the column under the condition of a second pH; (c)
amplifying the gene encoding the eluted antigen-binding molecule;
and (d) obtaining the eluted antigen-binding molecule. [33] the
screening method of [31] or [32], wherein the first pH is 6.7 to
10.0 and the second pH is 4.0 to 6.5; [34] the screening method of
any one of [28] to [33], wherein at least one or more amino acids
of the antigen-binding molecule has been substituted with
histidine, or at least one histidine has been inserted into the
antigen-binding molecule; [35] the screening method of any one of
[28] to [33], for obtaining an antigen-binding molecule that is
superior in retention in the plasma; [36] the screening method of
any one of [28] to [33], for obtaining an antigen-binding molecule
that is capable of binding to an antigen two or more times; [37]
the screening method of any one of [28] to [33], for obtaining an
antigen-binding molecule that is capable of binding to more
antigens as compared to the number of its antigen-binding sites;
[38] the screening method of any one of [28] to [33], for obtaining
an antigen-binding molecule that dissociates an
extracellularly-bound antigen within a cell. [39] the screening
method of any one of [28] to [33], for obtaining an antigen-binding
molecule that is bound to an antigen and internalized into a cell,
and released to the outside of the cell in an antigen-free form;
[40] the screening method of any one of [28] to [33], for obtaining
an antigen-binding molecule that has increased ability to eliminate
an antigen in plasma; [41] the screening method of any one of [28]
to [40], wherein the antigen-binding molecule is used as a
pharmaceutical composition; [42] the screening method of any one of
[28] to [41], wherein the antigen-binding molecule is an antibody;
[43] a method for producing an antigen-binding molecule, which
comprises the steps of: (a) determining the antigen-binding
activity of an antigen-binding molecule at pH 6.7 to pH 10.0; (b)
determining the antigen-binding activity of the antigen-binding
molecule at pH 4.0 to pH 6.5; (c) selecting the antigen-binding
molecule whose antigen-binding activity at pH 6.7 to pH 10.0 is
greater than that at pH 4.0 to pH 6.5; (d) obtaining the gene
encoding the antigen-binding molecule selected in (c); and (e)
producing the antigen-binding molecule using the gene obtained in
(d); [44] a method for producing an antigen-binding molecule, which
comprises the steps of: (a) binding an antigen-binding molecule to
an antigen under a condition of pH 6.7 to pH 10.0; (b) allowing the
antigen-binding molecule bound to the antigen of (a) to stand under
a condition of pH 4.0 to pH 6.5; (c) collecting the antigen-binding
molecule that dissociated under the condition of pH 4.0 to pH 6.5;
(d) obtaining the gene encoding the antigen-binding molecule
obtained in (c); and (e) producing the antigen-binding molecule
using the gene obtained in (d); [45] a method for producing an
antigen-binding molecule whose binding activity at a first pH is
greater than that at a second pH, which comprises the steps of: (a)
binding an antigen-binding molecule to an antigen-immobilized
column under the first pH condition; (b) eluting the
antigen-binding molecule, which is bound to the column at the first
pH, from the column under a second pH condition; (c) collecting the
eluted antigen-binding molecule; (d) obtaining the gene encoding
the antigen-binding molecule obtained in (c); and (e) producing the
antigen-binding molecule using the gene obtained in (d); [46] a
method for producing an antigen-binding molecule whose binding
activity at a first pH is greater than that at a second pH, which
comprises the steps of: (a) binding an antigen-binding molecule
library to an antigen-immobilized column under the first pH
condition; (b) eluting the antigen-binding molecule from the column
under the second pH condition; (c) amplifying the gene encoding the
eluted antigen-binding molecule; (d) collecting the eluted
antigen-binding molecule; (e) obtaining the gene encoding the
antigen-binding molecule collected in (d); and (f) producing the
antigen-binding molecule using the gene obtained in (e); [47] the
production method of [45] or [46], wherein the first pH is 6.7 to
10.0 and the second pH is 4.0 to 6.5. [48] the production method of
any one of [43] to [47], which further comprises the step of
substituting at least one amino acid of the antigen-binding
molecule with histidine, or inserting at least one histidine into
the antigen-binding molecule; [49] the production method of any one
of [43] to [48], wherein the antigen-binding molecule is an
antibody; [50] a pharmaceutical composition comprising an
antigen-binding molecule produced by the production method of any
one of [43] to [49].
Effects of the Invention
[0020] The present invention provides methods for making single
antigen-binding molecules to repeatedly bind to multiple antigen
molecules. When an antigen-binding molecule binds to multiple
antigen molecules, the pharmacokinetics of the antigen-binding
molecule can be improved and such molecule can exert more superior
in vivo effects than those of ordinary antigen-binding
molecules.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a diagram depicting a degradation pathway of
antibodies bound to membrane-bound antigen.
[0022] FIG. 2 is a diagram depicting a mechanism by which IgG
molecules are salvaged by FcRn.
[0023] FIG. 3 is a schematic diagram depicting the re-binding of
IgG molecules to new antigen following dissociation from
membrane-bound antigen within endosomes.
[0024] FIG. 4 is a schematic diagram depicting the re-binding of
IgG molecules to new antigen following dissociation from soluble
antigen within endosomes.
[0025] FIG. 5 is a diagram depicting the process of panning using
an antigen-immobilized column.
[0026] FIG. 6 presents graphs depicting the results of phage ELISA
for clones acquired by column panning. The upper graph depicts WT
and the lower graph depicts CL5.
[0027] FIG. 7 is a graph depicting the biological neutralization
activity of pH-dependently-binding anti-IL-6 receptor
antibodies.
[0028] FIG. 8 presents graphs depicting results of Biacore
sensorgram for binding of pH-dependently-binding anti-IL-6 receptor
antibodies to soluble IL-6 receptor at pH 7.4. The top graph
depicts WT; the second graph from the top depicts H3pI/L73; the
third graph from the top depicts H170/L82; and the bottom depicts
CLH5/L73.
[0029] FIG. 9 presents graphs depicting results of Biacore
sensorgram for binding of pH-dependently-binding anti-IL-6 receptor
antibodies to soluble IL-6 receptor at pH 5.8. The top graph
depicts WT; the second graph from the top depicts H3pI/L73; the
third graph from the top depicts H170/L82; and the bottom graph
depicts CLH5/L73.
[0030] FIG. 10 presents graphs depicting results of Biacore
sensorgram for association (pH 7.4) and dissociation (pH 5.8) of
pH-dependently-binding anti-IL-6 receptor antibodies to
membrane-type IL-6 receptor. The top graph depicts WT; the second
graph from the top depicts H3pI/L73; the third graph from the top
depicts H170/L82; and the bottom graph depicts CLH5/L73.
[0031] FIG. 11 is a Biacore sensorgram indicating repeated binding
of pH-dependently-binding anti-IL-6 receptor antibodies to
SR344.
[0032] FIG. 12 is a graph depicting the total amount of bound
antigen in a repetitive binding experiment of
pH-dependently-binding anti-IL-6 receptor antibodies to SR344.
[0033] FIG. 13 is a graph depicting time courses of antibody plasma
concentrations of pH-dependently-binding anti-IL-6 receptor
antibodies in human IL-6 receptor transgenic mice.
[0034] FIG. 14 is a graph depicting time courses of antibody plasma
concentrations of pH-dependently-binding anti-IL-6 receptor
antibodies in cynomolgus monkeys.
[0035] FIG. 15 is a graph depicting time courses of CRP
concentrations in cynomolgus monkeys, in relation to
pH-dependently-binding anti-IL-6 receptor antibodies.
[0036] FIG. 16 is a graph depicting time courses of unbound-type
cynomolgus monkey IL-6 receptor concentrations in cynomolgus
monkeys, in relation to pH-dependently-binding anti-IL-6 receptor
antibodies.
[0037] FIG. 17 presents graphs depicting results of Biacore
sensorgram of association (pH 7.4) and dissociation (pH 5.8) of
pH-dependently-binding anti-IL-6 receptor antibodies to
membrane-type IL-6 receptor. In order moving from the top, the
results for WT, H3pI/L73-IgG1, Fv2-IgG1, and Fv4-IgG1 are
shown.
[0038] FIG. 18 is a graph depicting time courses of plasma antibody
concentrations of pH-dependently-binding anti-IL-6 receptor
antibodies (WT, H3pI/L73-IgG1, Fv2-IgG1, and Fv4-IgG1) in human
IL-6 receptor transgenic mice.
[0039] FIG. 19 presents graphs depicting results of Biacore
sensorgram for association (pH 7.4) and dissociation (pH 5.8) of
pH-dependently-binding anti-IL-6 receptor antibodies to
membrane-type IL-6 receptor. Moving from the top down, the results
for WT, Fv4-IgG1, Fv4-IgG2, and Fv4-M58 are shown.
[0040] FIG. 20 is a graph showing time courses of antibody plasma
concentrations of pH-dependently-binding anti-IL-6 receptor
antibodies (WT, Fv4-IgG1, Fv4-IgG2, and Fv4-M58) in human IL-6
receptor transgenic mice.
[0041] FIG. 21 presents graphs depicting results of Biacore
sensorgram of association (pH 7.4) and dissociation (pH 5.8) of
pH-dependently-binding anti-IL-6 receptor antibodies to
membrane-type IL-6 receptor. Moving from the top down, the results
for Fv1-M71, Fv1-M73, Fv3-M71, and Fv3-M73 are shown.
[0042] FIG. 22 is a graph depicting time courses of antibody plasma
concentrations of pH-dependently-binding anti-IL-6 receptor
antibodies in cynomolgus monkeys, during administration of
H3pI/L73-IgG1, Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M73, and Fv4-M73 at
0.5 mg/kg and during administration of high affinity Ab at 1.0
mg/kg.
[0043] FIG. 23 is a graph depicting time courses of CRP
concentrations in cynomolgus monkeys, in relation to
pH-dependently-binding anti-IL-6 receptor antibodies
(H3pI/L73-IgG1-, Fv1-M71-, Fv1-M73-, Fv2-IgG1-, Fv3-M73-, Fv4-M73-,
and high-affinity-Ab-administered groups).
[0044] FIG. 24 is a graph depicting time courses of unbound-type
cynomolgus monkey IL-6 receptor concentrations in cynomolgus
monkeys, in relation to pH-dependently-binding anti-IL-6 receptor
antibodies (H3pI/L73-IgG1-, Fv1-M71-, Fv1-M73-, Fv2-IgG1-,
Fv3-M73-, Fv4-M73-, and high-affinity-Ab-administered groups).
[0045] FIG. 25 is a diagram depicting FR1, FR2, FR3, and FR4 along
with CDR1, CDR2, and CDR3 of heavy chains (VH1, VH2, VH3, VH4) and
light chains (VL1, VL2, VL3). Asterisks indicate locations where
amino acid mutations exist in the aligned sequences.
[0046] FIG. 26 presents a Biacore sensorgram depicting the
pH-dependent binding of an anti-IL-6 antibody, Anti-IL6 clone 2, to
IL-6 at pH 7.4 and pH 5.5. The curves in the sensorgram at pH 7.4
correspond to 100, 50, 25, 12.5, and 6.25 ng/mL IL-6, from
above.
[0047] FIG. 27 presents a Biacore sensorgram depicting the
pH-dependent binding of an anti-IL-31 receptor antibody, Anti-IL31R
clone 1, to the IL-31 receptor at pH 7.4 and pH 5.5. The curves in
the sensorgram at pH 5.5 correspond to 100, 50, 25, and 12.5 ng/mL
IL-31 receptor, from above.
[0048] FIG. 28 depicts the time course of plasma antibody
concentration after intravenous administration of a mixture
solution containing SR344 and an anti-human IL-6 receptor antibody
to mouse.
[0049] FIG. 29 depicts the time course of plasma SR344
concentration after intravenous administration of a mixture
solution containing SR344 and an anti-human IL-6 receptor antibody
to mouse.
MODE FOR CARRYING OUT THE INVENTION
[0050] The present invention provides methods for increasing the
number of times of antigen-binding in antigen-binding molecules.
More specifically, the present invention provides methods for
increasing the number of times of antigen-binding in
antigen-binding molecules by impairing the antigen-binding ability
of the antigen-binding molecules at acidic pH as compared to that
at neutral pH. Furthermore, the present invention provides methods
for increasing the number of times of antigen-binding in
antigen-binding molecules by substituting histidine for at least
one amino acid in the antigen-binding molecules or inserting at
least one histidine into the antigen-binding molecules. In
addition, the present invention provides methods for increasing the
number of times of antigen-binding in antigen-binding molecules by
substituting, deleting, adding, and/or inserting amino acids in the
antibody constant region of antigen-binding molecules.
[0051] The present invention also provides methods for increasing
the number of antigens that can be bound by an antigen-binding
molecule. More specifically, the present invention provides methods
for increasing the number of antigens that can be bound by an
antigen-binding molecule by impairing the antigen-binding ability
at acidic pH as compared to that at neutral pH. Furthermore, the
present invention provides methods for increasing the number of
antigens that can be bound by an antigen-binding molecule by
substituting histidine for at least one amino acid in the
antigen-binding molecules or inserting at least one histidine into
the antigen-binding molecules. In addition, the present invention
provides methods for increasing the number of antigens that can be
bound by an antigen-binding molecule through substituting,
deleting, adding, and/or inserting amino acids in the antibody
constant region of antigen-binding molecules.
[0052] The present invention also provides methods for dissociating
within a cell an antigen from an extracellularly-bound
antigen-binding molecule. More specifically, the present invention
provides methods for dissociating within a cell an antigen from an
extracellularly-bound antigen-binding molecule by impairing the
antigen-binding ability at acidic pH as compared to that at neutral
pH. Furthermore, the present invention provides methods for
dissociating within a cell an antigen from an extracellularly-bound
antigen-binding molecule by substituting histidine for at least one
amino acid in the antigen-binding molecule or inserting at least
one histidine into the antigen-binding molecule. In addition, the
present invention provides methods for dissociating within a cell
an antigen from an extracellularly-bound antigen-binding molecule
through substituting, deleting, adding, and/or inserting amino
acids in the antibody constant region of antigen-binding
molecule.
[0053] The present invention also provides methods for releasing an
antigen-binding molecule, which has been bound to an antigen and
internalized into a cell, in an antigen-free form to the outside of
the cell. More specifically, the present invention provides methods
for releasing an antigen-binding molecule, which has been bound to
an antigen and internalized into a cell, in an antigen-free form to
the outside of the cell, by impairing the antigen-binding ability
at acidic pH as compared to that at neutral pH. Furthermore, the
present invention provides methods for releasing an antigen-binding
molecule, which has been bound to an antigen and internalized into
a cell, in an antigen-free form to the outside of the cell, by
substituting histidine for at least one amino acid in the
antigen-binding molecule or inserting at least one histidine into
the antigen-binding molecule. In addition, the present invention
provides methods for releasing an antigen-binding molecule, which
has been bound to an antigen and internalized into a cell, in an
antigen-free form to the outside of the cell, through substituting,
deleting, adding, and/or inserting amino acids in the antibody
constant region of antigen-binding molecule.
[0054] The present invention also provides methods for increasing
the ability of an antigen-binding molecule to eliminate antigens in
plasma. More specifically, the present invention provides methods
for increasing the ability of an antigen-binding molecule to
eliminate antigens in plasma by impairing the antigen-binding
ability at acidic pH as compared to that at neutral pH.
Furthermore, the present invention provides methods for increasing
the ability of an antigen-binding molecule to eliminate antigens in
plasma by substituting histidine for at least one amino acid in the
antigen-binding molecules or inserting at least one histidine into
the antigen-binding molecules. In addition, the present invention
provides methods for increasing the ability of an antigen-binding
molecule to eliminate antigens in plasma through substituting,
deleting, adding, and/or inserting amino acids in the antibody
constant region of antigen-binding molecule.
[0055] The present invention also provides methods for improving
the pharmacokinetics of antigen-binding molecules. More
specifically, the present invention provides methods for improving
the pharmacokinetics of antigen-binding molecules (prolonging the
retention in plasma) by impairing the antigen-binding ability at
acidic pH as compared to that at neutral pH. Furthermore, the
present invention provides methods for improving the
pharmacokinetics of antigen-binding molecules by substituting
histidine for at least one amino acid in the antigen-binding
molecules or inserting at least one histidine into the
antigen-binding molecules. In addition, the present invention
provides methods for improving the pharmacokinetics of
antigen-binding molecules by substituting, deleting, adding, and/or
inserting amino acids in the antibody constant region of
antigen-binding molecules.
[0056] Further, the present invention provides methods for
increasing the ability of the antigen-binding molecules to
eliminate antigens in plasma. More specifically, the present
invention provides methods for increasing the ability of the
antigen-binding molecules to eliminate antigens in plasma by
impairing the antigen-binding ability of the antigen-binding
molecules at acidic pH as compared to that at neutral pH.
Furthermore, the present invention provides methods for increasing
the ability of the antigen-binding molecules to eliminate antigens
in plasma by substituting at least one amino acid in the
antigen-binding molecules with histidine or inserting at least one
histidine into the antigen-binding molecules. In addition, the
present invention provides methods for increasing the ability of
the antigen-binding molecules to eliminate antigens in plasma by
substituting, deleting, adding, and/or inserting amino acids in the
antibody constant region of antigen-binding molecules.
[0057] Herein, "improvement of the pharmacokinetics", "amelioration
of the pharmacokinetics", "superior pharmacokinetics" are
interchangeable with "improvement of the retention in plasma
(blood)", "amelioration of the retention in plasma (blood)", and
"superior retention in plasma (blood)", respectively, and these
phrases are synonymous.
[0058] Herein, impairing the antigen-binding activity at acidic pH
as compared to that at neutral pH means that the antigen-binding
ability of an antigen-binding molecule at pH 4.0 to pH 6.5 is
impaired as compared to that at pH 6.7 to pH 10.0, preferably 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,
and more preferably that the antigen-binding activity of an
antigen-binding molecule at pH 5.8 is impaired as compared to that
at pH 7.4. Accordingly, in the present invention, acidic pH is
typically pH 4.0 to pH 6.5, preferably pH 5.5 to pH 6.5, and more
preferably pH 5.8. Alternatively, in the present invention, neutral
pH is typically pH 6.7 to pH 10.0, preferably pH 7.0 to pH 8.0, and
more preferably pH 7.4.
[0059] Herein, the phrase "impairing the antigen-binding ability of
an antigen-binding molecule at acidic pH as compared to that at
neutral pH" is interchangable with the phrase "increasing the
antigen-binding ability of an antigen-binding molecule at neutral
pH as compared to that at acidic pH". In other words, in the
present invention, the difference in the antigen-binding ability of
an antigen-binding molecule should be increased between acidic and
neutral pHs. For example, the value of KD(pH5.8)/KD(pH7.4) should
be increased, as described below. The difference in the
antigen-binding ability of an antigen-binding molecule between
acidic and neutral pHs may be increased, for example, by either or
both, impairing the antigen-binding ability at acidic pH and
increasing the antigen-binding ability at neutral pH.
[0060] Conditions other than the pH for determining the
antigen-binding activity can be selected appropriately by those
skilled in the art, and the conditions are not particularly
limited. The antigen-binding activity can be determined, for
example, under conditions of MES buffer and 37.degree. C. as
described in the Examples herein. 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 (GE Healthcare) or the like, as described in the Examples
herein. When the antigen is a soluble antigen, the activity of
binding to the soluble antigen can be assessed by injecting the
antigen as an analyte onto a chip immobilized with the
antigen-binding molecule. Alternatively, when the antigen is a
membrane antigen, the activity of binding to the membrane antigen
can be assessed by injecting the antigen-binding molecule as an
analyte onto an antigen-immobilized chip.
[0061] In the present invention, the difference in the
antigen-binding activity between acidic and neutral pHs is not
particularly limited as long as the antigen-binding activity at
acidic pH is lower than that at neutral pH. However, the value of
KD(pH5.8)/KD(pH7.4), which is a ratio of dissociation constant (KD)
against an 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 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 the molecule can be produced by
technologies of those skilled in the art. When the antigen is a
soluble antigen, the antigen-binding activity can be presented in
terms of the dissociation constant (KD). Alternatively, when the
antigen is a membrane antigen, the antigen-binding activity can be
presented in terms of the apparent dissociation constant. The
dissociation constant (KD) and apparent dissociation constant
(apparent KD) can be determined by methods known to those skilled
in the art, for example, using Biacore (GE healthcare), Scatchard
plot, or FACS.
[0062] Alternatively, it is possible to use, for example, k.sub.d,
a dissociation rate constant, as an indicator for the difference in
the antigen-binding activity between acidic and neutral pHs. When
the dissociation rate constant (k.sub.d) is used as an indicator
for the difference in the binding activity instead of the
dissociation constant (KD), the value of
k.sub.d(pH5.8)/k.sub.d(pH7.4), which is a ratio of dissociation
rate constant (k.sub.d) against an antigen at pH 5.8 and that at pH
7.4, is preferably 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 k.sub.d(pH5.8)/k.sub.d(pH7.4) value is
not particularly limited, and may be any value, for example, 50,
100, or 200, as long as the molecule can be produced by
technologies common to those skilled in the art.
[0063] When the antigen is a soluble antigen, the antigen-binding
activity can be presented in terms of the dissociation rate
constant (k.sub.d). Alternatively, when the antigen is a membrane
antigen, the antigen-binding activity can be presented in terms of
the apparent dissociation rate constant. The dissociation rate
constant (k.sub.d) and apparent dissociation rate constant
(apparent k.sub.d) can be determined by methods known to those
skilled in the art, for example, using Biacore (GE healthcare) or
FACS.
[0064] In the present invention, when the antigen-binding activity
of an antigen-binding molecule is determined at different pHs, it
is preferred that the measurement conditions except for pH are
constant.
[0065] The methods for impairing the antigen-binding activity of an
antigen-binding molecule at pH 5.8 as compared to that at pH 7.4
(methods for conferring the pH-dependent binding ability) are not
particularly limited and may be any methods. Such methods include,
for example, methods for impairing the antigen-binding activity at
pH 5.8 as compared to that at pH 7.4 by substituting histidine for
amino acids in the antigen-binding molecule or inserting histidine
into the antigen-binding molecule. It is already known that an
antibody can be conferred with a pH-dependent antigen-binding
activity by substituting histidine for amino acids in the antibody
(FEBS Letter, 309(1), 8588 (1992)). Such histidine mutation
(substitution) or insertion sites are not particularly limited, and
any site is acceptable as long as the antigen-binding activity at
pH 5.8 is lowered than that at pH 7.4 (the value of
KD(pH5.8)/KD(pH7.4) gets greater) as compared to before mutation or
insertion. When the antigen-binding molecule is an antibody, such
sites include, for example, sites within an antibody variable
region. The appropriate number of histidine mutation or insertion
sites can be appropriately determined by those skilled in the art.
Histidine may be substituted or inserted at a single site, or two
or more sites. It is also possible to introduce non-histidine
mutation (mutation with amino acids other than histidine) at the
same time. Furthermore, histidine mutation may be introduced
simultaneously with histidine insertion. It is possible to
substitute or insert histidine at random using a method such as
histidine scanning, which uses histidine instead of alanine in
alanine scanning known to those skilled in the art. Alternatively,
antigen-binding molecules whose KD(pH5.8)/KD(pH7.4) is increased as
compared to before mutation can be selected from an antigen-binding
molecule library with random histidine mutation or insertion.
[0066] When histidine is substituted for amino acids of an
antigen-binding molecule or inserted between amino acids of the
molecule, it is preferred, but not required, that the
antigen-binding activity of the antigen-binding molecule at pH 7.4
after histidine substitution or insertion is comparable to that at
pH 7.4 before histidine substitution or insertion. The
"antigen-binding activity of the antigen-binding molecule at pH 7.4
after histidine substitution or insertion is comparable to that at
pH 7.4 before histidine substitution or insertion" means that even
after histidine substitution or insertion, the antigen-binding
molecule retains 10% or more, preferably 50% or more, more
preferably 80% or more, and still more preferably 90% or more of
the antigen-binding activity of before histidine substitution or
insertion. When the antigen-binding activity of the antigen-binding
molecule has been impaired due to histidine substitution or
insertion, the antigen-binding activity may be adjusted by
introducing substitution, deletion, addition, and/or insertion of
one or more amino acids into the antigen-binding molecule so that
the antigen-binding activity becomes comparable to that before
histidine substitution or insertion. The present invention also
includes such antigen-binding molecules having a comparable binding
activity as a result of substitution, deletion, addition, and/or
insertion of one or more amino acids after histidine substitution
or insertion.
[0067] Alternative methods for impairing the antigen-binding
activity of an antigen-binding molecule at pH 5.8 as compared to
that at pH 7.4 include methods of substituting non-natural amino
acids for amino acids in an antigen-binding molecule or inserting
non-natural amino acids into amino acids of an antigen-binding
molecule. It is known that the pKa can be artificially controlled
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). Thus, in the present invention, non-natural amino
acids can be used instead of histidine described above. Such
non-natural amino acid substitution and/or insertion may be
introduced simultaneously with the histidine substitution and/or
insertion described above. Any non-natural amino acids may be used
in the present invention. It is possible to use non-natural amino
acids known to those skilled in the art.
[0068] Furthermore, when the antigen-binding molecule is a
substance having an antibody constant region, alternative methods
for impairing the antigen-binding activity of the antigen-binding
molecule at pH 5.8 as compared to that at pH 7.4 include methods
for modifying the antibody constant region contained in the
antigen-binding molecule. Such methods for modifying the antibody
constant region include, for example, methods for substituting a
constant region described in the Examples herein.
[0069] Alternative methods for modifying the antibody constant
region include, for example, methods to assess various constant
region isotypes (IgG1, IgG2, IgG3, and IgG4) and select an isotype
that impairs the antigen-binding activity at pH 5.8 (increases the
dissociation rate at pH 5.8). Alternatively, methods include those
for impairing the antigen-binding activity at pH 5.8 (increasing
the dissociation rate at pH 5.8) by substituting amino acids in the
amino acid sequence of a wild-type isotype (the amino acid sequence
of wild type IgG1, IgG2, IgG3, or IgG4). The sequence of the hinge
region of an 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 impair the antigen-binding activity at pH
5.8 (to increase the dissociation rate at pH 5.8) by considering
the type of antigen or epitope. Furthermore, since the difference
in the hinge region amino acid sequence has a significant influence
on the antigen-binding activity, preferred amino acid substitution
sites in the amino acid sequence of a wild type isotype are assumed
to be within the hinge region.
[0070] When the antigen-binding activity of the antigen-binding
substance at pH 5.8 is weakened compared to that at pH 7.4 (when
KD(pH5.8)/KD(pH7.4) value is increased) by the above described
methods and such, it is generally preferable that the
KD(pH5.8)/KD(pH7.4) value be two times or more, more preferably
five times or more, and even more preferably ten times or more as
compared to that of the original antibody, although the invention
is not particularly limited thereto.
[0071] Herein, the "improvement of the pharmacokinetics" means
prolongation of the time required for the elimination of the
antigen-binding molecule from plasma (for example, reaching the
state where the antigen-binding molecule cannot return to the
plasma due to degradation in cells, or other reasons) after
administration to an animal such as human, mouse, rat, monkey,
rabbit, or dog, as well as prolongation of the plasma retention
time of the antigen-binding molecule being in a form capable of
binding to antigens (for example, being in an antigen-free form)
during the period until it is eliminated from the plasma after
administration. Even if an antigen-binding molecule is circulated
in plasma, it cannot bind to an antigen when it already binds to
another antigen. Accordingly, the period where the antigen-binding
molecule can newly binds to another antigen is prolonged (the
chance to bind another antigen increases) by prolonging the period
where the antigen-binding molecule is in an antigen-free form. This
makes it possible to shorten the period where the antigen is free
from antigen-binding molecules in vivo (in other words, to prolong
the period where the antigen is bound by an antigen-binding
molecule). For example, the ratio of antigens bound to
antigen-binding molecules against the antigens in the body in
plasma (total of antigen molecules bound to and free from the
antigen-binding molecules) generally decreases in a certain period
of time after the administration of the antigen-binding molecules.
However, such decrease can be suppressed (for example, the degree
of decrease can be made smaller) by prolonging the retention time
of the antigen-binding molecules in a form capable of binding to
antigens. This results in an increase in the ratio of antigens
bound to antigen-binding molecules against the antigens in the body
in a certain period of time after antibody administration.
[0072] Specifically, in the present invention, the "improvement of
the pharmacokinetics" does not necessarily mean the prolongation
(extension) of the time required for the elimination of the
antigen-binding molecule after administration. Even if the time
required for the elimination of the antigen-binding molecule after
administration remains unchanged, the pharmacokinetics can be said
"improved" in the present invention if
[0073] the plasma retention time of the antigen-binding molecule
being in a form capable of binding to an antigen (for example, the
antigen-binding molecule being in an antigen-free form) is
prolonged;
[0074] the period where the antigen is free from an antigen-binding
molecule in the body is shortened (in other words, the period where
the antigen-binding molecule is bound to an antigen is prolonged);
and
[0075] the ratio of antigens bound to antigen-binding molecules
against the antigens in the body is increased. Thus, in the present
invention, the "improvement of the pharmacokinetics" encompasses at
least:
[0076] (1) prolongation of the time required for the elimination of
the antigen-binding molecule from plasma after administration of
the antigen-binding molecule;
[0077] (2) prolongation of the plasma retention time of the
antigen-binding molecule in a form capable of binding to an antigen
after administration of the antigen-binding molecule;
[0078] (3) shortening of the period where the antigen is free from
an antigen-binding molecule in the body after administration of the
antigen-binding molecule (prolongation of the period where the
antigen-binding molecule is bound to an antigen in the body);
and
[0079] (4) increase in the ratio of antigens bound to
antigen-binding molecules to the antigen in the body.
[0080] When the antigen is a soluble antigen present in plasma,
even if the pharmacokinetics of the antigen-binding molecule (rate
of elimination from plasma) is equivalent, there are cases where
elimination of antigen bound to the antigen-binding molecule is
accelerated. Reducing the pharmacokinetics of the antigen
(accelerating elimination from plasma) results in the relative
improvement of the pharmacokinetics of the antigen-binding
molecule, and thus, leads to the prolongation of the time of the
antigen-binding molecule present in plasma in a form capable of
binding to antigens. Thus, in one embodiment, the "improvement of
the pharmacokinetics" of antigen-binding molecules of the present
invention includes increasing the rate of eliminating soluble
antigens from plasma after administration of the antigen-binding
molecules (the ability of the antigen-binding molecule to eliminate
antigens from plasma).
[0081] In the present invention, when the antigen is a membrane
antigen, whether a single antigen-binding molecule binds to
multiple antigens can be assessed by testing whether the
pharmacokinetics of the antigen-binding molecule is improved.
Whether the "pharmacokinetics is improved" can be assessed by the
following method. For example, whether the time required for the
elimination of an antigen-binding molecule after administration is
prolonged can be assessed by determining any one of parameters for
the antigen-binding molecule, such as half-life in plasma, mean
plasma retention time, and clearance in plasma ("Pharmacokinetics:
Enshu-niyoru Rikai (Understanding through practice)" Nanzando). For
example, when the half-life in plasma or mean plasma retention time
of an antigen-binding molecule administered to mice, rats, monkeys,
rabbits, dogs, humans, or other animals is prolonged, the
pharmacokinetics of the antigen-binding molecule is judged to be
improved. These parameters can be determined by methods known to
those skilled in the art. For example, the parameters can be
appropriately assessed by noncompartmental analysis using
pharmacokinetics analysis software WinNonlin (Pharsight) according
to the appended instruction manual.
[0082] Alternatively, whether the plasma retention time of an
antigen-binding molecule in a form capable of binding to antigens
after administration of the antigen-binding molecule is prolonged
can be assessed by measuring the plasma concentration of the
antigen-free antigen-binding molecule and determining any one of
parameters for the antigen-free antigen-binding molecule, such as
half-life in plasma, mean plasma retention time, and clearance in
plasma. The concentration of the antigen-free antigen-binding
molecule in plasma can be measured by methods known to those
skilled in the art. For example, such measurements are described in
Clin Pharmacol. 2008 April; 48(4):406-17.
[0083] Furthermore, whether the period where an antigen is free
from the antigen-binding molecules in the body after administration
of the antigen-binding molecules is shortened (the period where the
antigen-binding molecule is bound to an antigen in the body is
prolonged) can be assessed by determining the plasma concentration
of the unbound antigen that is free from antigen-binding molecules,
and considering the period where the concentration of free antigen
in plasm or the amount ratio of free antigen against the total
antigen remains low. The plasma concentration of the free antigen
or amount ratio of free antigen against total antigen can be
determined by methods known to those skilled in the art. For
example, such measurements are described in Pharm Res. 2006
January; 23(1):95-103. Alternatively, when the antigen exerts some
function in vivo, whether the antigen is bound by an
antigen-binding molecule that neutralizes the antigen's function
(antagonistic molecule) can be assessed by testing whether the
function of the antigen is neutralized. Whether the function of the
antigen is neutralized can be assessed by assaying an in vivo
marker that reflects the function of the antigen. Whether the
antigen is bound by an antigen-binding molecule that activates the
function of the antigen (agonistic molecule) can be assessed by
assaying an in vivo marker that reflects the function of the
antigen.
