U.S. patent application number 14/396521 was filed with the patent office on 2015-04-30 for anti-human cd69 antibody, and use thereof for medical purposes.
The applicant listed for this patent is GeneFrontier Corporation, National University Corporation Chiba University. Invention is credited to Yuki Hayami, Chiaki Iwamura, Shizue Katoh, Kanehisa Kojoh, Akira Miyakoshi, Mikiko Nakamura, Toshinori Nakayama, Kumiko Tsuihiji.
Application Number | 20150118237 14/396521 |
Document ID | / |
Family ID | 49483123 |
Filed Date | 2015-04-30 |
United States Patent
Application |
20150118237 |
Kind Code |
A1 |
Kojoh; Kanehisa ; et
al. |
April 30, 2015 |
ANTI-HUMAN CD69 ANTIBODY, AND USE THEREOF FOR MEDICAL PURPOSES
Abstract
The present invention provides an antibody that specifically
binds to human CD69, has an activity to suppress allergic
inflammation, and has cross-reactivity with mouse CD69. In
addition, the present invention provides an antibody having high
binding affinity for human CD69 and an activity to suppress
allergic inflammations. The antibody of the present invention can
be a human antibody.
Inventors: |
Kojoh; Kanehisa;
(Kashiwa-shi, Chiba, JP) ; Miyakoshi; Akira;
(Kashiwa-shi, Chiba, JP) ; Katoh; Shizue;
(Kashiwa-shi, JP) ; Tsuihiji; Kumiko;
(Kashiwa-shi, Chiba, JP) ; Hayami; Yuki;
(Kashiwa-shi, Chiba, JP) ; Nakamura; Mikiko;
(Kashiwa-shi, JP) ; Nakayama; Toshinori;
(Chiba-shi, JP) ; Iwamura; Chiaki; (Chiba-shi,
Chiba, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GeneFrontier Corporation
National University Corporation Chiba University |
Kashiwa-shi, Chiba
Chiba-shi, Chiba |
|
JP
JP |
|
|
Family ID: |
49483123 |
Appl. No.: |
14/396521 |
Filed: |
April 23, 2013 |
PCT Filed: |
April 23, 2013 |
PCT NO: |
PCT/JP2013/061918 |
371 Date: |
October 23, 2014 |
Current U.S.
Class: |
424/139.1 ;
435/252.31; 435/252.33; 435/254.2; 435/254.21; 435/320.1; 435/331;
530/387.9; 536/23.53 |
Current CPC
Class: |
C07K 2317/33 20130101;
C07K 2317/34 20130101; A61P 37/08 20180101; A61P 29/00 20180101;
C07K 2317/565 20130101; C07K 2317/92 20130101; C07K 2317/55
20130101; A61K 2039/505 20130101; C07K 2317/21 20130101; C07K
16/2851 20130101 |
Class at
Publication: |
424/139.1 ;
530/387.9; 536/23.53; 435/320.1; 435/252.33; 435/252.31; 435/254.2;
435/254.21; 435/331 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 23, 2012 |
JP |
2012-098243 |
Claims
1. An antibody that specifically binds to human CD69, has an
activity to suppress allergic inflammation, and has
cross-reactivity with mouse CD69.
2. The antibody according to claim 1, which binds to human CD69 at
an epitope comprising the amino acid sequence shown by SEQ ID NO:
33.
3. The antibody according to claim 1, comprising a light chain
variable region and a heavy chain variable region, wherein (1) the
light chain variable region comprises CDR1 comprising the amino
acid sequence shown in SEQ ID NO: 7, CDR2 comprising the amino acid
sequence shown in SEQ ID NO: 8 and CDR3 comprising the amino acid
sequence shown in SEQ ID NO: 9, 19 or 20, and the heavy chain
variable region comprises CDR1 comprising the amino acid sequence
shown in SEQ ID NO: 10, CDR2 comprising the amino acid sequence
shown in SEQ ID NO: 11 and CDR3 comprising the amino acid sequence
shown in SEQ ID NO: 12, or (2) the light chain variable region
comprises CDR1 comprising the amino acid sequence shown in SEQ ID
NO: 7, CDR2 comprising the amino acid sequence shown in SEQ ID NO:
8 and CDR3 comprising the amino acid sequence shown in SEQ ID NO:
9, 19 or 20, and the heavy chain variable region comprises CDR1
comprising the amino acid sequence shown in SEQ ID NO: 10, CDR2
comprising the amino acid sequence shown in SEQ ID NO: 11 and CDR3
comprising the amino acid sequence shown in SEQ ID NO: 12, except
that 1 to 3 amino acids are substituted, deleted, inserted, and/or
added in at least one amino acid sequence selected from the group
consisting of the amino acid sequences shown in SEQ ID NOs: 7-9, 19
and 20, and/or 1 to 3 amino acids are substituted, deleted,
inserted, and/or added in at least one amino acid sequence selected
from the group consisting of the amino acid sequences shown in SEQ
ID NOs: 10-12.
4. The antibody according to claim 3, wherein (1') the light chain
variable region comprises the amino acid sequence shown in SEQ ID
NO: 23, 27 or 28, and the heavy chain variable region comprises the
amino acid sequence shown by SEQ ID NO: 24.
5-7. (canceled)
8. The antibody according to claim 1, which has a K.sub.D value of
not more than 5.times.10.sup.-8 M relating to binding affinity to
human CD69.
9. The antibody according to claim 1, which is a human
antibody.
10. A pharmaceutical composition comprising the antibody according
to claim 1.
11. (canceled)
12. A polynucleotide encoding the antibody according to claim
1.
13. A vector comprising the polynucleotide according to claim
12.
14. A transformant comprising the vector according to claim 13.
15-17. (canceled)
18. A method for the prophylaxis or treatment of an allergic
disease or inflammatory disease in a mammal, comprising
administering an effective amount of the antibody according to
claim 1 to a mammal in need thereof, thereby preventing or treating
the allergic disease or inflammatory disease in the mammal.
19. (canceled)
20. The antibody according to claim 2, which is a human
antibody.
21. The antibody according to claim 3, which is a human
antibody.
22. The antibody according to claim 4, which is a human
antibody.
23. The antibody according to claim 8, which is a human
antibody.
24. A pharmaceutical composition comprising the antibody according
to claim 2.
25. A pharmaceutical composition comprising the antibody according
to claim 3.
26. A pharmaceutical composition comprising the antibody according
to claim 4.
27. A pharmaceutical composition comprising the antibody according
to claim 8.
Description
TECHNICAL FIELD
[0001] The present invention relates to an anti-human CD69
antibody, and pharmaceutical use thereof.
BACKGROUND ART
[0002] CD69 is a type II transmembrane protein belonging to the
C-type lectin family. Since the expression of CD69 increases within
a few hours after stimulation of T cells and B cells, it is widely
used as an early activation marker molecule to be an index of
lymphocyte activation (non-patent document 1). In addition, the
expression is also observed in T cells under selection during
differentiation in thymus (non-patent documents 2 and 3). While
CD69 is assumed to have a function as a coreceptor to potentiate
signal transduction from an antigen receptor, the detail is
unknown. Its ligand has not been identified to date. It is
constitutively expressed in platelet, and the expression is also
observed in activated neutrophils, eosinophils and the like.
Therefore, it is assumed to play a role in the expression of
function in platelets and topical inflammation reactions. Also, it
has been clarified that CD69 on the neutrophil plays an important
role in the onset of arthritis (non-patent document 4).
Furthermore, it has been reported that CD69 controls allergic
airway inflammation, and an antibody to mouse CD69 inhibits
allergic airway inflammation (non-patent document 5). It has also
been reported that COPD induced by cigarette smoke and lung
fibrogenesis induced by bleomycin are attenuated in CD69 deficient
mouse (non-patent document 6 and 7). Therefore, application of an
antibody to CD69 to the prophylaxis or treatment of such allergic
diseases and inflammatory diseases is expected. However, since the
ligand for CD69 is unknown, a method for efficiently evaluating in
vitro a pharmacological effect of an antibody to human CD69 is not
available. Moreover, since existing antibodies to human CD69 do not
cross-react with non-human CD69, evaluation of a pharmacological
effect of the existing antibodies in vivo is substantially
impossible, and even whether or not these antibodies afford a
useful pharmacological effect is not clear. As described above,
since a satisfactory method for evaluation of a pharmacological
effect of an anti-human CD69 antibody does not exist, the
development of an anti-human CD69 antibody applicable to the
prophylaxis or treatment of allergic diseases and inflammatory
diseases is delayed far behind.
[0003] The phage display method is one of the display techniques
that have realized an in vitro high-speed selection by forming a
one-to-one correspondence in the form of phage particle between a
functional peptide or protein and a DNA encoding same. This phage
display method has been applied to antibody selection, and
antibodies obtained by this method have been developed as
medicaments (non-patent document 8). Furthermore, a method of
obtaining a specific antibody by combining a human artificial
antibody library and a phage display method has been established,
and such methods have been practicalized by plural companies, as
evidenced by HuCAL (Human Combinatorial Antibody Library) of
MorphoSys.
DOCUMENT LIST
Non-Patent Documents
[0004] non-patent document 1: Testi, R. et al. Immunol. Today 15:
479-483, 1994. [0005] non-patent document 2: Yamashita, I. et al.
Int. Immunol. 5: 1139-1150, 1993. [0006] non-patent document 3:
Nakayama, T. et al. J. Immunol. 168: 87-94, 2002. [0007] non-patent
document 4: Murata, K. at al. Int. Immunol. 15: 987-992, 2003.
[0008] non-patent document 5: Miki-Hosokawa, T. et al. J. Immunol.
183: 8203-8215, 2009. [0009] non-patent document 6: Tsuyusaki, J.
et al. J. Recept. Signal Transduct. Res. 31: 434-439, 2011. [0010]
non-patent document 7: Yamauchi, K. et al. Respir. Res. 12: 131,
2011. [0011] non-patent document 8: Rothe, C. et al. J. Mol. Biol.
376: 1182-1200, 2008.
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0012] An object of the present invention is to provide an
anti-human CD69 antibody applicable to the prophylaxis or treatment
of allergic diseases and inflammatory diseases.
Means of Solving the Problems
[0013] To solve the above-mentioned problem, the present inventors
first produced an anti-CD69 antibody that binds to both human CD69
and mouse CD69. In addition, they intensively studied a method of
evaluating a pharmacological effect of anti-human CD69 antibody in
vivo. As a result, they have succeeded in reproducing an allergic
reaction in mouse, which is mediated by Th2 cells that express
human CD69, by forcibly expressing human CD69 in Th2 cells of CD69
deficient mouse immunized with a particular antigen, and returning
the Th2 cells into the body of the mouse. They have produced a
plurality of anti-human CD69 antibodies by HuCAL, and evaluated the
effect on the allergic reaction by using the aforementioned mouse.
As a result, they have found an anti-human CD69 antibody having
superior allergy-suppressing effect and inflammation-suppressing
effect. Furthermore, they have improved the affinity for human CD69
by modifying the light chain CDR3 of the obtained antibody, and
succeeded in potentiating the allergy-suppressing effect and
inflammation-suppressing effect, while maintaining the
cross-reactivity with mouse CD69. Based on the above-mentioned
findings, they have completed the present invention.
[0014] Accordingly, the present invention relates to the
following.
[1] An antibody that specifically binds to human CD69, has an
activity to suppress allergic inflammation, and has
cross-reactivity with mouse CD69. [2] The antibody of [1] that
binds to human CD69 at an epitope comprising the amino acid
sequence shown by SEQ ID NO: 33. [3] The antibody of [1],
comprising a light chain variable region and a heavy chain variable
region, wherein (1) the light chain variable region comprises CDR1
comprising the amino acid sequence shown in SEQ ID NO: 7, CDR2
comprising the amino acid sequence shown in SEQ ID NO: 8 and CDR3
comprising the amino acid sequence shown in SEQ ID NO: 9, 19 or 20,
and the heavy chain variable region comprises CDR1 comprising the
amino acid sequence shown in SEQ ID NO: 10, CDR2 comprising the
amino acid sequence shown in SEQ ID NO: 11 and CDR3 comprising the
amino acid sequence shown in SEQ ID NO: 12, or (2) the light chain
variable region comprises CDR1 comprising the amino acid sequence
shown in SEQ ID NO: 7, CDR2 comprising the amino acid sequence
shown in SEQ ID NO: 8 and CDR3 comprising the amino acid sequence
shown in SEQ ID NO: 9, 19 or 20, and the heavy chain variable
region comprises CDR1 comprising the amino acid sequence shown in
SEQ ID NO: 10, CDR2 comprising the amino acid sequence shown in SEQ
ID NO: 11 and CDR3 comprising the amino acid sequence shown in SEQ
ID NO: 12, except that 1 to 3 amino acids are substituted, deleted,
inserted, and/or added in at least one amino acid sequence selected
from the group consisting of the amino acid sequences shown in SEQ
ID NOs: 7-9, 19 and 20, and/or 1 to 3 amino acids are substituted,
deleted, inserted, and/or added in at least one amino acid sequence
selected from the group consisting of the amino acid sequences
shown in SEQ ID NOs: 10-12. [4] The antibody of [3], wherein (1')
the light chain variable region comprises the amino acid sequence
shown in SEQ ID NO: 23, 27 or 28, and the heavy chain variable
region comprises the amino acid sequence shown by SEQ ID NO: 24.
