U.S. patent application number 14/112590 was filed with the patent office on 2014-07-10 for diagnosis and treatment of cancer using anti-itm2a antibody.
This patent application is currently assigned to CHUGAI SEIYAKU KABUSHIKI KAISHA. The applicant listed for this patent is Hiroyuki Aburatani, Shumpei Ishikawa, Shigeto Kawai. Invention is credited to Hiroyuki Aburatani, Shumpei Ishikawa, Shigeto Kawai.
Application Number | 20140193420 14/112590 |
Document ID | / |
Family ID | 47041340 |
Filed Date | 2014-07-10 |
United States Patent
Application |
20140193420 |
Kind Code |
A1 |
Aburatani; Hiroyuki ; et
al. |
July 10, 2014 |
DIAGNOSIS AND TREATMENT OF CANCER USING ANTI-ITM2A ANTIBODY
Abstract
Disclosed is a monoclonal antibody binding to an ITM2A protein.
This antibody is useful in the diagnosis, prevention, and treatment
of cancer such as Ewing's sarcoma, T cell leukemia, T cell
lymphoma, acute myeloid leukemia, B cell tumor, and multiple
myeloma. The present invention also provides a pharmaceutical
composition, a cell growth inhibitor, and an anticancer agent
containing the antibody as an active ingredient, and a method for
treating cancer, a method for predicting the efficacy of cancer
treatment, and a method for determining the presence of cancer in a
test subject using the antibody.
Inventors: |
Aburatani; Hiroyuki; (Tokyo,
JP) ; Ishikawa; Shumpei; (Tokyo, JP) ; Kawai;
Shigeto; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Aburatani; Hiroyuki
Ishikawa; Shumpei
Kawai; Shigeto |
Tokyo
Tokyo
Tokyo |
|
JP
JP
JP |
|
|
Assignee: |
CHUGAI SEIYAKU KABUSHIKI
KAISHA
Kita-ku, Tokyo
JP
THE UNIVERSITY OF TOKYO
Bunkyo-ku, Tokyo
JP
|
Family ID: |
47041340 |
Appl. No.: |
14/112590 |
Filed: |
April 18, 2012 |
PCT Filed: |
April 18, 2012 |
PCT NO: |
PCT/JP12/02697 |
371 Date: |
January 16, 2014 |
Current U.S.
Class: |
424/139.1 ;
435/7.24; 530/387.3; 530/387.9 |
Current CPC
Class: |
G01N 2800/52 20130101;
A61P 35/02 20180101; C07K 16/3061 20130101; C07K 2317/732 20130101;
C07K 2317/73 20130101; C07K 2317/56 20130101; C07K 2317/565
20130101; A61P 35/00 20180101; G01N 33/57407 20130101; G01N
33/57426 20130101; C07K 16/28 20130101 |
Class at
Publication: |
424/139.1 ;
530/387.9; 530/387.3; 435/7.24 |
International
Class: |
C07K 16/30 20060101
C07K016/30; G01N 33/574 20060101 G01N033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 18, 2011 |
JP |
2011-092488 |
Claims
1. A monoclonal antibody binding to a fragment of an ITM2A protein
having the amino acid sequence represented by SEQ ID NO: 1.
2. The antibody according to claim 1, wherein the fragment is a
fragment consisting of amino acids 75 to 227 in the amino acid
sequence represented by SEQ ID NO: 1.
3. The antibody according to claim 1, wherein the antibody has a
cytotoxic activity.
4. The antibody according to claim 3, wherein the cytotoxic
activity is an antibody-dependent cell-mediated cytotoxicity (ADCC)
activity.
5. The antibody according to claim 3, wherein the cytotoxic
activity is a complement-dependent cytotoxicity (CDC) activity.
6. The antibody according to claim 1, wherein the antibody is
conjugated with a cytotoxic substance.
7. The antibody according to claim 6, wherein the antibody has an
internalization activity.
8. The antibody according to claim 1, wherein the antibody inhibits
cancer cell growth.
9. The antibody according to claim 8, wherein the cancer cell is a
Ewing's sarcoma cell.
10. The antibody according to claim 9, wherein the Ewing's sarcoma
cell is a cell having observable chromosomal translocation.