[0084] There is no particular limitation on the determination of
the plasma concentration of free antigen and amount ratio of free
antigen against total antigen, and in vivo marker assay, but the
determination is preferably carried out after a certain period
following administration of an antigen-binding substance. In the
present invention, such a period following administration of an
antigen-binding substance is not particularly limited, and an
appropriate period can be determined by those skilled in the art
depending on the properties of the administered antigen-binding
substance and the like. Examples of the period are: one day after
administration of an antigen-binding substance; three days after
administration of an antigen-binding substance, seven days after
administration of an antigen-binding substance, 14 days after
administration of an antigen-binding substance, and 28 days after
administration of an antigen-binding substance.
[0085] In the present invention, it is preferred to improve the
pharmacokinetics in human. Even when the plasma retention in human
is difficult to determine, it can be predicted based on the plasma
retention in mice (for example, normal mice, human
antigen-expressing transgenic mice, and human FcRn-expressing
transgenic mice) or monkeys (for example, cynomolgus monkeys).
[0086] Methods for determining the retention in plasma are not
particularly limited. The determination can be carried out, for
example, according to the methods described in the Examples
herein.
[0087] Whether an antigen-binding molecule is capable of binding to
antigens multiple times can be assessed by testing whether the
antigen bound to an antigen-binding molecule under the same neutral
condition as plasma dissociates under the same acidic condition as
endosome and how many antigens the antigen-binding molecule can
rebind to under the neutral condition. Specifically, the assessment
can be carried out by allowing the antigen-binding molecule and
antigen to form a complex under the neutral condition, exposing the
complex to an acidic condition for a predetermined period, and then
testing whether the antigen-binding molecule can rebind to an
antigen under the neutral condition, using a device for assaying
antigen-binding molecule-antigen reactions, such as Biacore. When
the antigen-binding capacity of the antigen-binding molecule
conferred with the pH-dependent binding ability has been improved
to twice that of the antigen-binding molecule before modification,
the number of times of binding of the antigen-binding molecule
conferred with the pH-dependent binding ability can be judged to be
increased to twice that of the antigen-binding molecule before
modification. Alternatively, when the antigen is a membrane antigen
and thus the antigen-binding molecule is eliminated from plasma
through antigen-mediated uptake and degradation in a lysosome,
whether the number of times of binding of the antigen-binding
molecule conferred with the pH-dependent binding ability is
increased as compared to that before modification can be assessed
by comparing the pharmacokinetics or duration of antigen binding
between the antigen-binding molecule conferred with the
pH-dependent binding ability and the antigen-binding molecule
before modification. For example, when the antigen-binding duration
of the antigen-binding molecule conferred with the pH-dependent
binding ability is prolonged twice that of the antigen-binding
molecule before modification, the number of times of binding of the
antigen-binding molecule conferred with the pH-dependent binding
ability is judged to be increased to twice that of the
antigen-binding molecule before modification. Alternatively, when
the plasma concentration of an unbound antigen, which is free from
the antigen-binding molecule, is determined and the period where
the plasma concentration of the free antigen or the amount ratio of
the free antigen against the total antigen remains low is prolonged
to twice, the number of times of binding of the antigen-binding
molecule conferred with the pH-dependent binding ability is judged
to be increased to twice that of the antigen-binding molecule
before modification.
[0088] When the antigen is a soluble antigen, if the antigen bound
to an antigen-binding molecule under the neutral condition in
plasma dissociates in an endosome, and the antigen-binding molecule
returns to the plasma, the antigen-binding molecule can again bind
to an antigen under the neutral condition in plasma. Thus, an
antigen-binding molecule that has the characteristics to dissociate
with an antigen in acidic condition of an endosome is capable of
binding to antigens multiple times. Compared to when the antigen
bound to an antigen-binding molecule does not dissociate in an
endosome (the antigen remains bound to the antigen-binding molecule
when returning to plasma), when the antigen bound to an
antigen-binding molecule dissociates in endosomes, the antigen is
delivered to a lysosome and then degraded, and thus, the rate of
elimination of the antigen from plasma increases. That is, it is
also possible to determine whether the antigen-binding molecule is
capable of binding to antigens multiple times using the rate of
elimination of antigen from plasma as an index. The rate of
elimination of the antigen from plasma can be determined, for
example, by administering the antigens (e.g., membrane antigen) and
antigen-binding molecules in vivo, and then measuring the
concentration of antigens in plasma. When an antigen (e.g.,
membrane antigen) is produced or secreted in vivo, the plasma
antigen concentration is reduced if the rate of elimination of the
antigen from plasma is increased. Thus, it is also possible to
determine whether the antigen-binding molecule is capable of
binding to antigens multiple times using the plasma antigen
concentration as an index.
[0089] Herein, "increasing the number of times of antigen-binding
of the antigen-binding molecule" means that the number of cycles is
increased when taking as one cycle the process where an
antigen-binding molecule administered to human, mouse, monkey, or
such binds to an antigen and is internalized into a cell.
Specifically, herein, "the antigen-binding molecule binds twice to
an antigen" means that the antigen-binding molecule bound by an
antigen is internalized into a cell and released in an antigen-free
form to the outside of the cell, and the released antigen-binding
molecule rebinds to another antigen and is internalized into a cell
again.
[0090] When the antigen-binding molecule is internalized into a
cell, it may be in a form bound by a single antigen, or two or more
antigens.
[0091] Herein, "the number of times of antigen-binding of an
antigen-binding molecule is increased" does not necessarily mean
that the number of times of antigen-binding increases in every
antigen-binding molecules. For example, among antigen-binding
molecules in an antigen-binding-molecule composition, the
proportion of antigen-binding molecules that bind to antigens twice
or more times may increase, or the average number of binding events
of antigen-binding molecules in an antigen-binding-molecule
composition may increase.
[0092] In the present invention, it is preferred that the number of
times of antigen-binding of an antigen-binding molecule increases
when the molecule is administered to a human. However, when it is
difficult to determine the number of times of antigen-binding in
human, the number in human may be predicted based on the results
obtained by in vitro assay or measurement using mice (for example,
antigen-expressing transgenic mice and human FcRn-expressing
transgenic mice) or monkeys (for example, cynomolgus monkeys).
[0093] In the present invention, it is preferred that an
antigen-binding molecule binds to antigens twice or more times. For
example, it is preferred that, of the antigen-binding molecules in
an antigen-binding-molecule composition, at least 10% or more,
preferably 30% or more, more preferably 50% or more, and still more
preferably 80% or more (for example, 90% or more, 95% or more, and
so on) bind to antigens twice or more times.
[0094] Herein, "increasing the number of antigens that can be bound
by an antigen-binding molecule" means increasing the number of
antigens that can be bound by an antigen-binding molecule during
the period until the antigen-binding molecule is degraded in a
lysosome of a cell after administration of the antigen-binding
molecule to an animal such as human, mouse, or monkey.
[0095] In general, antibodies such as IgG have two binding domains,
and thus a single antibody binds to a maximum of two antigens. An
antibody bound to antigen(s) is internalized into a cell, and the
antibody and antigen(s) are degraded in a lysosome. In general,
antibodies such as IgG can bind to a maximum of two antigens. When
the antigen-binding activity of an antigen-binding molecule such as
an antibody at the endosomal pH is impaired as compared to that at
the plasma pH by the methods of the present invention, the
antigen-binding molecule such as an antibody internalized into a
cell dissociates the antigen and is released to the outside of the
cell, and thus can bind to another antigen again. In other words,
the methods of the present invention enable for an antigen-binding
molecule to bind to more antigens than the number of its
antigen-binding sites. Specifically, by using the methods of the
present invention, for example, IgG having two antigen-binding
sites can bind to three or more antigens, preferably four or more
antigens, during a period until the antibody is degraded after
administration. For example, when the antibody is a neutralizing
antibody, "increasing the number of antigens that can be bound by
an antigen-binding molecule" is interchangable with "increasing the
number of antigens that the antigen-binding molecule can
neutralize". Thus, "bind" can be replaced with "neutralize" when
the antibody is a neutralizing antibody.
[0096] In the present invention, "increasing the number of antigens
that can be bound by an antigen-binding molecule" does not
necessarily mean increasing the number of antigens that can be
bound by every antigen-binding molecule. For example, the average
number of antigens that can be bound by an antigen-binding molecule
in an antigen-binding-molecule composition may increase, or the
proportion of antigen-binding molecules that can bind to more
antigens than the number of their antigen-binding sites may
increase.
[0097] In the present invention, it is preferred that the number of
antigens that can be bound by an antigen-binding molecule increases
when the molecule is administered to a human. However, when it is
difficult to determine the number in human, it may be predicted
based on the results obtained by in vitro assay or measurement
using mice (for example, antigen-expressing transgenic mice and
human FcRn-expressing transgenic mice) or monkeys (for example,
cynomolgus monkeys). When the antibody is a neutralizing antibody,
the above-described number of times of antigen-binding of the
antigen-binding molecule is generally assumed to correlates with
the number of antigens which can be neutralized by an
antigen-binding molecule. Thus, the number of antigens which can be
neutralized by an antigen-binding molecule can be determined by the
same methods described above for determining the number of times of
binding of an antigen-binding molecule.
[0098] Furthermore, the present invention provides methods for
binding an antigen-binding molecule to antigens twice or more times
in the body, by administering an antigen-binding molecule whose
antigen-binding activity at acidic pH is lower than that at neutral
pH.
[0099] The present invention also relates to methods for
neutralizing antigens that are greater in number than the number of
antigen-binding sites of an antigen-binding molecule having the
neutralizing activity, by administering the antigen-binding
molecule whose antigen-binding activity at acidic pH is lower than
that at neutral pH. Preferably, the present invention relates to
methods for neutralizing three or more antigens, preferably four or
more antigens by administering IgG whose antigen-binding activity
at acidic pH is lower than that at neutral pH.
[0100] The present invention also relates to methods for
dissociating within a cell an antigen from an extracellularly-bound
antigen-binding molecule by impairing the antigen-binding ability
of the antigen-binding molecule at acidic pH as compared to that at
neutral pH. In the present invention, the antigen may be
dissociated from the antigen-binding molecule anywhere within a
cell; however, it is preferred that the antigen is dissociated
within an early endosome. In the present invention, "an antigen is
dissociated within a cell from an extracellularly-bound
antigen-binding molecule" does not necessarily mean that every
antigen internalized into a cell via binding to the antigen-binding
molecule is dissociated from the antigen-binding molecule within
the cell. It is acceptable that the proportion of antigen that is
dissociated from the antigen-binding molecule within a cell
increases when compared to before impairing the antigen-binding
ability of the antigen-binding molecule at acidic pH as compared to
that at neutral pH.
[0101] Furthermore, the present invention relates to methods for
enhancing the intracellular binding of an antigen-binding molecule
free from an antigen to FcRn by impairing the antigen-binding
ability of the antigen-binding molecule at acidic pH as compared to
that at neutral pH. In general, FcRn binds to an antigen-binding
molecule within an endosome. However, an antigen-binding molecule
bound to a membrane antigen is assumed not to bind to FcRn. Thus,
in a preferred embodiment, when the antigen is a membrane-bound
antigen, the present invention includes methods for enhancing the
endosomal dissociation of antigens from antigen-binding molecules
and thus enhancing the FcRn binding of the antigen-binding
molecules, by impairing the antigen-binding ability of an
antigen-binding molecule at the endosomal pH (acidic pH) as
compared to that at the plasma pH (neutral pH). When the antigen is
a soluble antigen, the antigen-binding molecule can bind to FcRn in
the presence or absence of the antigen. If dissociation of the
antigen from the antigen-binding molecule within endosomes can be
promoted by impairing the antigen-binding ability of the
antigen-binding molecule at intraendosomal (acidic) pH as compared
to that at plasma (neutral) pH, the FcRn binding of the
antigen-binding molecule that is "free from an antigen" can be
enhanced by the methods of the present invention.
[0102] Regardless of whether an antigen is membrane-bound or
soluble, if an antigen-binding molecule free from an antigen can
return to plasma with FcRn, the antigen-binding molecule can bind
to the antigen again. By repeating this process, the
antigen-binding molecule can bind to the antigen multiple times. In
the present invention, "enhancing the FcRn binding of an
antigen-binding molecule within a cell" does not necessarily mean
that every antigen-binding molecule binds to FcRn. It is acceptable
that the proportion of an antigen-binding molecule free from an
antigen that binds to FcRn within a cell increases when compared to
before impairing the antigen-binding ability of the antigen-binding
molecule at the endosomal pH as compared to that at the plasma pH.
Preferred antigen-binding molecules in the methods of the present
invention for enhancing the intracellular binding between the
antigen-binding molecule and FcRn include, for example,
antigen-binding molecules that bind to membrane-bound antigens
(membrane antigens) such as membrane proteins. Other preferable
antigen-binding molecules include antigen-binding molecules that
bind to soluble antigens such as soluble proteins.
[0103] The methods of enhancing the binding of an antigen-binding
molecule and FcRn within a cell are alternatively expressed as the
methods of promoting the FcRn binding of an antigen-binding
molecule within a cell, for example, within endosomes.
[0104] Furthermore, the present invention relates to methods for
releasing an antigen-binding molecule, which has been bound to an
antigen and internalized into a cell, in an antigen-free form to
the outside of the cell, by impairing the antigen-binding ability
of the antigen-binding molecule at acidic pH as compared that at
neutral pH. In the present invention, "releasing an antigen-binding
molecule, which has been bound to an antigen and internalized into
a cell, in an antigen-free form to the outside of the cell" does
not necessarily mean that every antigen-binding molecule, which has
been bound to an antigen and internalized into a cell, is released
in an antigen-free form to the outside of the cell. It is
acceptable that the proportion of antigen-binding molecules that
are released to the outside of the cell increases when compared to
before impairing the antigen-binding ability of the antigen-binding
molecule at acidic pH as compared to that at neutral pH. It is
preferred that the antigen-binding molecule released to the outside
of a cell retains the antigen-binding ability. Furthermore, the
method of releasing an antigen-binding molecule, which has been
bound to an antigen and internalized into a cell, in an
antigen-free form to the outside of the cell can also be referred
to as a method of conferring to the antigen-binding molecule a
property that the antigen-binding molecule becomes more easily
released to the outside of the cell in an antigen-free form when
the antigen-binding molecule is bound to an antigen and
internalized into a cell.
[0105] Furthermore, the present invention relates to methods for
increasing the ability of the antigen-binding molecules to
eliminate antigens in plasma by impairing the antigen-binding
ability of the antigen-binding molecules at acidic pH as compared
to that at neutral pH. In the present invention "the ability to
eliminate antigens in plasma" refers to the ability to eliminate
from plasma antigens that are present in plasma, when the
antigen-binding molecules are administered in vivo or are secreted
in vivo. Thus, in the present invention, "increasing the ability of
the antigen-binding molecule to eliminate antigen in plasma" means
that the rate of elimination of antigens from plasma when the
antigen-binding molecules are administered in vivo is accelerated
as compared to that before lowering the antigen-binding ability of
the antigen-binding molecules at acidic pH as compared to that at
neutral pH. Whether the ability of the antigen-binding molecule to
eliminate antigens in plasma is increased can be determined by, for
example, administering soluble antigens and antigen-binding
molecules in vivo, and then measuring the concentration of soluble
antigens in plasma. When the concentration of soluble antigens in
plasma after the administration of soluble antigens and
antigen-binding molecules is reduced by lowering the
antigen-binding ability of the antigen-binding molecule at acidic
pH than that at neutral pH, it can be determined that the ability
of the antigen-binding molecule to eliminate antigens in plasma is
increased.
[0106] The present invention also relates to methods for improving
the pharmacokinetics of an antigen-binding molecule by substituting
histidine or non-natural amino acid for at least one amino acid in
the antigen-binding molecule or inserting histidine or non-natural
amino acid into the molecule.
[0107] Furthermore, the present invention provides methods for
increasing the number of times of antigen-binding of an
antigen-binding molecule by substituting histidine or non-natural
amino acid for at least one amino acid in the antigen-binding
molecule or inserting histidine or non-natural amino acid into the
molecule.
[0108] In addition, the present invention relates to methods for
increasing the number of antigens that can be bound by an
antigen-binding molecule by substituting histidine or non-natural
amino acid for at least one amino acid in the antigen-binding
molecule or inserting histidine or non-natural amino acid into the
molecule.
[0109] The present invention also provides methods for dissociating
an antigen within a cell from an extracellularly-bound
antigen-binding molecule by substituting at least one amino acid in
the antigen-binding molecule with histidine or non-natural amino
acid, or inserting histidine or non-natural amino acid into the
molecule.
[0110] The present invention also provides methods for releasing an
antigen-binding molecule, which has been bound to an antigen and
internalized into a cell, in an antigen-free form to the outside of
the cell by substituting at least one amino acid in the
antigen-binding molecule with histidine or non-natural amino acid,
or inserting histidine or non-natural amino acid into the
molecule.
[0111] The present invention also provides methods for increasing
the ability of the antigen-binding molecule to eliminate antigens
in plasma by substituting at least one amino acid in the
antigen-binding molecule with histidine or non-natural amino acid,
or inserting histidine or non-natural amino acid into the
molecule.
[0112] The site of histidine or non-natural amino acid mutation
(substitution, insertion, etc.) is not particularly limited. A
histidine or non-natural amino acid may be substituted or inserted
at any site. Preferred sites of histidine or non-natural amino acid
substitution or insertion include, for example, sites within a
region that has an impact on the antigen-binding ability of the
antigen-binding molecule. For example, when the antigen-binding
molecule is an antibody, such sites include an antibody variable
region or CDR. The number of histidine or non-natural amino acid
mutations is not particularly limited. Histidine or non-natural
amino acid may be substituted or inserted at a single site, or at
two or more sites. Furthermore, a deletion, addition, insertion,
and/or substitution of other amino acids may be introduced
simultaneously with the histidine or non-natural amino acid
substitution or insertion.
[0113] In the present invention, when the antigen-binding molecule
is an antibody, possible sites of histidine or non-natural amino
acid substitution include, for example, sites within the CDR
sequence or sequence responsible for the CDR structure of an
antibody. Such sites include, for example, the sites listed below.
The amino acid positions are numbered based on the Kabat numbering
(Kabat E A et al., (1991) Sequences of Proteins of Immunological
Interest, NIH).
[0114] Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61,
H62, H63, H64, H65, H99, H100b, and H102
[0115] Light chain: L24, L27, L28, L32, L53, L54, L56, L90, L92,
and L94
[0116] Among the above sites, H32, H61, L53, L90, and L94 could be
universal modification sites.
[0117] When the antigen is the IL-6 receptor (e.g., human IL-6
receptor), preferable modification sites include the following.
However, the modification sites are not particularly limited
thereto.
[0118] Heavy chain: H27, H31, H32, H35, H50, H58, H61, H62, H63,
H64, H65, H100b, and H102
[0119] Light chain: L24, L27, L28, L32, L53, L56, L90, L92, and
L94
[0120] When histidine or non-natural amino acid is substituted at
multiple sites, preferred combinations of substitution sites
include, for example, the combination of H27, H31, and H35;
combination of H27, H31, H32, H35, H58, H62, and H102; combination
of L32 and L53; and combination of L28, L32, and L53. In addition,
preferred combinations of substitution sites of heavy and light
chains include the combination of H27, H31, L32, and L53.
[0121] When the antigen is IL-6 (e.g., human IL-6), preferable
modification sites include the following. However, the modification
sites are not particularly limited thereto.
[0122] Heavy chain: H32, H59, H61, and H99
[0123] Light chain: L53, L54, L90, and L94
[0124] When the antigen is the IL-31 receptor (e.g., human IL-31
receptor), preferable modification sites include H33. However, the
modification sites are not particularly limited thereto.
[0125] Regarding the above sites, only one site may be substituted
with histidine or non-natural amino acid. Alternatively, multiple
sites may be substituted with histidine or non-natural amino
acid.
[0126] The methods of the present invention are applicable to any
antigen-binding molecules, regardless of the type of target
antigen.
[0127] The antigen-binding molecules of the present invention are
not particularly limited as long as they have the specific binding
activity to an antigen of interest. Preferred antigen-binding
molecules of the present invention include, for example, substances
having an antigen-binding domain of an antibody. The
antigen-binding domain of an antibody includes, for example, CDR
and variable region. When the antigen-binding domain of an antibody
is CDR, the antigen-binding molecule may include all of the six
CDRs of a whole antibody, or one, or two or more of them.
Alternatively, when an antigen-binding molecule includes CDR as a
binding domain of an antibody, the CDR may include amino acid
deletion, substitution, addition, and/or insertion, or may be a
partial CDR.
[0128] Furthermore, when the antigen-binding molecule includes an
antibody constant region, the present invention relates to methods
for improving the pharmacokinetics of antigen-binding molecules by
modification (for example, amino acid substitution, deletion,
addition, and/or insertion) of the antibody constant region in the
antigen-binding molecule.
[0129] In addition, when the antigen-binding molecule includes an
antibody constant region, the present invention provides methods
for increasing the number of times of antigen-binding of an
antigen-binding molecule by modification (for example, amino acid
substitution, deletion, addition, and/or insertion) of the antibody
constant region in the antigen-binding molecule.
[0130] Furthermore, when the antigen-binding molecule includes an
antibody constant region, the present invention relates to methods
for increasing the number of antigens that can be bound by an
antigen-binding molecule by modification (for example, amino acid
substitution, deletion, addition, and/or insertion) of the antibody
constant region in the antigen-binding molecule.
[0131] Furthermore, when the antigen-binding molecule includes an
antibody constant region, the present invention relates to methods
for dissociating within a cell an antigen from an
extracellularly-bound antigen-binding molecule by modification (for
example, amino acid substitution, deletion, addition, and/or
insertion) of the antibody constant region in the antigen-binding
molecule.
[0132] Furthermore, when the antigen-binding molecule includes an
antibody constant region, the present invention relates to methods
for releasing an antigen-binding molecule, which has been bound to
an antigen and internalized into a cell, in an antigen-free form to
the outside of the cell, by modification (for example, amino acid
substitution, deletion, addition, and/or insertion) of the antibody
constant region in the antigen-binding molecule.
[0133] Furthermore, when the antigen-binding molecule includes an
antibody constant region, the present invention relates to methods
for increasing the ability of an antigen-binding molecule to
eliminate antigens in plasma by modification (for example, amino
acid substitution, deletion, addition, and/or insertion) of the
antibody constant region in the antigen-binding molecule.
[0134] In a preferred embodiment, the antigen-binding substance of
the present invention includes antigen-binding substances including
an FcRn-binding region. After internalized into cells,
antigen-binding substances including an FcRn-binding region can
return to the plasma by the FcRn salvage pathway. The FcRn-binding
region is preferably a domain that directly binds to FcRn.
Preferred FcRn-binding region includes, for example, antibody Fc
regions. However, the FcRn-binding region of the present invention
may be a region that can bind to a polypeptide having the ability
to bind to FcRn such as albumin or IgG, since such region that can
bind to the polypeptide having FcRn-binding ability can binds
indirectly to FcRn via albumin, IgG, etc.
[0135] Antigens recognized by antigen-binding molecules such as
antibodies of interest in the methods of the present invention are
not particularly limited. Such antibodies of interest may recognize
any antigen. Antibodies whose pharmacokinetics is to be improved by
the methods of the present invention include, for example,
antibodies that recognize membrane antigens such as receptor
proteins (membrane-bound receptors and soluble receptors) and cell
surface markers, and antibodies that recognize soluble antigens
such as cytokines. Preferred examples of membrane antigens of the
present invention include membrane proteins. Examples of soluble
antigens of the present invention include soluble proteins.
Antigens recognized by antibodies whose pharmacokinetics is to be
improved by the methods of the present invention include, for
example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-11, IL-12, IL-15, IL-31, IL-23, IL-2 receptor, IL-6
receptor, OSM receptor, gp130, IL-5 receptor, CD40, CD4, Fas,
osteopontin, CRTH2, CD26, PDGF-D, CD20, monocyte chemotactic
factor, CD23, TNF-.alpha., HMGB-1, .alpha.4 integrin, ICAM-1, CCR2,
CD11a, CD3, IFN.gamma., BLyS, HLA-DR, TGF-.beta., CD52, and IL-31
receptor. Particularly preferred antigens include IL-6
receptor.
[0136] Furthermore, the antigen-binding molecule of interest in the
methods of the present invention includes antigen-binding molecules
having an antagonistic activity (antagonistic antigen-binding
molecules) and antigen-binding molecules having an agonistic
activity (agonistic antigen-binding molecules). In a preferred
embodiment, the antigen-binding molecule includes antagonistic
antigen-binding molecules, in particular, antagonistic
antigen-binding molecules that recognize membrane antigens such as
receptors, or soluble antigens such as cytokines. For example, an
antagonistic antigen-binding molecule that recognizes a receptor
inhibits the ligand-receptor binding by binding to the receptor,
and thus inhibits the signaling mediated via the receptor.
[0137] In the present invention, the antigen-binding molecule of
interest is not particularly limited, and may be any
antigen-binding molecules. The antigen-binding molecule of the
present invention preferably includes both antigen-binding activity
(antigen-binding region) and FcRn-binding region. In particular,
preferred antigen-binding molecule of the present invention
includes a region that binds to human FcRn. The antigen-binding
molecule including both antigen-binding activity and FcRn-binding
region includes, for example, antibodies. 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 limited; the IgG belonging to any isotype
(subclass) such as IgG1, IgG2, IgG3, or IgG4 can be used.
Furthermore, amino acid mutations (e.g., M73) may be introduced
into the constant region of any of these IgG isotypes. Amino acid
mutations to be introduced include, for example, those potentiate
or impair the binding to Fc.gamma. receptor (Proc Natl Acad Sci
USA. 2006 Mar. 14; 103(11):4005-10) and those potentiate or impair
the binding to FcRn (J Biol Chem. 2001 Mar. 2; 276(9):6591-604),
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.
[0138] When the antigen-binding molecule of interest of the present
invention is an antibody, it may be an antibody derived from any
animal, such as a mouse antibody, human antibody, rat antibody,
rabbit antibody, goat antibody, or camel antibody. Furthermore, the
antibody may be a modified antibody, for example, a chimeric
antibody, and in particular, a modified antibody including amino
acid substitution in the sequence of a humanized antibody, etc. The
antibodies also include bispecific antibodies, antibody
modification products linked with various molecules, and
polypeptides including antibody fragments.
[0139] "Chimeric antibodies" are antibodies prepared by combining
sequences derived from different animals. Specifically, the
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.
[0140] "Humanized antibodies", also referred to as reshaped human
antibodies, are 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).
[0141] Bispecific antibody 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.
[0142] Furthermore, polypeptides including antibody fragments
include, for example, Fab fragments, F(ab')2 fragments, scFv (Nat
Biotechnol. 2005 September; 23(9):1126-36), domain antibodies (dAb)
(WO 2004/058821, WO 2003/002609), scFv-Fc (WO 2005/037989), dAb-Fc,
and Fc fusion proteins. Of these, molecules including an Fc domain
have the activity of binding to FcRn, and are therefore suitable
for use in the methods discovered in the present invention.
[0143] Further, the antigen-binding molecules that are applicable
to the present invention may be antibody-like molecules. An
antibody-like 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 manner, it
is possible for a single molecule to bind multiple target
molecules.
[0144] Furthermore, the antigen-binding molecule may be a receptor
protein or a receptor-Fc fusion protein that binds to a target,
including, for example, TNFR-Fc fusion protein, IL1R-Fc fusion
protein, VEGFR-Fc fusion protein, and CTLA4-Fc fusion protein (Nat
Med. 2003 January; 9(1):47-52; BioDrugs. 2006; 20(3):151-60). If
such receptor proteins and receptor-Fc fusion proteins can bind to
target molecules in a pH-dependent manner, it is possible for a
single molecule to bind multiple target molecules.
[0145] Moreover, the antigen-binding molecule may be an artificial
ligand protein or artificial ligand fusion protein that binds to a
target and has the neutralizing effect, and includes, for example,
mutant IL-6 (EMBO J. 1994 Dec. 15; 13(24):5863-70). If such
artificial ligand proteins and artificial ligand fusion proteins
can bind to target molecules in a pH-dependent manner, it is
possible for a single molecule to bind multiple target
molecules.
[0146] Furthermore, the antibodies of the present invention may
include 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).
[0147] Although the methods of the present invention are not
limited to any specific theory, the relationship between making the
antigen-binding ability at acidic pH weaker as compared to that at
neutral pH, the improvement of the pharmacokinetics, and the
multiple-time binding to the antigen can be explained as follows,
for instance.
[0148] For example, when the antibody 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 and 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.
[0149] The relatively prolonged retention (slow elimination) of IgG
molecules in the plasma is due to the function of FcRn which is
known as a salvage receptor of IgG molecules. When taken up into
endosomes via pinocytosis, IgG molecules bind to FcRn expressed in
the endosomes under the acidic condition in the endosomes. While
IgG molecules that did not bind to FcRn transfer to lysosomes where
they are degraded, IgG molecules that bound to FcRn translocate to
the cell surface and return again in the plasma by dissociating
from FcRn under the neutral condition in the plasma.
[0150] Alternatively, when the antigen is an antigen 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 cells via FcRn. However, since the
antibodies are released to the outside of cells, 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.
[0151] The present inventors reasoned that, when antibodies that
bound to antigens such as membrane antigens are taken up into
endosomes by internalization, while the antibodies that are kept
bound to the antigens translocate to lysosomes and are degraded,
the IgG antibodies whose antigens dissociated in the endosomes
could bind to FcRn that are expressed in the endosomes.
Specifically, the present inventors discovered that an antibody
that strongly binds to an antigen in the plasma but weakly binds to
the antigen within the endosome can bind to an antigen in plasma
and be taken up while kept forming a complex with the antigen into
endosomes in the cells via internalization; dissociate from the
antigen in the endosome; then bind to FcRn and translocate to the
cell surface; and return again in the plasma in a state not bound
to antigens to neutralize multiple membrane-bound antigens.
Furthermore, the present inventors discovered that an antibody
having the property of strongly binding to antigens in the plasma
but weakly binding to antigens in the endosome can dissociate from
the antigens in the endosome even when the antibody had bound to
antigens such as soluble antigens; therefore, they are released
again into the plasma in a state not bound to antigens and can
neutralize multiple soluble antigens.
[0152] In particular, the present inventors noted that the pH in
the plasma was different from the pH in the endosomes, and thus
discovered that antibodies that strongly bind to antigens under
plasma pH condition but that weakly bind to antigens under
endosomal pH condition were superior in retention in the plasma,
because one antibody molecule could bind to multiple antigens.
[0153] The endosomes, which are membrane vesicles, form networks in
the cytoplasm of eukaryotic cells and are responsible for the
metabolism of macromolecules in the process from the cell membrane
to the lysosomes. The pH in the endosomes has been reported be
generally an acidic pH of 5.5 to 6.0 (Nat Rev Mol Cell Biol. 2004
February; 5(2):121-32). Meanwhile, the pH in the plasma is known to
be almost neutral (normally, pH 7.4).
[0154] Accordingly, an antigen-binding molecule whose
antigen-binding activity at acidic pH is weaker than the
antigen-binding activity at neutral pH binds to the antigen in the
plasma which have a neutral pH, is taken up into cells, and then
dissociates from the antigen in the endosomes which have an acidic
pH. The antigen-binding molecule that dissociated from the antigen
binds to FcRn, translocates to the cell surface, and returns again
in the plasma in a state not bound to antigens. As a result, the
antigen-binding molecule can bind to antigens multiple times, and
the pharmacokinetics is improved.
<Antigen-Binding Molecule Substances>
[0155] Furthermore, the present invention provides antigen-binding
molecules whose antigen-binding activity at pH 4.0 to pH 6.5 is
lower than that at pH 6.7 to pH 10.0, preferably antigen-binding
molecules whose antigen-binding activity at pH 5.0 to pH 6.0 is
lower than that at pH 7.0 to 8.0. Specifically, antigen-binding
molecules whose antigen-binding activity at pH 4.0 to pH 6.5 is
lower than that at pH 6.7 to pH 10.0 include, for example,
antigen-binding molecules whose antigen-binding activity at pH 5.8
is lower than that at pH 7.4. Antigen-binding molecules whose
antigen-binding activity at pH 5.8 is lower than that at pH 7.4 can
also be expressed as antigen-binding molecules whose
antigen-binding activity at pH 7.4 is higher than that at pH
5.8.