[5] An antibody that specifically binds to human CD69, has an
activity to suppress allergic inflammation, and binds to human CD69
at an epitope comprising the amino acid sequence shown by SEQ ID
NO: 59 or 78. [6] An antibody that specifically binds to human
CD69, has an activity to suppress allergic inflammation, and
comprises a light chain variable region and a heavy chain variable
region, wherein (3) the light chain variable region comprises CDR1
comprising the amino acid sequence shown in SEQ ID NO: 1, CDR2
comprising the amino acid sequence shown in SEQ ID NO: 2 and CDR3
comprising the amino acid sequence shown in SEQ ID NO: 3, and the
heavy chain variable region comprises CDR1 comprising the amino
acid sequence shown in SEQ ID NO: 4, CDR2 comprising the amino acid
sequence shown in SEQ ID NO: 5 and CDR3 comprising the amino acid
sequence shown in SEQ ID NO: 6; (4) the light chain variable region
comprises CDR1 comprising the amino acid sequence shown in SEQ ID
NO: 13, CDR2 comprising the amino acid sequence shown in SEQ ID NO:
14 and CDR3 comprising the amino acid sequence shown in SEQ ID NO:
15, and the heavy chain variable region comprises CDR1 comprising
the amino acid sequence shown in SEQ ID NO: 16, CDR2 comprising the
amino acid sequence shown in SEQ ID NO: 17 and CDR3 comprising the
amino acid sequence shown in SEQ ID NO: 18; (5) the light chain
variable region comprises CDR1 comprising the amino acid sequence
shown in SEQ ID NO: 1, CDR2 comprising the amino acid sequence
shown in SEQ ID NO: 2 and CDR3 comprising the amino acid sequence
shown in SEQ ID NO: 3, and the heavy chain variable region
comprises CDR1 comprising the amino acid sequence shown in SEQ ID
NO: 4, CDR2 comprising the amino acid sequence shown in SEQ ID NO:
5 and CDR3 comprising the amino acid sequence shown in SEQ ID NO:
6, except that 1 to 3 amino acids are substituted, deleted,
inserted, and/or added in at least one amino acid sequence selected
from the group consisting of the amino acid sequences shown in SEQ
ID NOs: 1 to 3, and/or 1 to 3 amino acids are substituted, deleted,
inserted, and/or added in at least one amino acid sequence selected
from the group consisting of the amino acid sequences shown in SEQ
ID NOs: 4 to 6; or (6) the light chain variable region comprises
CDR1 comprising the amino acid sequence shown in SEQ ID NO: 13,
CDR2 comprising the amino acid sequence shown in SEQ ID NO: 14 and
CDR3 comprising the amino acid sequence shown in SEQ ID NO: 15, and
the heavy chain variable region comprises CDR1 comprising the amino
acid sequence shown in SEQ ID NO: 16, CDR2 comprising the amino
acid sequence shown in SEQ ID NO: 17 and CDR3 comprising the amino
acid sequence shown in SEQ ID NO: 18, except that 1 to 3 amino
acids are substituted, deleted, inserted, and/or added in at least
one amino acid sequence selected from the group consisting of the
amino acid sequences shown in SEQ ID NOs: 13 to 15, and/or 1 to 3
amino acids are substituted, deleted, inserted, and/or added in at
least one amino acid sequence selected from the group consisting of
the amino acid sequences shown in SEQ ID NOs: 16 to 18. [7] The
antibody of [6], wherein (3') the light chain variable region
comprises the amino acid sequence shown in SEQ ID NO: 21, and the
heavy chain variable region comprises the amino acid sequence shown
by SEQ ID NO: 22; or (4') the light chain variable region comprises
the amino acid sequence shown in SEQ ID NO: 25, and the heavy chain
variable region comprises the amino acid sequence shown by SEQ ID
NO: 26. [8] The antibody of any of [1]-[7], which has a K.sub.D
value of not more than 5.times.10.sup.-8 M relating to binding
affinity to human CD69. [9] The antibody of any of [1]-[8], which
is a human antibody. [10] A pharmaceutical composition comprising
the antibody of any of [1]-[9]. [11] A prophylactic or therapeutic
agent for an allergic disease or inflammatory disease, comprising
the antibody of any of [1]-[9]. [12] A polynucleotide encoding the
antibody of any of [1]-[9]. [13] A vector comprising the
polynucleotide of [12]. [14] A transformant comprising the vector
of [13]. [15] A non-human mammal comprising transferred Th2 cells
of CD69 deficient non-human mammal immunized with a particular
antigen, wherein the Th2 cells express human CD69. [16] The
non-human mammal of [15], wherein the non-human mammal is a mouse.
[17] The antibody of any of [1]-[9], for use in the prophylaxis or
treatment of an allergic disease or inflammatory disease. [18] A
method for the prophylaxis or treatment of an allergic disease or
inflammatory disease in a mammal, comprising administering an
effective amount of the antibody of any of [1]-[9] to said mammal.
[19] Use of the antibody of any of [1]-[9] for producing an agent
for the prophylaxis or treatment of an allergic disease or
inflammatory disease.
Effect of the Invention
[0015] According to the present invention, an anti-human CD69
antibody applicable to the prophylaxis or treatment of allergic
diseases and inflammatory diseases is provided. According to the
present invention, moreover, an animal model permitting in vivo
evaluation of a pharmacological effect of an anti-human CD69
antibody can be provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows evaluation of binding to human CD69 and mouse
CD69 by cell staining.
[0017] FIG. 2 shows analysis of human CD69 expression by flow
cytometry.
[0018] FIG. 3 shows the effect of various anti-human CD69
antibodies on alveolar leukocyte infiltration.
[0019] FIG. 4 confirms cross-reactivity of various anti-human CD69
antibodies with mouse CD69 and human CD69 by flow cytometry.
[0020] FIG. 5 shows the effect of various anti-human CD69
antibodies against alveolar leukocyte infiltration.
[0021] FIG. 6 shows alignment of the amino acid sequences of mouse
CD69 (upper sequence) and human CD69 (lower sequence).
DESCRIPTION OF EMBODIMENTS
[0022] The present invention provides an antibody having a specific
binding activity to human CD69, and an activity to suppress
allergic inflammation.
[0023] CD69 is a known TYPE II membrane protein, and the amino acid
sequence thereof and the cDNA sequence thereof are also known. A
representative amino acid sequence of human CD69 is shown in SEQ ID
NO: 30, a representative cDNA sequence of human CD69 is shown in
SEQ ID NO: 29, a representative amino acid sequence of mouse CD69
is shown in SEQ ID NO: 32, and a representative cDNA sequence of
mouse CD69 is shown in SEQ ID NO: 31.
[0024] The antibody of the present invention has a specific binding
activity to an extracellular domain of human CD69. The
extracellular domain of human CD69 corresponds to the region of
62-199 in the amino acid sequence shown by SEQ ID NO: 30, and the
extracellular domain of mouse CD69 corresponds to the region of
62-199 in the amino acid sequence shown by SEQ ID NO: 32.
[0025] The "human CD69" means that the amino acid sequence or
nucleic acid sequence of CD69 has an amino acid sequence or nucleic
acid sequence which is the same as or substantially the same as the
amino acid sequence or nucleotide sequence of CD69 naturally
expressed in human. The "substantially the same" means that the
amino acid sequence or nucleic acid sequence of interest has 70% or
more (preferably 80% or more, more preferably 90% or more, further
preferably 95% or more, most preferably 99% or more) identity with
the amino acid sequence or nucleic acid sequence of a particular
CD69 naturally expressed in human, and has the function of the
particular human CD69. Biological species other than human,
proteins other than CD69, gene and fragments thereof are also
interpreted in the same manner.
[0026] The "specific binding" of an antibody to antigen X means
that the K.sub.D value of the binding affinity of an antibody to
antigen X in an antigen-antibody reaction is not more than
1.times.10.sup.-7 M.
[0027] In the present specification, the N.sub.D value relating to
the binding affinity of the antibody of the present invention to
human CD69 is calculated according to the principle described in
Immunoassays (OXFORD UNIVERSITY PRESS, 2000) using a scatchard plot
method. An antibody is incubated with various concentrations of
antigen (extracellular domain of human CD69) at room temperature
for 2 hr until equilibrium, and the amount of free antibody present
in the incubation solutions is measured by the ELISA method. The
binding constant and dissociation constant (K.sub.D value) are
determined based on the changes in the amount of free antibody in
each equilibrated sample. The antibody concentration during
equilibration reaction is to be 0.015 .mu.g/ml, and an ELISA plate
for the measurement of the amount of free antibody is to be
immobilized with the antigen at 1 .mu.g/ml.
[0028] In a preferable embodiment, the K.sub.D value relating to
the binding affinity of the antibody of the present invention to
human CD69 is not more than 5.times.10.sup.-8 M.
[0029] The antibody of the present invention has an activity to
suppress allergic inflammations. Allergic inflammation refers to
inflammations characterized by selective accumulation of
mononuclear cells in the target tissue, which occurs in association
with allergic reactions. Mononuclear cell encompasses Th2 cells,
eosinophils, basophils and mast cells. Whether or not an antibody
has an activity to suppress allergic inflammations can be confirmed
by evaluating whether or not it suppresses an allergic reaction
(e.g., leukocyte infiltration) induced by exposing the
below-mentioned non-human mammal of the present invention to an
antigen.
[0030] In the present specification, the "antibody" is used as one
encompassing a full-length antibody and any antigen-binding
fragment (i.e., "antigen-binding portion") thereof or a single
chain thereof. The "antibody" refers to a glycoprotein containing
at least two heavy chains (H) and two light chains (L), which are
linked by a disulfide bond, or an antigen-binding portion thereof.
Each heavy chain is constituted by a heavy chain variable region
(to be abbreviated as V.sub.H herein) and a heavy chain constant
region. The heavy chain constant region is constituted by 3 domains
of C.sub.H1, C.sub.H2 and C.sub.H3. Each light chain is constituted
by a light chain variable region (to be abbreviated as V.sub.L
herein) and a light chain constant region. The light chain constant
region is constituted by a single domain C.sub.L. V.sub.H and
V.sub.L regions are further subdivided into regions with higher
variability called complementarity determining regions (CDRs),
which contain more highly conservative regions called framework
regions (FRs) scattered therein. Each V.sub.H and V.sub.L is
constituted by 3 CDRs and 4 FRs, which are aligned in the following
order, i.e., FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino
terminus to the carboxy terminus. The variable regions of said
heavy chain and light chain contain binding domains that interact
with an antigen. The constant region of an antibody can mediate the
binding of immunoglobulin to host tissues or factors, including
various cells (e.g., effector cells) of the immune system and the
first component (C1q) of the conventional complement system.
[0031] In the present specification, the "antigen-binding portion"
of an antibody is used to refer to one or more fragments of an
antibody retaining an ability to specifically bind to an antigen
(e.g., human CD69). It has been clarified that the antigen binding
function of an antibody is performed by a fragment of a full-length
antibody. Examples of the binding fragment included in the term
"antigen binding portion" of an antibody include (i) Fab fragment,
a monovalent fragment constituted by V.sub.L, V.sub.H, C.sub.L and
C.sub.H1 domains, (ii) F(ab').sub.2 fragment, a divalent fragment
containing two Fab fragments linked by disulfide bond in the hinge
region, (iii) Fab' fragment, an inherent Fab having a hinge region
portion (see FUNDAMENTAL IMMUNOLOGY, Paul ed., 3. sup. rd ed.
1993), (iv) Fd fragment constituted by V.sub.H and C.sub.H1
domains, (v) Fv fragment constituted by V.sub.L and V.sub.H domains
in a single arm of an antibody, (vi) dAb fragment constituted by
V.sub.H domain (Ward et al., (1989) Nature 341:544-546), (vii)
isolated complementarity determining region (CDR) and (viii)
nanobody which is a heavy chain variable region containing single
variable domain and two constant regions. While V.sub.L and
V.sub.H, which are the two domains of Fv fragment, are encoded by
different genes, they can be linked by a synthetic linker to
produce a single protein chain from them by recombinant techniques,
wherein, in this chain, V.sub.L and V.sub.H regions pair with each
other to form a monovalent molecule (known as a single chain Fv
(scFv); see, for example, Bird et al. (1988) Science 242: 423-426;
and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:
5879-5883). Such single chain antibody is also encompassed in the
"antigen-binding portion" of an antibody. Such antibody fragments
are obtained by those of ordinary skill in the art by known
conventional techniques, and screened for usefulness in the same
manner as with unmodified antibody.
[0032] The antibody of the present invention is preferably a
monoclonal antibody. The "monoclonal antibody" refers to a
preparation of an antibody molecule of a single molecule
composition. The monoclonal antibody composition shows single
binding-specificity and affinity for a particular portion of an
antigen called epitope.
[0033] The antibody of the present invention is preferably a human
antibody. The "human antibody" refers to an antibody having
variable regions derived from a human germline immunoglobulin
sequence in both the framework and CDR regions. Furthermore, when
an antibody contains a constant region, the constant region also
derives from a human germline immunoglobulin sequence. In the
present specification, the "human antibody" also encompasses even
an embodiment including an amino acid residue not encoded by a
human germline immunoglobulin sequence (e.g., mutation introduced
by random or site-directed mutagenesis in vitro or somatic mutation
in vivo). In the present specification, however, the term of the
"human antibody" is not intended to include an antibody wherein a
CDR sequence derived from the germline of an animal species other
than human, such as mouse, is fused on the human framework
sequence.
[0034] In the present specification, the human antibody encompasses
a "reconstituted human antibody". The reconstituted human antibody
refers to a modified antibody wherein at least one CDR contained in
the first human donor antibody is used in the second human acceptor
antibody, instead of CDR of the second human acceptor antibody.
Preferably, all 6 CDRs are substituted. More preferably, the whole
antigen binding region (e.g., Fv, Fab or F(ab')2) of the first
human donor antibody is used instead of the corresponding region in
the second human acceptor antibody. More preferably, the Fab region
of the first human donor antibody is operably linked to an
appropriate constant region of the second human acceptor antibody
to form a full-length antibody.
[0035] The reconstituted human antibody can be produced by
conventional gene recombinant techniques disclosed in, for example,
EP125023, WO96/02576, non-patent document 8 and the like. To be
specific, for example, a DNA sequence designed to link a desired
CDR in a donor human antibody and a desired framework region (FR)
in an acceptor human antibody is synthesized by PCR method using,
as primers, several oligonucleotides produced to have a region
overlapping with the terminus regions of both CDR and FR (see the
method described in WO98/13388). The obtained DNA is linked to a
DNA encoding a human antibody constant region or a human antibody
constant region mutant, which is incorporated into a expression
vector and the vector is introduced into a host to allow for
production, whereby a reconstituted human antibody can be obtained
(see EP125023, WO96/02576).
[0036] In the present specification, moreover, the human antibody
encompasses an "artificial human antibody". The artificial human
antibody can be produced by conventional gene recombinant
techniques disclosed in, for example, non-patent document 8 and the
like.
[0037] The antibody of the present invention also includes a fusion
protein wherein the aforementioned antibody and other peptide or
protein are fused. The production method of a fusion protein
includes linking a polynucleotide encoding the antibody of the
present invention and a polynucleotide encoding other peptide or
polypeptide to match the frame, introducing same into an expression
vector, and allowing expression thereof in a host, and techniques
known to those of ordinary skill in the art can be used. As other
peptide to be fused with the antibody of the present invention,
known peptides such as FLAG (Hopp, T. P. et al., BioTechnology
(1988) 6, 1204-1210), 6.times.His consisting of six His (histidine)
residues, 10.times.His, human c-myc fragment, VSV-GP fragment,
p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment,
lck tag, .alpha.-tubulin fragment, B-tag, Protein C fragment and
the like can be used. Examples of other polypeptide to be fused
with the antibody of the present invention include GST
(glutathione-S-transferase), HA (influenza hemagglutinin),
immunoglobulin constant region, .beta.-galactosidase, MBP (maltose
binding protein) and the like. A commercially available
polynucleotide encoding such peptide or polypeptide is fused with a
polynucleotide encoding the antibody of the present invention, and
a fusion polynucleotide prepared thereby is expressed, whereby a
fusion polypeptide can be prepared.