11. The antibody according to claim 10, wherein the chromosomal
translocation is t(11;22)(q24;q12).
12. The antibody according to claim 8, wherein the cancer cell is a
blood cancer cell.
13. The antibody according to claim 12, wherein the blood cancer is
any of T cell leukemia, T cell lymphoma, acute myeloid leukemia, B
cell tumor, and multiple myeloma.
14. An antibody described in any of the following (1) to (26): (1)
an antibody comprising an H chain having the amino acid sequence
represented by SEQ ID NO: 3 as CDR1, the amino acid sequence
represented by SEQ ID NO: 4 as CDR2, and the amino acid sequence
represented by SEQ ID NO: 5 as CDR3; (2) an antibody comprising an
L chain having the amino acid sequence represented by SEQ ID NO: 6
as CDR1, the amino acid sequence represented by SEQ ID NO: 7 as
CDR2, and the amino acid sequence represented by SEQ ID NO: 8 as
CDR3; (3) an antibody comprising the H chain described in (1) and
the L chain described in (2); (4) an antibody comprising an H chain
having the amino acid sequence represented by SEQ ID NO: 9 as CDR1,
the amino acid sequence represented by SEQ ID NO: 10 as CDR2, and
the amino acid sequence represented by SEQ ID NO: 11 as CDR3; (5)
an antibody comprising an L chain having the amino acid sequence
represented by SEQ ID NO: 12 as CDR1, the amino acid sequence
represented by SEQ ID NO: 13 as CDR2, and the amino acid sequence
represented by SEQ ID NO: 14 as CDR3; (6) an antibody comprising
the H chain described in (4) and the L chain described in (5); (7)
an antibody comprising an H chain having the amino acid sequence
represented by SEQ ID NO: 15 as CDR1, the amino acid sequence
represented by SEQ ID NO: 16 as CDR2, and the amino acid sequence
represented by SEQ ID NO: 17 as CDR3; (8) an antibody comprising an
L chain having the amino acid sequence represented by SEQ ID NO: 18
as CDR1, the amino acid sequence represented by SEQ ID NO: 19 as
CDR2, and the amino acid sequence represented by SEQ ID NO: 20 as
CDR3; (9) an antibody comprising the H chain described in (7) and
the L chain described in (8); (10) an antibody comprising an H
chain having the amino acid sequence represented by SEQ ID NO: 21
as CDR1, the amino acid sequence represented by SEQ ID NO: 22 as
CDR2, and the amino acid sequence represented by SEQ ID NO: 23 as
CDR3; (11) an antibody comprising an L chain having the amino acid
sequence represented by SEQ ID NO: 24 as CDR1, the amino acid
sequence represented by SEQ ID NO: 25 as CDR2, and the amino acid
sequence represented by SEQ ID NO: 26 as CDR3; (12) an antibody
comprising the H chain described in (10) and the L chain described
in (11); (13) the antibody described in any of (1) to (12) which is
a chimeric antibody; (14) the antibody described in any of (1) to
(12) which is a humanized antibody; (15) the antibody described in
(1) or (3), comprising the amino acid sequence represented by SEQ
ID NO: 28; (16) the antibody described in (2) or (3), comprising
the amino acid sequence represented by SEQ ID NO: 30; (17) the
antibody described in (4) or (6), comprising the amino acid
sequence represented by SEQ ID NO: 32; (18) the antibody described
in (5) or (6), comprising the amino acid sequence represented by
SEQ ID NO: 34; (19) the antibody described in (7) or (9),
comprising the amino acid sequence represented by SEQ ID NO: 36;
(20) the antibody described in (8) or (9), comprising the amino
acid sequence represented by SEQ ID NO: 38; (21) the antibody
described in (10) or (12), comprising the amino acid sequence
represented by SEQ ID NO: 40; (22) the antibody described in (11)
or (12), comprising the amino acid sequence represented by SEQ ID
NO: 42; (23) the antibody described in any of (15) to (22) which is
a chimeric antibody; (24) an antibody that has an amino acid
sequence of an antibody described in any of (1) to (23) with a
substitution, deletion, addition, and/or insertion of one or more
amino acid(s) and has an activity equivalent to or a binding
activity equivalent to that of the antibody; (25) an antibody
capable of binding to an epitope to which a second antibody binds,
wherein the second antibody is the antibody described in any of (1)
to (23); and (26) an antibody capable of inhibiting the binding of
a second antibody to an ITM2A protein fragment consisting of amino
acids 75 to 227 in the amino acid sequence represented by SEQ ID
NO: 1, wherein the second antibody is the antibody described in any
of (1) to (23).