[0156] As for the antigen-binding molecules of the present
invention whose antigen-binding activity at pH 5.8 is lower than
that at pH 7.4, so long as the antigen-binding activity at pH 5.8
is lower than the binding at pH 7.4, there is no limitation on the
difference in binding activity, and the antigen-binding activity at
pH 5.8 only need to be lower, even slightly.
[0157] A preferred embodiment of an antigen-binding molecule of the
present invention whose antigen-binding activity at pH 5.8 is lower
than that at pH 7.4 includes antigen-binding molecules whose
antigen-binding activity at pH 7.4 is twice or greater than that at
pH 5.8. A more preferred embodiment of the antigen-binding molecule
includes antigen-binding molecules whose antigen-binding activity
at pH 7.4 is ten times or greater than that at pH 5.8. A still more
preferred embodiment of the antigen-binding molecule includes
antigen-binding molecules whose antigen-binding activity at pH 7.4
is 40 times or greater than that at pH 5.8.
[0158] Specifically, in a preferred embodiment, the antigen-binding
molecule of the present invention has 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, 1000, or 10000, as long as
production is possible using the technologies of those skilled in
the art.
[0159] 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
k.sub.d(pH5.8)/k.sub.d(pH7.4), which is a ratio of the k.sub.d for
the antigen at pH 5.8 and the k.sub.d 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 k.sub.d(pH5.8)/k.sub.d(pH7.4) value is not
particularly limited, and may be any value, for example, 50, 100,
or 200, as long as production is possible using the technologies of
those skilled in the art.
[0160] Conditions other than the pH at which the antigen-binding
activity is measured can be appropriately selected by those skilled
in the art, and the conditions are not particularly limited;
however, the measurements can be carried out, for example, under
conditions of MES buffer and 37.degree. 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.
[0161] It is presumed that such an antigen-binding molecule, which
weakly binds to an antigen at acidic pH, easily dissociates from
the antigen under the endosomal acidic condition, and that after
internalization into cells, it binds to FcRn and is easily released
to the outside of the cells. The antigen-binding molecule released
to the outside of the cells without being degraded inside the cells
can bind again to other antigens. Accordingly, when the
antigen-binding molecule is, for example, an antigen-binding
neutralizing molecule, the antigen-binding molecule that easily
dissociates from the antigen under the endosomal acidic condition
can bind and neutralize antigens multiple times. As a result,
antigen-binding molecules whose antigen-binding activity at pH 4.0
to pH 6.5 is lower than that at pH 6.7 to pH 10.0 are superior in
retention in the plasma.
[0162] In a preferred embodiment, the antigen-binding molecule
whose antigen-binding activity at pH 5.8 is lower than that at pH
7.4 includes antigen-binding molecules in which at least one amino
acid in the antigen-binding molecule is substituted with histidine
or a non-natural amino acid, 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, as long as the
antigen-binding activity at pH 5.8 is weaker than that at pH 7.4
(the KD(pH5.8)/KD(pH7.4) value is greater or the
k.sub.d(pH5.8)/k.sub.d(pH7.4) 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 substituted 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 substituted with histidine or
non-natural amino acid, or one amino acid may be inserted, or two
or more amino acids may be substituted with histidine or
non-natural amino acids, or two or more amino acids may be
inserted. Moreover, apart from the substitutions to histidine or to
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 to histidine or to 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 k.sub.d(pH5.8)/k.sub.d(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.
[0163] Preferred antigen-binding molecules with mutation to
histidine or to non-natural amino acid and whose antigen-binding
activity at pH 5.8 is lower than that at pH 7.4 include, for
example, antigen-binding molecules whose antigen-binding activity
at pH 7.4 after the mutation to histidine or to non-natural amino
acid is equivalent to the antigen-binding activity at pH 7.4 before
the mutation to histidine or to non-natural amino acid. In the
present invention, "an antigen-binding molecule after histidine or
non-natural amino acid mutation has an antigen-binding activity
that is equivalent to that of the antigen-binding molecule before
histidine or non-natural amino acid mutation" means that, 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 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
greater 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. The present invention also includes such antigen-binding
molecules whose binding activity has been made equivalent as a
result of substitution, deletion, addition, and/or insertion of one
or more amino acids after histidine substitution or insertion.
[0164] Further, when the antigen-binding molecule is a substance
including an antibody constant region, in another preferred
embodiment of the antigen-binding molecule whose antigen-binding
activity at pH 5.8 is lower than that at pH 7.4, the present
invention includes methods for modifying antibody constant regions
contained in the antigen-binding molecules. Specific examples of
antibody constant regions after modification include the constant
regions described in the Examples.
[0165] When the antigen-binding activity of the antigen-binding
substance at pH 5.8 is weakened compared to that at pH 7.4 (when
KD(pH5.8)/KD(pH7.4) value is increased) by the above described
methods and such, it is generally preferable that the
KD(pH5.8)/KD(pH7.4) value is two times or more, more preferably
five times or more, and even more preferably ten times or more as
compared to that of the original antibody, but is not particularly
limited thereto.
[0166] The antigen-binding molecules of the present invention may
further have any other property, as long as their antigen-binding
activity at pH 4.0 to pH 6.5 is lower than that at pH 6.7 to pH
10.0. For example, the antigen-binding molecules may be agonistic
or antagonistic antigen-binding molecules. Preferred
antigen-binding molecules of the present invention include, for
example, antagonistic antigen-binding molecules. In general, an
antagonistic antigen-binding molecule inhibits receptor-mediated
intracellular signaling by inhibiting the binding between a ligand
(agonist) and the receptor.
[0167] Furthermore, the present invention provides antibodies in
which amino acid on at least one site indicated below is
substituted with histidine or non-natural amino acid. Amino acid
positions are indicated based on the Kabat numbering (Kabat E A et
al., (1991) Sequences of Proteins of Immunological Interest,
NIH).
[0168] Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61,
H62, H63, H64, H65, H99, H100b, and H102
[0169] Light chain: L24, L27, L28, L32, L53, L54, L56, L90, L92,
and L94
[0170] Among the above sites, H32, H61, L53, L90, and L94 could be
universal modification sites.
[0171] When the antigen is the IL-6 receptor (e.g., human IL-6
receptor), preferable modification sites include the following.
However, the modification sites are not particularly limited
thereto.
[0172] Heavy chain: H27, H31, H32, H35, H50, H58, H61, H62, H63,
H64, H65, H100b, and H102
[0173] Light chain: L24, L27, L28, L32, L53, L56, L90, L92, and
L94
[0174] When histidine or non-natural amino acid is substituted at
multiple sites, preferred combinations of substitution sites
include, for example, the combination of H27, H31, and H35;
combination of H27, H31, H32, H35, H58, H62, and H102; combination
of L32 and L53; and combination of L28, L32, and L53. In addition,
preferred combinations of substitution sites of heavy and light
chains include the combination of H27, H31, L32, and L53.
[0175] When the antigen is IL-6 (e.g., human IL-6), preferable
modification sites include the following. However, the modification
sites are not particularly limited thereto.
[0176] Heavy chain: H32, H59, H61, and H99
[0177] Light chain: L53, L54, L90, and L94
[0178] When the antigen is the IL-31 receptor (e.g., human IL-31
receptor), preferable modification sites include H33. However, the
modification sites are not particularly limited thereto.
[0179] The antigen-binding molecules of the present invention may
recognize any antigen. Antigens recognized by antibodies of the
present invention specifically include the above-mentioned receptor
proteins (membrane-bound receptors or soluble receptors), membrane
antigens such as cell surface markers, and soluble antigens such as
cytokines, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-31, IL-23, IL-2
receptor, IL-6 receptor, OSM receptor, gp130, IL-5 receptor, CD40,
CD4, Fas, osteopontin, CRTH2, CD26, PDGF-D, CD20, monocyte
chemoattractant factor, CD23, TNF-.alpha., HMGB-1, .alpha.4
integrin, ICAM-1, CCR2, CD11a, CD3, IFN.gamma., BLyS, HLA-DR,
TGF-.beta., CD52, and IL-31 receptor.
[0180] Particularly preferred antigens include the IL-6
receptor.
[0181] The antigen-binding molecules of the present invention are
described above.
[0182] In a preferred embodiment of the present invention, the
antigen-binding molecules include antibodies. Antibodies having
antigen-binding activity and FcRn-binding region include, for
example, IgG antibodies. When the antibody used is an IgG antibody,
there is no limitation as to its type. It is possible to use IgG1,
IgG2, IgG3, IgG4, and such.
[0183] The origin of antibody of the present invention is not
particularly limited, and may be of any origin. It is possible to
use, for example, mouse antibodies, human antibodies, rat
antibodies, rabbit antibodies, goat antibodies, camel antibodies,
and others. Furthermore, the antibodies may be, for example, the
above-described chimeric antibodies, and in particular, modified
antibodies with amino acid sequence substitutions, such as
humanized antibodies. The antibodies may also be the
above-described bispecific antibodies, antibody modification
products to which various molecules have been linked, polypeptides
including antibody fragments, and antibodies with modified sugar
chains.
[0184] Generation of chimeric antibodies is 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.
[0185] "Humanized antibodies" are also referred to as reshaped
human antibodies, and are antibodies in which the complementarity
determining region (CDR) of a nonhuman mammal, for example a mouse,
is transplanted to the CDR 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 substituted so that the CDRs of the
reshaped human antibody can form a suitable antigen binding site
(Sato, K. et al., Cancer Res. (1993) 53:10.01-6). Amino acid
residues in the FRs that can be modified 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).
[0186] When the antibodies of the present invention are chimeric
antibodies or humanized antibodies, the C regions of these
antibodies are preferably derived from human antibodies. For
example, C.gamma.1, C.gamma.2, C.gamma.3, and C.gamma.4 can be used
for the H chain, while C.kappa. and C.lamda. 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 FcRn 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 an 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 of human IgG 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.
[0187] The variable and constant regions of chimeric and humanized
antibodies of the present invention may be modified by deletion,
substitution, insertion, and/or addition, and such, so long as the
binding specificity of the original antibodies is exhibited.
[0188] Since the immunogenicity in the human body is lowered,
chimeric and humanized antibodies using human-derived sequences are
thought to be useful when administered to humans for therapeutic
purposes or such.
[0189] The antibodies of the present invention may be prepared by
any method. For example, antibodies whose antigen-binding activity
at pH 5.8 is originally greater than or comparable to that at pH
7.4 may be artificially modified through histidine substitution
described above or the like so that their antigen-binding activity
at pH 5.8 becomes lower than that at pH 7.4. Alternatively,
antibodies whose antigen-binding activity at pH 5.8 is lower than
that at pH 7.4 may be selected by screening a number of antibodies
obtained from an antibody library or hybridomas as described
below.
[0190] When histidine is substituted for amino acids in an
antibody, known sequences may be used for the H chain or L chain
amino acid sequence of the antibody before introduction of
histidine mutations, or amino acid sequences of antibodies newly
obtained by methods known to those skilled in the art can also be
used. For example, the antibodies may be obtained from an antibody
library, or they may be obtained by cloning genes encoding
antibodies from hybridomas producing monoclonal antibodies.
[0191] 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 antibody 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.
[0192] 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).
[0193] More specifically, sensitizing antigens to obtain the
above-described antibody genes encoding the H chains and L chains
include both complete antigens with immunogenicity and incomplete
antigens including haptens and the like with no antigenicity;
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 antibodies 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.
[0194] Antibody-producing cells can be obtained by immunizing
animals using appropriate sensitizing antigens described above.
Alternatively, antibody-producing cells can be prepared by in vitro
immunization of lymphocytes that can produce antibodies. 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).
[0195] 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.
[0196] Antibody-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 antibody produced from these hybridomas
can be measured using known analysis methods, such as
immunoprecipitation, radioimmunoassay (RIA), and enzyme-linked
immunosorbent assay (ELISA). Thereafter, hybridomas producing
antibodies of interest whose specificity, affinity, or activity has
been determined can be subcloned by methods such as limiting
dilution.
[0197] Next, genes encoding the selected antibodies can be cloned
from hybridomas or antibody-producing cells (sensitized
lymphocytes, and such) using probes that can specifically bind to
the antibodies (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 antibodies are not particularly limited and
may originate from antibodies belonging to any of these classes or
subclasses; however, IgG is particularly preferred.
[0198] Herein, it is possible to modify H-chain-encoding genes and
L-chain-encoding genes using genetic engineering technologies.
Genetically modified antibodies, such as chimeric antibodies and
humanized antibodies, which have been artificially modified 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 substituted 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).
[0199] In addition to the humanization described above, antibodies
may be modified to improve their biological properties, for
example, the binding to the antigen. In the present invention, such
modifications 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:
[0200] (1) hydrophobic: alanine, isoleucine, valine, methionine,
and leucine;
[0201] (2) neutral hydrophilic: asparagine, glutamine, cysteine,
threonine, and serine;
[0202] (3) acidic: aspartic acid and glutamic acid;
[0203] (4) basic: arginine, histidine, and lysine;
[0204] (5) residues that affect the orientation of the chain:
glycine, and proline; and
[0205] (6) aromatic: tyrosine, tryptophan, and phenylalanine
[0206] In general, a total of six complementarity determining
regions (CDRs; hypervariable regions) present on the H chain and L
chain variable regions interact with each other to form an
antigen-binding site of an antibody. A variable region alone is
also known to be capable of recognizing and binding to an antigen,
although its affinity is lower than the affinity of the whole
binding site. Thus, antibody genes encoding the H chain and L chain
of the present invention may encode fragments each including the H
chain or L chain antigen-binding site, as long as the polypeptide
encoded by the gene retains the activity of binding to the desired
antigen.
[0207] As described above, the heavy chain variable region is in
general constituted by three CDRs and four FRs. In a preferred
embodiment of the present invention, amino acid residues to be
"modified" can be appropriately selected from amino acid residues,
for example, in a CDR or FR. In general, modifications of amino
acid residues in the CDRs may reduce the antigen-binding ability.
Thus, appropriate amino acid residues to be "modified" in the
present invention are preferably selected from amino acid residues
in the FRs, but are not limited thereto. It is possible to select
amino acids in a CDR as long as the modification has been confirmed
not to reduce the binding ability. Alternatively, by using public
databases or such, those skilled in the art can obtain appropriate
sequences that can be used as an FR of antibody variable region of
an organism such as human or mouse.
[0208] Furthermore, the present invention provides genes encoding
the antibodies of the present invention. The genes encoding the
antibodies of the present invention may be any genes, and may be
DNAs, RNAs, nucleic acid analogs, or the like.
[0209] 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.
[0210] 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 (J. Exp. Med. (1995)
108: 94.0), COS, HEK293, 3T3, myeloma, BHK (baby hamster kidney),
HeLa, and Vero; 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.
[0211] 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 antibodies 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 antibodies of the present invention.
[0212] 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 antibodies of the present invention.
<Screening Methods>
[0213] The present invention provides methods of screening for
antigen-binding molecules whose antigen-binding activity at acidic
pH is lower than that at neutral pH. The present invention also
provides methods of screening for antigen-binding molecules which
can individually bind to multiple antigens. The present invention
also provides methods of screening for antigen-binding molecules
which are superior in the retention in plasma. The present
invention also provides methods of screening for an antigen-binding
molecule that dissociates within a cell from an
extracellularly-bound antigen. The present invention also provides
methods of screening for an antigen-binding molecule that is bound
to an antigen and internalized into a cell, and released to the
outside of the cell in an antigen-free form. The present invention
also provides methods of screening for an antigen-binding molecule
that has increased ability to eliminate antigens in plasma.
Furthermore, the present invention also provides methods of
screening for antigen-binding molecules which are particularly
useful when used as pharmaceutical compositions.
[0214] Specifically, the present invention provides methods of
screening for antigen-binding molecules, which comprise the steps
of:
(a) determining the antigen-binding activity of an antigen-binding
molecule at pH 6.7 to pH 10.0; (b) determining the antigen-binding
activity of the antigen-binding molecule at pH 4.0 to pH 6.5; and
(c) selecting an antigen-binding molecule whose antigen-binding
activity at pH 6.7 to pH 10.0 is greater than the antigen-binding
activity at pH 4.0 to pH 6.5.
[0215] In the screening methods of the present invention, the
antigen-binding activity of the antigen-binding molecule at pH 6.7
to pH 10.0 is not particularly limited, as long as it is an
antigen-binding activity at a pH between pH 6.7 and pH 10.0.
However, for example, a preferred antigen-binding activity is an
antigen-binding activity at a pH between pH 7.0 and pH 8.0, and a
more preferred antigen-binding activity is an antigen-binding
activity at pH 7.4. Further, the antigen-binding activity of the
antigen-binding molecule at pH 4.0 to pH 6.5 is not particularly
limited, as long as it is an antigen-binding activity at a pH
between pH 4.0 and pH 6.5. However, for example, a preferred
antigen-binding activity is an antigen-binding activity at a pH
between pH 5.5 to pH 6.5, and a more preferred antigen-binding
activity is an antigen-binding activity at pH 5.8 or pH 5.5.
[0216] The antigen-binding activity of an antigen-binding molecule
can be determined by methods known to those skilled in the art.
Conditions other than the pH can be appropriately determined by
those skilled in the art. The antigen-binding activity of an
antigen-binding molecule can be assessed as dissociation constant
(KD), apparent dissociation constant (apparent KD), dissociation
rate (k.sub.d), apparent dissociation rate (apparent k.sub.d), or
such. These constants can be determined by methods known to those
skilled in the art, for example, using Biacore (GE healthcare),
Scatchard plot, or FACS.
[0217] Herein, "the step of selecting an antigen-binding molecule
whose antigen-binding activity at pH 6.7 to pH 10.0 is greater than
that at pH 4.0 to pH 6.5" has a same meaning as "the step of
selecting an antigen-binding molecule whose antigen-binding
activity at pH 4.0 to pH 6.5 is lower than that at pH 6.7 to pH
10.0".
[0218] The difference between the antigen-binding activity at pH
6.7 to pH 10.0 and that at pH 4.0 to pH 6.5 is not particularly
limited as long as the antigen-binding activity at pH 6.7 to pH
10.0 is greater than that at pH 4.0 to pH 6.5. However, the
antigen-binding activity at pH 6.7 to pH 10.0 is preferably twice
or greater, more preferably ten times or greater, and still more
preferably 40 times or greater than the antigen-binding activity at
pH 4.0 to pH 6.5.
[0219] Furthermore, the present invention also provides methods of
screening for antigen-binding molecules, which comprise the steps
of:
(a) binding an antigen-binding molecule to an antigen under a
condition of pH 6.7 to pH 10.0; (b) placing the antigen-binding
molecule that bound to the antigen of (a) under a condition of pH
4.0 to pH 6.5; and (c) obtaining the antigen-binding molecule that
dissociated under the condition of pH 4.0 to pH 6.5.
[0220] In addition, the present invention also provides methods of
screening for antigen-binding molecules, which comprise the steps
of:
(a) selecting an antigen-binding molecule that does not bind to an
antigen under a condition of pH 4.0 to pH 6.5; (b) binding the
antigen-binding molecule selected in (a) to an antigen under a
condition of pH 6.7 to pH 10.0; and (c) obtaining the
antigen-binding molecule that bound to the antigen under the
condition of pH 6.7 to pH 10.0.
[0221] Furthermore, the present invention also provides methods of
screening for antigen-binding molecules, which comprise the steps
of:
(a) binding an antigen-binding molecule to an antigen under a
condition of pH 6.7 to pH 10.0; (b) placing the antigen-binding
molecule that bound to the antigen of (a) under a condition of pH
4.0 to pH 6.5; (c) obtaining the antigen-binding molecule that
dissociated under the condition of pH 4.0 to pH 6.5; (d) amplifying
the gene encoding the antigen-binding molecule that dissociated;
and (e) obtaining the eluted antigen-binding molecule.
[0222] The steps of (a) to (d) may be repeated twice or more times.
Thus, the present invention provides the methods described above
further including a step of repeating the steps of (a) to (d) twice
or more times. The number for repeating the steps of (a) to (d) is
not particularly limited; however, the number is in general ten or
less.
[0223] Furthermore, the present invention also provides methods of
screening for antigen-binding molecules, which comprise the steps
of:
(a) selecting an antigen-binding molecule that does not bind to an
antigen under a condition of pH 4.0 to pH 6.5; (b) binding the
antigen-binding molecule selected in (a) to an antigen under a
condition of pH 6.7 to pH 10.0; (c) obtaining the antigen-binding
molecule that bound to the antigen under the condition of pH 6.7 to
pH 10.0; (d) amplifying the gene encoding the antigen-binding
molecule that dissociated; and (e) collecting the eluted
antigen-binding molecule.
[0224] The steps of (a) to (d) may be repeated twice or more times.
Thus, the present invention provides the methods described above
further including a step of repeating the steps of (a) to (d) twice
or more times. The number for repeating the steps of (a) to (d) is
not particularly limited; however, the number is in general ten or
less.
[0225] When a phage library or such is used in the screening
methods of the present invention, the step of amplifying the gene
encoding the antigen-binding molecule can also be a step of
amplifying phages.
[0226] In the methods of the present invention, binding of the
antigen-binding molecule and the antigen may be carried out under
any state, without particular limitation. For example, binding of
the antigen-binding molecule and the antigen may be carried out by
contacting an antigen with an immobilized antigen-binding molecule,
or by contacting an antigen-binding molecule with an immobilized
antigen. Alternatively, binding of the antigen-binding molecule and
the antigen may be carried out by contacting the antigen and
antigen-binding molecule in a solution.
[0227] Furthermore, the present invention also provides methods of
screening for antigen-binding molecules whose binding activity at a
first pH is greater than that at a second pH, which comprise the
steps of:
(a) binding an antigen-binding molecule to an antigen-immobilized
column under the condition of a first pH; (b) eluting the
antigen-binding molecule that had bound to the column at the first
pH from the column under the condition of a second pH; and (c)
obtaining the eluted antigen-binding molecule.
[0228] Furthermore, the present invention also provides methods of
screening for antigen-binding molecules whose binding activity at a
first pH is smaller than that at a second pH, which comprise the
steps of:
(a) passing an antigen-binding molecule through an
antigen-immobilized column under the condition of a first pH; (b)
collecting the antigen-binding molecule that eluted without binding
to the column in step (a); (c) binding the antigen-binding molecule
collected in (b) to a column under the condition of a second pH;
and (d) obtaining the antigen-binding molecule that bound to the
column in step (c).
[0229] Furthermore, the present invention also provides methods of
screening for antigen-binding molecules whose binding activity at a
first pH is greater than that at a second pH, which comprise the
steps of:
(a) binding an antigen-binding molecule library to an
antigen-immobilized column under the condition of a first pH; (b)
eluting the antigen-binding molecule from the column under the
condition of a second pH; (c) amplifying the gene encoding the
eluted antigen-binding molecule; and (d) obtaining the eluted
antigen-binding molecule.
[0230] The steps of (a) to (c) may be repeated twice or more times.
Thus, the present invention provides the methods described above
further including the step of repeating the steps of (a) to (c)
twice or more times. The number for repeating the steps of (a) to
(c) is not particularly limited; however, the number is in general
ten or less.
[0231] In the present invention, each of the first and second pHs
may be any pH, as long as they are not identical. In a preferred
combination of the first and second pHs, for example, the first pH
is between pH 6.7 and pH 10.0, and the second pH is between pH 4.0
and pH 6.5; in a more preferred combination, the first pH is
between pH 7.0 and pH 8.0, and the second pH is between pH 5.5 and
pH 6.5; and in a still more preferred combination, the first pH is
pH 7.4 and the second pH is pH 5.8 or pH5.5.
[0232] In another preferred combination of the first and second
pHs, for example, the first pH is between pH 4.0 and pH 6.5, and
the second pH is between pH 6.7 and pH 10.0; in a more preferred
combination, the first pH is between pH 5.5 and pH 6.5, and the
second pH is between pH 7.0 and pH 8.0; and in a still more
preferred combination, the first pH is pH 5.8 or pH5.5 and the
second pH is pH 7.4.
[0233] Antigen-binding molecules that are screened by the methods
of the present invention may be any antigen-binding molecules. For
example, it is possible to use the above-described antigen-binding
molecules in the screening of the present invention. For example,
it is possible to screen antigen-binding molecules including
natural sequences or antigen-binding molecules including amino acid
sequences with substitutions. Preferred antigen-binding molecules
that are screened in the present invention include, for example,
antigen-binding molecules in which at least one amino acid is
substituted with histidine or at least one histidine is inserted.
The site of introduction of histidine substitution or insertion is
not particularly limited, and may be introduced at any site.
Furthermore, histidine substitution or insertion may be introduced
at one site, or may be introduced at two or more sites.
Furthermore, preferred antigen-binding molecules that are screened
in the present invention include, for example, antigen-binding
molecules including modified antibody constant regions.
[0234] Antigen-binding molecules that are screened by the methods
of the present invention may be a number of different
antigen-binding molecules introduced with histidine substitutions
or insertions at different sites, for example, by histidine
scanning
[0235] Thus, the screening methods of the present invention may
further comprise the step of substituting at least one amino acid
in the antigen-binding molecule with histidine or inserting at
least one histidine into the antigen-binding molecule.
[0236] In the screening 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.
[0237] Moreover, the screening methods of the present invention may
further comprise the step of modifying amino acids of antibody
constant regions.
[0238] Antigen-binding substances that are screened by the
screening 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 (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) prepared by
introducing histidine mutations or non-natural amino acid mutations
into the above-described antibodies and libraries, and so on.
[0239] Antigen-binding molecules that bind to the antigen multiple
times, which are thus superior in the retention in plasma, can be
obtained by the screening methods of the present invention. Thus,
the screening methods of the present invention can be used as
screening methods for obtaining antigen-binding molecules that are
superior in the retention in plasma.
[0240] Furthermore, antigen-binding molecules that can bind to the
antigen two or more times when administered to animals such as
humans, mice, or monkeys can be obtained by the screening methods
of the present invention. Thus, the screening methods of the
present invention can be used as screening methods for obtaining
antigen-binding molecules that can bind to the antigen two or more
times.
[0241] Furthermore, antigen-binding molecules that are capable of
binding to more antigens as compared to the number of their
antigen-binding sites when administered to animals such as humans,
mice, or monkeys can be obtained by the screening methods of the
present invention. Thus, the screening methods of the present
invention can be used as screening methods for obtaining
antigen-binding molecules that are capable of binding to more
antigens as compared to the number of their antigen-binding sites.
For example, when the antibody is a neutralizing antibody, the
screening methods of the present invention can be used as screening
methods for obtaining antigen-binding molecules that can neutralize
more antigens as compared to the number of the antigen-binding
sites of the antigen-binding molecules.
[0242] Furthermore, antigen-binding molecules that are capable of
dissociating within a cell from an extracellularly-bound antigen
when administered to animals such as humans, mice, or monkeys can
be obtained by the screening methods of the present invention.
Thus, the screening methods of the present invention can be used as
screening methods for obtaining antigen-binding molecules that are
capable of dissociating within a cell from an extracellularly-bound
antigen.
[0243] Furthermore, antigen-binding molecules that are bound to an
antigen and internalized into a cell, and released to the outside
of the cell in an antigen-free form when administered to animals
such as humans, mice, or monkeys can be obtained by the screening
methods of the present invention. Thus, the screening methods of
the present invention can be used as screening methods for
obtaining antigen-binding molecules that are bound to an antigen
and internalized into a cell, and released to the outside of the
cell in an antigen-free form.
[0244] Furthermore, antigen-binding molecules that can rapidly
eliminate antigens in plasma when administered to animals such as
humans, mice, or monkeys can be obtained by the screening methods
of the present invention. Thus, the screening methods of the
present invention can be used as screening methods for obtaining
antigen-binding molecules with increased (high) ability to
eliminate antigens in plasma.
[0245] Furthermore, such antigen-binding molecules are expected to
be especially superior as pharmaceuticals, because the dose and
frequency of administration in patients can be reduced and as a
result the total dosage can be reduced. Thus, the screening methods
of the present invention can be used as methods of screening for
antigen-binding molecules for use as pharmaceutical
compositions.
[0246] In addition, the present invention provides libraries in
which the histidine content is increased as compared to the
original libraries. Libraries containing antigen-binding molecules
with increased histidine content can be used in the screening
methods described above and the production methods described
hereinafter.
[0247] Libraries with increased histidine content can be prepared
by methods known to those skilled in the art, which include the
following method. 20 types of triplet codons (trinucleotides)
encoding 20 types of amino acids can be incorporated at equal
frequency when synthesizing nucleic acids to prepare a library by
the trinucleotide-method (J Mol Biol. 2008 Feb. 29; 376(4):
1182-200). As a result, the position mutated for the library can be
made to contain 20 types of amino acids at equal probability. The
frequency of histidine in the position mutated for the library can
be increased by increasing the proportion of a histidine-encoding
trinucleotide as compared to the remaining amino acids among the 20
types in the synthesis.
<Methods for Producing Antigen-Binding Molecules>
[0248] The present invention provides methods for producing
antigen-binding molecules whose antigen-binding activity at the
endosomal pH is lower than that at the plasma pH. The present
invention also provides methods for producing antigen-binding
molecules that are superior in the retention in plasma. The present
invention also provides methods for producing antigen-binding
molecules that are especially useful when used as pharmaceutical
compositions.
[0249] Specifically, the present invention provides methods for
producing antigen-binding molecules, which comprise the steps
of:
(a) determining the antigen-binding activity of an antigen-binding
molecule at pH 6.7 to pH 10.0; (b) determining the antigen-binding
activity of the antigen-binding molecule at pH 4.0 to pH 6.5; (c)
selecting an antigen-binding molecule whose antigen-binding
activity at pH 6.7 to pH 10.0 is greater than that at pH 4.0 to pH
6.5; (d) obtaining the gene encoding the antigen-binding molecule
selected in (c); and (e) producing the antigen-binding molecule
using the gene obtained in (d).
[0250] The present invention also provides methods for producing
antigen-binding molecules, which comprise the steps of:
(a) binding an antigen-binding molecule to an antigen at pH 6.7 to
pH 10.0; (b) allowing the antigen-binding molecule bound to the
antigen of (a) to stand under the condition of pH 4.0 to pH 6.5;
(c) collecting the antigen-binding molecule that dissociated under
the condition of pH 4.0 to pH 6.5; (d) obtaining the gene encoding
the antigen-binding molecule obtained in (c); and (e) producing the
antigen-binding molecule using the gene obtained in (d).
[0251] Furthermore, the present invention provides methods for
producing antigen-binding molecules, which comprise the steps
of:
(a) selecting an antigen-binding molecule that does not bind to the
antigen under the condition of pH 4.0 to pH 6.5; (b) binding the
antigen under the condition of pH 6.7 to pH 10.0 to the
antigen-binding molecule selected in (a); (c) collecting the
antigen-binding molecule that bound to the antigen under the
condition of pH 6.7 to pH 10.0; (d) obtaining the gene encoding the
antigen-binding molecule collected in (c); and (e) producing the
antigen-binding molecule using the gene obtained in (d).
[0252] In addition, the present invention provides methods for
producing antigen-binding molecules, which comprise the steps
of:
(a) binding an antigen-binding molecule to an antigen under the
condition of pH 6.7 to 10.0; (b) allowing the antigen-binding
molecule that bound to the antigen in (a) to stand under the
condition of pH 4.0 to pH 6.5; (c) collecting the antigen-binding
molecule that dissociated under the condition of pH 4.0 to pH 6.5;
(d) amplifying the gene encoding the dissociated antigen-binding
molecule; (e) collecting the eluted antigen-binding molecule; (f)
obtaining the gene encoding the antigen-binding molecule collected
in (e); and (g) producing the antigen-binding molecule using the
gene obtained in (f).
[0253] Steps (a) to (d) may be repeated twice or more times. Thus,
the present invention provides the methods described above, which
further comprise the step of repeating steps (a) to (d) twice or
more times. The number of times steps (a) to (d) is repeated is not
particularly limited; however, it is generally ten times or
less.
[0254] Furthermore, the present invention provides methods of
screening for antigen-binding molecules, which comprise the steps
of:
(a) selecting an antigen-binding molecule that does not bind to the
antigen under the condition of pH 4.0 to pH 6.5; (b) binding the
antigen under the condition of pH 6.7 to pH 10.0 to the
antigen-binding molecule selected in (a); (c) collecting the
antigen-binding molecule that bound to the antigen under the
condition of pH 6.7 to pH 10.0; (d) amplifying the gene encoding
the dissociated antigen-binding molecule; (e) collecting the eluted
antigen-binding molecule; (f) obtaining the gene encoding the
antigen-binding molecule collected in (e); and (g) producing the
antigen-binding molecule using the gene obtained in (f).