[0038] The antibody of the present invention may be a conjugate
antibody bound with various molecules, for example, polymer
substances such as polyethylene glycol (PEG), hyaluronic acid and
the like, radioactive substance, fluorescent substance,
luminescence substance, enzyme, toxin and the like. Such conjugate
antibody can be obtained by chemically modifying the obtained
antibody. The modification method of antibody has already been
established in this field (e.g., U.S. Pat. No. 5,057,313, U.S. Pat.
No. 5,156,840).
[0039] The antibody of the present invention is preferably isolated
or purified. Being "isolated or purified" means that an operation
to remove components other than the component of interest has been
applied to the state of natural presence. The purity of the
isolated or purified antibody of the present invention (ratio of
the weight of the antibody of the present invention to the total
protein weight) is generally 50% or more, preferably 70% or more,
more preferably 90% or more, most preferably 95% or more (e.g.,
substantially 100%).
[0040] In one embodiment, the antibody of the present invention has
cross-reactivity with mouse CD69 (preferably extracellular domain
of mouse CD69). The "cross-reactivity" means that an antibody that
specifically binds to human CD69 also binds to mouse CD69
(preferably extracellular domain of mouse CD69) by antigen-antibody
reaction. The antibody of the present invention having
cross-reactivity with mouse CD69 is superior in that it can
evaluate efficacy even in mouse not expressing human CD69.
[0041] In a preferable embodiment, the antibody of the present
invention having cross-reactivity with mouse CD69 binds to human
CD69 at an epitope containing the amino acid sequence shown by SEQ
ID NO: 33 (YNCPG). The epitope containing the amino acid sequence
shown in SEQ ID NO: 33 includes, for example, an epitope consisting
of a continuous partial sequence of the amino acid sequence shown
in SEQ ID NO: 30, which contains the amino acid sequence shown in
SEQ ID NO: 33, and has an amino acid length of 12 or less. As the
epitope containing the amino acid sequence shown by SEQ ID NO: 33,
specifically, an epitope consisting of the amino acid sequence
shown by SEQ ID NO: 35, and an epitope consisting of the amino acid
sequence shown by SEQ ID NO: 36 can be mentioned.
[0042] As the antibody of the present invention having
cross-reactivity with mouse CD69, the antibodies described in the
following (1) and (2) can be mentioned:
(1) an antibody comprising a light chain variable region and a
heavy chain variable region, wherein the light chain variable
region comprises CDR1 comprising the amino acid sequence shown in
SEQ ID NO: 7, CDR2 comprising the amino acid sequence shown in SEQ
ID NO: 8 and CDR3 comprising the amino acid sequence shown in SEQ
ID NO: 9, 19 or 20, and the heavy chain variable region comprises
CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10,
CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11 and
CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12; and
(2) an antibody comprising a light chain variable region and a
heavy chain variable region, wherein the light chain variable
region comprises CDR1 comprising the amino acid sequence shown in
SEQ ID NO: 7, CDR2 comprising the amino acid sequence shown in SEQ
ID NO: 8 and CDR3 comprising the amino acid sequence shown in SEQ
ID NO: 9, 19 or 20, and the heavy chain variable region comprises
CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10,
CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11 and
CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12
except that 1 to 3 amino acids are substituted, deleted, inserted,
and/or added in at least one amino acid sequence selected from the
group consisting of the amino acid sequences shown in SEQ ID NOs:
7-9, 19 and 20, and/or 1 to 3 amino acids are substituted, deleted,
inserted, and/or added in at least one amino acid sequence selected
from the group consisting of the amino acid sequences shown in SEQ
ID NOs: 10-12.
[0043] The antibody described in the above-mentioned (1) or (2) can
bind to human CD69 at an epitope comprising the amino acid sequence
shown by SEQ ID NO: 33 (preferably, an epitope consisting of a
continuous partial sequence of the amino acid sequence shown in SEQ
ID NO: 30, which contains the amino acid sequence shown in SEQ ID
NO: 33, and has an amino acid length of 12 or less; more
preferably, epitope consisting of the amino acid sequence shown by
SEQ ID NO: 35 or SEQ ID NO: 36).
[0044] In one embodiment, the antibody of the present invention
binds to human CD69 at an epitope consisting of the amino acid
sequence shown by SEQ ID NO: 59.
[0045] In one embodiment, the antibody of the present invention
binds to human CD69 at an epitope containing the amino acid
sequence shown by SEQ ID NO: 78 (YAGREE). The epitope containing
the amino acid sequence shown in SEQ ID NO: 78 includes, for
example, an epitope consisting of a continuous partial sequence of
the amino acid sequence shown in SEQ ID NO: 30, which contains the
amino acid sequence shown in SEQ ID NO: 78, and has an amino acid
length of 12 or less. As the epitope containing the amino acid
sequence shown by SEQ ID NO: 78, specifically, an epitope
consisting of the amino acid sequence shown by SEQ ID NO: 57, an
epitope consisting of the amino acid sequence shown by SEQ ID NO:
58 and an epitope consisting of the amino acid sequence shown by
SEQ ID NO: 59 can be mentioned.
[0046] As other antibody of the present invention, the antibodies
described in the following (3)-(6) can be mentioned:
(3) an antibody comprising a light chain variable region and a
heavy chain variable region, wherein the light chain variable
region comprises CDR1 comprising the amino acid sequence shown in
SEQ ID NO: 1, CDR2 comprising the amino acid sequence shown in SEQ
ID NO: 2 and CDR3 comprising the amino acid sequence shown in SEQ
ID NO: 3, and the heavy chain variable region comprises CDR1
comprising the amino acid sequence shown in SEQ ID NO: 4, CDR2
comprising the amino acid sequence shown in SEQ ID NO: 5 and CDR3
comprising the amino acid sequence shown in SEQ ID NO: 6; (4) an
antibody comprising a light chain variable region and a heavy chain
variable region, wherein the light chain variable region comprises
CDR1 comprising the amino acid sequence shown in SEQ ID NO: 13,
CDR2 comprising the amino acid sequence shown in SEQ ID NO: 14 and
CDR3 comprising the amino acid sequence shown in SEQ ID NO: 15, and
the heavy chain variable region comprises CDR1 comprising the amino
acid sequence shown in SEQ ID NO: 16, CDR2 comprising the amino
acid sequence shown in SEQ ID NO: 17 and CDR3 comprising the amino
acid sequence shown in SEQ ID NO: 18; (5) an antibody comprising a
light chain variable region and a heavy chain variable region,
wherein the light chain variable region comprises CDR1 comprising
the amino acid sequence shown in SEQ ID NO: 1, CDR2 comprising the
amino acid sequence shown in SEQ ID NO: 2 and CDR3 comprising the
amino acid sequence shown in SEQ ID NO: 3, and the heavy chain
variable region comprises CDR1 comprising the amino acid sequence
shown in SEQ ID NO: 4, CDR2 comprising the amino acid sequence
shown in SEQ ID NO: 5 and CDR3 comprising the amino acid sequence
shown in SEQ ID NO: 6, except that 1 to 3 amino acids are
substituted, deleted, inserted, and/or added in at least one amino
acid sequence selected from the group consisting of the amino acid
sequences shown in SEQ ID NOs: 1 to 3, and/or 1 to 3 amino acids
are substituted, deleted, inserted, and/or added in at least one
amino acid sequence selected from the group consisting of the amino
acid sequences shown in SEQ ID NOs: 4 to 6; and (6) an antibody
comprising a light chain variable region and a heavy chain variable
region, wherein the light chain variable region comprises CDR1
comprising the amino acid sequence shown in SEQ ID NO: 13, CDR2
comprising the amino acid sequence shown in SEQ ID NO: 14 and CDR3
comprising the amino acid sequence shown in SEQ ID NO: 15, and the
heavy chain variable region comprises CDR1 comprising the amino
acid sequence shown in SEQ ID NO: 16, CDR2 comprising the amino
acid sequence shown in SEQ ID NO: 17 and CDR3 comprising the amino
acid sequence shown in SEQ ID NO: 18, except that 1 to 3 amino
acids are substituted, deleted, inserted, and/or added in at least
one amino acid sequence selected from the group consisting of the
amino acid sequences shown in SEQ ID NOs: 13 to 15, and/or 1 to 3
amino acids are substituted, deleted, inserted, and/or added in at
least one amino acid sequence selected from the group consisting of
the amino acid sequences shown in SEQ ID NOs: 16 to 18.
[0047] The antibody described in the above-mentioned (3) or (5) can
bind to human CD69 at an epitope consisting of the amino acid
sequence shown by SEQ ID NO: 59.
[0048] The antibody described in the above-mentioned (4) or (6) can
bind to human CD69 at an epitope consisting of the amino acid
sequence shown by SEQ ID NO: 78 (preferably, an epitope consisting
of a continuous partial sequence of the amino acid sequence shown
in SEQ ID NO: 30, which contains the amino acid sequence shown in
SEQ ID NO: 78, and has an amino acid length of 12 or less; more
preferably, an epitope consisting of the amino acid sequence shown
by SEQ ID NO: 57, SEQ ID NO: 58 or SEQ ID NO: 59).
[0049] The K.sub.D value of the antibody described in (1) relating
to the binding affinity to human CD69 is preferably not more than
3.times.10.sup.-8 M. When CDR3 in the light chain variable region
has the amino acid sequence shown by SEQ ID NO: 19, the K.sub.D
value of the antibody relating to the binding affinity to human
CD69 is preferably not more than 1.times.10.sup.-8 M, more
preferably not more than 5.times.10.sup.-9 M, further preferably
not more than 2.times.10.sup.-9 M. When CDR3 in the light chain
variable region has the amino acid sequence shown by SEQ ID NO: 20,
the K.sub.D value of the antibody relating to the binding affinity
to human CD69 is preferably not more than 1.times.10.sup.-8 M, more
preferably not more than 5.times.10.sup.-9 M, further preferably
not more than 3.times.10.sup.-9 M.
[0050] The K.sub.D value of the antibody described in (2) relating
to the binding affinity to human CD69 is preferably not more than
3.times.10.sup.-8 M.
[0051] When the antibody described in (2) is an antibody comprising
a light chain variable region and a heavy chain variable region,
wherein
the light chain variable region comprises CDR1 comprising the amino
acid sequence shown in SEQ ID NO: 7, CDR2 comprising the amino acid
sequence shown in SEQ ID NO: 8 and CDR3 comprising the amino acid
sequence shown in SEQ ID NO: 19, and the heavy chain variable
region comprises CDR1 comprising the amino acid sequence shown in
SEQ ID NO: 10, CDR2 comprising the amino acid sequence shown in SEQ
ID NO: 11 and CDR3 comprising the amino acid sequence shown in SEQ
ID NO: 12, except that 1 to 3 amino acids are substituted, deleted,
inserted, and/or added in at least one amino acid sequence selected
from the group consisting of the amino acid sequences shown in SEQ
ID NOs: 7, 8 and 19, and/or 1 to 3 amino acids are substituted,
deleted, inserted, and/or added in at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 10 to 12, the
K.sub.D value of the antibody relating to the binding affinity to
human CD69 is preferably not more than 1.times.10.sup.-8 M, more
preferably not more than 5.times.10.sup.-9 M, further preferably
not more than 2.times.10.sup.-9 M.
[0052] When the antibody described in (2) is an antibody comprising
a light chain variable region and a heavy chain variable region,
wherein
the light chain variable region comprises CDR1 comprising the amino
acid sequence shown in SEQ ID NO: 7, CDR2 comprising the amino acid
sequence shown in SEQ ID NO: 8 and CDR3 comprising the amino acid
sequence shown in SEQ ID NO: 20, and the heavy chain variable
region comprises CDR1 comprising the amino acid sequence shown in
SEQ ID NO: 10, CDR2 comprising the amino acid sequence shown in SEQ
ID NO: 11 and CDR3 comprising the amino acid sequence shown in SEQ
ID NO: 12, except that 1 to 3 amino acids are substituted, deleted,
inserted, and/or added in at least one amino acid sequence selected
from the group consisting of the amino acid sequences shown in SEQ
ID NOs: 7, 8 and 20, and/or 1 to 3 amino acids are substituted,
deleted, inserted, and/or added in at least one amino acid sequence
selected from the group consisting of the amino acid sequences SEQ
ID NOs: 10-12, the K.sub.D value relating to the binding affinity
of the antibody to human CD69 is preferably not more than
1.times.10.sup.-8 M, more preferably not more than
5.times.10.sup.-9 M, further preferably not more than
3.times.10.sup.-9 M.
[0053] The K.sub.D value of the antibody described in (3) or (5)
relating to the binding affinity to human CD69 is preferably not
more than 5.times.10.sup.-8 M.
[0054] The K.sub.D value of the antibody described in (4) or (6)
relating to the binding affinity to human CD69 is preferably not
more than 8.times.10.sup.-9 M.
[0055] In the embodiments of (2), (5) and (6), the number of amino
acids to be substituted, deleted, inserted and/or added is not
particularly limited as long as the antibody specifically binds to
human CD69, and has an activity to suppress allergic inflammation.
It is preferably within 2 amino acids, more preferably one amino
acid, per one CDR sequence. While the number of CDR sequences in
which amino acid is substituted, deleted, inserted and/or added is
not particularly limited as long as the antibody specifically binds
to human CD69, and has an activity to suppress allergic
inflammation. It is preferably within 2, more preferably one, per
one light chain variable region, and preferably within 2, more
preferably 1, per one heavy chain variable region. The
substitution, deletion, insertion and/or addition of amino acid may
be performed in both the light chain variable region and the heavy
chain variable region, or either one of them.
[0056] In the embodiments of (2), (5) and (6), 1-3 (preferably 1 or
2, more preferably 1) amino acids are preferably substituted,
deleted, inserted, and/or added only in the amino acid sequence of
CDR3 in the light chain variable region.
[0057] In the embodiment of (2), when 1 to 3 amino acids are
substituted, deleted, inserted and/or added in the amino acid
sequence of CDR3 in the light chain variable region, it is
preferable to maintain serine of the 2nd and tyrosine of the 3rd of
the amino acid sequence of the CDR3. The first amino acid of the
amino acid sequence of the CDR3 is mutually substitutable between
glutamine and glycine. The 4th amino acid of the amino acid
sequence of the CDR3 is mutually substitutable between aspartic
acid and threonine. The 5th amino acid of the amino acid sequence
of the CDR3 is mutually substitutable between serine and
threonine.