15. The antibody according to claim 1, wherein the antibody has a
human constant region.
16. The antibody according to claim 15, wherein the antibody is a
chimeric antibody, a humanized antibody, or a human antibody.
17. The antibody according to claim 1, wherein the antibody is
deficient in fucose added to its sugar chain or has a sugar chain
having bisecting GlcNAc.
18. A pharmaceutical composition comprising an antibody according
to claim 1 as an active ingredient.
19. A cell growth inhibitor comprising an antibody according to
claim 1 as an active ingredient.
20. An anticancer agent comprising an antibody according to claim 1
as an active ingredient.
21. The anticancer agent according to claim 20, wherein the cancer
to be treated is Ewing's sarcoma.
22. The anticancer agent according to claim 21, wherein the Ewing's
sarcoma has observable chromosomal translocation.
23. The anticancer agent according to claim 22, wherein the
chromosomal translocation is t(11;22)(q24;q12).
24. The anticancer agent according to claim 20, wherein the cancer
cell is a blood cancer cell.
25. The anticancer agent according to claim 24, wherein the blood
cancer is any of T cell leukemia, T cell lymphoma, acute myeloid
leukemia, B cell tumor, and multiple myeloma.
26. A method for treating cancer, comprising administering an
antibody according to claim 1.
27. The method according to claim 26, wherein the cancer to be
treated is Ewing's sarcoma.
28. The method according to claim 27, wherein the Ewing's sarcoma
has observable chromosomal translocation.
29. The method according to claim 28, wherein the chromosomal
translocation is t(11;22)(q24;q12).
30. The method according to claim 26, wherein the cancer cell is a
blood cancer cell.
31. The method according to claim 30, wherein the blood cancer is
any of T cell leukemia, T cell lymphoma, acute myeloid leukemia, B
cell tumor, and multiple myeloma.
32. A method for predicting the efficacy of cancer treatment by the
administration of an antibody according to claim 1, comprising the
step of detecting the expression level of an ITM2A in a biological
sample collected from a test subject.
33. The method according to claim 32, wherein an ITM2A protein in
the sample collected from a test subject is detected.
34. The method according to claim 33, wherein the detection of the
ITM2A protein is performed using an antibody binding to the ITM2A
protein.
35. The method according to claim 32, wherein the cancer to be
treated is Ewing's sarcoma.
36. The method according to claim 35, wherein the Ewing's sarcoma
has observable chromosomal translocation.
37. The method according to claim 36, wherein the chromosomal
translocation is t(11;22)(q24;q12).
38. The method according to claim 35, wherein the cancer cell is a
blood cancer cell.
39. The method according to claim 38, wherein the blood cancer is
any of T cell leukemia, T cell lymphoma, acute myeloid leukemia, B
cell tumor, and multiple myeloma.
40. A method for determining the presence of cancer in a test
subject, comprising detecting an ITM2A protein in a sample
collected from the test subject.
41. A method for determining the presence of cancer in a test
subject, comprising the following steps: (a) providing a sample
collected from the test subject; and (b) detecting an ITM2A protein
contained in the sample of step (a) using an antibody binding to
the ITM2A protein.
42. A method for determining the presence of cancer in a test
subject, comprising the following steps: (a) administering, to the
test subject, a radioisotope-labeled antibody having a binding
activity to an ITM2A protein; and (b) detecting the accumulation of
the radioisotope.
43. The diagnosis method according to claim 40, wherein the cancer
whose presence is to be determined is Ewing's sarcoma.
44. The method according to claim 43, wherein the Ewing's sarcoma
has observable chromosomal translocation.
45. The method according to claim 44, wherein the chromosomal
translocation is t(11;22)(q24;q12).