[0255] Steps (a) to (d) may be repeated twice or more times. Thus,
the present invention provides the methods described above, which
further comprise the step of repeating steps (a) to (d) twice or
more times. The number of times steps (a) to (d) is repeated is not
particularly limited; however, it is generally ten times or
less.
[0256] Furthermore, the present invention provides methods for
producing antigen-binding molecules whose binding activity at a
first pH is greater than that at a second pH, which comprise the
steps of:
(a) binding the antigen-binding molecule to a column immobilized
with antigen under the first pH condition; (b) eluting the
antigen-binding molecule, which is bound to the column under the
first pH condition from the column under a second pH condition; (c)
collecting the eluted antigen-binding molecule; (d) obtaining the
gene encoding the antigen-binding molecule collected in (c); and
(e) producing the antigen-binding molecule using the gene obtained
in (d).
[0257] Furthermore, the present invention provides methods for
producing antigen-binding molecules whose binding activity at a
first pH is greater than that at a second pH, which comprise the
steps of:
(a) binding an antigen-binding molecule library to a column
immobilized with antigen under the first pH condition; (b) eluting
the antigen-binding molecule from the column under the second pH
condition; (c) amplifying the gene encoding the eluted
antigen-binding molecule; (d) collecting the eluted antigen-binding
molecule; (e) obtaining the gene encoding the antigen-binding
molecule collected in (d); and (f) producing the antigen-binding
molecule using the gene obtained in (e).
[0258] Steps (a) to (c) may be repeated twice or more times. Thus,
the present invention provides the methods described above, which
further comprise the step of repeating steps (a) to (c) twice or
more times. The number of times steps (a) to (c) is repeated is not
particularly limited; however, it is generally ten times or
less.
[0259] When a phage library or such is used in the production
methods of the present invention, the step of amplifying the gene
encoding the antigen-binding molecule may be the step of amplifying
phages.
[0260] Antigen-binding substances 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 (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) prepared by
introducing histidine mutations or non-natural amino acid mutations
into the above-described antibodies and libraries, and so on.
[0261] In the above-described production methods, the
antigen-binding activity of the antigen-binding molecule at pH 6.7
to pH 10.0 is not particularly limited, as long as the
antigen-binding activity is that at a pH between pH 6.7 and pH
10.0. A preferred antigen-binding activity is that at a pH between
pH 7.0 and pH 8.0, and a more preferred antigen-binding activity is
that at pH 7.4. Alternatively, the antigen-binding activity of the
antigen-binding molecule at pH 4.0 to pH 6.5 is not particularly
limited, as long as the antigen-binding activity is that at a pH
between pH 4.0 and pH 6.5. A preferred antigen-binding activity is
that at a pH between pH 5.5 to pH 6.5, and a more preferred
antigen-binding activity is that at pH 5.8 or pH 5.5.
[0262] The antigen-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.
[0263] The step of selecting antigen-binding molecules whose
antigen-binding activity at pH 6.7 to pH 10.0 is greater than that
at pH 4.0 to pH 6.5 is synonymous with the step of selecting
antigen-binding molecules whose antigen-binding activity at pH 4.0
to pH 6.5 is lower than that at pH 6.7 to pH 10.0.
[0264] The difference in the antigen-binding activity at pH 6.7 to
pH 10.0 and at pH 4.0 to pH 6.5 is not particularly limited as long
as the antigen-binding activity at pH 6.7 to pH 10.0 is greater
than that at pH 4.0 to pH 6.5. The antigen-binding activity at pH
6.7 to pH 10.0 is preferably twice or greater, more preferably ten
times or greater, and still more preferably 40 times or greater
than that at pH 4.0 to pH 6.5.
[0265] In the production methods described above, the
antigen-binding molecule may be bound to the antigen under any
condition, and the condition is not particularly limited. For
example, the antigen-binding molecule may be bound to the antigen
by contacting the antigen with the immobilized antigen-binding
molecule, or by contacting the antigen-binding molecule with the
immobilized antigen. Alternatively, the antigen-binding molecule
may be bound to the antigen by contacting the antigen and
antigen-binding molecule in a solution.
[0266] In the production methods described above, each of the first
and second pHs may be any pH, as long as they are not identical. In
a preferred combination of the first and second pHs, for example,
the first pH is between pH 6.7 and pH 10.0, and the second pH is
between pH 4.0 and pH 6.5; in a more preferred combination, the
first pH is between pH 7.0 and pH 8.0, and the second pH is between
pH 5.5 and pH 6.5; and in a still more preferred combination, the
first pH is pH 7.4 and the second pH is pH 5.8 or pH 5.5.
[0267] In another preferred combination of the first and second
pHs, for example, the first pH is between pH 4.0 and pH 6.5, and
the second pH is between pH 6.7 and pH 10.0; in a more preferred
combination, the first pH is between pH 5.5 and pH 6.5, and the
second pH is between pH 7.0 and pH 8.0; and in a still more
preferred combination, the first pH is pH 5.8 or pH 5.5 and the
second pH is pH 7.4.
[0268] Antigen-binding molecules that are produced by the
production methods described above may be any antigen-binding
molecules. Preferred antigen-binding molecules include, for
example, antigen-binding molecules in which at least one amino acid
is substituted with histidine or at least one histidine has been
inserted. The site where such histidine mutation is introduced is
not particularly limited and may be introduced at any site.
Furthermore, histidine mutation may be introduced at one site or at
two or more sites.
[0269] Thus, the production methods of the present invention may
further comprise the step of substituting at least one amino acid
in an antigen-binding molecule with histidine or inserting at least
one histidine into antigen-binding molecules.
[0270] 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.
[0271] Furthermore, in another embodiment, the antigen-binding
molecules that are produced by the production methods described
above include, for example, antigen-binding molecules including
modified antibody constant regions. Accordingly, the production
methods of the present invention may further comprise the step of
modifying the amino acids of antibody constant regions.
[0272] The antigen-binding molecules that are produced by the
production methods of the present invention are superior in the
retention in plasma. Thus, the production methods of the present
invention can be used as methods for producing antigen-binding
molecules that are superior in the retention in plasma.
[0273] Furthermore, antigen-binding molecules produced by the
production methods are expected to be capable of binding to the
antigen two or more times when administered to animals such as
humans, mice, or monkeys. Thus, the production methods of the
present invention can be used as methods for producing
antigen-binding molecules that are capable of binding to the
antigen two or more times.
[0274] Furthermore, antigen-binding molecules produced by the
production methods of the present invention are expected to be
capable of binding to more antigens as compared to the number of
their antigen-binding sites when administered to animals such as
humans, mice, or monkeys. Thus, the production methods of the
present invention can be used as methods for producing
antigen-binding molecules that are capable of binding to more
antigens as compared to the number of their antigen-binding
sites.
[0275] Furthermore, antigen-binding molecules produced by the
production methods of the present invention are expected to be
capable of dissociating within a cell from an extracellularly-bound
antigen when administered to animals such as humans, mice, or
monkeys. Thus, the production methods of the present invention can
be used as methods for producing antigen-binding molecules that are
capable of dissociating within a cell from an extracellularly-bound
antigen.
[0276] Furthermore, antigen-binding molecules produced by the
production methods of the present invention are expected to be
capable of being bound to an antigen and internalized into a cell
as well as being released to the outside of the cell in an
antigen-free form, when administered to animals such as humans,
mice, or monkeys. Thus, the production methods of the present
invention can be used as methods for producing antigen-binding
molecules that are capable of being bound to an antigen and
internalized into a cell and being released to the outside of the
cell in an antigen-free form.
[0277] Furthermore, antigen-binding molecules that are produced by
the production methods of the present invention are expected to be
capable of rapidly eliminating antigens from plasma when
administered to animals such as humans, mice, or monkeys. Thus, the
production methods of the present invention can be used as method
for producing antigen-binding molecules with increased (high)
ability to eliminate antigens in plasma.
[0278] Furthermore, such antigen-binding molecules can reduce the
number of doses in patients and are expected to be especially
superior as pharmaceuticals. Thus, the production methods of the
present invention can be used as methods for producing
antigen-binding molecules for used as pharmaceutical
compositions.
[0279] 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 Escherichia coli (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.
8:466-472 (1988)) is preferred for bodies of organisms. 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).
[0280] 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.
[0281] The host cells can be cultured by known methods. For
example, when using animal cells as a host, DMEM, MEM, RPMI 1640,
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.degree. C. for about 15 to 200 hours. Medium
is exchanged, aerated, or agitated, as necessary.
[0282] 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.
[0283] 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.
[0284] 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.
[0285] 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).
[0286] 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.
[0287] 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).
[0288] 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.
[0289] Chromatography includes, for example, 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).
[0290] If needed, 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.
<Anti-IL-6 Receptor Antibodies>
[0291] Furthermore, the present invention provides the anti-IL-6
receptor antibodies of (a) to (m) below:
(a) an antibody that includes a heavy chain variable region
including an amino acid sequence, in which at least one of Tyr at
position 27, Asp at position 31, Asp at position 32, Trp at
position 35, Tyr at position 51, Asn at position 59, Ser at
position 63, Met at position 106, and Tyr at position 108 in the
amino acid sequence of SEQ ID NO: 1 (H53 variable region) has been
substituted with His; (b) an antibody that includes a heavy chain
variable region (H3pI) having an amino acid sequence, in which Tyr
at position 27, Asp at position 31, and Trp at position 35 in the
amino acid sequence of SEQ ID NO: 1 (H53 variable region) have been
substituted with His; (c) an antibody that includes a heavy chain
variable region having an amino acid sequence, in which Tyr at
position 27, Asp at position 31, Asp at position 32, Trp at
position 35, Asn at position 59, Ser at position 63, and Tyr at
position 108 in the amino acid sequence of SEQ ID NO: 1 (H53
variable region) have been substituted with His; (d) an antibody
that includes a heavy chain variable region (H170) having an amino
acid sequence, in which Tyr at position 27, Asp at position 31, Asp
at position 32, Trp at position 35, Asn at position 59, Ser at
position 63, and Tyr at position 108 have been substituted with
His, and in which Ser at position 99 has been substituted with Val
and Thr at position 103 has been substituted with Ile in the amino
acid sequence of SEQ ID NO: 1 (H53 variable region); (e) an
antibody that includes a heavy chain variable region having an
amino acid sequence, in which Asp at position 31, Tyr at position
51, Ser at position 63, Met at position 106, and Tyr at position
108 in the amino acid sequence of SEQ ID NO: 1 (H53 variable
region) have been substituted with His; (f) an antibody that
includes a heavy chain variable region (CLH5) having an amino acid
sequence, in which Asp at position 31, Tyr at position 51, Ser at
position 63, Met at position 106, and Tyr at position 108 have been
substituted with His, and in which Ser at position 99 has been
substituted with Phe and Thr at position 103 has been substituted
with Ile in the amino acid sequence of SEQ ID NO: 1 (H53 variable
region); (g) an antibody that includes a light chain variable
region having an amino acid sequence, in which at least one of Asp
at position 28, Tyr at position 32, Glu at position 53, Ser at
position 56, and Asn at position 92 in the amino acid sequence of
SEQ ID NO: 2 (PF1L variable region) has been substituted with His;
(h) an antibody that includes a light chain variable region (L73)
having an amino acid sequence, in which Asp at position 28, Tyr at
position 32, and Glu at position 53 in the amino acid sequence of
SEQ ID NO: 2 (PF1L variable region) have been substituted with His;
(i) an antibody that includes a light chain variable region (L82)
having an amino acid sequence, in which Tyr at position 32 and Glu
at position 53 in the amino acid sequence of SEQ ID NO: 1 (H53
variable region) have been substituted with His; (j) an antibody
that includes a light chain variable region (CLL5) having an amino
acid sequence, in which Tyr at position 32, Glu at position 53, Ser
at position 56, and Asn at position 92 in the amino acid sequence
of SEQ ID NO: 2 (PF1L variable region) have been substituted with
His; (k) an antibody that includes the heavy chain variable region
of (b) and the light chain variable region of (h); (l) an antibody
that includes the heavy chain variable region of (d) and the light
chain variable region of (i); and (m) an antibody that includes the
heavy chain variable region of (f) and the light chain variable
region of (h).
[0292] Specific examples of the heavy chain variable region having
an amino acid sequence in which at least one of Tyr at position 27,
Asp at position 31, Asp at position 32, Trp at position 35, Tyr at
position 51, Asn at position 59, Ser at position 63, Met at
position 106, and Tyr at position 108 in the amino acid sequence of
SEQ ID NO: 1 (H53 variable region) has been substituted with His
include, for example, the following heavy chain variable
regions.
[0293] a heavy chain variable region having the amino acid sequence
of SEQ ID NO: 3 (H3pI)
[0294] a heavy chain variable region having the amino acid sequence
of SEQ ID NO: 4 (H170)
[0295] a heavy chain variable region having the amino acid sequence
of SEQ ID NO: 5 (CLH5)
[0296] Specific examples of the light chain variable region having
an amino acid sequence in which at least one of Asp at position 28,
Tyr at position 32, Glu at position 53, Ser at position 56, and Asn
at position 92 in the amino acid sequence of SEQ ID NO: 2 (PF1L
variable region) has been substituted with His include, for
example, the following light chain variable regions.
[0297] a light chain variable region having the amino acid sequence
of SEQ ID NO: 6 (L73)
[0298] a light chain variable region having the amino acid sequence
of SEQ ID NO: 7 (L82)
[0299] a light chain variable region having the amino acid sequence
of SEQ ID NO: 8 (CLL5)
[0300] The amino acid positions and substitutions in each of the
above-described antibodies H3pI, H170, CLH5, L73, L82, and CLL5 are
shown below in Table 1. The amino acid positions are shown based on
the Kabat numbering.
TABLE-US-00001 TABLE 1 position 27 31 32 33 35 50 58 61 62 63 64 65
95 99 100B 102 H3pI H H H H H170 H H H H H H H V I H CLH5 H H H H F
I H H position 24 27 28 32 53 55 56 90 92 94 L73 H H H H L82 H H H
CLL5 H H H H H * In WT, the H chain has histidine at position 33,
while the L chain has histidine at position 55.
[0301] The present invention provides antibodies comprising at
least any one of the amino acid substitutions described above in
(a) to (j), and methods for producing the antibodies. Thus, the
antibodies of the present invention also include antibodies
comprising not only any of the amino acid substitutions described
above in (a) to (j) but also amino acid substitution(s) other than
those described above in (a) to (j). Amino acid substitutions other
than those described above in (a) to (j) include, for example,
substitution, deletion, addition, and/or insertion in the amino
acid sequence of CDRs and FRs.
[0302] Furthermore, the present invention provides the anti-IL-6
receptor antibodies of (1) to (28) below:
(1) an antibody that includes the heavy chain variable region
(VH1-IgG1 variable region) having the amino acid sequence from
positions 1 to 119 in SEQ ID NO: 21 (VH1-IgG1); (2) an antibody
that includes the heavy chain variable region (VH2-IgG1 variable
region) having the amino acid sequence from positions 1 to 119 in
SEQ ID NO: 22 (VH2-IgG1); (3) an antibody that includes the heavy
chain variable region (VH3-IgG1 variable region) having the amino
acid sequence from positions 1 to 119 in SEQ ID NO: 23 (VH3-IgG1);
(4) an antibody that includes the heavy chain variable region
(VH4-IgG1 variable region) having the amino acid sequence from
positions 1 to 119 in SEQ ID NO: 24 (VH4-IgG1); (5) an antibody
that includes the light chain variable region (VL1-CK variable
region) having the amino acid sequence from positions 1 to 107 in
SEQ ID NO: 25 (VL1-CK); (6) an antibody that includes the light
chain variable region (VL2-CK variable region) having the amino
acid sequence from positions 1 to 107 in SEQ ID NO: 26 (VL2-CK);
(7) an antibody that includes the light chain variable region
(VL3-CK variable region) having the amino acid sequence from
positions 1 to 107 in SEQ ID NO: 27 (VL3-CK); (8) an antibody
(Fv1-IgG1) that includes the heavy chain variable region of (2) and
the light chain variable region of (6); (9) an antibody (Fv2-IgG1)
that includes the heavy chain variable region of (1) and a light
chain variable region having the amino acid sequence of SEQ ID NO:
7 (L82); (10) an antibody (Fv3-IgG1) that includes the heavy chain
variable region of (4) and the light chain variable region of (5);
(11) an antibody (Fv4-IgG1) that includes the heavy chain variable
region of (3) and the light chain variable region of (7); (12) an
antibody (VH3-IgG2.DELTA.GK) that includes a heavy chain having the
amino acid sequence of SEQ ID NO: 33; (13) an antibody (VH3-M58)
that includes a heavy chain having the amino acid sequence of SEQ
ID NO: 34; (14) an antibody (VH3-M73) that includes a heavy chain
having the amino acid sequence of SEQ ID NO: 35; (15) an antibody
(Fv4-IgG2.DELTA.GK) that includes the heavy chain of (12) and a
light chain having the amino acid sequence of SEQ ID NO: 27
(VL3-CK); (16) an antibody (Fv4-M58) that includes the heavy chain
of (13) and a light chain having the amino acid sequence of SEQ ID
NO: 27 (VL3-CK); (17) an antibody (Fv4-M73) that includes the heavy
chain of (14) and a light chain having the amino acid sequence of
SEQ ID NO: 27 (VL3-CK); (18) an antibody (VH2-M71) that includes a
heavy chain having the amino acid sequence of SEQ ID NO: 36
(VH2-M71); (19) an antibody (VH2-M73) that includes a heavy chain
having the amino acid sequence of SEQ ID NO: 37 (VH2-M73); (20) an
antibody (VH4-M71) that includes a heavy chain having the amino
acid sequence of SEQ ID NO: 38 (VH4-M71); (21) an antibody
(VH4-M73) that includes a heavy chain having the amino acid
sequence of SEQ ID NO: 39 (VH4-M73); (22) an antibody (Fv1-M71)
that includes the heavy chain of (18) and a light chain having the
amino acid sequence of SEQ ID NO: 26 (VL2-CK); (23) an antibody
(Fv1-M73) that includes the heavy chain of (19) and a light chain
having the amino acid sequence of SEQ ID NO: 26 (VL2-CK); (24) an
antibody (Fv3-M71) that includes the heavy chain of (20) and a
light chain having the amino acid sequence of SEQ ID NO: 25
(VL1-CK); (25) an antibody (Fv3-M73) that includes the heavy chain
of (21) and a light chain having the amino acid sequence of SEQ ID
NO: 25 (VL1-CK); (26) an antibody that includes a light chain
having the amino acid sequence of SEQ ID NO: 25 (VL1-CK); (27) an
antibody that includes a light chain having the amino acid sequence
of SEQ ID NO: 26 (VL2-CK); and (28) an antibody that includes a
light chain having the amino acid sequence of SEQ ID NO: 27
(VL3-CK).
[0303] Furthermore, the present invention provides the FRs and CDRs
of (a) to (v) below:
(a) the heavy chain CDR1 of SEQ ID NO: 40 (VH1, 2, 3, 4); (b) the
heavy chain CDR2 of SEQ ID NO: 41 (VH1, 2); (c) the heavy chain
CDR2 of SEQ ID NO: 42 (VH3); (d) the heavy chain CDR2 of SEQ ID NO:
43 (VH4); (e) the heavy chain CDR3 of SEQ ID NO: 44 (VH1, 2); (f)
the heavy chain CDR3 of SEQ ID NO: 45 (VH3, 4); (g) the heavy chain
FR1 of SEQ ID NO: 46 (VH1, 2); (h) the heavy chain FR1 of SEQ ID
NO: 47 (VH3, 4): (i) the heavy chain FR2 of SEQ ID NO: 48 (VH1, 2,
3, 4); (j) the heavy chain FR3 of SEQ ID NO: 49 (VH1); (k) the
heavy chain FR3 of SEQ ID NO: 50 (VH2); (l) the heavy chain FR3 of
SEQ ID NO: 51 (VH3, 4); (m) the heavy chain FR4 of SEQ ID NO: 52
(VH1, 2, 3, 4); (n) the light chain CDR1 of SEQ ID NO: 53 (VL1, 2);
(o) the light chain CDR1 of SEQ ID NO: 54 (VL3); (p) the light
chain CDR2 of SEQ ID NO: 55 (VL1, VL3); (q) the light chain CDR2 of
SEQ ID NO: 56 (VL2); (r) the light chain CDR3 of SEQ ID NO: 57
(VL1, 2, 3); (s) the light chain FR1 of SEQ ID NO: 58 (VL1, 2, 3);
(t) the light chain FR2 of SEQ ID NO: 59 (VL1, 2, 3); (u) the light
chain FR3 of SEQ ID NO: 60 (VL1, 2, 3); and (v) the light chain FR4
of SEQ ID NO: 61 (VL1, 2, 3).
[0304] The respective sequences of the above (a) to (v) are shown
in FIG. 25. Furthermore, the present invention provides
polypeptides including any one of the FRs and CDRs of the above (a)
to (v).
[0305] The anti-IL-6 receptor antibodies of the present invention
also include fragments and modified products of antibodies
including any of the amino acid substitutions described above. Such
antibody fragments include, for example, Fab, F(ab')2, Fv, single
chain Fv (scFv) in which Fv of H and L chains are linked together
via an appropriate linker, single domain H chain and single domain
L chain (for example, Nat. Biotechnol. 2005 September;
23(9):1126-36), Unibody (WO 2007059782 A1), and SMIP (WO 2007014278
A2). The origin of antibodies is not particularly limited. The
antibodies include human, mouse, rat, and rabbit antibodies. The
antibodies of the present invention may also be chimeric,
humanized, fully humanized antibodies, or such.
[0306] Specifically, such antibody fragments are obtained by
treating antibodies with enzymes, for example, papain or pepsin, or
by constructing genes that encode such antibody fragments,
inserting them into expression vectors, and then expressing them in
appropriate host cells (see, for example, Co, M. S. et al., J.
Immunol. (1994) 152, 2968-2976; Better, M. & Horwitz, A. H.
Methods in Enzymology (1989) 178, 476-496; Plueckthun, A. &
Skerra, A. Methods in Enzymology (1989) 178, 497-515; Lamoyi, E.,
Methods in Enzymology (1989) 121, 652-663; Rousseaux, J. et al.,
Methods in Enzymology (1989) 121, 663-66; Bird, R. E. et al.,
TIBTECH (1991) 9, 132-137).
[0307] The present invention provides methods of producing (i) a
polypeptide of the present invention, or (ii) a polypeptide encoded
by a gene encoding the polypeptide of the present invention,
wherein the methods comprise the step of culturing a host cell
comprising a vector into which a polynucleotide encoding the
polypeptide of the present invention is introduced.
[0308] More specifically, the present invention provides methods of
producing a polypeptide of the present invention, which comprise
the steps of:
(a) culturing a host cell comprising a vector into which a gene
encoding the polypeptide of the present invention is introduced;
and (b) obtaining the polypeptide encoded by the gene.
[0309] scFv is obtained by linking the V regions of antibody H and
L chains. In such scFv, the H chain V region is linked to the L
chain V region via a linker, preferably a peptide linker (Huston,
J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 10.0,
5879-5883). The H chain and L chain V regions in an scFv may be
derived from any of the antibodies described above. The peptide
linker to link the V regions includes, for example, arbitrary
single chain peptides of 12 to 19 amino acid residues.
[0310] When an anti-IL-6 receptor antibody of the present invention
includes a constant region, the constant region may be of any type,
for example, IgG1, IgG2, or IgG4 may be used. The constant region
is preferably a human antibody constant region. Alternatively, the
constant region may be a modified form including substitution,
deletion, addition, and/or insertion in the amino acid sequence of
human IgG1, human IgG2, or human IgG4 constant regions.
[0311] Preferred IL-6 receptor to which an anti-IL-6 receptor
antibody of the present invention binds is human IL-6 receptor.
[0312] The anti-IL-6 receptor antibodies of the present invention
are superior in the retention in plasma, and they exist for a
prolonged period in the plasma in a form capable of binding to the
antigen, i.e., soluble or membrane-associated IL-6 receptors. Thus,
the anti-IL-6 receptor antibodies bind in vivo to soluble or
membrane-associated IL-6 receptors for a prolonged period.
Furthermore, the anti-IL-6 receptor antibodies are capable of
binding to IL-6 receptors twice or more times, and thus assumed to
be able to neutralize three or more IL-6 receptors.
<Pharmaceutical Compositions>
[0313] The present invention also relates to pharmaceutical
compositions that include antigen-binding molecules of the present
invention, antigen-binding molecules isolated by the screening
methods 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 are superior in the retention in plasma, and
thus, expected to reduce the administration frequency of the
antigen-binding molecules, and are therefore useful as
pharmaceutical compositions. The pharmaceutical composition of the
present invention may include pharmaceutically acceptable
carriers.
[0314] In the present invention, pharmaceutical compositions
ordinarily refer to agents for treating or preventing, or testing
and diagnosing diseases.
[0315] 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 is adjusted
to obtain an appropriate amount in a pre-determined range.
[0316] Sterile compositions for injection can be formulated using
vehicles such as distilled water for injection, according to
standard formulation practice.
[0317] 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).
[0318] 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.
[0319] 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. For example, they can be administered systemically
or locally by intravenous injection, intramuscular injection,
intraperitoneal injection, subcutaneous injection, or such.
[0320] 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 1000 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.
[0321] 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 (Gln) residue
into a pyroglutamic acid (pGlu) residue 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.
[0322] All prior art documents cited in the specification are
incorporated herein by reference.
EXAMPLES
[0323] Herein below, the present invention will be specifically
described with reference to Examples, but it is not to be construed
as being limited thereto.
Example 1
Production of Modified Humanized PM1 Antibody
Preparation of Recombinant Soluble Human IL-6 Receptor (SR344)
[0324] A recombinant human IL-6 receptor of the human IL-6
receptor, which served an antigen, was prepared as described below.
A CHO cell line that constantly expresses a soluble human IL-6
receptor (hereinafter referred to as SR344) (Yamasaki, et al.,
Science 1988; 241: 825-828 (GenBank #X12830)) consisting of the
amino acid sequence from the 1st amino acid to the 344th amino acid
on the N-terminal side as reported in J. Biochem., 108, 673-676
(1990), was produced.
[0325] SR344 was purified from culture supernatant obtained from
the SR344-expressing CHO cells using three column chromatographies:
Blue Sepharose 6 FF column chromatography, affinity chromatography
using a column in which an antibody specific to SR344 is
immobilized, and gel filtration column chromatography. The fraction
that eluted as the main peak was used as the final purified
product.
Preparation of Recombinant Cynomolgus Monkey Soluble IL-6 Receptor
(cIL-6R)
[0326] Oligo DNA primers Rhe6Rf1 (SEQ ID NO: 16) and Rhe6Rr2 (SEQ
ID NO: 17) were produced based on the publicly-available rhesus
monkey IL-6 receptor gene sequence (Birney et al., Ensemble 2006,
Nucleic Acids Res., 2006, Jan. 1; 34 (Database issue): D556-61).
Using cDNA prepared from cynomolgus pancreas as a template, a DNA
fragment encoding the entire length of cynomolgus monkey IL-6
receptor gene was prepared by PCR using primers Rhe6Rf1 and
Rhe6Rr2. Using the resulting DNA fragment as a template, a 1131 bp
DNA fragment (SEQ ID NO: 20) encoding a protein in which
6.times.His is added to the C terminal of the soluble region
(Met1-Pro363) containing a signal region of cynomolgus monkey IL-6
receptor gene, was amplified by PCR using the oligo DNA primers
CynoIL6R N-EcoRI (SEQ ID NO: 18) and CynoIL6R C-NotI-His (SEQ ID
NO: 19). The resulting DNA fragment was digested with EcoRI and
NotI and inserted into a mammalian cell expression vector, and this
was then used to produce a stable expression CHO line
(cyno.sIL-6R-producing CHO cells).
[0327] A culture medium of cyno.sIL-6R-producing CHO cells was
purified with a HisTrap column (GE Healthcare Biosciences),
concentrated using Amicon Ultra-15 Ultracel-10k (Millipore), and
further purified with a Superdex 200 pg 16/60 gel filtration column
(GE Healthcare Biosciences) to obtain the final purified product of
soluble cynomolgus monkey IL-6 receptor (hereinafter referred to as
cIL-6R).
Preparation of Recombinant Cynomolgus Monkey IL-6 (cIL-6)
[0328] Cynomolgus monkey IL-6 was prepared as follows. A nucleotide
sequence encoding the 212 amino acids registered under SWISSPROT
Accession No. P79341 was produced, cloned into a mammalian cell
expression vector, and introduced into CHO cells to produce a
stable expression cell line (cyno.IL-6-producing CHO cells). A
culture medium of cyno.IL-6-producing CHO cells was purified with
an SP-Sepharose/FF column (GE Healthcare Biosciences), concentrated
using Amicon Ultra-15 Ultracel-5k (Millipore), and then further
purified with a Superdex 75 pg 26/60 gel filtration column (GE
Healthcare Biosciences). This was concentrated using Amicon
Ultra-15 Ultracel-5k (Millipore) to obtain a final purified product
of cynomolgus monkey IL-6 (hereinafter referred to as cIL-6).
Establishment of Human gp130-Expressing BaF3 Cell Line
[0329] A BaF3 cell line expressing human gp130 was established as
indicated below in order to obtain a cell line exhibiting
IL-6-dependent growth.
[0330] Full-length human gp130 cDNA (Hibi et al., Cell 1990; 63:
1149-1157 (GenBank #NM.sub.--002184)) was amplified by PCR and
cloned into the expression vector pCOS2Zeo, which was prepared by
removing the DHFR gene expression site from pCHOI (Hirata, et al.,
FEBS Letter 1994; 356: 244-248) and inserting a Zeocin resistance
gene expression site, to construct pCOS2Zeo/gp130. Full-length
human IL-6R cDNA was amplified by PCR and cloned into pcDNA3.1(+)
(Invitrogen) to construct hIL-6R/pcDNA3.1(+). 10 .mu.g of
pCOS2Zeo/gp130 was mixed into BaF3 cells (0.8.times.10.sup.7 cells)
suspended in PBS, and pulsed using a Gene Pulser (Bio-Rad) at a
voltage of 0.33 kV and capacitance of 950 .mu.FD. BaF3 cells having
undergone gene introduction by electroporation treatment were
cultured one whole day and night in RPMI1640 medium (Invitrogen)
containing 0.2 ng/mL of mouse interleukin-3 (Peprotech) and 10%
fetal bovine serum (hereinafter referred to as FBS, HyClone), and
then screened by adding RPMI1640 medium containing 100 ng/mL of
human interleukin-6 (R&D Systems), 100 ng/mL of human
interleukin-6 soluble receptor (R&D Systems) and 10% FBS to
establish a human gp130-expressing BaF3 cell line (hereinafter
referred to as BaF3/gp130). Since this BaF3/gp130 proliferates in
the presence of human interleukin-6 (R&D Systems) and SR344, it
can be used to evaluate the growth inhibitory activity (namely,
IL-6 receptor-neutralizing activity) of anti-IL-6 receptor
antibody.
Production of Humanized Anti-IL-6 Receptor Antibody
[0331] In the context of the instant example and elsewhere herein,
the term "wild type" is abbreviated as WT, the term "wild type H
chain" is abbreviated as H(WT) (amino acid sequence of SEQ ID NO:
9), and the term "wild type L chain is abbreviated as L(WT) (amino
acid sequence: SEQ ID NO. 10). In this context, mutations were
introduced into the framework sequence and CDR sequence of
humanized mouse PM1 antibody described in Cancer Res. 1993, Feb.
15; 53(4): 851-6, to produce modified H chains H53 (amino acid
sequence: SEQ ID NO: 1) and PF1H (amino acid sequence: SEQ ID NO:
11), and modified L chains L28 (amino acid sequence: SEQ ID NO: 12)
and PF1L (amino acid sequence: SEQ ID NO: 2). More specifically,
the mutants were produced using the QuikChange Site-Directed
Mutagenesis Kit (Stratagene) according to the method described in
the instructions provided, and the resulting plasmid fragments were
inserted into a mammalian cell expression vector to produce the
desired H chain expression vectors and L chain expression vectors.
The nucleotide sequences of the obtained expression vectors were
determined using conventional methodologies known to persons
skilled in the art.
Expression and Purification of Humanized Anti-IL-6 Receptor
Antibody
[0332] The antibodies were expressed by the method described below.