[0058] Examples of the method for substituting one or plural amino
acid residues with other desired amino acid include site-directed
mutagenesis method (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y,
and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual
amber method for site-directed mutagenesis. Gene 152, 271-275;
Zoller, M J, and Smith, M. (1983) Oligonucleotide-directed
mutagenesis of DNA fragments cloned into M13 vectors. Methods
Enzymol. 100, 468-500; Kramer, W, Drutsa, V, Jansen, H W, Kramer,
B, Pflugfelder, M, and Fritz, H J (1984) The gapped duplex DNA
approach to oligonucleotide-directed mutation construction. Nucleic
Acids Res. 12, 9441-9456; Kramer W, and Fritz H J (1987)
Oligonucleotide-directed construction of mutations via gapped
duplex DNA Methods. Enzymol. 154, 350-367, Kunkel, T A (1985) Rapid
and efficient site-specific mutagenesis without phenotypic
selection. Proc Natl Acad Sci USA. 82, 488-492). Using these
methods, desired amino acid in an antibody can be substituted by
other amino acid of interest. Also, using the library technique
such as framework shuffling (Mol Immunol. 2007 April;
44(11):3049-60) and CDR repair (US2006/0122377) and the like, an
amino acid in a framework or CDR can also be substituted by other
appropriate amino acid.
[0059] In the antibody of the present invention, as a framework
region (FR) of the antibody to be linked to a CDR, a framework
which enables the CDR to form a good antigen binding site is
selected. While FR to be used for the antibody of the present
invention is not particularly limited and any FR can be used, FR of
a human antibody is preferably used. As the FR of a human antibody,
one having a natural sequence may be used, or one or plural amino
acids in the framework region having a natural sequence may be
substituted, deleted, added and/or inserted and the like as
necessary, so that CDR will form an appropriate antigen binding
site. For example, a mutant FR sequence having desired properties
can be selected by measuring and evaluating the binding activity of
an antibody having FR with substituted amino acid to an antigen
(Sato, K. et al., Cancer Res. (1993)53, 851-856).
[0060] In the antibodies of (1) and (2), FR of V13 (Kabat database)
of human antibody is preferably used for the light chain, and FR of
VH3 (Kabat database) of human antibody is preferably used for the
heavy chain.
[0061] In the antibodies of (3) and (5), FR of Vk1 (Kabat database)
of human antibody is preferably used for the light chain, and FR of
VH1B (Kabat database) of human antibody is preferably used for the
heavy chain.
[0062] In the antibodies of (4) and (6), FR of Vk3 (Kabat database)
of human antibody is preferably used for the light chain, and FR of
VH3 (Kabat database) of human antibody is preferably used for the
heavy chain.
[0063] The constant region used for the antibody of the present
invention is not particularly limited, and any constant region may
be used. Preferable examples of the constant region used for the
antibody of the present invention include constant regions of human
antibody (constant regions derived from IgG1, IgG2, IgG3, IgG4,
IgA, IgM and the like). For example, C.gamma.1, C.gamma.2,
C.gamma.3, C.gamma.4, C.mu., C.delta., C.alpha.1, C.alpha.2,
C.epsilon. can be used in H chain, and C.kappa., C.lamda. can be
used in L chain.
[0064] In the antibodies of (1) and (2), the constant region of
C.lamda. of human antibody is preferably used for the light chain,
and the constant region of C.gamma.4 of human antibody is
preferably used for the heavy chain.
[0065] In the antibodies of (3) and (5), the constant region of
C.kappa. of human antibody is preferably used for the light chain,
and the constant region of C.gamma.4 of human antibody is
preferably used for the heavy chain.
[0066] In the antibodies of (4) and (6), the constant region of
C.kappa. of human antibody is preferably used for the light chain,
and the constant region of C.gamma.4 of human antibody is
preferably used for the heavy chain.
[0067] Preferable antibody of the present invention includes the
following:
(1') An antibody comprising a light chain variable region and a
heavy chain variable region, wherein the light chain variable
region comprises the amino acid sequence shown in SEQ ID NO: 23, 27
or 28 and the heavy chain variable region comprises the amino acid
sequence shown in SEQ ID NO: 24; (3') an antibody comprising a
light chain variable region and a heavy chain variable region,
wherein the light chain variable region comprises the amino acid
sequence shown in SEQ ID NO: 21 and the heavy chain variable region
comprises the amino acid sequence shown in SEQ ID NO: 22; and (4')
an antibody comprising a light chain variable region and a heavy
chain variable region, wherein the light chain variable region
comprises the amino acid sequence shown in SEQ ID NO: 25 and the
heavy chain variable region comprises the amino acid sequence shown
in SEQ ID NO: 26.
[0068] The antibody of the above-mentioned (1') corresponds to a
preferable embodiment of the antibody of the above-mentioned (1),
the antibody of the above-mentioned (3') corresponds to a
preferable embodiment of the antibody of the above-mentioned (3),
and the antibody of the above-mentioned (4') corresponds to a
preferable embodiment of the antibody of the above-mentioned (4),
respectively.
[0069] The antibody described in the above-mentioned (1') can bind
to human CD69 at an epitope comprising the amino acid sequence
shown by SEQ ID NO: 33 (preferably, an epitope consisting of a
continuous partial sequence of the amino acid sequence shown in SEQ
ID NO: 30, which contains the amino acid sequence shown in SEQ ID
NO: 33, and has an amino acid length of 12 or less; more
preferably, epitope consisting of the amino acid sequence shown by
SEQ ID NO: 35 or SEQ ID NO: 36).
[0070] The antibody described in the above-mentioned (3') can bind
to human CD69 at an epitope consisting of the amino acid sequence
shown by SEQ ID NO: 59.
[0071] The antibody described in the above-mentioned (4') can bind
to human CD69 at an epitope comprising the amino acid sequence
shown by SEQ ID NO: 78 (preferably, an epitope consisting of a
continuous partial sequence of the amino acid sequence shown in SEQ
ID NO: 30, which contains the amino acid sequence shown in SEQ ID
NO: 78, and has an amino acid length of 12 or less; more
preferably, an epitope consisting of the amino acid sequence shown
by SEQ ID NO: 57, SEQ ID NO: 58 or SEQ ID NO: 59).
[0072] The present invention provides a polynucleotide containing a
nucleotide sequence encoding the above-mentioned antibody of the
present invention. The polynucleotide may be a DNA or RNA, or a
DNA/RNA chimera. The polynucleotide may be double stranded or
single stranded. When the polynucleotide is double stranded, it may
be a double stranded DNA, a double stranded RNA or a DNA:RNA
hybrid.
[0073] The polynucleotide of the present invention encompasses a
polynucleotide containing a nucleotide sequence encoding both the
heavy chain variable region and the light chain variable region of
the antibody of the present invention, and a combination of a
polynucleotide containing a nucleotide sequence encoding the heavy
chain variable region of the antibody of the present invention and
a polynucleotide containing a nucleotide sequence encoding the
light chain variable region of the antibody of the present
invention.
[0074] The polynucleotide of the present invention can be easily
produced based on the information of the amino acid sequence of the
antibody of the present invention, known sequence information and
sequence information described in the Sequence Listing in the
present specification, and by utilizing known gene recombination
techniques. For example, suitable primers are designed based on the
sequence information, a DNA encoding the elements constituting the
antibody of the present invention is amplified by the PCR reaction,
DNA fragments are ligated by appropriate enzymes such as ligase and
the like, whereby the polynucleotide of the present invention can
be produced. Alternatively, a polynucleotide encoding each element
may be synthesized by a polynucleotide synthesizer, based on the
information of the amino acid sequence of the antibody of the
present invention.
[0075] The obtained polynucleotide encoding the antibody of the
present invention may be, depending on the object, directly used,
or used after digestion with a restriction enzyme when desired, or
addition of a linker. The polynucleotide may have ATG as a
translation initiation codon on the 5' terminal side, and may have
TAA, TGA or TAG as a translation stop codon on the 3' terminal
side. These translation initiation codon and translation stop codon
can be added using a suitable synthesized DNA adapter.
[0076] The polynucleotide of the present invention is preferably
isolated or purified. The isolated or purified polynucleotide of
the present invention has a purity (ratio of the weight of the
polynucleotide of the present invention to the total polynucleotide
weight) of generally 50% or more, preferably 70% or more, more
preferably 90% or more, most preferably 95% or more (e.g.,
substantially 100%).
[0077] The present invention provides a vector comprising the
above-mentioned polynucleotide of the present invention. The vector
of the present invention encompasses a vector comprising a
polynucleotide comprising a nucleotide sequence encoding both the
heavy chain variable region and the light chain variable region of
the antibody of the present invention, and a combination of a
vector comprising a polynucleotide comprising a nucleotide sequence
encoding the heavy chain variable region of the antibody of the
present invention and a vector comprising a polynucleotide
comprising a nucleotide sequence encoding the light chain variable
region of the antibody of the present invention. The vector is
preferably isolated or purified. Examples of the vector include
expression vector, cloning vector and the like, which can be
selected according to the object. Preferably, the vector is an
expression vector. The expression vector can express the antibody
of the present invention. The expression vector can be produced by
operably linking the polynucleotide of the present invention to the
downstream of a promoter in a suitable expression vector. The kind
of the vector includes, for example, plasmid vector, virus vector
and the like, which can be appropriately selected according to the
host to be used.
[0078] As the host, the genus Escherichia (Escherichia coli etc.),
the genus Bacillus (Bacillus subtilis etc.), yeast (Saccharomyces
cerevisiae etc.), insect cell (established cell line derived from
larva of Mamestra brassicae (Spodoptera frugiperda cell; Sfcell)
etc.), insect (larva of Bombyx mori etc.), mammalian cells (rat
nerve cell, monkey cell (COS-7 etc.), Chinese hamster cell (CHO
cell etc.) etc.) and the like are used.
[0079] Examples of the mammal include, but are not limited to,
experiment animals such as rodents such as mouse, rat, hamster and
guinea pig and the like, rabbit and the like, domestic animals such
as swine, bovine, goat, horse, sheep, mink and the like, companion
animals such as dog, cat and the like, primates such as human,
monkey, Macaca fascicularis, Macaca mulatta, marmoset, orangutan,
chimpanzee and the like, and the like.
[0080] Examples of the plasmid vector include plasmid vectors
derived from Escherichia coli (e.g., pBR322, pBR325, pUC12, pUC13),
plasmid vectors derived from Bacillus subtilis (e.g., pUB110, pTP5,
pC194), plasmid vectors derived from yeast (e.g., pSH19, pSH15) and
the like, which can be appropriately selected according to the kind
of the host to be used and the object of use.
[0081] The kind of the virus vector can be appropriately selected
according to the kind of the host to be used and object of use. For
example, when an insect cell is used as a host, baculovirus vector
and the like can be used. When a mammalian cell is used as a host,
retrovirus vectors such as moloney murine leukemia virus vector,
lentivirus vector, sindbis virus vector and the like, adenovirus
vector, herpes virus vector, adeno-associated virus vector,
parvovirus vector, vaccinia virus vector, sendai virus vector and
the like can be used.
[0082] The promoter can be selected according to the kind of the
host to be used, and one capable of initiating transcription in the
host can be selected. For example, when the host is the genus
Escherichia, trp promoter, lac promoter, T7 promoter and the like
are preferable. When the host is the genus Bacillus, SPO1 promoter,
SPO2 promoter, penP promoter and the like are preferable. When the
host is yeast, PHO5 promoter, PGK promoter and the like are
preferable. When the host is an insect cell, polyhedrin promoter,
P10 promoter and the like are preferable. When the host is a
mammalian cell, subgenomic (26S) promoter, CMV promoter, SR.alpha.
promoter and the like are preferable.
[0083] The vector of the present invention may contain a signal
sequence for antibody secretion. As the signal sequence for
antibody secretion when it is produced in the periplasm of
Escherichia coli, pelB signal sequence (Lei, S. P. et al J.
Bacteriol. (1987) 169, 4379) may be used.
[0084] When desired, the vector of the present invention may
contain enhancer, splicing signal, polyA addition signal, selection
marker, SV40 replication origin (hereinafter iv sometimes to be
abbreviated as SV40ori) and the like each in an operable manner.
Examples of the selection marker include dihydrofolate reductase
(hereinafter sometimes to be abbreviated as dhfr) gene
[methotrexate (MTX) resistance], ampicillin resistance gene
(sometimes to be abbreviated as Amp.sup.r), neomycin resistance
gene (sometimes to be abbreviated as Neo.sup.r, G418 resistance)
and the like.
[0085] By introducing the above-mentioned vector of the present
invention into the above-mentioned host by gene transfer methods
known per se (e.g., lipofection method, calcium phosphate method,
microinjection method, proplast fusion method, electroporation
method, DEAE dextran method, gene transfer method by Gene Gun
etc.), a transformant with the vector introduced thereinto
(transformant of the present invention) can be produced. When an
expression vector is used as the vector to be introduced, the
transformant can express the antibody of the present invention. The
transformant of the present invention is useful for the production
of the antibody of the present invention and the like.
[0086] The antibody of the present invention can be produced by
culturing the transformant of the present invention by a method
known per se according to the kind of the host, and isolating the
antibody of the present invention from the culture. When the host
is the genus Escherichia, the transformant is cultured in an
appropriate medium such as LB medium, M9 medium and the like at
generally about 15-43.degree. C. for about 3-24 hr. When the host
is the genus Bacillus, the transformant is cultured in an
appropriate medium generally at about 30-40.degree. C. for about
6-24 hr. When the host is yeast, the transformant is cultured in an
appropriate medium such as Burkholder's medium and the like
generally at about 20.degree. C.-35.degree. C. for about 24-72 hr.
When the host is an insect cell or insect, the transformant is
cultured in an appropriate medium such as Grace's Insect medium
added with about 10% of bovine serum and the like generally at
about 27.degree. C. for about 3-5 days. When the host is an animal
cell, the transformant is cultured in an appropriate medium such as
MEM medium added with about 10% of bovine serum and the like
generally at about 30.degree. C.-40.degree. C. for about 15-60 hr.
In any culture, aeration and stirring may be performed as
necessary.
[0087] As for the production method of antibody by genetic
engineering, for example, Co, M. S. et al., J. Immunol. (1994) 152,
2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol. (1989)
178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989)
178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663;
Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird,
R. E. and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137 and
the like can be referred to.
[0088] The separation and purification of the antibody of the
present invention from a culture is not limited in any manner, and
the separation and purification methods generally used for
purification of antibody can be employed. For example, antibody can
be separated and purified by appropriately selecting and combining
chromatography column, filter, ultrafiltration, salting out,
solvent precipitation, solvent extraction, distillation,
immunoprecipitation, SDS-polyacrylamide gel electrophoresis,
isoelectric focusing, dialysis, recrystallization and the like.