46. The method according to claim 40, wherein the cancer is blood
cancer.
47. The method according to claim 46, wherein the blood cancer is
any of T cell leukemia, T cell lymphoma, acute myeloid leukemia, B
cell tumor, and multiple myeloma.
Description
TECHNICAL FIELD
Related Application
[0001] The present application claims the priority based on
Japanese Patent Application No. 2011-092488 (filed on Apr. 18,
2011). The contents thereof are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates to an antibody binding to an
ITM2A protein, a method for diagnosing cancer, a method for
treating cancer, and an anticancer agent.
BACKGROUND ART
[0003] The ITM2A molecule is a type II membrane protein that is
expressed in precursor cells involved in chondrogenesis or
osteogenesis (Non Patent Literature 1). This protein is known to be
expressed at the early stage of chondrogenesis (Non Patent
Literature 2) to inhibit the chondrogenesis (Non Patent Literature
3). ITM2A is also expressed in T cells in the thymus gland (Non
Patent Literature 4). The inhibition of T cell activation by an
anti-ITM2A polyclonal antibody is disclosed (Patent Literature 1).
Patent Literature 1 claims the treatment of T cell
leukemia/lymphoma using an anti-ITM2A antibody, but does not
specifically discuss the expression of ITM2A in T cell
leukemia/lymphoma or the treatment of these diseases. According to
the reports, the expression of ITM2A at the gene level in Ewing's
sarcoma or acute myeloid leukemia has been confirmed by microarray
analysis (Non Patent Literatures 5, 6, and 7). Nonetheless, it has
not been specifically confirmed so far that T cell
leukemia/lymphoma, Ewing's sarcoma, or acute myeloid leukemia can
be treated using an anti-ITM2A antibody.
[0004] References cited herein are as shown below. The contents
described in these literatures are incorporated herein by reference
in their entirety.
CITATION LIST
Patent Literature
[0005] Patent Literature 1: WO2008137500
Non Patent Literature
[0005] [0006] Non Patent Literature 1: J Biol Chem (1996) 271:
19475 [0007] Non Patent Literature 2: Biol Cell (2004) 96: 463
[0008] Non Patent Literature 3: Differentiation (2009) 78: 108
[0009] Non Patent Literature 4: J Exp Med (1999) 190: 217 [0010]
Non Patent Literature 5: Cancer Res (2004) 64: 8213 [0011] Non
Patent Literature 6: Cancer Res (2008) 68: 2176 [0012] Non Patent
Literature 7: PLoS One (2010) 5: e9466
SUMMARY OF INVENTION
Technical Problem
[0013] An object of the present invention is to provide a novel
antibody binding to an ITM2A protein, a novel method for diagnosing
cancer, a novel method for treating cancer, a novel cell growth
inhibitor, and an anticancer agent.
Solution to Problem
[0014] The present inventors have found that ITM2A mRNA is
expressed in Ewing's sarcoma having EWS-FLI1 translocation. The
present inventors have prepared an anti-ITM2A monoclonal antibody
and also found that ITM2A protein is expressed in Ewing's sarcoma,
acute myeloid leukemia, T cell lymphoma, and T cell acute
lymphocytic leukemia cell lines. The present inventors have further
found that the anti-ITM2A monoclonal antibody exerts an
antibody-dependent cell-mediated cytotoxicity (ADCC) activity and
inhibits the growth of the Ewing's sarcoma cells, acute myeloid
leukemia, and T cell lymphoma cell lines, and T cell acute
lymphocytic leukemia cells in the presence of a toxin-conjugated
secondary antibody. From these results, the present inventors have
found that the anti-ITM2A antibody is useful in the treatment and
diagnosis of cancer involving ITM2A expression, such as Ewing's
sarcoma, acute myeloid leukemia, T cell lymphoma, and T cell acute
lymphocytic leukemia, and consequently completed the present
invention.
[0015] Specifically, the present invention provides a monoclonal
antibody binding to an ITM2A protein. The present invention further
provides a monoclonal antibody which binds to an ITM2A protein and
has a cytotoxic activity against cells expressing the ITM2A
protein. Preferably, the cytotoxic activity is an ADCC activity.