Human embryonic kidney cancer-derived HEK293H cell line
(Invitrogen) was suspended in DMEM (Invitrogen) supplemented with
10% Fetal Bovine Serum (Invitrogen). The cells were plated at 10 ml
per dish in dishes for adherent cells (10 cm in diameter; CORNING)
at a cell density of 5 to 6.times.10.sup.5 cells/ml and cultured in
a CO.sub.2 incubator (37.degree. C., 5% CO.sub.2) for one whole day
and night. Then, the medium was removed by aspiration, and 6.9 ml
of CHO-S-SFM-II medium (Invitrogen) was added. The prepared plasmid
was introduced into the cells by the lipofection method. The
resulting culture supernatants were collected, centrifuged
(approximately 2000 g, 5 min, room temperature) to remove cells,
and sterilized by filtering through 0.22-.mu.m filter
MILLEX.RTM.-GV (Millipore) to obtain the supernatants. Antibodies
were purified from the obtained culture supernatants by a method
known to those skilled in the art using rProtein A Sepharose.TM.
Fast Flow (Amersham Biosciences). To determine the concentration of
the purified antibody, absorbance was measured at 280 nm using a
spectrophotometer. Antibody concentrations were calculated from the
determined values using an absorbance coefficient calculated by the
PACE method (Protein Science 1995; 4:2411-2423).
Example 2
Production of pH-Dependently-Binding Antibody H3pI/L73
Method for Creating Antibody Capable of Neutralizing Antigen
Multiple Times
[0333] Since IgG molecules are divalent, a single IgG molecule can
neutralize up to two antigen molecules when the two sites bind to
the antigens; however, it cannot neutralize three or more antigen
molecules. Therefore, to maintain the neutralizing effect of a
neutralizing antibody over a certain period, it is necessary to
administer an amount of the antibody equal to or greater than the
amount of antigen produced during the period. Thus, there is a
limitation on the extent to which the required dose of antibody can
be reduced by improving the pharmacokinetics or affinity of
antibody. Therefore, if it were possible to neutralize two or more
antigen molecules with a single IgG molecule, the same dose could
improve the duration of neutralizing effect, or alternatively the
dose of antibody required to achieve the same duration could be
reduced.
[0334] For neutralizing antibodies, there are two types of target
antigens: soluble-type antigens, which are present in plasma, and
membrane-bound antigens, which are expressed on the surface of
cells.
[0335] When the antigen is a membrane-bound antigen, an
administered antibody binds to the membrane antigen on the cellular
surface, and the antibody is subsequently taken up into endosomes
within the cell by internalization together with the membrane
antigen bound to the antibody. Then, the antibody which is kept
bound to the antigen moves to a lysosome where it is degraded by
lysosome together with the antigen. The elimination of antibody
from the plasma mediated by internalization by membrane antigen is
referred to as antigen-dependent elimination, and this has been
reported for numerous antibody molecules (Drug Discov. Today, 2006
January; 11(1-2): 81-8). Since a single IgG antibody molecule binds
to two antigen molecules when it divalently binds to antigens, and
is then internalized and directly degraded by lysosome, a single
ordinary IgG antibody cannot neutralize two or more antigen
molecules (FIG. 1).
[0336] The reason for the long retention (slow elimination) of IgG
molecules in plasma is that FcRn, known as an IgG molecule salvage
receptor, functions (Nat. Rev. Immunol. 2007 September; 7(9):
715-25). IgG molecules that have been taken up into endosomes by
pinocytosis bind to FcRn expressed in endosomes under
intraendosomal acidic conditions. IgG molecules bound to FcRn move
to the cell surface where they dissociate from FcRn under neutral
conditions in plasma and return to plasma, while IgG molecules
unable to bind to FcRn proceed into lysosomes where they are
degraded (FIG. 2).
[0337] IgG molecules bound to a membrane antigen are taken up into
intracellular endosomes by internalization, move into lysosomes
while bound to the antigen, and undergo degradation. When an IgG
antibody divalently binds to antigens, it neutralizes two antigen
molecules and undergoes degradation together with the antigens. If
the IgG antibody, when taken up into intracellular endosomes by
internalization, can dissociate from the antigen under
intraendosomal acidic conditions, the dissociated antibody may be
able to bind to FcRn expressed in the endosomes. The IgG molecule
dissociated from the antigen and bound to FcRn is transferred to
the cell surface and then dissociated from FcRn under neutral
conditions in the plasma, thereby return to the plasma again. The
IgG molecule that has returned to the plasma is able to bind to a
new membrane antigen again. The repetition of this process allows a
single IgG molecule to repeatedly bind to membrane antigens,
thereby enabling neutralization of a multiple antigens with a
single IgG molecule (FIG. 3).
[0338] In the case of a soluble antigen, an antibody administered
binds to the antigen in the plasma, and remains in the plasma in
the form of an antigen-antibody complex. Normally, while the
retention of antibody in plasma is very long (elimination rate is
very slow) due to the function of FcRn as described above, the
retention of antigen in plasma is short (elimination rate is fast).
Thus, antibody-bound antigens show retention in plasma comparable
to that of antibody (elimination rate is very slow). Antigens are
produced in the body at a constant rate and, in the absence of
antibody, present in plasma at a concentration at which the antigen
production rate and the antigen elimination rate are under
equilibrium. In the presence of antibody, most of the antigens are
bound to antibodies, resulting in the very slow elimination of
antigens. Thus, the antigen concentration in plasma increases as
compared with that in the absence of antibody (Kidney Int. 2003,
64, 697-703; J. National Cancer Institute 2002, 94(19), 1484-1493;
J. Allergy and Clinical Immunology 1997, 100(1), 110-121; Eur. J.
Immunol. 1993, 23; 2026-2029). Even if the affinity of antibody for
antigen is infinite, antigen concentration elevates as antibody is
slowly eliminated from the plasma, and the neutralizing effect of
antibody terminates after the concentrations of antibody and
antigen become equal. Although antibodies with a stronger
dissociation constant (KD) can neutralize soluble antigens at a
lower antibody concentration, antibodies at a concentration half or
less than the concentration of antigen present are unable to
neutralize antigens regardless of how strong the affinity of
antibody is (Biochem. Biophys. Res. Commun. 2005 Sep. 9; 334(4):
1004-13). As is the case with IgG molecules not bound to antigens,
IgG molecules bound to antigens in the plasma are also taken up
into endosomes by pinocytosis, and bind to FcRn expressed in
endosomes under intraendosomal acidic conditions. The IgG molecules
bound to FcRn moves to the cell surface while the antibody is kept
bound to the antigen and then dissociate from the FcRn under
neutral conditions in the plasma. Since the IgG molecules return to
the plasma while bound to the antigen, they cannot bind to new
antigens in the plasma. In this case, if IgG molecules can
dissociate from the antigen under intraendosomal acidic conditions,
the dissociated antigen will not be able to bind to FcRn and
thereby may be degraded by lysosomes. On the other hand, the IgG
molecules can return to the plasma again by binding to FcRn. Since
the IgG molecules that have returned to the plasma have already
dissociated from the antigen in endosomes, they are able to bind to
a new antigen again in the plasma. The repetition of this process
allows a single IgG molecule to repeatedly bind to soluble
antigens. This enables a single IgG molecule to neutralize multiple
antigens (FIG. 4).
[0339] Thus, regardless of whether the antigen is a membrane
antigen or soluble antigen, if the dissociation of IgG antibody
from the antigen is possible under intraendosomal acidic
conditions, a single IgG molecule would be able to repeatedly
neutralize antigens. In order for IgG antibodies to dissociate from
antigens under intraendosomal acidic conditions, it is necessary
that antigen-antibody binding be considerably weaker under acidic
conditions than under neutral conditions. Since membrane antigens
on the cell surface need to be neutralized, antibodies have to
strongly bind to antigens at the cell surface pH, namely pH 7.4.
Since the intraendosomal pH has been reported to be typically pH
5.5 to 6.0 (Nat. Rev. Mol. Cell. Biol. 2004 February; 5(2):
121-32), an antibody that weakly binds to an antigen at pH 5.5 to
6.0 is considered to dissociate from the antigen under
intraendosomal acidic conditions. More specifically, a single IgG
molecule that strongly binds to an antibody at the cell surface pH
of 7.4 and weakly binds to the antigen at the intraendosomal pH of
5.5 to 6.0 may be able to neutralize a multiple antigens and
thereby improve the pharmacokinetics.
[0340] In general, protein-protein interactions consist of
hydrophobic interaction, electrostatic interaction and hydrogen
bonding, and the binding strength is typically expressed as a
binding constant (affinity) or apparent binding constant (avidity).
pH-dependent binding, whose binding strength varies between neutral
conditions (pH 7.4) and acidic conditions (pH 5.5 to 6.0), is
present in naturally-occurring protein-protein interactions. For
example, the above-mentioned binding between IgG molecules and FcRn
known as a salvage receptor for IgG molecules is strong under
acidic conditions (pH 5.5 to 6.0) but remarkably weak under neutral
conditions (pH 7.4). Most of such pH-dependently changing
protein-protein interactions are associated with histidine
residues. Since the pKa of histidine residue is in the vicinity of
pH 6.0 to 6.5, the proton dissociation state of histidine residues
varies between neutral conditions (pH 7.4) and acidic conditions
(pH 5.5 to 6.0). Specifically, histidine residues are not charged
and function as hydrogen atom acceptors under neutral conditions
(pH 7.4), while they become positively charged and function as
hydrogen atom donors under acidic conditions (pH 5.5 to 6.0). It
has been reported that the pH-dependent binding of the
above-described IgG-FcRn interaction is also associated with
histidine residues present in IgG (Mol. Cell. 2001 April; 7(4):
867-77).
[0341] Therefore, pH-dependence can be imparted to protein-protein
interactions by substituting an amino acid residue involved in
protein-protein interactions with a histidine residue, or by
introducing a histidine into an interaction site. Such attempts
have also been made in protein-protein interactions between
antibodies and antigens, and a mutant antibody with antigen-binding
ability decreased under acidic conditions has been successfully
acquired by introducing histidine into the CDR sequence of an
anti-egg white lysozyme antibody (FEBS Letter (vol. 309, No. 1,
85-88, 1992)). In addition, an antibody that is prepared by
introducing histidine into its CDR sequence and specifically binds
to an antigen at the low pH of cancer tissues but weakly binds
under neutral conditions has been reported (WO 2003-105757).
[0342] Although methods for introducing pH dependency into
antigen-antibody reactions have been reported as described above,
an IgG molecule that neutralizes multiple antigens by strongly
binding to antigens at the body fluid pH of 7.4 but weakly binding
to antigens at the intraendosomal pH of pH 5.5 to 6.0 has not been
reported. In other words, there have been no reports relating to
modifications that significantly reduce the binding under acidic
conditions while maintaining the binding under neutral conditions
such that, as compared to an unmodified antibody, a modified
antibody binds to antigens multiple times in vivo and thereby shows
improved pharmacokinetics as well as improved duration of the
neutralizing effect at the same dose.
[0343] The IL-6 receptor is present in the body in the form of
either soluble IL-6 receptor or membrane IL-6 receptor (Nat. Clin.
Pract. Rheumatol. 2006 November; 2(11): 619-26). Anti-IL-6 receptor
antibodies bind to both the soluble IL-6 receptor and membrane IL-6
receptor, and neutralize their biological action. It is considered
that, after binding to the membrane IL-6 receptor, anti-IL-6
receptor antibodies are taken up into intracellular endosomes by
internalization while bound to the membrane IL-6 receptor, then
move into lysosomes while the antibodies are kept bound to the
membrane IL-6 receptor, and undergo degradation by lysosomes
together with the membrane IL-6 receptor. In fact, it has been
reported that a humanized anti-IL-6 receptor antibody exhibits
non-linear clearance, and its antigen-dependent elimination greatly
contributes to the elimination of the humanized anti-IL-6 receptor
antibody (The Journal of Rheumatology, 2003, 30; 71426-1435). Thus,
one humanized anti-IL-6 receptor antibody binds to one or two
membrane IL-6 receptors (monovalently or divalently), and is then
internalized and degraded in lysosomes. Therefore, if it is
possible to produce modified antibodies that exhibit greatly
reduced binding ability under acidic conditions but retain the same
binding ability as the wild type humanized anti-IL-6 receptor
antibody under neutral conditions (pH-dependent binding anti-IL-6
receptor antibody), multiple IL-6 receptors can be neutralized with
a single humanized anti-IL-6 receptor antibody. Thus, in comparison
with wild type humanized anti-IL-6 receptor antibodies,
pH-dependent binding anti-IL-6 receptor antibodies may improve the
duration of the neutralizing effect in vivo at the same dosage.
Production of pH-Dependently Binding Humanized Anti-IL-6 Receptor
Antibody H3pI/L73:
[0344] Introduction of histidine into a CDR has been reported as a
method for introducing pH-dependent binding to antigen-antibody
reaction (FEBS Letter (vol. 309, No. 1, 85-88, 1992)). In order to
find amino acid residues exposed on the surface of the variable
region of the H53/PF1L produced in Example 1 and possible residues
interacting with the antigen, a Fv region model of H53/PF1L was
created by homology modeling using MOE software (Chemical Computing
Group Inc.). A three-dimensional model constructed on the basis of
the sequence information of H53/PF1L was used to select H27, H31,
H35, L28, L32 and L53 (Kabat numbering, Kabat, E. A. et al., 1991,
Sequences of Proteins of Immunological Interest, NIH) as mutation
sites that may introduce pH-dependent antigen-binding by histidine
introduction. The product in which the residues at H27, H31 and H35
in H53 produced in Example 1 were substituted with histidines was
designated as H3pI (amino acid sequence: SEQ ID NO: 3), and the
product in which the residues at L28, L32 and L53 in PF1L produced
in Example 1 were substituted with histidines was designated as L73
(amino acid sequence: SEQ ID NO: 6).
Production, Expression and Purification of H3pI/L73 Expression
Vector
[0345] Amino acid modification was carried out to produce
antibodies modified at the selected sites. Mutations were
introduced into H53 (nucleotide sequence: SEQ ID NO: 13) and PF1L
(nucleotide sequence: SEQ ID NO: 14) produced in Example 1 to
produce H3pI (amino acid sequence: SEQ ID NO: 3) and L73 (amino
acid sequence: SEQ ID NO: 6). More specifically, the QuikChange
Site-Directed Mutagenesis Kit (Stratagene) was used according to
the method described in the instructions provided, and the
resulting plasmid fragments were inserted into a mammalian cell
expression vector to produce the desired H chain expression vector
and L chain expression vector. The nucleotide sequences of the
resulting expression vectors were determined using a method known
to persons skilled in the art. H3pI/L73 which uses H3pI for the H
chain and L73 for the L chain was expressed and purified by the
method described in Example 1.
Example 3
Conferring pH-Dependent Antigen Binding Ability by His Modification
of CDR Using Phage Display Technology
[0346] Production of scFv Molecule of Humanized PM1 Antibody
[0347] The humanized PM1 antibody, which is a humanized anti-IL-6R
antibody (Cancer Res. 1993 Feb. 15; 53(4): 851-6), was converted
into scFv. The VH and VL regions were amplified by PCR, and
humanized PM1 HL scFv having the linker sequence GGGGSGGGGSGGGGS
(SEQ ID NO. 15) between VH and VL was produced.
Selection of Histidine-Introducible Positions by Histidine
Scanning
[0348] PCR was performed using the produced humanized PM1 HL scFv
DNA as a template to produce a histidine library in which any one
of the CDR amino acids is replaced with histidine. The library
portions were constructed by PCR using primers in which the codon
of an amino acid desired to be mutated for the library was replaced
with CAT, a codon corresponding to histidine, and other portions
were constructed by normal PCR. These portions were then linked by
assemble PCR. The constructed library was digested with SfiI,
inserted into a phagemide vector pELBG lad that was also digested
with SfiI, and then used to transform XL1-Blue (Stratagene). The
resulting colonies were used to evaluate antigen binding by phage
ELISA and analyze the sequence of HL scFv. Phage ELISA was carried
out using a plate coated with SR344 at 1 .mu.g/mL in accordance
with J. Mol. Biol. 1992; 227: 381-388. Clones that were found to
bind to SR344 were subjected to sequence analysis using specific
primers.
[0349] Phage titer was determined by ELISA with an anti-Etag
antibody (GE Healthcare) and anti-M13 antibody (GE Healthcare).
This value was then used to select positions where substitution of
the CDR residue with histidine did not significantly alter the
binding ability as compared to humanized PM1 HL scFv, based on the
results of phage ELISA for SR344. The selected positions are shown
in Table 2. Numbering of each residue was in accordance with the
Kabat numbering (Kabat, et al., 1991, Sequences of Proteins of
Immunological Interest, NIH).
TABLE-US-00002 TABLE 2 Positions of Histidine Substitution Not
Significantly Affecting Binding Ability H31, H50, H54, H56, H57,
H58, H59, H60, H61, H62, H63, H64, H65, H100a, H100b, H102 L24,
L26, L27, L28, L30, L31, L32, L52, L53, L54, L56, L90, L92, L93,
L94
Construction of Histidine-Modified CDR Library
[0350] A library was designed in which the amino acids of CDR
residues that did not significantly alter the binding ability when
substituted with histidine as shown in Table 2
(histidine-introducible positions) are their original sequence
(wild type sequence) or histidine. The library was constructed
based on the sequences of the H chain PF1H and the L chain PF1L
produced in Example 1 such that the mutated positions for the
library have the original sequences or histidines (either the
original sequence or histidines).
[0351] The library portions were constructed by PCR using primers
that were designed such that a position desired to be mutated for
the library has the original amino acid codon or histidine codon,
and other portions were produced by normal PCR, or by PCR using
synthetic primers as in the library portions. These portions were
then linked by assemble PCR (J. Mol. Biol. 1996; 256: 77-88).
[0352] This library was used to construct a ribosome display
library in accordance with J. Immunological Methods 1999; 231:
119-135. In order to carry out Escherichia coli cell-free in vitro
translation, an SDA sequence (ribosome binding site) and T7
promoter were added to the 5' side, and a gene3 partial sequence
serving as a linker for ribosome display was ligated to the 3' side
using SfiI.
Acquisition of pH-Dependent Binding scFv from Library by Bead
Panning
[0353] In order to concentrate only scFv having the ability to bind
to SR344, panning was carried out twice by the ribosome display
method in accordance with Nature Biotechnology 2000 December; 18:
1287-1292. The prepared SR344 was biotinylated using
NHS-PEO4-Biotin (Pierce) to obtain an antigen. Panning was carried
out using 40 nM of the biotinylated antigen.
[0354] Using the resulting DNA pool as a template, HL scFv was
restored by PCR using specific primers. After digesting with SfiI,
the digested HL scFv was inserted into a phagemide vector pELBG lad
that was also digested with SfiI, and then used to transform
XL1-Blue (Stratagene).
[0355] Escherichia coli cells carrying the desired plasmid were
grown to 0.4 to 0.6 O.D./mL in 2YT medium containing 100 .mu.g/mL
ampicillin and 2% glucose. A helper phage (M13KO7,
4.5.times.10.sup.11 pfu) was added thereto, statically cultured for
30 minutes at 37.degree. C., and then cultured with shaking for 30
minutes at 37.degree. C. The culture was transferred to a 50 mL
Falcon tube, centrifuged for 10 minutes at 3000 rpm, resuspended in
2YT medium containing 100 .mu.g/mL ampicillin, 25 kanamycin, and
0.5 mM IPTG, and then incubated overnight at 30.degree. C.
[0356] The culture incubated overnight was precipitated with 2.5 M
NaCl and 10% PEG, and then diluted with PBS to obtain a phage
library solution. 10% M-PBS (PBS containing 10% skim milk) and 1 M
Tris-HCl were added to the phage library solution to the final
concentration of 2.5% M-PBS and pH 7.4. Panning was carried out by
a typical panning method using an antigen immobilized on magnetic
beads (J. Immunol. Methods 2008 Mar. 20; 332(1-2): 2-9; J. Immunol.
Methods 2001 Jan. 1; 247(1-2): 191-203; Biotechnol. Prog. 2002
March-April; 18(2): 212-20). More specifically, 40 pmol of
biotin-labeled SR344 was added to the prepared phage library and
the library was contacted with the antigen for 60 minutes at
37.degree. C. Streptavidin-coated beads (Dynal M-280) washed with
5% M-PBS (PBS containing 5% skim milk) were added and allowed to
bind for 15 minutes at 37.degree. C. The beads were washed five
times with both 0.5 ml of PBST (PBS containing 0.1% Tween-20, pH
7.4) and PBS (pH 7.4). The beads were then suspended in 1 mL of PBS
(pH 5.5) at 37.degree. C., and the phage was recovered immediately.
The recovered phage solution was added to 10 mL of
logarithmic-growth-phase XL1-Blue (OD600 of 0.4 to 0.5) and allowed
to stand for 30 minutes at 37.degree. C. for infection. The
infected E. coli were plated onto a 225 mm.times.225 mm plate
containing 2 YT, 100 .mu.g/mL ampicillin and 2% glucose. These E.
coli were used to begin additional phage culture in the same manner
as described above and repeat the panning 8 times.
Evaluation by Phage ELISA
[0357] The above single colonies were inoculated in 100 .mu.L of
2YT, 100 .mu.g/mL ampicillin, 2% glucose and 12.5 .mu.g/mL
tetracycline and cultured overnight at 30.degree. C. 2 .mu.L of
this culture was inoculated into 300 .mu.L of 2YT, 100 .mu.g/mL
ampicillin and 2% glucose, and then cultured for 4 hours at
37.degree. C. A helper phage (M13KO7) was added to the culture at
9.times.10.sup.8 pfu, allowed to stand for 30 minutes at 37.degree.
C. and then shaken for 30 minutes at 37.degree. C. for infection.
Subsequently, the medium was replaced with 300 .mu.L of 2 YT, 100
.mu.g/mL ampicillin, 25 kanamycin, and 0.5 mM IPTG. After culturing
overnight at 30.degree. C., the centrifuged supernatant was
recovered. 360 .mu.L of 50 mM PBS (pH 7.4) was added to 40 .mu.L of
the centrifuged supernatant and subjected to ELISA. A StreptaWell
96-well microtiter plate (Roche) was coated overnight with 100
.mu.L of PBS containing 62.5 ng/mL of biotin-labeled SR344. After
removing the antigen by washing with PBST, blocking was carried out
with 250 .mu.L of 2% BSA-PBS for 1 hour or more. After removing the
2% BSA-PBS, the prepared culture supernatant was added and allowed
to stand for 1 hour at 37.degree. C. for antibody binding. After
washing, 50 mM PBS (pH 7.4) or 50 mM PBS (pH 5.5) was added and
incubated by standing for 30 minutes at 37.degree. C. After
washing, detection was carried out with an HRP-conjugated anti-M13
antibody (Amersham Pharmacia Biotech) diluted with 2% BSA-PBS and
TMB single solution (Zymed), followed by the addition of sulfuric
acid to stop the reaction, and the measurement of absorbance at 450
nm.
[0358] However, no clones exhibiting potent pH-dependent binding
ability were obtained by this panning using the antigen immobilized
on the magnetic beads. Clones that were found to show weak
pH-dependent binding ability were subjected to sequence analysis
using specific primers. The positions in these clones where
histidine was present at a high rate are shown in Table 3.
TABLE-US-00003 TABLE 3 Positions of Histidine Substitution Detected
Using Phage Library (Magnetic Bead Panning) H50, H58, H61, H62,
H63, H64, H65, H102 L24, L27, L28, L32, L53, L56, L90, L92, L94
Acquisition of pH-Dependently Binding scFv from Library by Column
Panning
[0359] No clones having strong pH-dependent binding ability were
obtained by typical panning using the magnetic bead-immobilized
antigen. This may be due to the following reasons. In the panning
using an antigen immobilized on magnetic beads or a plate, all
phages dissociated from the magnetic beads or plate under acidic
conditions are collected. Thus, phage clones having weak pH
dependency recovered together reduce the likelihood that clones
having strong pH dependency are included in the finally
concentrated clones.
[0360] Therefore, panning using a column immobilized with an
antigen was examined as a more stringent panning method (FIG. 5).
There have been no previous reports on the acquisition of clones
having pH-dependent binding ability by using panning with an
antigen-immobilized column. In the panning using an
antigen-immobilized column, when phages that have been bound under
neutral conditions are eluted under acidic conditions, clones
having weak pH dependency rebind to the antigen within the column
and are thereby less eluted, allowing strongly pH-dependent clones
that less rebind within the column to be selectively eluted from
the column. In addition, although "all" phages that have
dissociated under acidic conditions are recovered in the panning
using the antigen immobilized on magnetic beads or a plate, the
panning using a column immobilized with the antigen enables
selective recovery of phages having strong pH-dependent binding
ability by allowing an acidic buffer to flow through the column to
begin the elution and recovering only "appropriate fractions".
[0361] First, a column to which the antigen SR344 was immobilized
was prepared. 200 .mu.l of Streptavidin Sepharose (GE Healthcare)
was washed with 1 ml of PBS, suspended in 500 .mu.L of PBS, and
contacted with 400 pmol of biotin-labeled SR344 for 1 hour at room
temperature. Subsequently, an empty column (Amersham Pharmacia
Biotech) was filled with the above sepharose and washed with about
3 mL of PBS. The above-mentioned PEG-precipitated library phages
were diluted to 1/25 with 0.5% BSA-PBS (pH 7.4), passed through a
0.45 nm filter, and then added to the column. After washing with
about 6 mL of PBS (pH 7.4), 50 mM MES-NaCl (pH5.5) was allowed to
flow through the column to elute antibodies that dissociate under
low pH. The appropriate eluted fractions were collected, and the
recovered phage solution was added to 10 mL of
logarithmic-growth-phase XL1-Blue (OD600 of 0.4 to 0.5) and allowed
to stand for 30 minutes at 37.degree. C.
[0362] The infected E. coli were plated onto a 225 mm.times.225 mm
plate containing 2YT, 100 .mu.g/mL ampicillin, and 2% glucose.
These E. coli were used to begin additional phage culture in the
same manner as described above and repeat the panning 6 times.
Evaluation by Phage ELISA
[0363] The resulting phages were evaluated by phage ELISA. Clones
that were found to have strong pH dependency were subjected to
sequence analysis using specific primers. As a result, several
clones showing strong pH-dependent binding as compared to WT were
obtained. As shown in FIG. 6, clone CL5 (H chain: CLH5, L chain:
CLL5) (CLH5: amino acid sequence of SEQ ID NO: 5, CLL5: amino acid
sequence of SEQ ID NO: 8) was found to exhibit particularly strong
pH-dependent binding as compared to WT. It was thus confirmed that
antibodies exhibiting strong pH-dependent binding, while being
unable to be obtained by typical panning using the antigen
immobilized onto magnetic beads, can be obtained by panning using a
column immobilized with the antigen. Therefore, panning using an
antigen-immobilized column was found to be a highly effective
method for obtaining pH-dependently binding antibodies from a
library. The amino acid sequences of the clones showing
pH-dependent binding were analyzed, and the positions where
histidine was present at a high probability in the concentrated
clones are shown in Table 4.
TABLE-US-00004 TABLE 4 Positions of Histidine Substitution found by
Phage Library (Column Panning) H31, H50, H58, H62, H63, H65, H100b,
H102 L24, L27, L28, L32, L53, L56, L90, L92, L94
Example 4
Expression and Purification of Histidine-Modified Humanized IL-6
Receptor Antibody Production, Expression and Purification of
Expression Vector of Histidine-Modified Humanized IL-6 Receptor
Antibody
[0364] In order to convert clones showing strong pH dependency in
phage ELISA to IgG, VH and VL were respectively amplified by PCR,
digested with XhoI/NheI and EcoRI, and inserted to a mammalian cell
expression vector. The nucleotide sequence of each DNA fragment was
determined by a method known to persons skilled in the art.
CLH5/L73, in which CLH5 was used for the H chain and L73 obtained
in Example 2 was used for the L chain, was expressed and purified
as IgG. Expression and purification were carried out by the method
described in Example 1.
[0365] Antibody having even higher pH dependency was produced by
combining mutation sites. Based on the locations where His was
concentrated in the phage library as well as the structural
information and the like, H32, H58, H62 and H102 in H3pI which was
obtained as an H chain in Example 2 were substituted with
histidine, and H95 and H99 were further substituted with valine and
isoleucine, respectively, to produce H170 (SEQ ID NO: 4). The
variant production was carried out using the method described in
Example 1. In addition, L82 (SEQ ID NO: 7) was produced by
substituting the 28th histidine of L73, which was produced as an L
chain in Example 2, with aspartic acid. The variant production was
carried out using the method described in Example 1. H170/L82, in
which H170 was used for the H chain and L82 was used for the L
chain, was expressed and purified as IgG using the method described
in Example 1.
Example 5
Evaluation of IL-6R-Neutralizing Activity of pH-Dependent Binding
Antibody
Evaluation of Human IL-6 Receptor-Neutralizing Activity of Clones
Converted to IgG
[0366] The IL-6 receptor-neutralizing activity was evaluated for
four antibodies: humanized PM1 antibody (WT) and H3pI/L73, CLH5/L73
and H170/L82 produced in Examples 2 and 4.
[0367] More specifically, the IL-6 receptor-neutralizing activity
was evaluated using BaF3/gp130 exhibiting IL-6/IL-6
receptor-dependent growth. BaF3/gp130 was washed three times with
RPMI1640 medium containing 10% FBS, then suspended at
5.times.10.sup.4 cells/mL in RPMI1640 medium containing 60 ng/mL of
human interleukin-6 (Toray), 60 ng/mL of recombinant soluble human
IL-6 receptor (SR344) and 10% FBS. 50 .mu.L of the suspension was
dispensed into each of the wells of a 96-well plate (Corning).
Next, the purified antibody was diluted with RMPI1640 containing
10% FBS, and 50 .mu.l of the antibody was mixed into each well.
After culturing for 3 days at 37.degree. C. and 5% CO.sub.2, WST-8
reagent (Cell Counting Kit-8, Dojindo Laboratories) diluted
two-fold with PBS was added at 20 .mu.L/well, and then immediately
measured for absorbance at 450 nm (reference wavelength: 620 nm)
using the Sunrise Classic (Tecan). After culturing for 2 hours,
absorbance at 450 nm was measured again (reference wavelength: 620
nm). The IL-6 receptor-neutralizing activity was evaluated based on
the change in absorbance after 2 hours. As a result, as shown in
FIG. 7, H3pI/L73, CLH5/L73 and H170/L82 were shown to have
equivalent biological neutralization activity in comparison with
the humanized PM1 antibody (WT).
Example 6
Biacore Analysis of pH-Dependently Binding Antibody
[0368] Analysis of Binding of pH-Dependently Binding Clones to
Soluble IL-6 Receptor
[0369] Kinetic analyses of antigen-antibody reactions at pH 5.8 and
pH 7.4 were carried out using Biacore T100 (GE Healthcare) on the
four antibodies: humanized PM1 antibody (WT) and H3pI/L73,
CLH5/L73, and H170/L82 produced in Examples 2 and 4 (buffer: 10 mM
MES (pH 7.4 or pH 5.8), 150 mM NaCl, and 0.05% Tween 20). Various
antibodies were bound onto a sensor chip immobilized with
recomb-protein A/G (Pierce) by amine coupling. SR344 adjusted to
concentrations of 9.8 to 400 nM was injected to the chip as an
analyte. Association and dissociation of the pH-dependent binding
clones to SR344 was observed in real time (FIGS. 8 and 9). All the
measurements were carried out at 37.degree. C. Association rate
constants k.sub.a (1/Ms) and dissociation rate constants k.sub.d
(1/s) were calculated using Biacore T100 Evaluation Software (GE
Healthcare), and dissociation constants KD (M) were calculated on
the basis of those values (Table 5). Moreover, the ratio of the
affinities at pH 5.8 and pH 7.4 were calculated for each clone to
evaluate pH-dependent binding. All the measurements were carried
out at 37.degree. C.
[0370] As a result of calculating the affinity ratio between pH 5.8
and pH 7.4 for each clone, the pH-dependent binding (affinity) of
H3pI/L73, H170/L82 and CLH5/L73 to SR344 was 41-fold, 394-fold and
66-fold, respectively, each showing the pH-dependent binding more
than 15 times higher than WT.
[0371] Anti-IL-6 receptor antibodies that strongly bind to the
antigen at the plasma pH of 7.4 but weakly bind to the antigen at
the intraendosomal pH of 5.5 to 6.0 have not been reported yet. In
this study, antibodies were obtained that retain the biological
neutralization activity equivalent to the WT humanized IL-6
receptor antibody and the affinity at pH 7.4, but exhibit the
affinity at pH 5.8 that has been specifically lowered more than 10
times.