[0089] Examples of the chromatography include affinity
chromatography, ion exchange chromatography, hydrophobic
chromatography, gelfiltration, reversed-phase chromatography,
adsorption chromatography and the like (Strategies for Protein
Purification and Characterization: A Laboratory Course Manual. Ed
Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press,
1996). These chromatographys can be performed by using liquid phase
chromatography, for example, liquid phase chromatography such as
HPLC, FPLC and the like. Examples of the column to be used for
affinity chromatography include protein A column and protein G
column. For example, as a column using protein A, Hyper D, POROS,
Sepharose FF (manufactured by GE Amersham Biosciences) and the like
can be mentioned. The present invention also encompasses an
antibody highly purified by these purification methods.
[0090] In addition, the present invention provides a pharmaceutical
composition containing the above-mentioned antibody of the present
invention as an active ingredient. The pharmaceutical composition
of the present invention can also be used for the prophylaxis or
treatment of allergic diseases and inflammatory diseases involving
human CD69. That is, the present invention also provides a
prophylactic agent or a therapeutic agent for allergic diseases or
inflammatory diseases, which contains the aforementioned antibody
as an active ingredient. Examples of the allergic disease include,
but are not limited to, allergic asthma, allergic rhinitis,
pollinosis, atopic dermatitis, urticaria, food allergy, allergic
conjunctivitis, and the like. Examples of the inflammatory disease
include, but are not limited to, chronic obstructive pulmonary
disease (COPD), emphysema, bronchitis, interstitial pneumonia, lung
fibrosis, lung edema, adult respiratory distress syndrome,
rheumatoid arthritis, septic shock, ulcerative colitis, Crohn's
disease, reperfusion damage, chronic glomerulonephritis, endotoxin
shock, osteoarthritis, multiple sclerosis and the like. The
above-mentioned allergic disease or inflammatory disease is
preferably an allergic disease or inflammatory disease in the
respiratory organs (lung, bronchus, airway etc.). Examples of the
allergic disease in the respiratory organs (lung, bronchus, airway
etc.) include, but are not limited to, allergic asthma and the
like. Examples of the inflammatory disease in the respiratory
organs (lung, bronchus, airway etc.) include, but are not limited
to, chronic obstructive pulmonary disease (COPD), emphysema,
bronchitis, interstitial pneumonia, lung fibrosis, lung edema,
adult respiratory distress syndrome and the like.
[0091] When the antibody of the present invention is "contained as
an active ingredient", it means that the antibody of the present
invention is contained as at least one of the active ingredients,
and does not limit the content thereof. The pharmaceutical
composition of the present invention may contain other active
ingredient(s) together with the antibody of the present
invention.
[0092] The antibody of the present invention can be formulated
according to a conventional method (e.g., Remington's
Pharmaceutical Science, latest edition, Mark Publishing Company,
Easton, U.S.A). Where necessary, moreover, it may contain a
pharmaceutically acceptable carrier and/or additive. For example,
it can contain surfactant (PEG, Tween etc.), excipient, antioxidant
(ascorbic acid etc.), colorant, flavor, preservative, stabilizer,
buffering agent (phosphate, citrate, other organic acid etc.),
chelating agent (EDTA etc.), suspending agent, isotonizing agent,
binder, disintegrant, lubricant, glidant, corrigent and the like.
Not being limited to these, the pharmaceutical composition of the
present invention may contain other conventional carriers as
appropriate. Specific examples include light anhydrous silicic
acid, lactose, crystalline cellulose, mannitol, starch, carmellose
calcium, carmellose sodium, hydroxypropylcellulose,
hydroxypropylmethylcellulose, polyvinyl acetaldiethylaminoacetate,
polyvinylpyrrolidone, gelatin, medium-chain fatty acid
triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose,
carboxymethylcellulose, cornstarch, inorganic salts and the like.
It may also contain other low-molecular-weight polypeptide, serum
albumin, gelatin and protein such as immunoglobulin and the like,
as well as amino acid. When an aqueous solution for injection is
formulated, the antibody of the present invention is dissolved in,
for example, isotonic solution containing saline, glucose or other
auxiliary agent. Examples of the auxiliary agent include
D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and may be
used in combination with suitable solubilizing agents, for example,
alcohol (ethanol etc.), polyalcohol (propylene glycol, PEG etc.),
non-ionic surfactant (polysorbate 80, HCO-50) and the like.
[0093] Where necessary, polypeptide may also be included in a
microcapsule (microcapsules made of hydroxymethylcellulose,
gelatin, poly[methylmethacrylate] and the like), or formulated as a
colloid drug delivery system (liposome, albumin microsphere,
microemulsion, nanoparticles and nanocapsule etc.) (see Remington's
Pharmaceutical Science 16th edition &, Oslo Ed. (1980) etc.).
Furthermore, a method of formulating a drug as a sustained-release
medicament is also known, and applicable to polypeptide (Langer et
al., J. Biomed. Mater. Res. (1981)15: 167-277; Langer, Chem. Tech.
(1982)12: 98-105; U.S. Pat. No. 3,773,919; EP-A-58,481; Sidman et
al., Biopolymers (1983) 22: 547-56; EP No. 133,988). Furthermore,
it is also possible to increase the liquid amount to be
subcutaneously administered by adding or blending hyaluronidase to
or with the present agent (e.g., WO 2004/078140 etc.).
[0094] The content of the antibody of the present invention in a
pharmaceutical composition is, for example, about 0.01-100 wt %,
preferably 0.1-99.9 wt %, of the whole pharmaceutical
composition.
[0095] While the pharmaceutical composition of the present
invention can be administered both orally and parenterally, it is
preferably administered parenterally. Specifically, it is
administered to patients by injection or transdermal
administration. As an example of the dosage form of injection, it
can be administered systemically or topically by intravenously
injection, intramuscular injection, subcutaneous injection and the
like. It may also be administered to the treatment site or in the
vicinity thereof by topical injection, particularly intramuscular
injection. Examples of the dosage form of transdermal
administration include ointment, gel, cream, plaster, patch and the
like, which can be administered systemically or topically. In
addition, the administration method can be appropriately selected
according to the age and symptom of the patients. The dose can be
selected from, for example, the range of 0.5 mg-2.5 mg/kg body
weight as the antibody of the present invention. However, the
pharmaceutical composition of the present invention is not limited
by these doses.
[0096] The present invention provides a non-human mammal useful for
the analysis of the function of human CD69 in allergic diseases,
inflammatory diseases and the like. Specifically, the present
invention provides a non-human mammal comprising a transferred Th2
cells of CD69 deficient non-human mammal immunized with a
particular antigen, which express human CD69.
[0097] Examples of the non-human mammal include experiment animals
such as mouse, rat, hamster, guinea pig, rabbit and the like,
domestic animals such as swine, bovine, goat, horse, sheep and the
like, pets such as dog, cat and the like, primates such as monkey,
orangutan, chimpanzee and the like. The non-human mammal is
preferably mouse.
[0098] The genotype of the CD69 deficient non-human mammal, from
which the Th2 cells are derived, may be immunologically self (i.e.,
syngenic) or immunologically nonself (i.e., allogeneic or
xenogeneic) to the animal species of the non-human mammal of the
present invention. Since the transferred Th2 cells are engrafted in
the body for a long term, it is preferably immunologically
self.
[0099] Being CD69 deficient refers to the state where sufficient
operation of the normal function that CD69 gene intrinsically has
is prevented. Examples thereof include a complete absence of the
expression of CD69 gene, a decreased expression level at which the
normal function that CD69 gene intrinsically has cannot be
exhibited sufficiently, complete loss of the function of CD69 gene
product, and a decreased function of CD69 gene product at which the
normal function that CD69 gene intrinsically has cannot be
exhibited.
[0100] The CD69 deficient non-human mammal is preferably an animal
accompanying modification of genome DNA, i.e., transgenic animal.
The CD69 deficient non-human mammal may be CD69 gene deficient
heterozygote, or CD69 gene deficient homozygote, preferably CD69
gene deficient homozygote.
[0101] A CD69 deficient non-human mammal can be obtained by, for
example, transfecting ES cells with a targeting vector inducing
homologous recombination of CD69 gene to prepare ES cells
introduced with deficiency in one of the alleles of CD69 gene,
preparing, from the obtained ES cells, offspring animals introduced
with deficiency in one of the alleles of CD69 gene derived
therefrom, and crossing the offspring animals. For the production
of CD69 deficient mouse, for example, Murata, K. et al. 2003. Int.
Immunol. 15: 987-992 can be referred to.
[0102] The kind of the antigen is not particularly limited as long
as it has antigenicity to the CD69 deficient non-human mammal, and
a desired antigen can be selected. Examples of the antigen include
protein, peptide, lipid, sugar chain and the like having
antigenicity to the CD69 deficient non-human mammal. The CD69
deficient non-human mammal can be immunized with an antigen by
injecting the antigen in an amount sufficient for immunizing the
CD69 deficient non-human mammal. For example, the CD69 deficient
non-human mammal is immunized with the antigen at a frequency of
once in 1-3 weeks, 2-6 times in total. For immunization, the
antigen may be administered together with an adjuvant to the CD69
deficient non-human mammal. While the adjuvant is not particularly
limited as long as it can enhance immunogenicity, for example,
aluminum hydroxide, keyhole limpet hemocyanin, dextran, BCG,
aluminum phosphate, TLR ligand (e.g., lipopolysaccharide (LPS),
CpG) and the like can be mentioned. Aluminum hydroxide and the like
are preferably used for efficient induction of Th2 cells.
[0103] Th2 cell refers to a CD4T cell which is differentiated from
naive CD4T cells by antigen stimulation, and predominantly
producing IL-4.
[0104] Th2 cells can be obtained by, for example, isolating CD4T
cells from the spleen or peripheral blood of a CD69 deficient
non-human mammal immunized with a particular antigen, and
cultivating the CD4T cells in the presence of the antigen, antigen
presenting cells, IL-2 and IL-4. An immobilized anti-TCR antibody
or anti-CD3 antibody may also be used instead of the antigen. Th2
cells can be induced more potently by using an immobilized
anti-CD28 antibody in combination.
[0105] Whether the obtained cells are Th2 cells can be confirmed by
stimulating the obtained cells with the antigen, and evaluating
whether it predominantly produces IL-4 as compared to IFN-.gamma.
by flow cytometry.
[0106] To achieve expression of human CD69 in Th2 cells of a CD69
deficient non-human mammal, generally, Th2 cells of the CD69
deficient non-human mammal are transfected with an expression
vector capable of expressing human CD69 in Th2 cells of the
non-human mammal. As the vector, plasmid vector, virus vector,
retrovirus vector and the like can be mentioned. When a retrovirus
vector is used, transgene can be easily incorporated into the
chromosome and the expression of human CD69 can be stably continued
even when Th2 cells are proliferated. Thus, retrovirus vector is
preferably used in the present invention. For the detail of gene
transfer into Th2 cells by retrovirus, see, for example, Kimura, M.
et al. 2001. Immunity 15: 275-287.
[0107] The expression of human CD69 in Th2 cell can be confirmed by
flow cytometry using an anti-human CD69 antibody.
[0108] Th2 cells of a CD69 deficient non-human mammal immunized
with a particular antigen, that express human CD69, can be
transferred into a recipient non-human mammal by intravenously or
intraperitoneally injecting the Th2 cells to the recipient
non-human mammal. The number of the Th2 cells to be transferred is
not particularly limited as long as the response reaction of the
transferred Th2 cells to said antigen can be observed in the
recipient non-human mammal. When the recipient non-human mammal is
a mouse, for example, 1,000,000-3,000,000 Th2 cells are preferably
transferred.
[0109] The transferred Th2 cells are activated by stimulation with
the particular antigen to produce a large amount of IL-4.
Therefore, when the non-human mammal of the present invention is
exposed to said antigen, an allergic reaction mediated by the Th2
cells expressing human CD69 and an inflammation reaction associated
therewith occur. The present invention also provides such non-human
mammal allergy model. Using the non-human mammal of the present
invention, the role of human CD69 in allergic reactions and
inflammation reactions can be easily analyzed in vivo. In addition,
efficacy evaluation of an anti-human CD69 antibody for allergic
diseases and inflammatory diseases can be performed in non-human
mammals.
[0110] All references cited in the present specification, including
publication, patent document and the like, are hereby incorporated
individually and specifically by reference, to the extent that the
entireties thereof have been specifically disclosed herein.
EXAMPLES
[0111] The present invention is explained in more detail in the
following by referring to Examples, which are not to be construed
as limitative. Various gene manipulations in the Examples followed
the method described in Molecular cloning third. ed. (Cold Spring
Harbor Lab. Press, 2001).
Example 1
Production of Antigen and Antibody
(1) Production of Human CD69 Recombinant Protein
[0112] Since human CD69 forms a homodimer via cysteine 68, cDNA
(NM.sub.--001781) encoding the extracellular region (amino acid
sequences; 62-199) containing cysteine 68 was inserted into a
vector for Escherichia coli periplasm expression. Competent cells
of Escherichia coli TG1F(-) strain prepared in advance (Z-competent
E. Coli Transformation Buffer Set: manufactured by ZYMO RESEARCH)
were transformed with this expression vector, and cultured on a LB
agar plate containing chloramphenicol (final concentration 34
.mu.g/mL) at 37.degree. C. overnight. This Escherichia coli cells
were inoculated in a 2.times.YT medium, and cultured at 37.degree.
C. for 3-5 hr (OD600=0.5-0.8). IPTG (final concentration 0.1 mM)
was added, and the mixture was cultured at 25.degree. C. overnight.
The cultured Escherichia coli cells were collected by
centrifugation, and lysed with lysis buffer (200 mM borate, 160 mM
NaCl, 2 mM EDTA, 1 mg/ml lysozyme, pH 8.0), and the lysate was
centrifuged to give a soluble fraction. From this soluble fraction,
human CD69 homodimer protein was purified according to the standard
method of Strep-Tactin column (manufactured by IBA). In addition,
the purity of the purified human CD69 recombinant protein was
confirmed to be not less than 95% by SDS-PAGE, and the protein
concentration was determined by using BCA Protein Assay Kit
(manufactured by PIERCE).
(2) Biotinylation of Human CD69 Recombinant Protein
[0113] The purified human CD69 recombinant protein was biotinylated
according to the standard protocol of EZ-Link NHS-PEO.sub.4-Biotin
(Thermo Scientific), and the concentration was determined by using
BCA Protein Assay Kit (manufactured by PIERCE).