The present invention also provides an anti-ITM2A monoclonal
antibody conjugated with a cytotoxic substance.
[0016] The present invention further provides a pharmaceutical
composition comprising the monoclonal antibody binding to an ITM2A
protein as an active ingredient. The present invention further
provides a cell growth inhibitor comprising the monoclonal antibody
binding to an ITM2A protein as an active ingredient. The present
invention further provides an anticancer agent comprising the
monoclonal antibody binding to an ITM2A protein as an active
ingredient.
[0017] The present invention further provides a pharmaceutical
composition comprising the monoclonal antibody binding to an ITM2A
protein and a pharmaceutically acceptable carrier. More
specifically, the present invention provides the following [1] to
[47]:
[1] a monoclonal antibody binding to a fragment of an ITM2A protein
having the amino acid sequence represented by SEQ ID NO: 1; [2] the
antibody according to [1], wherein the fragment is a fragment
consisting of amino acids 75 to 227 in the amino acid sequence
represented by SEQ ID NO: 1; [3] the antibody according to [1] or
[2], wherein the antibody has a cytotoxic activity; [4] the
antibody according to [3], wherein the cytotoxic activity is an
antibody-dependent cell-mediated cytotoxicity (ADCC) activity; [5]
the antibody according to [3], wherein the cytotoxic activity is a
complement-dependent cytotoxicity (CDC) activity; [6] the antibody
according to any of [1] to [5], wherein the antibody is conjugated
with a cytotoxic substance; [7] the antibody according to [6],
wherein the antibody has an internalization activity; [8] the
antibody according to any of [1] to [7], wherein the antibody
inhibits cancer cell growth; [9] the antibody according to [8],
wherein the cancer cell is a Ewing's sarcoma cell; [10] the
antibody according to [9], wherein the Ewing's sarcoma cell is a
cell having observable chromosomal translocation; [11] the antibody
according to [10], wherein the chromosomal translocation is
t(11;22)(q24;q12); [12] the antibody according to [8], wherein the
cancer cell is a blood cancer cell; [13] the antibody according to
[12], wherein the blood cancer is any of T cell leukemia, T cell
lymphoma, acute myeloid leukemia, B cell tumor, and multiple
myeloma; [14] an antibody described in any of the following (1) to
(27): (1) an antibody comprising an H chain having the amino acid
sequence represented by SEQ ID NO: 3 as CDR1, the amino acid
sequence represented by SEQ ID NO: 4 as CDR2, and the amino acid
sequence represented by SEQ ID NO: 5 as CDR3; (2) an antibody
comprising an L chain having the amino acid sequence represented by
SEQ ID NO: 6 as CDR1, the amino acid sequence represented by SEQ ID
NO: 7 as CDR2, and the amino acid sequence represented by SEQ ID
NO: 8 as CDR3; (3) an antibody comprising the H chain described in
(1) and the L chain described in (2); (4) an antibody comprising an
H chain having the amino acid sequence represented by SEQ ID NO: 9
as CDR1, the amino acid sequence represented by SEQ ID NO: 10 as
CDR2, and the amino acid sequence represented by SEQ ID NO: 11 as
CDR3; (5) an antibody comprising an L chain having the amino acid
sequence represented by SEQ ID NO: 12 as CDR1, the amino acid
sequence represented by SEQ ID NO: 13 as CDR2, and the amino acid
sequence represented by SEQ ID NO: 14 as CDR3; (6) an antibody
comprising the H chain described in (4) and the L chain described
in (5); (7) an antibody comprising an H chain having the amino acid
sequence represented by SEQ ID NO: 15 as CDR1, the amino acid
sequence represented by SEQ ID NO: 16 as CDR2, and the amino acid
sequence represented by SEQ ID NO: 17 as CDR3; (8) an antibody
comprising an L chain having the amino acid sequence represented by
SEQ ID NO: 18 as CDR1, the amino acid sequence represented by SEQ
ID NO: 19 as CDR2, and the amino acid sequence represented by SEQ
ID NO: 20 as CDR3; (9) an antibody comprising the H chain described
in (7) and the L chain described in (8); (10) an antibody
comprising an H chain having the amino acid sequence represented by