TABLE-US-00005 TABLE 5 Comparison of Binding of pH-Dependently
Binding Clones Directed Against SR344 to Soluble IL-6 Receptor pH
7.4 pH 5.8 ka(1/Ms) kd(1/s) KD(M) ka(1/Ms) kd(1/s) KD(M) KD(pH
5.8)/KD(pH 7.4) WT 5.1E+05 1.0E-03 2.1E-09 7.6E+05 3.8E-03 5.0E-09
2.4 H3pI/L73 5.4E+05 7.4E-04 1.4E-09 1.7E+05 9.7E-03 5.7E-08 41.3
H170/L82 6.8E+05 1.1E-03 1.6E-09 2.6E+04 1.7E-02 6.4E-07 393.5
CLH5/L73 7.1E+05 7.9E-04 1.1E-09 3.8E+05 2.8E-02 7.4E-08 66.1
Analysis of Binding of pH-Dependently Binding Clones to Membrane
IL-6 Receptor
[0372] Antigen-antibody reactions to membrane IL-6 receptor at pH
5.8 and pH 7.4 were observed for the above produced pH-dependent
binding clones, using Biacore T100 (GE Healthcare). The binding to
membrane IL-6 receptor was evaluated by evaluating the binding to
the IL-6 receptor immobilized onto a sensor chip. SR344 was
biotinylated according to a method known to persons skilled in the
art, and the biotinylated SR344 was immobilized on the sensor chip
via streptavidin by utilizing the affinity between streptavidin and
biotin. All the measurements were carried out at 37.degree. C., and
the mobile phase buffer contained 10 mM MES (pH 5.8), 150 mM NaCl
and 0.05% Tween 20. The pH-dependent binding clones were injected
therein under the condition of pH 7.4 and allowed to bind to SR344
(injection sample buffer: 10 mM MES (pH 7.4), 150 mM NaCl, 0.05%
Tween 20), and the pH-dependent dissociation of each clone at the
mobile phase pH of 5.8 was observed (FIG. 10).
[0373] The dissociation rate (k.sub.d(1/s)) at pH 5.8 was
calculated using Biacore T100 Evaluation Software (GE Healthcare)
by fitting only the dissociation phase at pH 5.8, where 0.5
.mu.g/mL of the sample was bound in 10 mM MES (pH 7.4), 150 mM
NaCl, and 0.05% Tween 20, and dissociated in 10 mM MES (pH 5.8),
150 mM NaCl, and 0.05% Tween 20. Similarly, the dissociation rate
(k.sub.d(1/s)) at pH 7.4 was calculated using Biacore T100
Evaluation Software (GE Healthcare) by fitting only the
dissociation phase at pH 7.4, where 0.5 .mu.g/mL of the sample was
bound in 10 mM MES (pH 7.4), 150 mM NaCl, and 0.05% Tween 20, and
dissociated in 10 mM MES (pH 7.4), 150 mM NaCl, and 0.05% Tween 20.
The pH-dependent dissociation rate constant of each clone is shown
in Table 6.
TABLE-US-00006 TABLE 6 Comparison of Rate Constant of Dissociation
of pH-Dependent Binding Clones directed against SR344 from Membrane
IL-6 Receptor kd(1/s) kd ratio pH 7.4 pH 5.8 pH 5.8/pH 7.4 WT
4.84E-04 7.15E-04 1.5 H3pI/L73 3.44E-04 3.78E-03 11.0 H170/L82
7.70E-04 1.44E-03 1.9 CLH5/L73 1.04E-03 5.67E-03 5.5
[0374] The highest pH dependency of the dissociation ratio was
observed in H3pI/L73 followed by CLH5/L73 and H170/L82 in
descending order, and each clone demonstrated higher pH-dependent
dissociation from the membrane IL-6 receptor than WT. However, the
rank of pH-dependent association/dissociation was different between
the soluble IL-6 receptor and membrane IL-6 receptor. It was
revealed that H170/L82, which exhibited the highest pH-dependent
binding in the analysis of binding to soluble IL-6 receptor, showed
the lowest pH-dependent binding in the analysis of binding to the
membrane IL-6 receptor. In general, it is known that while IgG
molecules monovalently bind to a soluble antigen (affinity), they
divalently bind to membrane antigens (avidity). It is suggested
that this difference in the binding mode between soluble antigens
and membrane antigens influenced the pH-dependent binding of
H170/L82.
Example 7
Confirmation of Multiple Binding to Antigen by pH-Dependent Binding
Antibody
[0375] As described in Example 2, pH-dependent binding antibodies
may be able to bind to antigens multiple times. Specifically, a
pH-dependent binding antibody that has bound to an antigen is
non-specifically taken up into endosomes, but dissociated from the
soluble antigen under the intraendosomal acidic conditions. The
antibody binds to FcRn and thereby returns to the plasma. Since the
antibody that has returned to the plasma is not bound to antigen,
it is able to bind to a new antigen again. The repetition of this
process enables pH-dependent binding antibodies to bind to antigens
multiple times. However, for IgG antibodies that do not have
pH-dependent binding ability, not all antigens are dissociated from
the antibodies under the intraendosomal acidic conditions. Thus,
such antibodies that have been returned to the plasma by FcRn
remain bound to antigen, and therefore cannot bind to new antigens.
Consequently, in nearly all cases, each single molecule of IgG
antibodies is able to neutralize only two antigens (in the case of
divalent binding).
[0376] Therefore, it was evaluated whether the three pH-dependently
binding antibodies (H3pI/L73, CLH5/L73, and H170/L82) constructed
in Examples 2 and 4 were able to bind to the antigen SR344 multiple
times as compared to the humanized PM1 antibody (wild type,
WT).
[0377] Biacore (GE Healthcare) was used to evaluate that the
antibodies binding at pH 7.4 and dissociating at pH 5.8 were able
to bind to the antigen multiple times. The antibody to be evaluated
was bound to a recomb-protein A/G (Pierce)-immobilized sensor chip
by the amine coupling method, and a mobile phase of pH 7.4 was
allowed to flow (step 1). SR344 solution adjusted to pH 7.4 was
then allowed to flow as an analyte to bind SR344 to the antibody at
pH 7.4 (step 2). This binding at pH 7.4 mimics the antigen binding
in plasma. Subsequently, buffer adjusted to pH 5.8 alone (not
containing SR344) was added as an analyte to expose the antigen
bound to the antibody to acidic conditions (step 3). This
dissociation at pH 5.8 mimics the binding state of antigen-antibody
complexes in endosomes. Subsequently, step 2 was repeated. This
mimics the rebinding of antibody that has been returned to plasma
by FcRn to a new antigen. Subsequently, step 2 was repeated to
expose the antibody-antigen complex to acidic conditions. Repeating
"step 2 to step 3" multiple times at 37.degree. C. as described
above can mimic the in vivo state in which antibodies are
repeatedly taken up from the plasma into endosomes by pinocytosis
and returned to the plasma by FcRn (Nat. Rev. Immunol. 2007
September; 7(9): 715-25).
[0378] The produced pH-dependent binding clones described above
were analyzed using Biacore T100 (GE Healthcare) for their ability
to bind to the antigen SR344 multiple times at pH 5.8 and pH 7.4.
More specifically, the analysis was carried out as follows. All the
measurements were carried out at 37.degree. C. First, the sample
antibodies described above were bound onto a recomb-protein A/G
(Pierce)-immobilized sensor chip by amine-coupling, where the
mobile phase buffer was 10 mM MES (pH 5.8), 150 mM NaCl, and 0.05%
Tween 20 (step 1). SR344 adjusted to a concentration of about 40 nM
was injected as an analyte for 3 minutes at pH 7.4 and allowed to
bind (the buffer for injected SR344 was 10 mM MES (pH 7.4), 150 mM
NaCl, and 0.05% Tween 20) (step 2). Subsequently, the injection of
SR344 was discontinued and a mobile phase of pH 5.8 was allowed to
flow for about 70 seconds to expose the antibody/SR344 complex
under acidic conditions (step 3). Ten sets of this binding (step
2)/acidity exposure (step 3) process were continuously repeated to
observe the sensorgram in real-time, which is shown in FIG. 11. WT
showed less dissociation of SR344 during the acidic exposure in
step 3, and consequently the proportion of antibody capable of
binding to new antigens in the subsequent step 2 was extremely low.
In contrast, it was found that the pH-dependent binding clones,
particularly H170/L82 and CLH5/L73, demonstrated so strong
dissociation during the acidic exposure in step 3 that most of the
bound SR344 was dissociated, and therefore nearly all antibodies
were able to bind to new antigens in the subsequent step 2. In the
10-set repetition of the binding (step 2) and acidic exposure (step
3), almost all H170/L82 and CLH5/L73 antibodies were able to bind
to new antigens in each set.
[0379] The obtained sensorgrams were used to calculate the binding
amount of SR344 in each set for each sample using Biacore T100
Evaluation Software (GE Healthcare). The integrated values in the
time course of the 10 sets are shown in FIG. 12. The integrated RU
values obtained at the 10th set are equivalent to the total amount
of antigens bound during the ten cycles. The pH-dependent binding
clones, particularly H170/L82 and CLH5/L73, showed the largest
total amounts of bound antigens in comparison with WT, and were
demonstrated to be able to repeatedly bind to roughly four times
the amount of antigens bound by WT. Accordingly, it was revealed
that by conferring pH-dependent binding ability to WT, such
antibodies can repeatedly bind to antigens and thereby neutralize
multiple antigens.
Example 8
PK/PD Test of pH-Dependently-Binding Antibody Using Human IL-6
Receptor Transgenic Mice
[0380] IL-6 receptors are present in the body in both soluble IL-6
receptor form and membrane-type IL-6 receptor form (Nat. Clin.
Pract. Rheumatol. 2006 November; 2(11): 619-26). Anti-IL-6 receptor
antibodies bind to soluble IL-6 receptors and membrane-type IL-6
receptors and neutralize their biological action. It is believed
that an anti-IL-6 receptor antibody binds to a membrane-type IL-6
receptor, is subsequently taken up into an endosome within a cell
by internalization while the antibody is kept bound to the
membrane-type IL-6 receptor, and then moves to a lysosome while
still kept bound to the membrane-type IL-6 receptor where it is
degraded by lysosome together with the membrane-type IL-6 receptor.
If H3pI/L73, CLH5/L73, and H170/L82, which are the
pH-dependent-binding IL-6 receptor antibodies evaluated in Example
6, are able to return to plasma via FcRn as a result of
dissociation under acidic conditions within endosomes, the
antibodies that have returned to the plasma can bind to antigens
again. This enables neutralization of multiple membrane-type IL-6
receptors with a single antibody molecule. Whether or not the
return to the plasma via FcRn as a result of dissociation under
acidic conditions within endosomes is achieved with the constructed
pH-dependent-binding anti-IL-6 receptor antibodies can be
determined by evaluating whether the pharmacokinetics of these
antibodies are improved as compared with that of WT.
[0381] Thus, the pharmacokinetics in human-IL-6-receptor transgenic
mice (hIL-6R tg mice, Proc. Natl. Acad. Sci. USA 1995 May 23;
92(11): 4862-6) was evaluated for the four types of antibodies,
that is, humanized PM1 antibody (wild type: WT) and H3pI/L73,
CLH5/L73, and H170/L82 constructed in Examples 2 and 4. WT,
H3pI/L73, CLH5/L73, or H170/L82 was administered by single-dose
intravenous administration to hIL-6R tg mice at 25 mg/kg, and blood
samples were collected, before administration and over time.
Collected blood was immediately centrifuged for 15 minutes at
15,000 rpm and 4.degree. C. to obtain plasma. Separated plasma was
stored in a freezer set to -20.degree. C. or lower until
measurements were carried out.
[0382] The measurement of concentration in mouse plasma was carried
out by ELISA. Samples for calibration curve were prepared at plasma
concentrations of 6.4, 3.2, 1.6, 0.8, 0.4, 0.2 and 0.1 .mu.g/mL.
The calibration curve samples and mouse plasma measurement samples
were dispensed into an immunoplate (Nunc-Immuno Plate, MaxiSorp
(Nalge Nunc International)) immobilized with anti-human IgG
(.gamma.-chain specific) F(ab')2 (Sigma), and allowed to stand
undisturbed for one hour at room temperature. Goat anti-human
IgG-BIOT (Southern Biotechnology Associates) and
streptavidin-alkaline phosphatase conjugate (Roche Diagnostics)
were sequentially allowed to react, and a chromogenic reaction was
carried out by using BluePhos Microwell Phosphatase Substrates
System (Kirkegaard & Perry Laboratories) as substrate.
Absorbance at 650 nm was measured with a microplate reader. The
concentrations in mouse plasma were calculated from the absorbance
of the calibration curve using the analytical software SOFTmax PRO
(Molecular Devices). Time courses of plasma concentrations of WT as
well as H3pI/L73, CLH5/L73, and H170/L82 are shown in FIG. 13.
[0383] The pharmacokinetics was improved for all of H3pI/L73,
CLH5/L73, and H170/L82 as compared with WT. In particular, the
pharmacokinetics of H3pI/L73 and CLH5/L73 were improved
considerably. A wild type anti-IL-6 receptor antibody (WT) bound to
membrane-type IL-6 receptor is taken up into an endosome within a
cell by internalization, moves to a lysosome while the antibody is
kept bound to the antigen, and then degraded; therefore, it has a
short residence time in the plasma. In contrast, since the
pharmacokinetics of the pH-dependent-binding anti-IL-6 receptor
antibodies were improved considerably, the pH-dependent-binding
anti-IL-6 receptor antibodies were thought to return to the plasma
again via FcRn as a result of dissociation from the antigen,
membrane-type IL-6 receptor, under acidic conditions within
endosomes.
[0384] Although the pharmacokinetics was improved for all of
H3pI/L73, CLH5/L73, and H170/L82 as compared with WT, the effect of
prolonging plasma persistence time of H170/L82 was weaker than that
of H3pI/L73 and CLH5/L73. Since IgG molecules are thought to
normally bind divalently to membrane-bound antigen, it is thought
that anti-IL-6 receptor antibodies also bind divalently (avidity)
to membrane-type IL-6 receptors and then are internalized. As
indicated in Example 6, the analysis using Biacore revealed that
H170/L82 rapidly dissociated from the IL-6 receptor at pH 5.8 when
binding to soluble IL-6 receptor (FIG. 9), but the dissociation
rate thereof from the IL-6 receptor at pH 5.8 when binding to
membrane-type IL-6 receptor was extremely slow (FIG. 10). From this
result, the reason for the weak effect of prolonging residence time
in plasma of H170/L82 is thought to be that the antibody was unable
to adequately dissociate within endosomes after having been
internalized due to its slow dissociation at pH 5.8 when binding to
membrane-type IL-6 receptor. Namely, as for the case relating to
membrane antigens, it was determined that in order for a single IgG
molecule to neutralize multiple membrane antigens, the pH
dependency of dissociation from divalent binding (avidity) is more
important than the pH dependency of monovalent binding
(affinity).
Example 9
PK/PD Test of pH-Dependently-Binding Antibody Using Cynomolgus
Monkeys
[0385] Since the pharmacokinetics of the pH-dependently-binding
anti-IL-6 receptor antibodies were improved considerably in Example
8, the pH-dependently-binding anti-IL-6 receptor antibodies were
thought to return to plasma via FcRn as a result of dissociation
from the antigen, membrane-type IL-6 receptor, under acidic
conditions within endosomes. If antibodies that have returned to
the plasma can bind to membrane-type IL-6 receptors again,
neutralization of an antigen, the membrane-type IL-6 receptor, by
pH-dependently-binding anti-IL-6 receptor antibodies is thought to
persist longer than that by the wild-type anti-IL-6 receptor
antibody, at the same dosage. In addition, since the soluble IL-6
receptor is also present among IL-6 receptors, the duration of
neutralization is thought to be longer for the same dosage with
respect to the soluble IL-6 receptor as well.
[0386] The pharmacokinetics in cynomolgus monkeys was evaluated for
WT and H3pI/L73. WT or H3pI/L73 was administered to cynomolgus
monkeys by single-dose intravenous administration at 1 mg/kg, and
blood samples were collected, before administration and over time.
Collected blood was immediately centrifuged for 15 minutes at
15,000 rpm and 4.degree. C. to obtain plasma. The separated plasma
was stored in a freezer set to -20.degree. C. or lower until
measurements were carried out.
[0387] The measurement of concentration in cynomolgus monkey plasma
was carried out by ELISA. First, anti-human IgG (.gamma.-chain
specific) F(ab')2 fragment of antibody (Sigma) was dispensed into a
Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and allowed to
stand undisturbed overnight at 4.degree. C. to prepare plates
immobilized with anti-human IgG. Calibration curve samples having
plasma concentrations of 3.2, 1.6, 0.8, 0.4, 0.2, 0.1 and 0.05
.mu.g/mL and cynomolgus monkey plasma measurement samples diluted
100-fold or more were prepared; 200 of 20 ng/mL cynomolgus monkey
IL-6R was added to 100 .mu.L of the calibration curve samples and
plasma measurement samples; and then, they were allowed to stand
undisturbed for one hour at room temperature. Subsequently, the
samples were dispensed into the anti-human IgG-immobilized plate
and allowed to stand undisturbed for another one hour at room
temperature. Biotinylated Anti-Human IL-6R Antibody (R&D) was
allowed to react for one hour at room temperature, and then
Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was
allowed to react for one hour. A chromogenic reaction was carried
out by using TMP One Component HRP Microwell Substrate (BioFX
Laboratories) as substrate, then, the reaction was stopped with 1N
sulfuric acid (Showa Chemical) and the absorbance at 450 nm was
measured with a microplate reader. The concentrations in cynomolgus
monkey plasma were calculated from the absorbance of the
calibration curve using the analytical software SOFTmax PRO
(Molecular Devices). The time courses of plasma concentrations of
WT and H3pI/L73 after the intravenous administration are shown in
FIG. 14. As a result, the pharmacokinetics of H3pI/L73 was improved
considerably in comparison with WT in cynomolgus monkeys in the
same manner as in human IL-6 receptor transgenic mice. Since the
pharmacokinetics of a pH-dependent-binding anti-IL-6 receptor
antibody, H3pI/L73, was improved considerably, H3pI/L73 was thought
to return to the plasma via FcRn as a result of dissociation from
the antigen, membrane-type IL-6 receptor, under acidic conditions
within endosomes.
[0388] In order to evaluate the degree to which cynomolgus monkey
membrane-type IL-6 receptor is neutralized by the intravenous
administration of WT and H3pI/L73, the effects of sample antibodies
on plasma C-reactive protein (CRP) induced by cynomolgus monkey
IL-6 were studied. Since CRP is secreted when IL-6 binds to
membrane-type IL-6 receptors, CRP serves as an indicator of
neutralization of membrane-type IL-6 receptors. Cynomolgus monkey
IL-6 (cyno.IL-6 prepared in Example 1) containing 1% inactivated
cynomolgus monkey plasma was administered subcutaneously into lower
backs of the animals daily at 5 .mu.g/kg from day 3 to day 10 after
the administration of WT or H3pI/L73. Blood samples were collected
from the saphenous vein immediately before the start of cynomolgus
monkey IL-6 administration (day 3) and after the administration at
24-hour intervals (day 4 to day 11), then were separated into
plasma. The CRP concentrations of individual animals were measured
with Cias R CRP (Kanto Chemical) using an automated analyzer
(TBA-120FR, Toshiba Medical Systems). The time courses of CRP
concentration upon induction with cynomolgus IL-6 with respect to
WT and H3pI/L73 are shown in FIG. 15. As a result, the duration of
CRP suppression was prolonged considerably by H3pI/L73 in
comparison with WT. On the basis of this finding, a
pH-dependent-binding anti-IL-6 receptor antibody, H3pI/L73, was
thought to return to the plasma via FcRn as a result of
dissociation from its antigen, membrane-type IL-6 receptor, under
acidic conditions within endosomes; and neutralize the
membrane-type IL-6 receptor by re-binding thereto; and thereby
suppress production of CRP for a longer period of time than WT. In
other words, H3pI/L73 was shown to be able to bind to and
neutralize the membrane-type IL-6 receptor more than once, as a
single antibody molecule. Since the duration of suppression of CRP
production by H3pI/L73 is prolonged in comparison to that by WT,
the duration of time where an antigen, the membrane-type IL-6
receptor, is bound by antibodies was indicated to be prolonged for
H3pI/L73 than WT.
[0389] In order to evaluate the degree to which cynomolgus monkey
soluble IL-6 receptor is neutralized by the intravenous
administration of WT and H3pI/L73, the concentration of unbound
cynomolgus monkey soluble IL-6 receptor in cynomolgus monkey plasma
was measured. All IgG-type antibodies (cynomolgus monkey IgG,
anti-human IL-6 receptor antibody, and a complex of anti-human-IL-6
receptor antibody and cynomolgus monkey soluble IL-6 receptor)
present in the plasma were adsorbed to Protein A by adding 30 .mu.L
of cynomolgus monkey plasma to an appropriate amount of rProtein A
Sepharose Fast Flow (GE Healthcare) resin dried in a 0.22 .mu.m
filter cup (Millipore). After spinning down with a high-speed
centrifuge, the solution that passed through (hereinafter referred
to as "pass solution") was recovered. Since the pass solution does
not contain the complex of anti-human IL-6 receptor antibody and
cynomolgus monkey soluble IL-6 receptor which is bound to protein
A, the concentration of unbound soluble IL-6 receptor can be
measured by measuring the concentration of cynomolgus monkey
soluble IL-6 receptor in the pass solution. Monoclonal Anti-human
IL-6R Antibody (R&D) that was ruthenium-labeled with Sulfo-Tag
NHS Ester (Meso Scale Discovery) and Biotinylated Anti-human IL-6R
Antibody (R&D) were mixed with cynomolgus monkey IL-6 receptor
calibration curve samples adjusted to concentrations of 4000, 2000,
1000, 500, 250, 125, and 62.5 pg/mL and the plasma samples treated
with Protein A as described above. The mixtures were allowed to
react for one hour at room temperature. Subsequently, the mixtures
were dispensed into an SA-Coated Standard MA2400 96-well plate
(Meso Scale Discovery). After allowing to react for another one
hour and washing, Read Buffer T (x4) (Meso Scale Discovery) was
dispensed. Immediately thereafter, the measurement with Sector
Imager 2400 (Meso Scale Discovery) was conducted. The
concentrations of cynomolgus monkey IL-6 receptor were calculated
from the response of the calibration curve by using the analytical
software, SOFTmax PRO (Molecular Devices). The time courses of
concentrations of unbound cynomolgus monkey soluble IL-6 receptor
for WT and H3pI/L73 are shown in FIG. 16. As a result, the duration
of neutralization of cynomolgus monkey soluble IL-6 receptor by
H3pI/L73 was considerably prolonged as compared to that by WT. On
the basis of this finding, the pH-dependent-binding anti-IL-6
receptor antibody H3pI/L73 was thought to dissociate from its
antigen, soluble IL-6 receptor, under acidic conditions in
endosomes; and return to the plasma via FcRn; and bind to and
neutralize the soluble IL-6 receptor again. Since the duration of
suppression of unbound cynomolgus monkey soluble IL-6 receptor by
H3pI/L73 is prolonged in comparison to that by WT, the duration of
time where an antigen, the soluble IL-6 receptor, is bound by
antibodies was indicated to be prolonged for H3pI/L73 than WT.
[0390] From these findings, the time until the antibody disappears
from the plasma as well as the time where soluble and membrane-type
IL-6 receptors are bound by the antibody in the body were found to
be considerably elongated for the pH-dependent-binding anti-IL-6
receptor antibodies that were made to bind strongly to the antigen
at pH 7.4, which is the pH in the plasma, but bind weakly to the
antigen at pH 5.8, which is the pH within endosomes, as compared to
the wild-type anti-IL-6 receptor antibody. This makes it possible
to reduce the dosage and frequency of administration to patients,
and in consequence, the total administration dosage. Therefore, the
pH-dependent-binding anti-IL-6 receptor antibody is thought to be
particularly advantageous as a pharmaceutical for use as an IL-6
antagonist.
Example 10
Improvement of pH-Dependent Binding to Membrane-Type IL-6 Receptor
by Optimization of Variable Region
[0391] Optimization of Variable Regions H3pI/L73 and CLH5/L82
[0392] Antibodies having pH-dependent binding abilities were shown
to demonstrate superior effects in Example 9. Therefore, to further
improve the pH-dependent binding abilities, mutations were
introduced into the CDR sequence of CLH5 obtained in Example 3 to
construct VH1-IgG1 (SEQ ID NO: 21) and VH2-IgG1 (SEQ ID NO: 22). In
addition, mutations were introduced into the framework sequence and
CDR sequence of H3pI to construct the modified H chains VH3-IgG1
(SEQ ID NO: 23) and VH4-IgG1 (SEQ ID NO: 24). Mutations were
introduced into the CDR sequences of L73 and L82 to construct the
modified L chains VL1-CK (SEQ ID NO: 25), VL2-CK (SEQ ID NO: 26),
and VL3-CK (SEQ ID NO: 27). More specifically, the mutants were
constructed using the QuikChange Site-Directed Mutagenesis Kit
(Stratagene) according to the method described in the appended
instructions, and the resulting plasmid fragments were inserted
into an mammalian cell expression vector to construct the desired H
chain expression vectors and L chain expression vectors. The
nucleotide sequences of the resulting expression vectors were
determined by methods known to persons with ordinary skill in the
art.
[0393] The antibody having VH2-IgG1 (SEQ ID NO: 22) as H chain and
VL2-CK (SEQ ID NO: 26) as L chain was denoted as Fv1-IgG1, the
antibody having VH1-IgG1 (SEQ ID NO: 21) as H chain and L82 as L
chain was denoted as Fv2-IgG1, the antibody having VH4-IgG1 (SEQ ID
NO: 24) as H chain and VL1-CK (SEQ ID NO: 25) as L chain was
denoted as Fv3-IgG1, and the antibody having VH3-IgG1 (SEQ ID NO:
23) as H chain and VL3-CK (SEQ ID NO: 27) as L chain was denoted as
Fv4-IgG1. Of these, Fv2-IgG1 and Fv4-IgG1 were expressed and
purified. The expression and purification were carried out by the
method described in Example 1.
Analysis of Binding of pH-Dependent-Binding Clones to Soluble IL-6
Receptor
[0394] Kinetic analyses of antigen-antibody reactions at pH 7.4
were carried out on the four types of antibodies, i.e., the
humanized PM1 antibody (wild type: WT), and WT, H3pI/L73-IgG1,
Fv2-IgG1, and Fv4-IgG1 constructed in Examples 2 and 10, by using
Biacore T100 (GE Healthcare) (buffer: 10 mM MES (pH 7.4), 150 mM
NaCl, 0.05% Tween 20). Each antibody was bound on a sensor chip on
which an anti-IgG .gamma. chain specific F(ab).sub.2 (Pierce) was
immobilized by amine coupling, and then, SR344 adjusted to a
concentration of 9.8 to 40 nM was injected thereto as an analyte.
The association to and dissociation from SR344 were observed on a
real-time basis for the pH-dependent-binding clones. All the
measurements were carried out at 37.degree. C. Association rate
constants k.sub.a (1/Ms) and dissociation rate constants k.sub.d
(1/s) were calculated using Biacore T100 Evaluation Software (GE
Healthcare), and dissociation constants KD (M) were calculated on
the basis of those values (Table 7).
TABLE-US-00007 TABLE 7 Comparison of Dissociation Rate Constants of
pH-Dependent- Binding Clones from Soluble IL-6 Receptor, SR344
Sample k.sub.a (1/Ms) k.sub.d (1/s) KD (M) WT 4.0E+05 1.1E-03
2.7E-09 H3pI/L73 4.1E+05 5.9E-04 1.4E-09 Fv2-IgG1 3.9E+05 7.7E-04
2.0E-09 Fv4-IgG1 7.2E+05 1.0E-03 1.4E-09
[0395] As a result of calculating the affinity at pH 7.4 for each
clone, the dissociation constants (affinity, KD value) of WT,
H3pI/L73-IgG1, Fv2-IgG1, and Fv4-IgG1 to SR344 were, respectively,
2.7 nM, 1.4 nM, 2.0 nM, and 1.4 nM, and they are nearly equivalent.
Fv2-IgG1 and Fv4-IgG1 were demonstrated to have binding ability to
the soluble IL-6 receptor that is equal to or greater than that of
WT.
Analysis of Binding of pH-Dependent-Binding Clones to Membrane-Type
IL-6 Receptor
[0396] Antigen-antibody reactions to membrane-type IL-6 receptor
were observed at pH 5.8 and pH 7.4 for the four types of
constructed clones, WT, H3pI/L73-IgG1, Fv2-IgG1, and Fv4-IgG1 by
using Biacore T100 (GE Healthcare). Binding to the membrane-type
IL-6 receptor was evaluated by evaluating binding to the IL-6
receptor immobilized on a sensor chip. SR344 was biotinylated in
accordance with a method known among persons with ordinary skill in
the art, and the biotinylated SR344 was immobilized on the sensor
chip via streptavidin using the affinity between streptavidin and
biotin. All the measurements were carried out at 37.degree. C. The
mobile phase buffer was 10 mM MES (pH 5.8), 150 mM NaCl and 0.05%
Tween 20. The pH-dependent-binding clones were injected therein
under conditions of pH 7.4 to allow them to bind to SR344
(injection sample buffer: 10 mM MES (pH 7.4), 150 mM NaCl, 0.05%
Tween 20), then, pH-dependent dissociation of each clone was
observed at the pH of the mobile phase of 5.8 (FIG. 17).
[0397] Sample concentrations were adjusted to 0.25 .mu.g/mL.
Binding was carried out in 10 mM MES (pH 7.4), 150 mM NaCl, and
0.05% Tween 20. Dissociation was carried out in 10 mM MES (pH 5.8),
150 mM NaCl, and 0.05% Tween 20. For this case, the dissociation
rate constants (k.sub.d(1/s)) at pH 5.8 were calculated by fitting
only the dissociation phase at pH 5.8 using Biacore T100 Evaluation
Software (GE Healthcare). In a similar manner, sample
concentrations were adjusted to 0.25 .mu.g/mL, binding was carried
out in 10 mM MES (pH 7.4), 150 mM NaCl, and 0.05% Tween 20,
dissociation was carried out in 10 mM MES (pH 7.4), 150 mM NaCl,
and 0.05% Tween 20, and the dissociation rate constants
(k.sub.d(1/s)) at pH 7.4 were calculated by fitting only the
dissociation phase at pH 7.4 using Biacore T100 Evaluation Software
(GE Healthcare). The pH-dependent dissociation rate constants of
each clone are shown in Table 8.
TABLE-US-00008 TABLE 8 Comparison of Dissociation Rate Constants of
pH-Dependent- Binding Clones from Membrane-Type IL-6 Receptor,
SR344 pH 7.4 pH 5.8 pH Dependency Sample kd (1/s) kd (1/s) kd(pH
5.8)/kd(pH 7.4) WT 2.5E-04 2.5E-04 1.00 H3pI/L73 2.6E-04 6.7E-04
2.59 Fv2-IgG1 3.4E-04 2.4E-03 7.18 Fv4-IgG1 4.7E-04 2.6E-03
5.56
[0398] As a result of calculating pH dependency for each clone, the
pH dependencies of binding to the membrane-type IL-6 receptor of
the four clones, WT, H3pI/L73-IgG1, Fv2-IgG1, and Fv4-IgG1 with
respect to SR344 were 1.0-fold, 2.59-fold, 7.18-fold, and
5.56-fold, respectively. Fv2-IgG1 and Fv4-IgG1 demonstrated higher
pH-dependency in dissociation from the membrane-type IL-6 receptor
than H3pI/L73-IgG1.
[0399] On the basis of the above, Fv2-IgG1 and Fv4-IgG1 were shown
to demonstrate stronger pH-dependent binding to the membrane-type
IL-6 receptor than H3pI/L73-IgG1 while maintaining the affinity for
the soluble IL-6 receptor equal to or stronger than that of WT.
Example 11
PK/PD Test of pH-Dependent-Binding Antibodies with Optimized
Variable Regions Using Human IL-6 Receptor Transgenic Mice
[0400] The pharmacokinetics of Fv2-IgG1 and Fv4-IgG1, as well as WT
and H3pI/L73-IgG1 prepared and evaluated in Example 10 were
evaluated using the human IL-6 receptor transgenic mice used in
Example 8. WT, H3pI/L73-IgG1, Fv2-IgG1, or Fv4-IgG1 was
administered by single-dose intravenous administration to hIL-6R tg
mice at 25 mg/kg, and the concentration of each antibody in the
plasma was measured in the same manner as in Example 8. The time
courses of the plasma concentrations of WT, H3pI/L73-IgG1,
Fv2-IgG1, and Fv4-IgG1 are shown in FIG. 18.