(3) Selection of Human CD69 Specific Antibody Clone by Phage
Display Method
[0114] The biotinylated human CD69 recombinant protein was
immobilized on streptavidin-coated magnetic beads (Dynabeads MyOne
Streptavidin T1 magnetic beads, manufactured by Invitrogen, 100
.mu.l) at 4.degree. C. for 1 hr, and washed 5 times with 1 ml PEST
(PBS containing 0.05% Tween 20). Using HuCAL GOLD (manufactured by
MorphoSys) for human antibody phage library, antibody selection was
performed according to the method described in WO 2007/042309, WO
2006/122797 and the like. Human CD69-immobilized beads were added
to the phage library to bind an antigen-specific antibody. The
magnetic beads were recovered and washed several times, and the
phage was eluted from the magnetic beads. Escherichia coli cells
were infected with the eluted phage and cultured at 37.degree. C.
overnight. An operation of phage-rescue from the phage-infected
Escherichia coli cells followed a general method (Molecular cloning
third. Ed. Cold Spring Harbor Lab. Press, 2001). The selection
round described above was repeated several times to concentrate a
phage presenting an antibody specific to the antigen.
(4) Screening for Antigen-Specific Antibody by ELISA
[0115] The pool of Fab genes obtained after the concentration
operation was subcloned to Escherichia coli expression vector.
According to the method described in WO 2006/122797 and the like,
the Fab antibody was expressed, and the antigen-specific antibody
was screened for by the ELISA method. The Fab antibody was purified
from a soluble fraction of Escherichia coli lysate according to the
standard method of Strep-Tactin column (manufactured by IBA). In
addition, the purity of the purified antibody was confirmed by
SDS-PAGE, and the concentration was determined by using BCA Protein
Assay Kit (manufactured by PIERCE).
(5) Screening for Antibody Clone by Cell Staining Evaluation
[0116] The purified ELISA-positive Fab antibody clones were further
evaluated for antigen reactivity by cell staining of human CD69 and
mouse CD69 over-expressing cells. As the antigen, CHO-S cells fixed
with 4% PFA 48 hr after the transfection with human CD69 or mouse
CD69 expression vector by the standard method using FreeStyle MAX
Reagent (manufactured by Invitrogen) were used. With 50 .mu.g/ml
purified Fab antibody as the primary antibody for staining, cells
were incubated at room temperature for 1 hr, and washed 3 times
with PBS. With 500-fold diluted Alexa555-labeled anti-human IgG
(manufactured by Invitrogen) as the secondary antibody, cells were
incubated at room temperature for 1 hr, and washed 3 times with
PBS. These cells were observed under a fluorescence microscope
(IX71, manufactured by OLYMPUS), and the presence or absence of
staining was evaluated. As a result, it was confirmed that 160-c76
clone binds to both human CD69 (hCD69) and mouse CD69 (mCD69) (FIG.
1), and 160-c7 and 160-c103 bind only to human CD69, and 3 clones
in total were finally obtained as anti-human CD69 antibody clones
that specifically bind to native-form human CD69 on the cell
surface.
(6) Analysis of Base Sequence of Anti-Human CD69 Antibody Clone
[0117] The obtained three clones (160-c7, 160-c76 and 160-c103) of
Escherichia coli were cultured, and plasmids were recovered
(QIAprep Spin MiniPrep kit: manufactured by QIAGEN) and used for
the base sequence analysis. Table 1 shows the amino acid sequences
of CDRs (complementarity determining regions) of the respective
clones.
TABLE-US-00001 TABLE 1 light chain LCDR1 LCDR2 LCDR3 160-
RASQDISSYLN YGASNLQS QQYSDYPH c7 (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ
ID NO: 3) 160- SGDSLGSKYVY VVIYGDSKRPS QSYDSNIM c76 (SEQ ID NO: 7)
(SEQ ID NO: 8) (SEQ ID NO: 9) 160- RASQSVSSYLA YGTSIRAT QQEYSSPP
c103 (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 15) heavy chain
HCDR1 HCDR2 HCDR3 160- YTFTSYDMH WINPYSGNTNYAQK MYYDKDYLSWGTDS c7
(SEQ ID NO: 4) FQG (SEQ ID NO: 6) (SEQ ID NO: 5) 160- FTFSNFVMH
SISGSSSSTYYADS YYYASFDY c76 (SEQ ID NO: 10) VKG (SEQ ID NO: 12)
(SEQ ID NO: 11) 160- FTFSNYYMS VISYDGISTHYADS YIGNSLYMDF c103 (SEQ
ID NO: 16) VKG (SEQ ID NO: 18) (SEQ ID NO: 17)
(7) Production of IgG Antibody of Anti-Human CD69 Antibody
Clones
[0118] Fab antibody genes of the obtained 3 clones were subcloned
to construct IgG expression vectors (constant region of heavy chain
was IgG4). HEK293T cells were transfected with these expression
vectors according to the standard method of Lipofectamine
(manufactured by Invitrogen), and the culture supernatant after
culture for 72 hr was recovered. As the medium, DMEM (Sigma)
supplemented with 10% Ultra Low IgG FBS (manufactured by
Invitrogen) was used. From the culture supernatant, IgG antibody
was purified by the standard method using rProteinA Sepharose Fast
Flow (manufactured by GE healthcare). Protein after purification
was confirmed to show a single band by SDS-PAGE, and the
concentration was determined by using BCA Protein Assay Kit
(manufactured by PIERCE).
Example 2
Effect of Anti-Human CD69 Antibody on Intraalveolar Mononuclear
Cell Infiltration
[0119] Mice obtained by crossing BALB/c mouse or CD69 deficient
(CD69KO) mouse back-crossed not less than 10 times onto BALB/c
(Murata, K. et al. 2003. Int. Immunol. 15: 987-992) with D011.10
transgenic mouse were used. The spleen CD4T cells of these mice
were purified by AutoMACS sorter (Miltenyi Biotec) to a purity of
>98%. The isolated CD4T cells were cultured with stimulation
with immobilized anti-TCR and anti-CD28 monoclonal antibodies under
Th2 conditions (IL-2 10 u/ml, IL-4 100 U/ml). Two days after the
start of the culture, and human CD69 gene was introduced by a
retrovirus vector containing human CD69 (hCD69) gene. The method of
introducing human CD69 gene followed the method described
previously (Kimura, M. et al. 2001. Immunity 15: 275-287). Five
days after the start of the culture, the cultured cells were
recovered, and the expression of human CD69 was confirmed by flow
cytometry (FIG. 2). 50.2% of the cells were human CD69
positive.
[0120] 3,000,000 cells of CD69 KO mouse Th2 cells that overexpress
human CD69 (hCD69) obtained by the above-mentioned culture, or
wild-type BALB/c mouse Th2 cells were intravenously injected into
wild-type BALB/c mice (day 0). After cell transfer, on day 1 and
day 3, the mice were exposed to allergen challenge via airway by
inhaling 1% OVA solution in aerosolized saline for 30 min using an
ultrasonication nebulizer (NE-U07; manufactured by Omron).
[0121] On day 1, 2 hr before OVA inhalation, the following
antibodies were intraperitoneally injected at a dose of 100
.mu.g/mouse:
control antibody (anti-"TSLYKKAG" peptide, IgG4, self-developed)
mouse anti-human CD69 monoclonal antibody (FN50 manufactured by
BioLegend) 160-c7 160-c76 160-c103
[0122] On day 5, according to the report described previously,
bronchoalveolar lavage (BAL) was performed (Kamata, T. et al.,
2003, J. Clin. Invest. 111: 109-119). All bronchoalveolar lavage
was collected, and the cells in 150 .mu.l of the fractionated
liquid were counted. Viable BAL cells (100,000 cells) were
cytocentrifuged onto a slide by Cytospin 4 (manufactured by Thermo
Fisher Scientific), and stained with May-Gruenwald Giemsa solution
(manufactured by Merck). 500 leukocytes were counted on each slide,
and the cell type was identified using the morphological criteria.
The percentage of each cell type was calculated.
[0123] The results are shown in FIG. 3. The anti-hCD69 antibodies
suppressed intraalveolar infiltration of mononuclear cells,
particularly infiltration of eosinophils, which is caused by OVA
inhalation.
Example 3
Selection of High Affinity Anti-Human CD69 Antibody
[0124] Selection of an antibody having higher affinity for human
CD69 was tried by introducing a mutation into the light chain CDR3
of the antibodies selected in Example 1. To be specific, the
methods described in Prassler J, Steidl S, Urlinger S.
Immunotherapy. 2009 July; 1(4):571-83. and Hillig R C, Urlinger S,
Fanghanel J, Brocks B, Haenel C, Stark Y, Sulzle D, Svergun D I,
Baesler S, Malawski G, Moosmayer D, Menrad A, Schirner M, Licha K.
J Mol Biol. 2008 Mar. 14; 377(1):206-19 were employed. The antibody
selection round was repeated twice, the base sequences of the light
chain CDR3 of the obtained antibody clones were examined and
antibody clones having a novel sequence were identified. They were
expressed in Escherichia coli, and ELISA was performed for the
antigen by using the lysate and the amount of the antibody in the
lysate was simultaneously measured by sandwich ELISA. The relative
specific binding activity of each clone was calculated from the
absorbance of ELISA against the antigen and the antibody amount,
and high affinity clones were selected. In addition, IgG of top 10
clones having high affinity were prepared and K.sub.D values were
measured.
[0125] In the same manner as in Example 1, Fab antibody gene was
subcloned to construct IgG expression vector (constant region of
heavy chain was IgG4). HEK293T cells were transfected with the
expression vector by Lipofectamine (manufactured by Invitorogen),
cultured for 72 hr, and IgG antibody was purified from the
recovered culture supernatant.
[0126] The affinity of the prepared IgG clones for human CD69 was
evaluated by scatchard plot. To be specific, it was calculated
according to the principle described in Immunoassays (OXFORD
UNIVERSITY PRESS, 2000). An antibody was incubated with various
concentrations of antigen at room temperature for 2 hr until it
reaches equilibrium, and the amount of free antibody present in the
incubation liquids was measured by the ELISA method. The binding
constant and dissociation constant (K.sub.D value) were determined
based on the changes in the amount of free antibody in each
equilibrate sample. The concentration of the antibody in the
equilibration reaction was set to 0.015 .mu.g/ml, and an ELISA
plate immobilized with the antigen at 1 .mu.g/ml was used for the
measurement of the amount of free antibody.
[0127] As a result, a plurality of high affinity clones which bind
to human CD69 while maintaining the cross-reactivity with mouse
CD69 were selected from clones with the same CDRs as 160-c76 other
than light chain CDR3. Table 2 shows the amino acid sequences of
light chain CDR3 and affinity of two clones, 234-61 and 234-83,
that showed particularly high affinity for human CD69. The affinity
of these two clones for human CD69 increased to 9-fold or more that
of 160-c76 clone having the same sequence other than light chain
CDR3.
TABLE-US-00002 TABLE 2 cross-reactivity clone K.sub.D (M) with
mCD69 LCDR3 160-c7 4.76E-08 - QQYSDYPH (SEQ ID NO: 3) 160-c103
7.58E-09 - QQEYSSPP (SEQ ID NO: 15) 160-c76 2.78E-08 ++ QSYDSNIM
(SEQ ID NO: 9) 234-61 1.13E-09 + QSYTSFTTKI (SEQ ID NO: 19) 234-83
2.83E-09 +++ GSYTTGAKSH (SEQ ID NO: 20)
Example 4
Cross-Reactivity with Mouse CD69
[0128] The reactivity with mouse CD69 was evaluated as follows.
Splenocytes were isolated from wild-type mouse and CD69 KO mouse
(both Balb/c), and stimulated with Phorbol 12-myristate 13-acetate
(PMA) for 4 hr to induce expression of CD69 on the cell surface.
Each anti-human CD69 antibody (160-c76, 234-61 and 234-83) (1
.mu.g) was added to 1.times.10.sup.6 splenocytes, and the mixture
was incubated on ice for 30 min. The cells were washed, anti-human
IgG-Alexa488 (.times.200 diluted) was added as a secondary
antibody, and the mixture was incubated on ice for 20 min. After
washing the cells, the intensity of staining with each anti-human
CD69 antibody was evaluated by flow cytometry (FACS Calibur:
manufactured by Becton, Dickinson). As a positive control, hamster
anti-mouse CD69 monoclonal antibody (H1.2F3)-FITC (manufactured by
Becton, Dickinson) was used.
[0129] On the other hand, the reactivity with human CD69 was
evaluated in the same manner as for mouse CD69 by using peripheral
blood mononuclear cells (PBMCs) of a healthy subject, which were
induced to express CD69 on the cell surface by stimulating with PMA
for 4 hr. As a positive control, mouse anti-human CD69 monoclonal
antibody (FN50) (manufactured by BioLegend) was used.
[0130] As a result, all 160-c76, 234-61 and 234-83 clones bound to
activated mouse splenocytes and activated human peripheral blood
mononuclear cells, and cross-reaction with both mouse CD69 (mCD69)
and human CD69 (hCD69) was observed (FIG. 4). As compared to
160-c76, the intensity of staining of mouse CD69 by 234-61 was
weak. On the other hand, 234-83 strongly bound to mouse CD69.
Example 5
Effect of Anti-Human CD69 Antibody on Intraalveolar Mononuclear
Infiltration
[0131] The effect of the following anti-human CD69 antibodies on
intraalveolar mononuclear infiltration was evaluated according to a
similar protocol as in Example 2.
control antibody (anti-"TSLYKKAG" peptide, IgG4, self-developed)
mouse anti-human CD69 monoclonal antibody (FN50 manufactured by
BioLegend) 160-c76 234-61 234-83
[0132] CD69 deficient (CD69 KO) mice back-crossed not less than 10
times onto BALB/c were intraperitoneally immunized with 250 .mu.g
of OVA (hen egg albumin from Sigma-Aldrich) in 4 mg of aluminum
hydroxide gel (alum). Spleen CD4T cells of the OVA-immunized CD69
deficient mouse were purified using CD4+T cell isolation kit
manufactured by Miltenyi Biotec) and AutoMACS sorter (manufactured
by Miltenyi Biotec) to a purity of >98%. The isolated CD4T cells
were cultured with stimulating with immobilized anti-TCR and
anti-CD28 mAbs under Th2 conditions. Two days after the start of
the culture, hCD69 gene was introduced by a retrovirus vector
containing human CD69 (hCD69) gene. Five days after the start of
the culture, the cultured cells were recovered, and the expression
of hCD69 was confirmed by flow cytometry. 58.7% of the cells were
hCD69 positive.
[0133] 3,000,000 cells of CD69 KO mouse Th2 cells that overexpress
hCD69 obtained by the above-mentioned culture, or wild-type BALE/c
mouse Th2 cells were intravenously injected into wild-type BALB/c
mice (day 0). After cell transfer, on is day 1 and day 3, the mice
were exposed to allergen challenge via airway by inhaling 1% OVA
solution in aerosolized saline for 30 min using an ultrasonication
nebulizer (NE-U07; manufactured by Omron).