SEQ ID NO: 21 as CDR1, the amino acid sequence represented by SEQ
ID NO: 22 as CDR2, and the amino acid sequence represented by SEQ
ID NO: 23 as CDR3; (11) an antibody comprising an L chain having
the amino acid sequence represented by SEQ ID NO: 24 as CDR1, the
amino acid sequence represented by SEQ ID NO: 25 as CDR2, and the
amino acid sequence represented by SEQ ID NO: 26 as CDR3; (12) an
antibody comprising the H chain described in (10) and the L chain
described in (11); (13) the antibody described in any of (1) to
(12) which is a chimeric antibody; (14) the antibody described in
any of (1) to (12) which is a humanized antibody; (15) the antibody
described in (1) or (3), comprising the amino acid sequence
represented by SEQ ID NO: 28; (16) the antibody described in (2) or
(3), comprising the amino acid sequence represented by SEQ ID NO:
30; (17) the antibody described in (4) or (6), comprising the amino
acid sequence represented by SEQ ID NO: 32; (18) the antibody
described in (5) or (6), comprising the amino acid sequence
represented by SEQ ID NO: 34; (19) the antibody described in (7) or
(9), comprising the amino acid sequence represented by SEQ ID NO:
36; (20) the antibody described in (8) or (9), comprising the amino
acid sequence represented by SEQ ID NO: 38; (21) the antibody
described in (10) or (12), comprising the amino acid sequence
represented by SEQ ID NO: 40; (22) the antibody described in (11)
or (12), comprising the amino acid sequence represented by SEQ ID
NO: 42; (23) the antibody described in any of (15) to (22) which is
a chimeric antibody; (24) an antibody that has an amino acid
sequence of an antibody described in any of (1) to (23) with a
substitution, deletion, addition, and/or insertion of one or more
amino acid(s) and has an activity equivalent to or a binding
activity equivalent to that of the antibody; (25) an antibody
capable of binding to an epitope to which a second antibody binds,
wherein the second antibody is the antibody described in any of (1)
to (23); and (26) an antibody capable of inhibiting the binding of
a second antibody to an ITM2A protein fragment consisting of amino
acids 75 to 227 in the amino acid sequence represented by SEQ ID
NO: 1, wherein the second antibody is the antibody described in any
of (1) to (23); [15] the antibody according to any of [1] to [14],
wherein the antibody has a human constant region; [16] the antibody
according to [15], wherein the antibody is a chimeric antibody, a
humanized antibody, or a human antibody; [17] the antibody
according to any of [1] to [16], wherein the antibody is deficient
in fucose added to its sugar chain or has a sugar chain having
bisecting GlcNAc; [18] a pharmaceutical composition comprising an
antibody according to any of [1] to [17] as an active ingredient;
[19] a cell growth inhibitor comprising an antibody according to
any of [1] to [17] as an active ingredient; [20] an anticancer
agent comprising an antibody according to any of [1] to [17] as an
active ingredient; [21] the anticancer agent according to [20],
wherein the cancer to be treated is Ewing's sarcoma; [22] the
anticancer agent according to [21], wherein the Ewing's sarcoma has
observable chromosomal translocation; [23] the anticancer agent
according to [22], wherein the chromosomal translocation is
t(11;22)(q24;q12); [24] the anticancer agent according to [20],
wherein the cancer cell is a blood cancer cell; [25] the anticancer
agent according to [24], wherein the blood cancer is any of T cell
leukemia, T cell lymphoma, acute myeloid leukemia, B cell tumor,
and multiple myeloma; [26] a method for treating cancer, comprising
administering an antibody according to any of [1] to [17]; [27] the
method according to [26], wherein the cancer to be treated is
Ewing's sarcoma; [28] the method according to [27], wherein the
Ewing's sarcoma has observable chromosomal translocation; [29] the
method according to [28], wherein the chromosomal translocation is
t(11;22)(q24;q12); [30] the method according to [26], wherein the
cancer cell is a blood cancer cell; [31] the method according to