[0401] The pharmacokinetics of H3pI/L73-IgG1 improved in comparison
with WT in the same manner as in Example 8, while the
pharmacokinetics of Fv2-IgG1 and Fv4-IgG1 was further improved than
H3pI/L73-IgG1. Measurement as to the unbound IL-6 receptor
concentrations, as measured in cynomolgus monkeys in Example 9, was
carried out in the hIL-6R tg mice in this test using the same
method. As a result, prolongation of the duration of neutralization
of the soluble IL-6 receptor was confirmed for Fv2-IgG1 and
Fv4-IgG1 in comparison to that for H3pI/L73-IgG1 (data not shown).
As indicated in Example 10, the pH-dependent binding to the
membrane-type IL-6 receptor was improved for Fv2-IgG1 and Fv4-IgG1
as compared with H3pI/L73-IgG1. Therefore, it was indicated that
further improvement in the pharmacokinetics and duration of
neutralization of the soluble IL-6 receptor over those of
H3pI/L73-IgG1 is possible by improving the pH-dependent binding to
the membrane-type IL-6 receptor.
Example 12
Improvement of pH-Dependent Binding to Membrane-Type IL-6 Receptor
by Optimization of Constant Region
Optimization of Constant Region of Fv4-IgG1
[0402] Generally, binding to membrane-bound antigens has been
reported to vary depending on the constant region of the antibody
(J. Immunol. Methods 1997 Jun. 23; 205(1): 67-72). The constant
regions of the pH-dependent-binding antibodies prepared above were
of the IgG1 isotype. Therefore, a study was made for optimization
of the constant region in order to improve the pH-dependent binding
to the membrane-type IL-6 receptor.
[0403] A mutation was introduced into a naturally-occurring
constant region, i.e., constant region IgG2 (SEQ ID NO: 28), to
construct constant region IgG2AGK (SEQ ID NO: 29). Another mutation
was introduced into the constant region IgG2AGK to construct
constant region M58 (SEQ ID NO: 30). Mutations were further
introduced into the constant regions IgG2 and M58 to construct
constant regions M71 (SEQ ID NO: 31) and M73 (SEQ ID NO: 32).
[0404] VH3-IgG2.DELTA.GK (SEQ ID NO: 33) was constructed by
substituting the constant region of VH3-IgG1 prepared in Example 10
with IgG2.DELTA.GK, VH3-M58 (SEQ ID NO: 34) was constructed by
substituting the constant region with M58, and VH3-M73 (SEQ ID NO:
35) was constructed by substituting the constant region with M73.
More specifically, expression vectors in which the constant region
portion of VH3 used in Example 10 was substituted by a desired
constant region by NheI/NotI digestion and ligation were
constructed. The nucleotide sequences of the resulting expression
vectors were determined using a method known among persons with
ordinary skill in the art.
[0405] Expression and purification were carried out for the
following: Fv4-IgG2 using VH3-IgG2.DELTA.GK (SEQ ID NO: 33) for the
H chain and VL3-CK (SEQ ID NO: 27) for the L chain; Fv4-M58 using
VH3-M58 (SEQ ID NO: 34) for the H chain and VL3-CK (SEQ ID NO: 27)
for the L chain; and Fv4-M73 using VH3-M73 (SEQ ID NO: 35) for the
H chain and VL3-CK (SEQ ID NO: 27) for the L chain. Expression and
purification were carried out using the method described in Example
1.
Analysis of Binding of Fv4 Having Optimized Constant Region to
Soluble IL-6 Receptor
[0406] The association with and dissociation from SR344 were
observed on a real-time basis using the same method as Example 10
for thus-prepared Fv4-IgG1, Fv4-IgG2, Fv4-M58, and Fv4-M73 as well
as WT. The association rate constants k.sub.a (1/Ms) and
dissociation rate constants k.sub.d (1/s) were calculated after
analysis in the same manner, and then, dissociation constants KD
(M) were calculated on the basis of those values (Table 9).
TABLE-US-00009 TABLE 9 Comparison of Dissociation Rate Constants of
pH-Dependently- Binding Clones from Soluble IL-6 Receptor, SR344
Sample ka (1/Ms) kd (1/s) KD (M) Fv4-IgG1 7.2E+05 1.0E-03 1.4E-09
Fv4-IgG2 9.6E+05 1.2E-03 1.3E-09 Fv4-M58 8.3E+05 1.1E-03 1.4E-09
Fv4-M73 7.5E+05 1.0E-03 1.4E-09
[0407] As a result of calculating the affinity at pH 7.4 for each
clone, the dissociation constants (affinity, KD value) of Fv4-IgG1,
Fv4-IgG2, Fv4-M58, and Fv4-M73 to SR344 were 1.4 nM, 1.3 nM, 1.4
nM, and 1.4 nM, respectively, and they are almost equivalent. This
indicates that the binding ability of pH-dependent-binding clones
to the soluble IL-6 receptor, SR344, does not change even after
modifying the constant region. On the basis of this finding, the
binding ability to the soluble IL-6 receptor was thought to not
change for Fv1, Fv2, and Fv3 even if the constant region was
similarly modified.
Analysis of Binding of Fv4 Having Optimized Constant Region to
Membrane-Type IL-6 Receptor
[0408] Antigen-antibody reactions to the membrane-type IL-6
receptor at pH 5.8 and pH 7.4 were observed for thus-prepared
Fv4-IgG1, Fv4-IgG2, Fv4-M58, and Fv4-M73 as well as WT, in the same
manner as in Example 10 using Biacore T100 (GE Healthcare). The
results obtained by injecting the pH-dependent-binding clones under
the conditions of pH 7.4 to allow binding to SR344, and by
observing the pH-dependent dissociation of each clone in the pH 5.8
mobile phase, are shown in FIG. 19. Further analyses were conducted
in the same manner as in Example 10, and the pH-dependent
dissociation rates for each clone are shown in Table 10.
TABLE-US-00010 TABLE 10 Comparison of Dissociation Rate Constants
of pH-Dependently- Binding Clones from Membrane-Type IL-6 Receptor,
SR344 pH 7.4 pH 5.8 pH Dependency Sample kd (1/s) kd (1/s) kd(pH
5.8)/kd(pH 7.4) Fv4-IgG1 4.7E-04 2.6E-03 5.56 Fv4-IgG2 1.0E-03
1.8E-02 16.99 Fv4-M58 5.4E-04 9.5E-03 17.64 Fv4-M73 5.1E-04 5.1E-03
10.06
[0409] As a result of calculating the pH dependency for each clone,
the pH dependencies of Fv4-IgG1, Fv4-IgG2, Fv4-M58, and Fv4-M73 to
SR344 were 5.6-fold, 17.0-fold, 17.6-fold, and 10.1-fold,
respectively; thus, Fv4-IgG2, Fv4-M58, and Fv4-M73 all demonstrated
higher pH-dependent dissociation from the membrane-type IL-6
receptor than Fv4-IgG1.
[0410] Based on the results of analyzing binding to the soluble
IL-6 receptor and binding to the membrane-type IL-6 receptor using
the variable region of Fv4, it was found that substitution of the
constant region from IgG1 to IgG2, M58, or M73 could improve the
pH-dependent binding to the membrane-type IL-6 receptor, without
causing a change in the affinity to the soluble IL-6 receptor. This
was considered to similarly hold for Fv1, Fv2, and Fv3.
Example 13
PK/PD Test of pH-Dependently-Binding Antibodies Having Optimized
Constant Region Using Human IL-6 Receptor Transgenic Mice
[0411] The pharmacokinetics of Fv4-IgG1, Fv4-IgG2, and Fv4-M58
prepared in Example 13 were evaluated using the human IL-6 receptor
transgenic mice (hIL-6R tg mice) used in Example 8 to examine the
effects of the constant region on the pharmacokinetics. WT,
Fv4-IgG1, Fv4-IgG2, or Fv4-M58 was administered to the hIL-6R tg
mice by single-dose intravenous administration at 25 mg/kg, and
then, measurement of the plasma concentrations of each antibody was
carried out in the same manner as in Example 8. The time courses of
plasma concentrations of WT, Fv4-IgG1, Fv4-IgG2, and Fv4-M58 are
shown in FIG. 20.
[0412] Similar to Example 11, the pharmacokinetics of Fv4-IgG1 was
improved in comparison with WT, and the pharmacokinetics of
Fv4-IgG2 and Fv4-M58 was further improved in comparison with
Fv4-IgG1. Measurement as to the unbound IL-6 receptor
concentrations, as measured in cynomolgus monkeys in Example 9, was
carried out in the hIL-6R tg mice in this test using the same
method. As a result, prolongation of the duration of neutralization
of the soluble IL-6 receptor was confirmed for Fv4-IgG2 and Fv4-M58
in comparison to that of Fv4-IgG1 (data not shown). As indicated in
Example 10, the pH-dependent binding to the membrane-type IL-6
receptor was improved for Fv4-IgG2 and Fv4-M58 as compared with
Fv4-IgG1. Therefore, it was shown that improvement in the
pH-dependent binding to the membrane-type IL-6 receptor and
improvement in the pharmacokinetics and duration of neutralization
of the soluble IL-6 receptor are possible by substituting the
constant region from IgG1 to IgG2 or M58. On the basis of this
finding, it was thought that the pharmacokinetics and duration of
neutralization of the soluble IL-6 receptor, not only in the case
of Fv4, but also in the cases of Fv1, Fv2, and Fv3, can be improved
as compared to IgG1 by substituting the constant region from IgG1
to IgG2 or M58.
Example 14
Construction of pH-Dependently-Binding Antibodies Having Optimized
Variable and Constant Regions
[0413] VH2-M71 (SEQ ID NO: 36) and VH2-M73 (SEQ ID NO: 37), having
M71 and M73 for the constant region of VH2-IgG1, and VH4-M71 (SEQ
ID NO: 38) and VH4-M73 (SEQ ID NO: 39), having M71 and M73 for the
constant region of VH4-IgG1, were constructed using the same method
as described above.
[0414] Fv1-M71 using VH2-M71 for the H chain and VL2-CK for the L
chain, Fv1-M73 using VH2-M73 for the H chain and VL2-CK for the H
chain, Fv3-M71 using VH4-M71 for the H chain and VL1-CK for the L
chain, and Fv3-M73 using VH4-M73 for the H chain and VL1-CK for the
L chain, were expressed and purified. Expression and purification
were carried out using the method described in Example 1.
Analyses of Binding of pH-Dependent-Binding Antibodies Having
Optimized Variable and Constant Regions to Soluble IL-6
Receptor
[0415] The association to and dissociation from SR344 were observed
on a real-time basis using the same method as Example 10 for the
eleven types of antibodies, humanized PM1 antibody (wild type: WT)
and H3pI/L73-IgG1, Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M71, Fv3-M73,
Fv4-IgG1, Fv4-IgG2, Fv4-M58, and Fv4-M73, constructed as described
above. The association rate constants k.sub.a (1/Ms) and
dissociation rate constants k.sub.d (1/s) were calculated by
analysis in the same manner, and the dissociation constants KD (M)
were calculated on the basis of those values (Table 11).
TABLE-US-00011 TABLE 11 Comparison of Dissociation Rate Constants
of pH-Dependently- Binding Clones from Soluble IL-6 Receptor, SR344
Sample k.sub.a (1/Ms) k.sub.d (1/s) KD (M) WT 4.0E+05 1.1E-03
2.7E-09 H3pI/L73 4.1E+05 5.9E-04 1.4E-09 Fv1-M71 5.5E+05 5.4E-04
9.7E-10 Fv1-M73 6.1E+05 5.5E-04 9.1E-10 Fv2-IgG1 3.9E+05 7.7E-04
2.0E-09 Fv3-M71 7.8E+05 8.2E-04 1.1E-09 Fv3-M73 8.5E+05 8.7E-04
1.0E-09 Fv4-IgG1 7.2E+05 1.0E-03 1.4E-09 Fv4-IgG2 9.6E+05 1.2E-03
1.3E-09 Fv4-M58 8.3E+05 1.1E-03 1.4E-09 Fv4-M73 7.5E+05 1.0E-03
1.4E-09
[0416] All of the resulting ten types of pH-dependent-binding
clones were found to have dissociation constants (affinity, KD
values) to the soluble IL-6 receptor equal to or stronger than that
of WT.
Analyses of Binding of pH-Dependently-Binding Antibodies Having
Optimized Variable and Constant Regions to Membrane-Type IL-6
Receptor
[0417] Antigen-antibody reactions to the membrane-type IL-6
receptor at pH 5.8 and pH 7.4 were observed in the same manner as
in Example 10 using Biacore T100 (GE Healthcare) for the eleven
types of antibodies, humanized PM1 antibody (wild type: WT) and
H3pI/L73-IgG1, Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M71, Fv3-M73,
Fv4-IgG1, Fv4-IgG2, Fv4-M58, and Fv4-M73, prepared as described
above. The pH-dependently-binding clones were injected under the
condition of pH 7.4 to allow them to bind to SR344, and then, the
pH-dependent dissociation of each clone at the pH of the mobile
phase, pH 5.8, was observed. The results are shown in FIG. 21
(results for Fv1-M71, Fv1-M73, Fv3-M71, and Fv3-M73 are shown in
FIG. 21, while results for other clones are shown in FIGS. 17 and
19). Analyses were carried out in the same manner as in Example 10
and the pH dependencies of the dissociation rate constants of all
of the eleven types of clones are shown in Table 12.
TABLE-US-00012 TABLE 12 pH Dependencies of Dissociation Rate
Constants of pH-Dependently- Binding Clones from Membrane-Type IL-6
Receptor, SR344 pH 7.4 pH 5.8 pH Dependency Sample kd (1/s) kd
(1/s) kd(pH 5.8)/kd(pH 7.4) WT 2.5E-04 2.5E-04 1.00 H3pI/L73
2.6E-04 6.7E-04 2.59 Fv1-M71 6.1E-04 6.9E-03 11.29 Fv1-M73 3.7E-04
3.2E-03 8.80 Fv2-IgG1 3.4E-04 2.4E-03 7.18 Fv3-M71 9.1E-04 9.7E-03
10.74 Fv3-M73 4.9E-04 5.3E-03 10.88 Fv4-IgG1 4.7E-04 2.6E-03 5.56
Fv4-IgG2 1.0E-03 1.8E-02 16.99 Fv4-M58 5.4E-04 9.5E-03 17.64
Fv4-M73 5.1E-04 5.1E-03 10.06
[0418] The ten types of obtained pH-dependently-binding clones
demonstrated pH-dependent binding ability to the membrane-type IL-6
receptor. Moreover, all of Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M71,
Fv3-M73, Fv4-IgG1, Fv4-IgG2, Fv4-M58, and Fv4-M73 were found to
demonstrate improved pH-dependent binding to the membrane-type IL-6
receptor, in comparison with H3pI/L73-IgG1, for which the time
until the antibody disappears from the plasma as well as the time
where the soluble IL-6 receptor and membrane-type IL-6 receptor are
bound by the antibody in the body were found to be prolonged
considerably in comparison with WT, as shown in cynomolgus monkeys
in Example 9.
Example 15
PK/PD Test of pH-Dependently-Binding Antibodies Having Optimized
Variable and Constant Regions Using Cynomolgus Monkeys
Construction of Known High-Affinity Anti-IL-6 Receptor Antibody
[0419] A mammalian cell expression vector was constructed to
express the high-affinity anti-IL-6 receptor antibody VQ8F11-21
hIgG1 described in US 2007/0280945 A1 (US 2007/0280945 A1, amino
acid sequences 19 and 27), as a known high-affinity anti-IL-6
receptor antibody. An antibody variable region was constructed by
PCR combining synthetic oligo-DNAs (assembly PCR). The constant
region was amplified by PCR from the expression vector used in
Example 1. The antibody variable region and antibody constant
region were ligated by assembly PCR and inserted into a vector for
expression in mammals. The resulting H chain and L chain DNA
fragments were inserted into mammalian cell expression vectors to
construct the H chain expression vector and L chain expression
vector of interest. The nucleotide sequences of the resulting
expression vectors were determined by a method known among persons
with ordinary skill in the art. Expression and purification were
carried out using the constructed expression vectors. Expression
and purification were carried out using the method described in
Example 1 to obtain the high-affinity anti-IL-6 receptor antibody
(high affinity Ab).
PK/PD Test in Cynomolgus Monkeys
[0420] The pharmacokinetics and pharmacological efficacy were
evaluated in cynomolgus monkeys for the pH-dependently-binding
antibodies H3pI/L73-IgG1 and Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M73,
and Fv4-M73 and the known high-affinity anti-IL-6 receptor antibody
(high affinity Ab). H3pI/L73-IgG1, Fv1-M71, Fv1-M73, Fv2-IgG1,
Fv3-M73, or Fv4-M73 was administered to cynomolgus monkeys by
single-dose intravenous administration at 0.5 mg/kg, while the high
affinity Ab was administered by single-dose intravenous
administration at 1.0 mg/kg. Blood samples were collected before
administration and over time. The plasma concentration of each
antibody was measured in the same manner as in Example 9. The time
courses of plasma concentrations of H3pI/L73-IgG1, Fv1-M71,
Fv1-M73, Fv2-IgG1, Fv3-M73, and Fv4-M73, as well as the high
affinity Ab are shown in FIG. 21. In order to evaluate the
pharmacological efficacy in terms of the degree to which the
cynomolgus monkey membrane-type IL-6 receptor is neutralized,
cynomolgus monkey IL-6 was administered subcutaneously into the
lower backs of the animals daily at 5 .mu.g/kg from day 3 to day 10
(from day 6 to day 10 with respect to the high affinity Ab) after
the administration of antibody, in the same manner as in Example 9.
The CRP concentration of each animal was measured 24 hours after
each administration. The time courses of CRP concentrations with
the administration of each antibody are shown in FIG. 22. In order
to evaluate the pharmacological efficacy in terms of the degree of
neutralization of cynomolgus monkey soluble IL-6 receptor, the
concentration of the unbound cynomolgus monkey soluble IL-6
receptor in cynomolgus monkey plasma was measured in the same
manner as Example 9. The time courses of the unbound cynomolgus
monkey soluble IL-6 receptor concentrations with the administration
of each antibody are shown in FIG. 23.
[0421] As a result, the antibody concentrations in plasma were
maintained high for each of Fv1-M71, Fv1-M73, Fv2-IgG1, Fv3-M73 and
Fv4-M73 in comparison with H3pI/L73-IgG1, while the concentrations
of CRP and the unbound cynomolgus monkey soluble IL-6 receptor were
maintained at low levels. Namely, this result showed that the time
where the membrane-type and soluble IL-6 receptors are bound by the
antibody (or in other words, the duration of neutralization) was
prolonged by the antibodies in comparison with H3pI/L73-IgG1.
[0422] In addition, these pH-dependently-binding anti-IL-6 receptor
antibodies were confirmed for their neutralization effects and
sustained efficacy equal to or greater than that of the known
high-affinity anti-IL-6 receptor antibody (high affinity Ab)
administered at 1.0 mg/kg, at only half the dosage thereof, i.e.,
at 0.5 mg/kg. Therefore, the pH-dependently-binding antibodies were
elucidated to have the neutralization effects and sustained
efficacy superior to those of the known high-affinity anti-IL-6
receptor antibody.
[0423] Those antibodies shown in Table 12 for which PK/PD tests
were not carried out using cynomolgus monkeys as in this test, have
also been confirmed to demonstrate improved pH-dependent binding to
the membrane-type IL-6 receptor in comparison with H3pI/L73-IgG1.
Therefore, the time during which the membrane-type and soluble IL-6
receptor are bound by the antibodies (or in other words, the
duration of neutralization and sustained neutralization effects)
are also thought to be prolonged for the antibodies in comparison
with H3pI/L73-IgG1.
[0424] In Example 9, for H3pI/L73-IgG1, the time until the antibody
disappears from the plasma as well as the time where the soluble
IL-6 receptor and membrane-type IL-6 receptor are bound by the
antibody in the body (sustained neutralization effects) were found
to be prolonged considerably as compared with WT. Fv1-M71, Fv1-M73,
Fv2-IgG1, Fv3-M71, Fv3-M73, Fv4-IgG1, Fv4-IgG2, Fv4-M58, and
Fv4-M73, having superior sustained neutralization effects to
H3pI/L73-IgG1, are therefore thought to have remarkably improved
sustained neutralization effects as compared with WT.
[0425] In contrast to anti-IL-6 receptor antibodies, the
pH-dependent-binding anti-IL-6 receptor antibodies that are made to
strongly bind to the antigen at the pH in the plasma of pH 7.4 but
only weakly bind to the antigen at the pH in endosomes of pH 5.8,
make it possible to reduce the patient dosage and administration
frequency of the anti-IL-6 receptor antibody, and as a result, they
can considerably reduce the total administration amount. Therefore,
the pH-dependently-binding anti-IL-6 receptor antibodies are
thought to be extremely superior as a pharmaceutical for use as an
IL-6 antagonist.
Example 16
Construction of pH-Dependently Binding Anti-IL-6 Antibody
Expression and Purification of Anti-IL-6 Antibody
[0426] In Examples 1 to 15, a plurality of humanized anti-IL-6
receptor antibodies that pH-dependently bind to the IL-6 receptor
were successfully created by imparting the dependency through
introducing histidine substitutions and the like into the variable
region, in particular, the CDR sequences of the humanized anti-IL-6
receptor antibodies. It was found that all of these antibodies
repeatedly bind to the IL-6 receptor and demonstrate a considerable
improvement in PK/PD.
[0427] Therefore, it was confirmed that the pH-dependent ability of
an antibody to bind to an antigen could be confered to another
antibody that binds to an antigen other than the IL-6 receptor
using a similar method. Human IL-6 was selected as the antigen, and
an anti-IL-6 antibody including the H chain (WT) (amino acid
sequence: SEQ ID NO: 62) and L chain (WT) (amino acid sequence: SEQ
ID NO: 63), which binds to IL-6 as described in WO 2004/039826,
("Anti-IL6 wild type") was constructed. Using a method known to
those skilled in the art, gene fragments encoding the antibody
amino acid sequences of interest were inserted into mammalian cell
expression vectors to construct the H chain expression vector and L
chain expression vector of interest. The nucleotide sequences of
the resulting expression vectors were determined using a method
known to a skilled person. Anti-IL6 wild type was expressed and
purified by the method described in Example 1.
Construction of pH-Dependent Anti-IL-6 Antibody
[0428] To confer the pH-dependent ability of the antibody to bind
to IL-6, histidine substitutions were introduced into the amino
acids in CDR of the anti-IL-6 antibody (Anti-IL6 wild type)
including the H chain (WT) (amino acid sequence: SEQ ID NO: 62) and
L chain (WT) (amino acid sequence: SEQ ID NO: 63). By substituting
histidine in the CDR amino acids and subsequently screening,
several clones that demonstrate the pH-dependent binding were
obtained. The binding at pH 5.5 was significantly reduced as
compared with the binding at pH 7.4. The positions of histidine
substitution in the pH-dependent clones are shown in Table 13. The
examples include "Anti-IL6 clone 1" including the H chain (c1)
(amino acid sequence: SEQ ID NO: 64) and L chain (c1) (amino acid
sequence: SEQ ID NO: 65), and "Anti-IL-6 clone 2" including the H
chain (c1) (amino acid sequence: SEQ ID NO: 64) and L chain (c2)
(amino acid sequence: SEQ ID NO: 66). Anti-IL6 clone 1 and
Anti-IL-6 clone 2 were expressed and purified by the method
described in Example 1.
TABLE-US-00013 TABLE 13 Positions of Histidine Substitution in
pH-dependent clones H32, H59, H61, H99 L53, L54, L90, L94
Analysis of Binding of pH-Dependent Clones to Human IL-6
[0429] Kinetic analysis of antigen-antibody reactions at pH 5.5 and
pH 7.4 was carried out using Biacore T100 (GE Healthcare) for the
three types of antibodies prepared as mentioned above: Anti-IL6
wild type, Anti-IL6 clone 1, and Anti-IL-6 clone 2 (buffer: DPBS(-)
(pH 7.4 or pH 5.5), 150 mM NaCl). The antibodies were bound to a
sensor chip on which recomb-protein A/G (Pierce) was immobilized by
amine coupling, and then human IL-6 (Toray) adjusted to an
appropriate concentration was injected on the chip as an analyte.
All the measurements were carried out at 37.degree. C. Association
rate constants k.sub.a (1/Ms) and dissociation rate constants
k.sub.d (1/s) were calculated using Biacore T100 Evaluation
Software (GE Healthcare), and dissociation constants KD (M) were
calculated based on those values (Table 14). Furthermore, the ratio
of affinity at pH 5.5 and pH 7.4 was calculated for each clone to
evaluate the pH-dependent binding.
TABLE-US-00014 TABLE 14 Comparison of Binding of pH-Dependent
Clones to IL-6 KD(pH 5.5)/ Sample pH ka(1/Ms) kd(1/s) KD(M) KD(pH
7.4) Wild type pH 7.4 2.05E+07 3.91E-04 1.91E-11 0.8 pH 5.5
1.52E+07 2.45E-04 1.61E-11 clone1 pH 7.4 1.07E+07 4.71E-03 4.38E-10
10.3 pH 5.5 2.05E+06 9.26E-03 4.52E-09 clone2 pH 7.4 8.96E+06
2.63E-03 2.94E-10 13.5 pH 5.5 2.76E+06 1.10E-02 3.98E-09
[0430] The ratio of affinity at pH 5.5 and pH 7.4 ((KD)(pH
5.5)/(KD)(pH 7.4)) calculated, which indicates the pH-dependent
binding to human IL-6, was 0.8, 10.3, and 13.5 for Anti-IL6 wild
type, Anti-IL6 clone 1, and Anti-IL6 clone 2, respectively. That
is, the pH-dependent binding ability of each clone is more than 10
times greater than that of WT. The sensorgrams of Anti-IL-6 clone 2
at pH 7.4 and pH 5.5 are shown in FIG. 26.
[0431] Thus, it was shown that, as in the case of the anti-IL-6
receptor antibodies, pH-dependently binding anti-IL-6 antibodies
that bind to the antigen strongly under plasma neutral conditions,
but weakly under intraendosomal acidic conditions can be
constructed by introducing histidine substitutions and the like
mainly into the CDR amino acid sequences. As indicated in Examples
1 to 15, an anti-IL-6 receptor antibody that has the pH-dependent
binding ability repeatedly binds to the IL-6 receptor and PK/PD is
remarkably improved. That is, it was suggested that Anti-IL-6 clone
1 and Anti-IL-6 clone 2, which have the pH-dependent binding
ability, repeatedly bind to more antigens with significantly
improved PK/PD, as compared with Anti-IL6 wild type.
Example 17
Construction of pH-Dependently Binding Anti-IL-31 Receptor
Antibody
Expression and Purification of Anti-IL-31 Receptor Antibody
[0432] In Examples 1 to 15, a plurality of humanized anti-IL-6
receptor antibodies that pH-dependently bind to the IL-6 receptor
were successfully created by conferring the pH dependency through
introducing histidine substitutions and the like into the variable
region, in particular, the CDR sequences of the humanized anti-IL-6
receptor antibodies. It was found that all of these antibodies
repeatedly bind to the IL-6 receptor and demonstrate a considerable
improvement of PK/PD.
[0433] Therefore, it was confirmed that the pH dependent ability of
an antibody to bind to an antigen could be confered to another
antibody that binds to an antigen other than the IL-6 receptor
using a similar method. Mouse IL-31 receptor was selected as the
antigen, and an anti-IL-31 receptor antibody including the H chain
(WT) (amino acid sequence: SEQ ID NO: 67) and L chain (WT) (amino
acid sequence: SEQ ID NO: 68), which binds to the mouse IL-31
receptor as described in WO 2007/142325, ("Anti-IL31R wild type")
was constructed. Using a method known to those skilled in the art,
gene fragments encoding the amino acid sequences of interest were
inserted into mammalian cell expression vectors to construct the H
chain expression vector and L chain expression vector of interest.
The nucleotide sequences of the resulting expression vectors were
determined using a method known to a skilled person. Anti-IL31R
wild type was expressed and purified by the method described in
Example 1.
Construction of pH-Dependent Anti-IL-31 Receptor Antibody
[0434] To confer the pH-dependent ability of the antibody to bind
to the IL-31 receptor, histidine substitutions were introduced into
the amino acids of CDR of the anti-IL-31 receptor antibody
(Anti-IL31R wild type) including the H chain (WT) (amino acid
sequence: SEQ ID NO: 67) and L chain (WT) (amino acid sequence: SEQ
ID NO: 68). By histidine substitutions in the CDR amino acids and
subsequent screening, several clones that demonstrate the
pH-dependent binding were obtained. The binding at pH 5.5 was
significantly reduced as compared with the binding at pH 7.4. The
position of histidine substitution in the pH-dependent clones is
shown in Table 15. An example is "Anti-IL31R clone 1" including the
H chain (c1) (amino acid sequence: SEQ ID NO: 69) and L chain (WT).
Anti-IL31R clone 1 was expressed and purified using the method
described in Example 1.
TABLE-US-00015 TABLE 15 Position of Histidine Substitution in
pH-dependent clones H33
Analysis of Binding of pH-Dependent Clones to Soluble IL-31
Receptor
[0435] Kinetic analysis of antigen-antibody reactions at pH 5.5 and
pH 7.4 was carried out using Biacore T100 (GE Healthcare) for the
two types of antibodies prepared as mentioned above: Anti-IL31R
wild type and Anti-IL31R clone 1 (buffer: DPBS(-) (pH 7.4 or pH
5.5), 150 mM NaCl, 0.01% Tween 20, 0.02% NaN.sub.3). The antibodies
were bound to a sensor chip on which recomb-protein A/G (Pierce)
was immobilized by amine coupling, and then the soluble mouse IL-31
receptor (prepared according to the method described in WO
2007/142325) adjusted to an appropriate concentration was injected
therein as an analyte. All the measurements were carried out at
25.degree. C. Association rate constants k.sub.a (1/Ms) and
dissociation rate constants k.sub.d (1/s) were calculated using
Biacore T100 Evaluation Software (GE Healthcare), and dissociation
constants KD (M) were calculated based on those values (Table 16).
Furthermore, the ratio of affinity at pH 5.5 and pH 7.4 was
calculated for each clone to evaluate the pH-dependent binding.
TABLE-US-00016 TABLE 16 Comparison of Binding of pH-Dependent
Clones to Mouse IL-31 Receptor KD(pH 5.5)/ Sample pH ka(1/Ms)
kd(1/s) KD(M) KD(pH 7.4) Wild type pH 7.4 1.40E+05 3.40E-03
2.30E-08 3.2 pH 5.5 5.10E+05 3.80E-03 7.40E-08 clone1 pH 7.4
1.70E+05 3.30E-03 2.20E-08 1000.0 pH 5.5 1.10E+03 2.40E-02
2.20E-05
[0436] The ratio of affinity at pH 5.5 and pH 7.4 ((KD)(pH
5.5)/(KD)(pH 7.4)) calculated, which indicates the pH-dependent
binding to the mouse IL-31 receptor, was 3.2 and 1000 for
Anti-IL31R wild type and Anti-IL31R clone 1, respectively. That is,
the pH-dependent binding ability of Anti-IL31R clone 1 is about 300
times greater than that of WT. The sensorgrams for Anti-IL31R clone
1 at pH 7.4 and pH 5.5 are shown in FIG. 27.
[0437] Thus, it was shown that, as in the cases of the anti-IL-6
receptor antibodies and anti-IL-6 antibodies, pH-dependent binding
anti-IL-31 receptor antibodies that bind to the antigen strongly
under plasma neutral conditions, but weakly under intraendosomal
acidic conditions can be constructed by introducing histidine
substitutions and the like mainly into the CDR amino acid
sequences. As indicated in Examples 1 to 15, an anti-IL-6 receptor
antibody that has the pH-dependent binding ability repeatedly binds
to the IL-6 receptor and PK/PD is remarkably improved. That is, it
was suggested that Anti-IL31R clone 1, which has the pH-dependent
binding ability, repeatedly binds to more antigens with
significantly improved PK/PD, as compared with Anti-IL31R wild
type.
Example 18
Repetitive Binding to Antigen by pH-Dependently Binding
Antibody
Expression and Purification of Antibody Administered to Mice
[0438] The four types of humanized IL-6 receptor antibodies
described below were prepared. As the antibodies that do not
pH-dependently bind to the IL-6 receptor, WT-IgG1 including H (WT)
(amino acid sequence: SEQ ID NO: 9) and L (WT) (amino acid
sequence: SEQ ID NO: 10), and H54/L28-IgG1 including H54 (amino
acid sequence: SEQ ID NO: 70) and L28 (amino acid sequence: SEQ ID
NO: 12) were expressed and purified using the method indicated in
Example 1. As the antibodies that pH-dependently bind to the IL-6
receptor, H170/L82-IgG1 of Example 3 including H170 (amino acid
sequence: SEQ ID NO: 4) and L82 (amino acid sequence: SEQ ID NO:
7), and Fv4-IgG1 of Example 10 including VH3-IgG1 (SEQ ID NO: 23)
and VL3-CK (SEQ ID NO: 27), were expressed and purified using the
method indicated in Example 1.