[0134] On day 1, 2 hr before OVA inhalation, the evaluation target
antibodies were intraperitoneally injected at a dose of 100
.mu.g/mouse. On day 4, according to the report described
previously, bronchoalveolar lavage (BAL) was performed (Kamata, T.
et al., 2003, J. Clin. Invest. 111: 109-119). All bronchoalveolar
lavage was collected, and the cells in 150 .mu.l of the
fractionated liquid were counted. Viable BAL cells (100,000 cells)
were centrifuged onto a slide by Cytospin 4 (manufactured by Thermo
Fisher Scientific), and stained with May-Gruenwald Giemsa solution
(manufactured by Merck). 500 leukocytes were counted on each slide,
and the cell type was identified using the morphological criteria.
The percentage of each cell type was calculated.
[0135] The results are shown in FIG. 5. All evaluated anti-hCD69
antibody suppressed intraalveolar infiltration of leukocytes
(eosinophils, neutrophils, lymphocytes, macrophages) caused by OVA
inhalation. The leukocyte infiltration suppressing capacity was
FN50<160-c76<234-61<234-83, whereby enhancement of
leukocyte infiltration suppressing capacity by affinity improvement
was confirmed.
Example 6
Identification of Epitope
[0136] Using a peptide array on which partial peptides of human
CD69 were immobilized, epitope mapping of anti-human CD69
antibodies 234-83, 160-c7 and 160-c103 was performed. To be
specific, as shown in the following Table, a peptide array
consisting of peptides having the residue number of 12 amino acid
residues and an offset of 3 amino acid residues was produced for a
sequence covering the extracellular domain of human CD69 (60-199)
(PepSpots, manufactured by JPT). The peptide array and the
anti-human CD69 antibody were reacted according to the manual of
JPT. Anti-human CD69 antibodies labeled with HRP (Peroxidase
Labeling Kit--NH2, manufactured by Dojindo) were used.
TABLE-US-00003 TABLE 3 1 ALSVGQYNCPGQ 16 SWTSAQNACSEH 31
GHPWKWSNGKEF (SEQ ID (SEQ ID (SEQ ID NO: 34) NO: 49) NO: 64) 2
VGQYNCPGQYTF 17 SAQNACSEHGAT 32 WKWSNGKEFNNW (SEQ ID (SEQ ID (SEQ
ID NO: 35) NO: 50) NO:65) 3 YNCPGQYTFSMP 18 NACSEHGATLAV 33
SNGKEFNNWFNV (SEQ ID (SEQ ID (SEQ ID NO: 36) NO: 51) NO: 66) 4
PGQYTFSMPSDS 19 SEHGATLAVIDS 34 KEFNNWFNVTGS (SEQ ID (SEQ ID (SEQ
ID NO: 37) NO: 52) NO: 67) 5 YTFSMPSDSHVS 20 GATLAVIDSEKD 35
NNWFNVTGSDKC (SEQ ID (SEQ ID (SEQ ID NO: 38) NO: 53) NO: 68) 6
SMPSDSHVSSCS 21 LAVIDSEKDMNF 36 FNVTGSDKCVFL (SEQ ID (SEQ ID (SEQ
ID NO: 39) NO: 54) NO: 69) 7 SDSHVSSCSEDW 22 IDSEKDMNFLKR 37
TGSDKCVFLKNT (SEQ ID (SEQ ID (SEQ ID NO: 40) NO: 55) NO: 70) 8
HVSSCSEDWVGY 23 EKDMNFLKRYAG 38 DKCVFLKNTEVS (SEQ ID (SEQ ID (SEQ
ID NO: 41) NO: 56) NO: 71) 9 SCSEDWVGYQRK 24 MNFLKRYAGREE 39
VFLKNTEVSSME (SEQ ID (SEQ ID (SEQ ID NO: 42) NO: 57) NO: 72) 10
EDWVGYQRKCYF 25 LKRYAGREEHWV 40 KNTEVSSMECEK (SEQ ID (SEQ ID (SEQ
ID NO: 43) NO: 58) NO: 73) 11 VGYQRKCYFIST 26 YAGREEHWVGLK 41
EVSSMECEKNLY (SEQ ID (SEQ ID (SEQ ID NO: 44) NO: 59) NO: 74) 12
QRKCYFISTVKR 27 REEHWVGLKKEP 42 SMECEKNLYWIC (SEQ ID (SEQ ID (SEQ
ID NO: 45) NO: 60) NO: 75) 13 CYFISTVKRSWT 28 HWVGLKKEPGHP 43
CEKNLYWICNKP (SEQ ID (SEQ ID (SEQ ID NO: 46) NO: 61) NO: 76) 14
ISTVKRSWTSAQ 29 GLKKEPGHPWKW 44 ENLYWICNKPYK (SEQ ID (SEQ ID (SEQ
ID NO: 47) NO: 62) NO: 77) 15 VKRSWTSAQNAC 30 KEPGHPWKWSNG (SEQ ID
(SEQ ID NO: 48) NO: 63)
[0137] As a result, 234-83 specifically bound to the
above-mentioned peptides #2 and #3, particularly strongly bound to
peptide #3. The results suggest that the epitope of 234-83 contains
the amino acid sequence shown in SEQ ID NO: 33 (YNCPG) which is
common to peptide #2 and peptide #3, and to human CD69 and mouse
CD69 (FIG. 6).
[0138] 160-c7 specifically bound to the above-mentioned peptide
#26. The results show that 160-c7 binds to human CD69 at an epitope
consisting of the amino acid sequence shown in SEQ ID NO: 59.
[0139] 160-c103 specifically bound to the above-mentioned peptides
#24, #25 and #26. The results suggest that the epitope of 160-c103
contains the amino acid sequence shown in SEQ ID NO: 78 (YAGREE)
which is common to peptides #24, #25 and #26.
INDUSTRIAL APPLICABILITY
[0140] According to the present invention, an anti-human CD69
antibody applicable to the prophylaxis or treatment of allergic
diseases and inflammatory diseases is provided. According to the
present invention, moreover, an animal model permitting in vivo
evaluation of a pharmacological effect of an anti-human CD69
antibody can be provided.
[0141] This application is based on a patent application No.
2012-098243 filed in Japan (filing date: Apr. 23, 2012), the
contents of which are incorporated in full herein.
Sequence CWU 1
1
78111PRTArtificial SequenceSynthetic 160-c7 LCDR1 1Arg Ala Ser Gln
Asp Ile Ser Ser Tyr Leu Asn 1 5 10 28PRTArtificial
SequenceSynthetic 160-c7 LCDR2 2Tyr Gly Ala Ser Asn Leu Gln Ser 1 5
38PRTArtificial SequenceSynthetic 160-c7 LCDR3 3Gln Gln Tyr Ser Asp
Tyr Pro His 1 5 49PRTArtificial SequenceSynthetic 160-c7 HCDR1 4Tyr
Thr Phe Thr Ser Tyr Asp Met His 1 5 517PRTArtificial
SequenceSynthetic 160-c7 HCDR2 5Trp Ile Asn Pro Tyr Ser Gly Asn Thr
Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 614PRTArtificial
SequenceSynthetic 160-c7 HCDR3 6Met Tyr Tyr Asp Lys Asp Tyr Leu Ser
Trp Gly Thr Asp Ser 1 5 10 711PRTArtificial SequenceSynthetic
160-c76 LCDR1 7Ser Gly Asp Ser Leu Gly Ser Lys Tyr Val Tyr 1 5 10
811PRTArtificial SequenceSynthetic 160-c76 LCDR2 8Val Val Ile Tyr
Gly Asp Ser Lys Arg Pro Ser 1 5 10 98PRTArtificial
SequenceSynthetic 160-c76 LCDR3 9Gln Ser Tyr Asp Ser Asn Ile Met 1
5 109PRTArtificial SequenceSynthetic 160-c76 HCDR1 10Phe Thr Phe
Ser Asn Phe Val Met His 1 5 1117PRTArtificial SequenceSynthetic
160-c76 HCDR2 11Ser Ile Ser Gly Ser Ser Ser Ser Thr Tyr Tyr Ala Asp
Ser Val Lys 1 5 10 15 Gly 128PRTArtificial SequenceSynthetic
160-c76 HCDR3 12Tyr Tyr Tyr Ala Ser Phe Asp Tyr 1 5
1311PRTArtificial SequenceSynthetic 160-c103 LCDR1 13Arg Ala Ser
Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 148PRTArtificial
SequenceSynthetic 160-c103 LCDR2 14Tyr Gly Thr Ser Ile Arg Ala Thr
1 5 158PRTArtificial SequenceSynthetic 160-c103 LCDR3 15Gln Gln Glu
Tyr Ser Ser Pro Pro 1 5 169PRTArtificial SequenceSynthetic 160-c103
HCDR1 16Phe Thr Phe Ser Asn Tyr Tyr Met Ser 1 5 1717PRTArtificial
SequenceSynthetic 160-c103 HCDR2 17Val Ile Ser Tyr Asp Gly Ile Ser
Thr His Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 1810PRTArtificial
SequenceSynthetic 160-c103 HCDR3 18Tyr Ile Gly Asn Ser Leu Tyr Met
Asp Phe 1 5 10 1910PRTArtificial SequenceSynthetic 234-61 LCDR3
19Gln Ser Tyr Thr Ser Phe Thr Thr Lys Ile 1 5 10 2010PRTArtificial
SequenceSynthetic 234-83 LCDR3 20Gly Ser Tyr Thr Thr Gly Ala Lys
Ser His 1 5 10 21109PRTArtificial SequenceSynthetic 160-c7 VL 21Asp
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 Gly Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Tyr Ser Asp Tyr Pro His 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Thr 100 105 22123PRTArtificial
SequenceSynthetic 160-c7 VH 22Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile
Asn Pro Tyr Ser Gly Asn 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 Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Met Tyr Tyr Asp Lys Asp Tyr Leu Ser Trp Gly
Thr Asp Ser 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 23107PRTArtificial SequenceSynthetic 160-c76 VL 23Asp Ile
Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Ser Leu Gly Ser Lys Tyr Val 20
25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile
Tyr 35 40 45 Gly Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe
Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
Gly Thr Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ser
Tyr Asp Ser Asn Ile Met Val 85 90 95 Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 105 24107PRTArtificial SequenceSynthetic
160-c76 VH 24Gln Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly 1 5 10 15 Phe Thr Phe Ser Asn Phe Val Met His Trp Val
Arg Gln Ala Pro Gly 20 25 30 Lys Gly Leu Glu Trp Val Ser Ser Ile
Ser Gly Ser Ser Ser Ser Thr 35 40 45 Tyr Tyr Ala Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn 50 55 60 Ser Lys Asn Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 65 70 75 80 Thr Ala Val
Tyr Tyr Cys Ala Arg Tyr Tyr Tyr Ala Ser Phe Asp Tyr 85 90 95 Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 25109PRTArtificial
SequenceSynthetic 160-c103 VL 25Asp Ile Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Thr
Ser Ile Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Glu Tyr Ser Ser Pro
Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
100 105 26119PRTArtificial SequenceSynthetic 160-c103 VH 26Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20
25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser Val Ile Ser Tyr Asp Gly Ile Ser Thr His Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe 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 Tyr Ile Gly Asn Ser
Leu Tyr Met Asp Phe Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val
Ser Ser 115 27109PRTArtificial SequenceSynthetic 234-61 VL 27Asp
Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10
15 Thr Ala Arg Ile Ser Cys Ser Gly Asp Ser Leu Gly Ser Lys Tyr Val
20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val
Ile Tyr 35 40 45 Gly Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg
Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile
Ser Gly Thr Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln
Ser Tyr Thr Ser Phe Thr Thr Lys 85 90 95 Ile Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu Gly 100 105 28109PRTArtificial
SequenceSynthetic 234-83 VL 28Asp Ile Glu Leu Thr Gln Pro Pro Ser
Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Ser Cys Ser
Gly Asp Ser Leu Gly Ser Lys Tyr Val 20 25 30 Tyr Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Val Val Val Ile Tyr 35 40 45 Gly Asp Ser
Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn
Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu 65 70
75 80 Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Tyr Thr Thr Gly Ala Lys
Ser 85 90 95 His Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 29600DNAHomo sapiensCDS(1)..(600) 29atg agc tct gaa aat tgt
ttc gta gca gag aac agc tct ttg cat ccg 48Met Ser Ser Glu Asn Cys
Phe Val Ala Glu Asn Ser Ser Leu His Pro 1 5 10 15 gag agt gga caa
gaa aat gat gcc acc agt ccc cat ttc tca aca cgt 96Glu Ser Gly Gln
Glu Asn Asp Ala Thr Ser Pro His Phe Ser Thr Arg 20 25 30 cat gaa
ggg tcc ttc caa gtt cct gtc ctg tgt gct gta atg aat gtg 144His Glu
Gly Ser Phe Gln Val Pro Val Leu Cys Ala Val Met Asn Val 35 40 45
gtc ttc atc acc att tta atc ata gct ctc att gcc tta tca gtg ggc
192Val Phe Ile Thr Ile Leu Ile Ile Ala Leu Ile Ala Leu Ser Val Gly
50 55 60 caa tac aat tgt cca ggc caa tac aca ttc tca atg cca tca
gac agc 240Gln Tyr Asn Cys Pro Gly Gln Tyr Thr Phe Ser Met Pro Ser
Asp Ser 65 70 75 80 cat gtt tct tca tgc tct gag gac tgg gtt ggc tac
cag agg aaa tgc 288His Val Ser Ser Cys Ser Glu Asp Trp Val Gly Tyr
Gln Arg Lys Cys 85 90 95 tac ttt att tct act gtg aag agg agc tgg
act tca gcc caa aat gct 336Tyr Phe Ile Ser Thr Val Lys Arg Ser Trp
Thr Ser Ala Gln Asn Ala 100 105 110 tgt tct gaa cat ggt gct act ctt
gct gtc att gat tct gaa aag gac 384Cys Ser Glu His Gly Ala Thr Leu
Ala Val Ile Asp Ser Glu Lys Asp 115 120 125 atg aac ttt cta aaa cga
tac gca ggt aga gag gaa cac tgg gtt gga 432Met Asn Phe Leu Lys Arg
Tyr Ala Gly Arg Glu Glu His Trp Val Gly 130 135 140 ctg aaa aag gaa
cct ggt cac cca tgg aag tgg tca aat ggc aaa gaa 480Leu Lys Lys Glu
Pro Gly His Pro Trp Lys Trp Ser Asn Gly Lys Glu 145 150 155 160 ttt
aac aac tgg ttc aac gtt aca ggg tct gac aag tgt gtt ttt ctg 528Phe
Asn Asn Trp Phe Asn Val Thr Gly Ser Asp Lys Cys Val Phe Leu 165 170
175 aaa aac aca gag gtc agc agc atg gaa tgt gag aag aat tta tac tgg
576Lys Asn Thr Glu Val Ser Ser Met Glu Cys Glu Lys Asn Leu Tyr Trp
180 185 190 ata tgt aac aaa cct tac aaa taa 600Ile Cys Asn Lys Pro
Tyr Lys 195 30199PRTHomo sapiens 30Met Ser Ser Glu Asn Cys Phe Val
Ala Glu Asn Ser Ser Leu His Pro 1 5 10 15 Glu Ser Gly Gln Glu Asn
Asp Ala Thr Ser Pro His Phe Ser Thr Arg 20 25 30 His Glu Gly Ser
Phe Gln Val Pro Val Leu Cys Ala Val Met Asn Val 35 40 45 Val Phe
Ile Thr Ile Leu Ile Ile Ala Leu Ile Ala Leu Ser Val Gly 50 55 60
Gln Tyr Asn Cys Pro Gly Gln Tyr Thr Phe Ser Met Pro Ser Asp Ser 65
70 75 80 His Val Ser Ser Cys Ser Glu Asp Trp Val Gly Tyr Gln Arg
Lys Cys 85 90 95 Tyr Phe Ile Ser Thr Val Lys Arg Ser Trp Thr Ser
Ala Gln Asn Ala 100 105 110 Cys Ser Glu His Gly Ala Thr Leu Ala Val
Ile Asp Ser Glu Lys Asp 115 120 125 Met Asn Phe Leu Lys Arg Tyr Ala
Gly Arg Glu Glu His Trp Val Gly 130 135 140 Leu Lys Lys Glu Pro Gly
His Pro Trp Lys Trp Ser Asn Gly Lys Glu 145 150 155 160 Phe Asn Asn
Trp Phe Asn Val Thr Gly Ser Asp Lys Cys Val Phe Leu 165 170 175 Lys
Asn Thr Glu Val Ser Ser Met Glu Cys Glu Lys Asn Leu Tyr Trp 180 185
190 Ile Cys Asn Lys Pro Tyr Lys 195 31600DNAMus
musculusCDS(1)..(600) 31atg gat tct gaa aac tgt tct ata acg gaa aat
agc tct tca cat ctg 48Met Asp Ser Glu Asn Cys Ser Ile Thr Glu Asn
Ser Ser Ser His Leu 1 5 10 15 gag aga ggg cag aag gac cat ggc acc
agt ata cat ttt gag aag cat 96Glu Arg Gly Gln Lys Asp His Gly Thr
Ser Ile His Phe Glu Lys His 20 25 30 cat gaa gga tcc att caa gtt
tct atc cct tgg gct gtg tta ata gtg 144His Glu Gly Ser Ile Gln Val
Ser Ile Pro Trp Ala Val Leu Ile Val 35 40 45 gtc ctc atc acg tcc
tta ata ata gct ctc att gcc tta aat gtg ggc 192Val Leu Ile Thr Ser
Leu Ile Ile Ala Leu Ile Ala Leu Asn Val Gly 50 55 60 aag tac aat
tgc cca ggc ttg tac gag aag ttg gaa tca tct gac cac 240Lys Tyr Asn
Cys Pro Gly Leu Tyr Glu Lys Leu Glu Ser Ser Asp His 65 70 75 80 cat
gtt gct acc tgc aag aat gag tgg att tca tac aag agg aca tgt 288His
Val Ala Thr Cys Lys Asn Glu Trp Ile Ser Tyr Lys Arg Thr Cys 85 90
95 tac ttc ttc tcc acc aca acc aag agt tgg gcc ttg gcc caa cgc tct
336Tyr Phe Phe Ser Thr Thr Thr Lys Ser Trp Ala Leu Ala Gln Arg Ser
100 105 110 tgt tct gaa gat gct gct act ctt gct gta att gat tca gaa
aag gac 384Cys Ser Glu Asp Ala Ala Thr Leu Ala Val Ile Asp Ser Glu
Lys Asp 115 120 125 atg acg ttt ctg aag cga tat tct ggt gaa ctg gaa
cat tgg att ggg 432Met Thr Phe Leu Lys Arg Tyr Ser Gly Glu Leu Glu
His Trp Ile Gly 130 135 140 ctg aaa aat gaa gct aat cag aca tgg aaa
tgg gca aat ggc aaa gaa 480Leu Lys Asn Glu Ala Asn Gln Thr Trp Lys
Trp Ala Asn Gly Lys Glu 145 150 155 160 ttt aac agc tgg ttc aac ttg
acg ggg tct ggg agg tgc gtg tcc gtg 528Phe Asn Ser Trp Phe Asn Leu
Thr Gly Ser Gly Arg Cys Val Ser Val 165 170 175 aac cac aaa aat gtt
acc gct gtg gac tgt gag gca aac ttc cac tgg 576Asn His Lys Asn Val
Thr Ala Val Asp Cys Glu Ala Asn Phe His Trp 180 185 190 gtc tgc agc
aag ccc tcc aga tga 600Val Cys Ser Lys Pro Ser Arg 195 32199PRTMus
musculus 32Met Asp Ser Glu Asn Cys Ser Ile Thr Glu Asn Ser Ser Ser
His Leu 1 5 10 15 Glu Arg Gly Gln Lys Asp His Gly Thr Ser Ile His
Phe Glu Lys His 20 25 30 His Glu Gly Ser Ile Gln Val Ser Ile Pro
Trp Ala Val Leu Ile Val 35 40 45 Val Leu Ile Thr Ser Leu Ile Ile
Ala Leu Ile Ala Leu Asn Val Gly 50 55 60 Lys Tyr Asn Cys Pro Gly
Leu Tyr Glu Lys Leu Glu Ser Ser Asp His 65 70 75
80 His Val Ala Thr Cys Lys Asn Glu Trp Ile Ser Tyr Lys Arg Thr Cys
85 90 95 Tyr Phe Phe Ser Thr Thr Thr Lys Ser Trp Ala Leu Ala Gln
Arg Ser 100 105 110 Cys Ser Glu Asp Ala Ala Thr Leu Ala Val Ile Asp
Ser Glu Lys Asp 115 120 125 Met Thr Phe Leu Lys Arg Tyr Ser Gly Glu
Leu Glu His Trp Ile Gly 130 135 140 Leu Lys Asn Glu Ala Asn Gln Thr
Trp Lys Trp Ala Asn Gly Lys Glu 145 150 155 160 Phe Asn Ser Trp Phe
Asn Leu Thr Gly Ser Gly Arg Cys Val Ser Val 165 170 175 Asn His Lys
Asn Val Thr Ala Val Asp Cys Glu Ala Asn Phe His Trp 180 185 190 Val
Cys Ser Lys Pro Ser Arg 195 335PRTArtificial SequenceSynthetic
human CD69 partial peptide 33Tyr Asn Cys Pro Gly 1 5
3412PRTArtificial SequenceSynthetic human CD69 partial peptide
34Ala Leu Ser Val Gly Gln Tyr Asn Cys Pro Gly Gln 1 5 10
3512PRTArtificial SequenceSynthetic human CD69 partial peptide
35Val Gly Gln Tyr Asn Cys Pro Gly Gln Tyr Thr Phe 1 5 10
3612PRTArtificial SequenceSynthetic human CD69 partial peptide
36Tyr Asn Cys Pro Gly Gln Tyr Thr Phe Ser Met Pro 1 5 10
3712PRTArtificial SequenceSynthetic human CD69 partial peptide
37Pro Gly Gln Tyr Thr Phe Ser Met Pro Ser Asp Ser 1 5 10
3812PRTArtificial SequenceSynthetic human CD69 partial peptide
38Tyr Thr Phe Ser Met Pro Ser Asp Ser His Val Ser 1 5 10
3912PRTArtificial SequenceSynthetic human CD69 partial peptide
39Ser Met Pro Ser Asp Ser His Val Ser Ser Cys Ser 1 5 10
4012PRTArtificial SequenceSynthetic human CD69 partial peptide
40Ser Asp Ser His Val Ser Ser Cys Ser Glu Asp Trp 1 5 10
4112PRTArtificial SequenceSynthetic human CD69 partial peptide
41His Val Ser Ser Cys Ser Glu Asp Trp Val Gly Tyr 1 5 10
4212PRTArtificial SequenceSynthetic human CD69 partial peptide
42Ser Cys Ser Glu Asp Trp Val Gly Tyr Gln Arg Lys 1 5 10
4312PRTArtificial SequenceSynthetic human CD69 partial peptide
43Glu Asp Trp Val Gly Tyr Gln Arg Lys Cys Tyr Phe 1 5 10
4412PRTArtificial SequenceSynthetic human CD69 partial peptide
44Val Gly Tyr Gln Arg Lys Cys Tyr Phe Ile Ser Thr 1 5 10
4512PRTArtificial SequenceSynthetic human CD69 partial peptide
45Gln Arg Lys Cys Tyr Phe Ile Ser Thr Val Lys Arg 1 5 10
4612PRTArtificial SequenceSynthetic human CD69 partial peptide
46Cys Tyr Phe Ile Ser Thr Val Lys Arg Ser Trp Thr 1 5 10
4712PRTArtificial SequenceSynthetic human CD69 partial peptide
47Ile Ser Thr Val Lys Arg Ser Trp Thr Ser Ala Gln 1 5 10
4812PRTArtificial SequenceSynthetic human CD69 partial peptide
48Val Lys Arg Ser Trp Thr Ser Ala Gln Asn Ala Cys 1 5 10
4912PRTArtificial SequenceSynthetic human CD69 partial peptide
49Ser Trp Thr Ser Ala Gln Asn Ala Cys Ser Glu His 1 5 10
5012PRTArtificial SequenceSynthetic human CD69 partial peptide
50Ser Ala Gln Asn Ala Cys Ser Glu His Gly Ala Thr 1 5 10
5112PRTArtificial SequenceSynthetic human CD69 partial peptide
51Asn Ala Cys Ser Glu His Gly Ala Thr Leu Ala Val 1 5 10
5212PRTArtificial SequenceSynthetic human CD69 partial peptide
52Ser Glu His Gly Ala Thr Leu Ala Val Ile Asp Ser 1 5 10
5312PRTArtificial SequenceSynthetic human CD69 partial peptide
53Gly Ala Thr Leu Ala Val Ile Asp Ser Glu Lys Asp 1 5 10
5412PRTArtificial SequenceSynthetic human CD69 partial peptide
54Leu Ala Val Ile Asp Ser Glu Lys Asp Met Asn Phe 1 5 10
5512PRTArtificial SequenceSynthetic human CD69 partial peptide
55Ile Asp Ser Glu Lys Asp Met Asn Phe Leu Lys Arg 1 5 10
5612PRTArtificial SequenceSynthetic human CD69 partial peptide
56Glu Lys Asp Met Asn Phe Leu Lys Arg Tyr Ala Gly 1 5 10
5712PRTArtificial SequenceSynthetic human CD69 partial peptide
57Met Asn Phe Leu Lys Arg Tyr Ala Gly Arg Glu Glu 1 5 10
5812PRTArtificial SequenceSynthetic human CD69 partial peptide
58Leu Lys Arg Tyr Ala Gly Arg Glu Glu His Trp Val 1 5 10
5912PRTArtificial SequenceSynthetic human CD69 partial peptide
59Tyr Ala Gly Arg Glu Glu His Trp Val Gly Leu Lys 1 5 10
6012PRTArtificial SequenceSynthetic human CD69 partial peptide
60Arg Glu Glu His Trp Val Gly Leu Lys Lys Glu Pro 1 5 10
6112PRTArtificial SequenceSynthetic human CD69 partial peptide
61His Trp Val Gly Leu Lys Lys Glu Pro Gly His Pro 1 5 10
6212PRTArtificial SequenceSynthetic human CD69 partial peptide
62Gly Leu Lys Lys Glu Pro Gly His Pro Trp Lys Trp 1 5 10
6312PRTArtificial SequenceSynthetic human CD69 partial peptide
63Lys Glu Pro Gly His Pro Trp Lys Trp Ser Asn Gly 1 5 10
6412PRTArtificial SequenceSynthetic human CD69 partial peptide
64Gly His Pro Trp Lys Trp Ser Asn Gly Lys Glu Phe 1 5 10
6512PRTArtificial SequenceSynthetic human CD69 partial peptide
65Trp Lys Trp Ser Asn Gly Lys Glu Phe Asn Asn Trp 1 5 10
6612PRTArtificial SequenceSynthetic human CD69 partial peptide
66Ser Asn Gly Lys Glu Phe Asn Asn Trp Phe Asn Val 1 5 10
6712PRTArtificial SequenceSynthetic human CD69 partial peptide
67Lys Glu Phe Asn Asn Trp Phe Asn Val Thr Gly Ser 1 5 10
6812PRTArtificial SequenceSynthetic human CD69 partial peptide
68Asn Asn Trp Phe Asn Val Thr Gly Ser Asp Lys Cys 1 5 10
6912PRTArtificial SequenceSynthetic human CD69 partial peptide
69Phe Asn Val Thr Gly Ser Asp Lys Cys Val Phe Leu 1 5 10
7012PRTArtificial SequenceSynthetic human CD69 partial peptide
70Thr Gly Ser Asp Lys Cys Val Phe Leu Lys Asn Thr 1 5 10
7112PRTArtificial SequenceSynthetic human CD69 partial peptide
71Asp Lys Cys Val Phe Leu Lys Asn Thr Glu Val Ser 1 5 10
7212PRTArtificial SequenceSynthetic human CD69 partial peptide
72Val Phe Leu Lys Asn Thr Glu Val Ser Ser Met Glu 1 5 10
7312PRTArtificial SequenceSynthetic human CD69 partial peptide
73Lys Asn Thr Glu Val Ser Ser Met Glu Cys Glu Lys 1 5 10
7412PRTArtificial SequenceSynthetic human CD69 partial peptide
74Glu Val Ser Ser Met Glu Cys Glu Lys Asn Leu Tyr 1 5 10
7512PRTArtificial SequenceSynthetic human CD69 partial peptide
75Ser Met Glu Cys Glu Lys Asn Leu Tyr Trp Ile Cys 1 5 10
7612PRTArtificial SequenceSynthetic human CD69 partial peptide
76Cys Glu Lys Asn Leu Tyr Trp Ile Cys Asn Lys Pro 1 5 10
7712PRTArtificial SequenceSynthetic human CD69 partial peptide
77Lys Asn Leu Tyr Trp Ile Cys Asn Lys Pro Tyr Lys 1 5 10
786PRTArtificial SequenceSynthetic human CD69 partial peptide 78Tyr
Ala Gly Arg Glu Glu 1 5
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