Analysis of Binding of Each Type of Antibody to Soluble IL-6
Receptor
[0439] Kinetic analysis of antigen-antibody reactions at pH 7.4 and
pH 5.8 was carried out using Biacore T100 (GE Healthcare) for the
four types of antibodies prepared: WT-IgG1, H54/L28-IgG1,
H170/L82-IgG1, and Fv4-IgG1 (buffer: 10 mM MES (pH 7.4 or pH 5.8),
150 mM NaCl, 0.05% Surfactant-P20). The antibodies were bound to a
sensor chip on which recomb-protein A/G (Pierce) was immobilized by
amine coupling, and SR344 adjusted to an appropriate concentration
was injected therein as an analyte. The association with and
dissociation from SR344 of each type of antibody were observed on a
real-time basis. All the measurements were carried out at
37.degree. C. Association rate constants k.sub.a (1/Ms) and
dissociation rate constants k.sub.d (1/s) were calculated using
Biacore T100 Evaluation Software (GE Healthcare), and dissociation
constants KD (M) were calculated based on those values (Table
17).
TABLE-US-00017 TABLE 17 Comparison of Association Rates (k.sub.a),
Dissociation Rates (k.sub.d), and Dissociation Constants of Each
Type of Antibody Against Soluble IL-6 Receptor (SR344) pH
Dependency pH 7.4 pH 5.8 KD(pH 5.8)/ Sample ka(1/Ms) kd(1/s) KD(M)
ka(1/Ms) kd(1/s) KD(M) kd(pH 5.8)/kd(pH 7.4) KD(pH 7.4) WT-IgG1
4.9E+05 9.4E-04 1.9E-09 8.9E+05 2.7E-03 3.1E-09 2.9 1.6
H54/L28-IgG1 8.3E+05 1.4E-03 1.7E-09 2.4E+06 2.7E-03 1.1E-09 2.0
0.7 H170/L82-IgG1 6.7E+05 1.1E-03 1.6E-09 1.2E+05 1.3E-02 1.0E-07
11.4 61.9 Fv4-IgG1 9.8E+05 9.5E-04 9.7E-10 1.4E+06 3.7E-02 2.6E-08
38.8 27.3
[0440] The ratio of affinity (KD) at pH 5.8 and pH 7.4 for each
antibody was calculated. The KD ratio, which indicates the
pH-dependent binding to SR344, was 1.6, 0.7, 61.9, and 27.3 for
WT-IgG1, H54/L28-IgG1, H170/L82-IgG1, and Fv4-IgG1, respectively.
In addition, the ratio of dissociation rate (k.sub.d) at pH 5.8 and
pH 7.4 for each antibody was calculated. The k.sub.d ratio, which
indicates the pH-dependent dissociation rate for SR344, was 2.9,
2.0, 11.4, and 38.8 for WT-IgG1, H54/L28-IgG1, H170/L82-IgG1, and
Fv4-IgG1, respectively. Thus, it was confirmed that H170/L82-IgG1
and Fv4-IgG1 demonstrate the pH-dependent binding, while the
conventional antibodies WT-IgG1 and H54/L28-IgG1 hardly exhibit the
ability. In addition, since the affinity (KD) of these antibodies
at pH 7.4 was nearly equal, their ability to bind to SR344 in the
plasma was thought to be equivalent.
[0441] In Vivo Pharmacokinetics Test Using Mice
[0442] The pharmacokinetics of SR344 and the anti-human IL-6
receptor antibody were evaluated following administration of SR344
(human IL-6 receptor, prepared in Example 1) only or simultaneous
administration of SR344 and the anti-human IL-6 receptor antibody
to mice that do not express the human IL-6 receptor (C57BL/6J; the
anti-human IL-6 receptor antibodies do not bind to the mouse IL-6
receptor). An SR344 solution (5 .mu.g/mL) or a mixed solution
containing SR344 and the anti-human IL-6 receptor antibody (5
.mu.g/mL and 0.1 mg/mL, respectively) was administered into a
caudal vein by single-dose administration at 10 mL/kg. Since the
anti-human IL-6 receptor antibody was present in an adequate excess
amount relative to SR344, it was thought that nearly all of the
SR344 molecules were bound by the antibody. Blood samples were
collected at 15 minutes, 2 hours, 8 hours, 1 day, 2 days, 3 days, 4
days, 7 days, 14 days, 21 days, and 28 days after administration.
The collected blood samples were immediately centrifuged for 15
minutes at 15,000 rpm and 4.degree. C. to obtain the plasma. The
plasma separated was stored in a freezer set to -20.degree. C. or
lower until the time of measurement. Above-described WT-IgG1,
H54/L28-IgG1, H170/L82-IgG1, and Fv4-IgG1 were used as the
anti-human IL-6 receptor antibodies.
Measurement of Anti-Human IL-6 Receptor Antibody Plasma
Concentration by ELISA
[0443] The concentration of 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.degree. 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
.mu.g/mL, and mouse plasma samples diluted 100-fold or more were
prepared. 200 .mu.L of 20 ng/mL SR344 was added to 100 .mu.L of the
calibration curve samples and plasma samples, and then the samples
were allowed to stand for 1 hour at room temperature. Subsequently,
the samples were dispensed into the anti-human IgG-immobilized
plates, and allowed to stand for 1 hour at room temperature. Then,
Biotinylated Anti-Human IL-6R Antibody (R&D) was added to react
for 1 hour at room temperature. Subsequently,
Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was
added to react for 1 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). The time course of plasma concentration after intravenous
administration as measured by this method is shown in FIG. 28.
Measurement of SR344 Plasma Concentration by
Electrochemiluminescence
[0444] The concentration of SR344 in mouse plasma was measured by
electrochemiluminescence. SR344 calibration curve samples adjusted
to concentrations of 2000, 1000, 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), and WT-IgG1, and then allowed to react
overnight at 37.degree. C. The final concentration of WT-IgG1 was
333 .mu.g/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 SR344 molecules in the samples to
WT-IgG1. Subsequently, the samples were dispensed into an MA400 PR
Streptavidin Plate (Meso Scale Discovery), and allowed to react for
1 hour at room temperature, and washing was performed. Immediately
after Read Buffer T (x4) (Meso Scale Discovery) was dispensed, the
measurement was performed by the Sector PR 400 Reader (Meso Scale
Discovery). The SR344 concentration was calculated based on the
response of the calibration curve using the analytical software
SOFTmax PRO (Molecular Devices). The time course of SR344 plasma
concentration after intravenous administration as measured by this
method is shown in FIG. 29.
Effects of pH-Dependent Binding
[0445] With respect to the time course of antibody concentration of
WT-IgG1 and H54/L28-IgG1, which do not demonstrate the pH-dependent
binding, and H170/L82-IgG1 and Fv4-IgG1, which demonstrate the
pH-dependent binding, the time course of concentration was roughly
identical for WT-IgG1, H54/L28-IgG1 and Fv4-IgG1, while
H170/L82-IgG1 was eliminated slightly more rapidly. The data of the
time course of plasma concentration was analyzed by the
pharmacokinetics analysis software WinNonlin (Pharsight). The
half-lives in the plasma of WT-IgG1, H54/L28-IgG1, Fv4-IgG1, and
H170/L28-IgG1 were 21.0, 28.8, 26.2, and 7.5 days,
respectively.
[0446] As described in Example 2, when the antigen is a soluble
antigen, an administered antibody binds to the antigen in the
plasma, and is retained in the plasma in the form of an
antigen-antibody complex. Generally, in contrast to extremely long
plasma retention time of an antibody (the elimination rate is
extremely low) due to the function of FcRn, the plasma retention
time of an antigen is short (the elimination rate is high). Thus,
an antigen that is bound to an antibody has a prolonged plasma
retention time similar to that of an antibody (the elimination rate
is extremely low). Similarly, when the antigen of humanized IL-6
receptor antibody, SR344 (soluble human IL-6 receptor), was
administered alone, SR344 was extremely rapidly eliminated (plasma
half-life: 0.2 days). However, in the case of concurrent
administration of SR344 with a conventional antibody, WT-IgG1 or
H54/L28-IgG1, which does not demonstrate the pH-dependent binding,
the elimination rate of SR344 was considerably reduced, and the
plasma retention time of SR344 was prolonged (plasma half-life: 5.3
days for WT-IgG1, 6.3 days for H54/L28-IgG1). This is because
nearly all of the SR344 molecules were bound by the antibodies
administered together, and thus SR344 bound by the antibodies had a
prolonged plasma retention time similar to that of the antibody,
due to the function of FcRn as described above.
[0447] In the case of concurrent administration of SR344 with the
H170/L82-IgG1 or Fv4-IgG1 antibody, which demonstrates the
pH-dependent binding, the elimination of SR344 was significantly
rapid (plasma half-life: 1.3 days for H170/L82-IgG1, 0.6 days for
Fv4-IgG1), as compared to the case of concurrent administration
with WT-IgG1 or H54/L28-IgG1. This tendency was particularly
prominent for Fv4-IgG1. Since the affinity of Fv4-IgG1 at pH 7.4 is
equivalent to or stronger than that of WT-IgG1 and H54/L28-IgG1, it
is thought that nearly all of the SR344 molecules were bound to
Fv4-IgG1. Even though Fv4-IgG1 demonstrates equivalent or slightly
longer plasma retention and slower elimination compared to WT-IgG1
and H54/L28-IgG1, the elimination of SR344 bound to Fv4-IgG1 was
extremely rapid. This can be explained by the concept of the
present technology shown in FIG. 4. In the case of conventional
antibodies that do not demonstrate the pH-dependent binding, an
antibody-soluble antigen complex is taken up into endosomes by
pinocytosis in the plasma, and binds to FcRn expressed in endosomes
under intraendosomal acidic conditions. Since the antibody-soluble
antigen complex bound to FcRn transfers to the cell surface as it
is, and again returns to the plasma, the antigen bound to the
antibody has a prolonged plasma retention time similar to that of
the antibody (the elimination is extremely slow). On the other
hand, in the case of antibodies that demonstrate the pH-dependent
binding, the antigen dissociates from the antibody under
intraendosomal acidic conditions, and thus only the antibody binds
to FcRn and returns again to the plasma. Since the antigen
dissociated from the antibody is degraded in lysosomes without
returning to the plasma, the elimination of antigen is extremely
rapid as compared to the case of antibodies that do not demonstrate
the pH-dependent binding. Namely, in the case of concurrent
administration of SR344 with the WT-IgG1 or H54/L28-IgG1 antibody,
which does not demonstrate the pH-dependent binding, the
elimination of SR344 is slow to the similar degree as the antibody,
since SR344 binds to WT-IgG1or H54/L28-IgG1 both in plasma and
endosomes. In contrast, in the case of concurrent administration of
SR344 with the H170/L82-IgG1 or Fv4-IgG1 antibody, which
demonstrates the pH-dependent binding, the elimination of SR344 is
extremely rapid, since SR344 dissociates from the antibody in the
intraendosomal low-pH environment. That is, since the H170/L28-IgG1
and Fv4-IgG1 antibodies, which demonstrate the pH-dependent
binding, dissociate from SR344 in the intraendosomal low-pH
environment, the majority of H170/L82-IgG1 or Fv4-IgG1 that has
returned again to the plasma with FcRn is thought to be not bound
to SR344. Thus, as shown in FIG. 4, it was revealed that, by
dissociating from an antigen in the intraendosomal low-pH
environment and returning to the plasma with FcRn without binding
to the antigen, an antibody that demonstrates the pH-dependent
binding can again bind to an antigen in the plasma. It was also
shown that, by repeating this process, the antibody that
demonstrates the pH-dependent binding can repeatedly bind to
multiple antigens. This is consistent with the Biacore data shown
in Example 7, demonstrating that pH-dependent antibodies can
repeatedly bind to antigens. Thus, by enhancing the pH-dependent
binding of an antibody to an antigen, the number of times of
repetitive antigen binding can be increased.
[0448] When the antigen is a soluble antigen, and the antigen binds
to an antibody under plasma neutral conditions, but dissociates
from the antibody in endosomes and the antibody returns to the
plasma with FcRn, the antibody can again bind to an antigen under
plasma neutral conditions. Thus, an antibody that has the ability
to dissociate from an antigen under intraendosomal acidic
conditions can bind to antigens multiple times. As compared to when
an antigen bound to an antibody does not dissociate from the
antibody in endosomes (i.e., the antigen bound to the antibody
returns to the plasma), if an antigen bound to an antibody
dissociates from the antibody in endosomes, the plasma elimination
rate of the antigen is increased, since the antigen is transported
to lysosomes and degraded. Thus, the plasma elimination rate of an
antigen can be used as an index to determine whether an antibody
can bind to the antigen multiple times. Determination of the plasma
elimination rate of an antigen can be performed, for example, by
administering an antigen and an antibody in vivo, and then
measuring the plasma antigen concentration after the
administration, as shown in the Examples.
[0449] An antibody that demonstrates the pH-dependent binding can
repeatedly bind to multiple antigens in contrast to the case of a
conventional antibody that does not demonstrate the pH-dependent
binding. Thus, the amount of antibody administered can be
considerably reduced, and the administration intervals can be
greatly prolonged.
[0450] The repetitive binding to multiple antigens of this
mechanism is based on pH-dependent antigen-antibody reaction. Thus,
regardless of the type of antigen, if an antibody demonstrating the
pH-dependent binding that binds to an antigen at pH 7.4 of plasma
but dissociates from the antigen at intraendosomal acidic pH can be
constructed, then such an antibody can repeatedly bind to multiple
antigens. Accordingly, the present technology is useful in that it
can be applied not only to antibodies to the IL-6 receptor, IL-6,
and the IL-31 receptor, but generally to any antibody to any
antigen, regardless of the type of antigen.
Sequence CWU 1
1
701119PRTArtificialAn artificially synthesized peptide sequence
1Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp
Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 2107PRTArtificialAn artificially synthesized
peptide sequence 2Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser
Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Tyr Gly Ser Glu Leu
His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp
Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr 85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu 100 105
3119PRTArtificialAn artificially synthesized peptide sequence 3Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10
15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp
20 25 30 His Ala His Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu
Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr
Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr
Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg
Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 4119PRTArtificialAn artificially synthesized
peptide sequence 4Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly
His Ser Ile Ser His His 20 25 30 His Ala His Ser Trp Val Arg Gln
Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr
Ser Gly Ile Thr His Tyr Asn Pro His Leu 50 55 60 Lys Gly Arg Val
Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95
Ala Arg Val Leu Ala Arg Ile Thr Ala Met Asp His Trp Gly Glu Gly 100
105 110 Thr Leu Val Thr Val Ser Ser 115 5119PRTArtificialAn
artificially synthesized peptide sequence 5Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu
Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser His Asp 20 25 30 His Ala
Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45
Ile Gly His Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro His Leu 50
55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe
Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala
Tyr Tyr Cys 85 90 95 Ala Arg Phe Leu Ala Arg Ile Thr Ala His Asp
His Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
6107PRTArtificialAn artificially synthesized peptide sequence 6Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln His Ile Ser Ser His
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu
Leu Ile 35 40 45 Tyr Tyr Gly Ser His Leu His Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr
Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys
Gly Gln Gly Asn Arg Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Glu 100 105 7107PRTArtificialAn artificially
synthesized peptide sequence 7Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys
Gln Ala Ser Gln Asp Ile Ser Ser His 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Tyr Gly
Ser His Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala 65 70
75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro
Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu 100 105
8107PRTArtificialAn artificially synthesized peptide sequence 8Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser His
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu
Leu Ile 35 40 45 Tyr Tyr Gly Ser His Leu His His Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr
Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys
Gly Gln Gly His Arg Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Glu 100 105 9448PRTArtificialAn artificially
synthesized peptide sequence 9Gln Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp
Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr
Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys
Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70
75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp
Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195
200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315
320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 10214PRTArtificialAn artificially synthesized peptide sequence
10Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr
Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
11119PRTArtificialAn artificially synthesized peptide sequence
11Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp
Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Val Leu Ala
Arg Ile Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 12107PRTArtificialAn artificially synthesized
peptide sequence 12Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser
Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Tyr Gly Ser Glu Leu
His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp
Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Leu Pro Tyr 85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu 100 105
131404DNAArtificialAn artificially synthesized nucleotide sequence
13atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt ccactcccag
60gtccaactgc aggagagcgg tccaggtctt gtgaaaccta gcgagaccct gagcctgacc
120tgcgccgtgt ctggctactc aattagcgac gatcatgcct ggagctgggt
tcgccagcca 180cctggagaag gtcttgagtg gattggatac attagttata
gtggaatcac aaactataat 240ccatctctca aaggcagagt gacaatatcg
agagacacca gcaagaacca gttcagcctg 300aaactcagca gcgtgacagc
cgccgacacc gcggcttatt attgtgcaag atccctagct 360cggactacgg
ctatggacta ctggggtgaa ggcaccctcg tcacagtctc ctcagcctcc
420accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc
tgggggcaca 480gcggccctgg gctgcctggt caaggactac ttccccgaac
cggtgacggt gtcgtggaac 540tcaggcgccc tgaccagcgg cgtgcacacc
ttcccggctg tcctacagtc ctcaggactc 600tactccctca gcagcgtggt
gaccgtgccc tccagcagct tgggcaccca gacctacatc 660tgcaacgtga
atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct
720tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg
gggaccgtca 780gtcttcctct tccccccaaa acccaaggac accctcatga
tctcccggac ccctgaggtc 840acatgcgtgg tggtggacgt gagccacgaa
gaccctgagg tcaagttcaa ctggtacgtg 900gacggcgtgg aggtgcataa
tgccaagaca aagccgcggg aggagcagta caacagcacg 960taccgtgtgg
tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac
1020aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat
ctccaaagcc 1080aaagggcagc cccgagaacc acaggtgtac accctgcccc
catcccggga tgagctgacc 1140aagaaccagg tcagcctgac ctgcctggtc
aaaggcttct atcccagcga catcgccgtg 1200gagtgggaga gcaatgggca
gccggagaac aactacaaga ccacgcctcc cgtgctggac 1260tccgacggct
ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag
1320gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta
cacgcagaag 1380agcctctccc tgtctccggg taaa 140414321DNAArtificialAn
artificially synthesized nucleotide sequence 14gacatccaga
tgacccagag cccaagcagc ctgagcgcca gcgtgggtga cagcgtgacc 60atcacctgtc
aagccagcca ggacatcagc agttacctga attggtacca gcagaagcca
120ggaaaggctc cagagctgct gatctactac ggctccgaac tgcactctgg
tgtgccaagc 180agattcagcg gtagcggtag cggtaccgac ttcaccttca
ccatcagcag cctcgaggca 240gaggacgccg ctacctacta ctgcgggcag
ggtaaccggc ttccatacac gttcggccaa 300gggaccaagg tggaaatcga a
3211515PRTArtificialAn artificially synthesized peptide sequence
15Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5
10 15 1624DNAArtificialAn artificially syntheisized primer sequence
16ttcccaccag cctgtccgcc tctg 241724DNAArtificialAn artificially
syntheisized primer sequence 17cgtgaagcca aaggccccac tccc
241832DNAArtificialAn artificially syntheisized primer sequence
18tagaattcca ccatgctggc cgtcggctgc gc 321957DNAArtificialAn
artificially syntheisized primer sequence 19attgcggccg cttatcagtg
gtgatgatga tgatgtggta ccgaagaaga atcttgc 57201131DNAArtificialAn
artificially synthesized nucleotide sequence
20gaattccacc atgctggccg tcggctgcgc gctgctggct gccctgctgg ccgcgccggg
60ggcggcgctg gccccggggg gctgccctgc acaggaggtg gcgagaggtg tgctgaccag
120tctgccagga gacagcgtga ctctgacctg cccaggggga gagccggaag
acaatgccac 180tgttcactgg gttctcagga agccagctgt aggctcccac
ctcagcagat gggctggcgt 240gggaaggagg ctgctgctga ggtcggtgca
gctccatgac tctggaaact attcatgcta 300ccgggccggc cgcccggctg
gaactgtgca cttgctggtg gatgttcccc ccgaggagcc 360ccagctctcc
tgcttccgga agagccccct cagcaacgtt gtttgtgagt ggggtcctcg
420gagcacccca tctccgacga ccaaggctgt gctgttggtg aggaagtttc
agaacagtcc 480ggccgaagac ttccaggagc cgtgccagta ttcccaggag
tcccagaagt tctcctgcca 540gttggcagtc ccggagggag acagctcttt
ctacatagtg tccatgtgcg tcgccagtag 600tgtcgggagc aagctcagca
aaactcagac ctttcagggt tgtggaatct tgcagcctga 660tccgcctgcc
aacatcacag tcactgccgt ggccagaaac ccccgctggc tcagtgtcac
720ctggcaagac ccccactcct ggaactcatc tttctacaga ctacggtttg
agctcagata 780tcgagctgaa cggtcaaaga cattcacaac atggatggtc
aaggacctcc agcatcactg 840tgtcatccac gacgcctgga gcggcctgag
gcacgtggtg cagcttcgtg cccaggagga 900gttcgggcaa ggcgagtgga
gcgagtggag cccggaggcc atgggcacgc cttggacaga 960atccaggagt
cctccagctg agaacgaggt gtccaccccc acgcaggcac ctactactaa
1020taaagatgat gataatattc tctccagaga ttctgcaaat gcgacaagcc
tcccagtgca 1080agattcttct tcggtaccac atcatcatca tcaccactga
taagcggccg c 113121449PRTArtificialAn artificially synthesized
peptide sequence 21Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly
Tyr Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln
Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly His Ile Ser Tyr
Ser Gly Ile Thr Asn Tyr Asn Pro His Leu 50 55 60 Gln Asp Arg Val
Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95
Ala Arg Phe Leu Ala Arg Ile Thr Ala His Asp His Trp Gly Glu Gly 100
105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225
230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345
350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys
22449PRTArtificialAn artificially synthesized peptide sequence
22Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly His Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro His Leu 50 55 60 Gln Asp Arg Val Thr Ile Ser Arg Asp
Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Leu Ala
Arg Ile Thr Ala His Asp His Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 445 Lys 23449PRTArtificialAn
artificially synthesized peptide sequence 23Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu
Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala
Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45
Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50
55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp
Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305
310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425
430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445 Lys 24449PRTArtificialAn artificially synthesized
peptide sequence 24Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly
His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln
Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr
Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu 50 55 60 Gln Gly Arg Val
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100
105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225
230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345
350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys
25214PRTArtificialAn artificially synthesized peptide sequence
25Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Arg Asp Ile Ser Ser
His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu
Leu Leu Ile 35 40 45 Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr
Cys Gly Gln Gly Asn Arg Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
26214PRTArtificialAn artificially synthesized peptide sequence
26Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Arg Asp Ile Ser Ser
His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu
Leu Leu Ile 35 40 45 Tyr Tyr Gly Ser His Leu Glu Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala
65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu
Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg
Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205 Phe Asn Arg Gly Glu Cys 210 27214PRTArtificialAn
artificially synthesized peptide sequence 27Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr
Ile Thr Cys Gln Ala Ser Thr Asp Ile Ser Ser His 20 25 30 Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45
Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu
Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg
Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu
Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180
185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 28326PRTHomo sapiens
28Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1
5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys
Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135
140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260
265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325
29324PRTArtificialAn artificially synthesized peptide sequence
29Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1
5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys
Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135
140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260
265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro
30324PRTArtificialAn artificially synthesized peptide sequence
30Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1
5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys
Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135
140 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260
265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro
31326PRTArtificialAn artificially synthesized peptide sequence
31Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1
5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys
Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135
140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260
265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Ala His Tyr
Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325
32324PRTArtificialAn artificially synthesized peptide sequence
32Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1
5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys
Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135
140 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260
265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Ala His Tyr
Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro
33443PRTArtificialAn artificially synthesized peptide sequence
33Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Asn Phe Gly Thr Gln Thr Tyr Thr
Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Thr Val Glu Arg Lys Cys Cys Val Glu 210 215 220 Cys Pro Pro Cys Pro
Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
Arg Val Val Ser Val Leu 290 295 300 Thr Val Val His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Thr Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360
365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 34443PRTArtificialAn artificially
synthesized peptide sequence 34Gln Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala
Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp
Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe
Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln
Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp
Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195
200 205 Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val
Glu 210 215 220 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser
Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu
Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300 Thr Val
Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315
320 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335 Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Glu Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
35443PRTArtificialAn artificially synthesized peptide sequence
35Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Asn Phe Gly Thr Gln Thr Tyr Thr
Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Thr Val Glu Arg Lys Ser Cys Val Glu 210 215 220 Cys Pro Pro Cys Pro
Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260
265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val
Ser Val Leu 290 295 300 Thr Val Val His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Thr Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385
390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln 405 410 415 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Ala 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 435 440 36445PRTArtificialAn artificially synthesized peptide
sequence 36Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser
Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro
Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly His Ile Ser Tyr Ser Gly
Ile Thr Asn Tyr Asn Pro His Leu 50 55 60 Gln Asp Arg Val Thr Ile
Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Phe Leu Ala Arg Ile Thr Ala His Asp His Trp Gly Glu Gly 100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Asn Phe Gly Thr Gln
Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys
Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu 210 215 220 Cys Pro
Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 225 230 235
240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
Arg Val Val Ser Val Leu 290 295 300 Thr Val Val His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Thr Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360
365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Ala 420 425 430 His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 435 440 445 37443PRTArtificialAn
artificially synthesized peptide sequence 37Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu
Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser His Asp 20 25 30 His Ala
Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45
Ile Gly His Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro His Leu 50
55 60 Gln Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe
Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Phe Leu Ala Arg Ile Thr Ala His Asp
His Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190 Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys
Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser
Cys Val Glu 210 215 220 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305
310 315 320 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys 325 330 335 Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala 420 425
430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
38445PRTArtificialAn artificially synthesized peptide sequence
38Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Thr Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Asn Phe Gly Thr Gln Thr Tyr Thr
Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Thr Val Glu Arg Lys Cys Cys Val Glu 210 215 220 Cys Pro Pro Cys Pro
Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln 260
265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val
Ser Val Leu 290 295 300 Thr Val Val His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Thr Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360
365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Ala 420 425 430 His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 435 440 445 39443PRTArtificialAn
artificially synthesized peptide sequence 39Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu
Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala
Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45
Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu 50
55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp
Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190 Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys
Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser
Cys Val Glu 210 215 220 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305
310 315 320 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys 325 330 335 Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala 420 425
430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
406PRTArtificialAn artificially synthesized peptide sequence 40His
Asp His Ala Trp Ser 1 5 4116PRTArtificialAn artificially
synthesized peptide sequence 41His Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro His Leu Gln Asp 1 5 10 15 4216PRTArtificialAn
artificially synthesized peptide sequence 42Phe Ile Ser Tyr Ser Gly
Ile Thr Asn Tyr Asn Pro Ser Leu Gln Gly 1 5 10 15
4316PRTArtificialAn artificially synthesized peptide sequence 43Phe
Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu Gln Gly 1 5 10
15 4410PRTArtificialAn artificially synthesized peptide sequence
44Phe Leu Ala Arg Ile Thr Ala His Asp His 1 5 10
4510PRTArtificialAn artificially synthesized peptide sequence 45Ser
Leu Ala Arg Thr Thr Ala Met Asp Tyr 1 5 10 4630PRTArtificialAn
artificially synthesized peptide sequence 46Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu
Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser 20 25 30
4730PRTArtificialAn artificially synthesized peptide sequence 47Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10
15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser 20 25 30
4814PRTArtificialAn artificially synthesized peptide sequence 48Trp
Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp Ile Gly 1 5 10
4932PRTArtificialAn artificially synthesized peptide sequence 49Arg
Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10
15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys Ala Arg
20 25 30 5032PRTArtificialAn artificially synthesized peptide
sequence 50Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser
Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys Ala Arg 20 25 30 5132PRTArtificialAn artificially
synthesized peptide sequence 51Arg Val Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 5211PRTArtificialAn
artificially synthesized peptide sequence 52Trp Gly Glu Gly Thr Leu
Val Thr Val Ser Ser 1 5 10 5311PRTArtificialAn artificially
synthesized peptide sequence 53Gln Ala Ser Arg Asp Ile Ser Ser His
Leu Asn 1 5 10 5411PRTArtificialAn artificially synthesized peptide
sequence 54Gln Ala Ser Thr Asp Ile Ser Ser His Leu Asn 1 5 10
557PRTArtificialAn artificially synthesized peptide sequence 55Tyr
Gly Ser His Leu Leu Ser 1 5 567PRTArtificialAn artificially
synthesized peptide sequence 56Tyr Gly Ser His Leu Glu Ser 1 5
579PRTArtificialAn artificially synthesized peptide sequence 57Gly
Gln Gly Asn Arg Leu Pro Tyr Thr 1 5 5823PRTArtificialAn
artificially synthesized peptide sequence 58Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr
Ile Thr Cys 20 5915PRTArtificialAn artificially synthesized peptide
sequence 59Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile
Tyr 1 5 10 15 6032PRTArtificialAn artificially synthesized peptide
sequence 60Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr 1 5 10 15 Phe Thr Ile Ser Ser Leu Glu Ala Glu Asp Ala Ala
Thr Tyr Tyr Cys 20 25 30 6110PRTArtificialAn artificially
synthesized peptide sequence 61Phe Gly Gln Gly Thr Lys Val Glu Ile
Glu 1 5 10 62449PRTArtificialAn artificially synthesized peptide
sequence 62Glu Val Gln Leu Val Glu Ser Gly Gly Lys Leu Leu Lys Pro
Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Phe 20 25 30 Ala Met Ser Trp Phe Arg Gln Ser Pro Glu
Lys Arg Leu Glu Trp Val 35 40 45 Ala Glu Ile Ser Ser Gly Gly Ser
Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Ser
Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg
Gly Leu Trp Gly Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110
Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235
240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360
365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys
63213PRTArtificialAn artificially synthesized peptide sequence
63Gln Ile Val Leu Ile Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1
5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu
Leu Ile Tyr 35 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Trp Ser Gly Tyr Pro Tyr Thr 85 90 95 Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135
140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210
64449PRTArtificialAn artificially synthesized peptide sequence
64Glu Val Gln Leu Val Glu Ser Gly Gly Lys Leu Leu Lys Pro Gly Gly 1
5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
His 20 25 30 Ala Met Ser Trp Phe Arg Gln Ser Pro Glu Lys Arg Leu
Glu Trp Val 35 40 45 Ala Glu Ile Ser Ser Gly Gly Ser Tyr Thr Tyr
His Pro His Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Ser Ser Leu Arg
Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Trp
Gly His Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 445 Lys 65213PRTArtificialAn
artificially synthesized peptide sequence 65Gln
Ile Val Leu Ile Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10
15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu
Ile Tyr 35 40 45 Asp Thr Ser Asn His Ala Ser Gly Val Pro Val Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln
His Trp Ser Gly His Pro Tyr Thr 85 90 95 Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145
150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210
66213PRTArtificialAn artificially synthesized peptide sequence
66Gln Ile Val Leu Ile Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1
5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu
Leu Ile Tyr 35 40 45 Asp Thr Ser His His Ala Ser Gly Val Pro Val
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys
Gln His Trp Ser Gly His Pro Tyr Thr 85 90 95 Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135
140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210
67456PRTArtificialAn artificially synthesized peptide sequence
67Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly
Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp
Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Val
Val Pro Ala Ala Met Ser Phe Tyr Tyr Gly Met 100 105 110 Asp Val Trp
Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135
140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260
265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385
390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys Gly
450 455 68214PRTArtificialAn artificially synthesized peptide
sequence 68Leu Pro Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro
Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly
Ser Lys Thr Val 20 25 30 Tyr Trp Tyr Gln Gln Glu Pro Gly Gln Ala
Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Thr Asp Arg Pro Ala Gly
Ile Pro Glu Arg Phe Ser Gly Ala 50 55 60 Asn Ser Gly Asn Thr Ala
Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp
Tyr Tyr Cys Gln Val Trp Asp Ser Ser Thr Asp His 85 90 95 Gly Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110
Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115
120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro
Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val
Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Lys Pro Ser Lys Gln Ser
Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro
Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr
His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu
Cys Ser 210 69456PRTArtificialAn artificially synthesized peptide
sequence 69Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Gly Tyr 20 25 30 His Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly
Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Asp Val Val Pro Ala Ala Met Ser Phe Tyr Tyr Gly Met 100 105 110
Asp Val Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115
120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235
240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360
365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro
Gly Lys Gly 450 455 70449PRTArtificialAn artificially synthesized
peptide sequence 70Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly
Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln
Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr
Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val
Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100
105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225
230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345
350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys
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