[30], wherein the blood cancer is any of T cell leukemia, T cell
lymphoma, acute myeloid leukemia, B cell tumor, and multiple
myeloma; [32] a method for predicting the efficacy of cancer
treatment by the administration of an antibody according to any of
[1] to [17], comprising the step of detecting the expression level
of an ITM2A in a biological sample collected from a test subject;
[33] the method according to [32], wherein an ITM2A protein in the
sample collected from a test subject is detected; [34] the
diagnosis method according to [33], wherein the detection of the
ITM2A protein is performed using an antibody binding to the ITM2A
protein; [35] the method according to any of [32] to [34], wherein
the cancer to be treated is Ewing's sarcoma; [36] the method
according to [35], wherein the Ewing's sarcoma has observable
chromosomal translocation; [37] the method according to [36],
wherein the chromosomal translocation is t(11;22)(q24;q12); [38]
the method according to [35], wherein the cancer cell is a blood
cancer cell; [39] the method according to [38], wherein the blood
cancer is any of T cell leukemia, T cell lymphoma, acute myeloid
leukemia, B cell tumor, and multiple myeloma; [40] a method for
determining the presence of cancer in a test subject, comprising
detecting an ITM2A protein in a sample collected from the test
subject; [41] a method for determining the presence of cancer in a
test subject, comprising the following steps: (a) providing a
sample collected from the test subject; and (b) detecting an ITM2A
protein contained in the sample of step (a) using an antibody
binding to the ITM2A protein; [42] a method for determining the
presence of cancer in a test subject, comprising the following
steps: (a) administering, to the test subject, a
radioisotope-labeled antibody having a binding activity to an ITM2A
protein; and (b) detecting the accumulation of the radioisotope;
[43] the method according to any of [40] to [42], wherein the
cancer whose presence is to be determined is Ewing's sarcoma; [44]
the method according to [43], wherein the Ewing's sarcoma has
observable chromosomal translocation; [45] the method according to
[44], wherein the chromosomal translocation is t(11;22)(q24;q12);
[46] the method according to [43], wherein the cancer cell is a
blood cancer cell; and [47] the method according to [46], wherein
the blood cancer is any of T cell leukemia, T cell lymphoma, acute
myeloid leukemia, B cell tumor, and multiple myeloma.
BRIEF DESCRIPTION OF DRAWINGS
[0018] FIG. 1 shows the expression profile of ITM2A mRNA in normal
tissues, Ewing's sarcoma cell lines, Ewing's sarcoma tissues, and
blood cancer cell lines obtained using Human Exon 1.0 ST Array.
FIG. 1(A) shows the expression profile of ITM2A mRNA in various
normal tissues. FIG. 1(B) shows the expression profile of ITM2A
mRNA in blood cancer cell lines, Ewing's sarcoma cell lines, and
Ewing's sarcoma tissues.
[0019] FIG. 2 shows the binding activity of an isolated anti-ITM2A
antibody to GST-ITM2A-L and GST-ITM2A-S obtained using ELISA assay.
FIG. 2(A) shows the binding activity of the anti-ITM2A antibody to
GST-ITM2A-L. FIG. 2(B) shows the binding activity of the anti-ITM2A
antibody to GST-ITM2A-S.
[0020] FIG. 3 shows results of examining binding domains of
isolated anti-ITM2A antibodies obtained using FACS. FIG. 3(A) shows
the binding activity of the anti-ITM2A antibodies to
ITM2A-expressing CHO cells. FIG. 3(B) shows the binding activity of
the anti-ITM2A antibodies to ITM2A-furin-expressing CHO cells. FIG.
3(C) shows the binding activity of the anti-ITM2A antibodies to CHO
cells. FIG. 3(D) shows the binding activity of an anti-HA antibody
to ITM2A- or ITM2A-furin-expressing CHO cells.
[0021] FIG. 4 shows results of examining interspecies cross
reactivity of the isolated anti-ITM2A antibodies. FIG. 4(A) shows
the binding activity of the anti-ITM2A antibodies to human
ITM2A-expressing CHO cells. FIG. 4(B) shows the binding activity of
the anti-ITM2A antibodies to mouse ITM2A-expressing CHO cells.
[0022] FIG. 5 shows results of evaluating the binding activity of
the isolated anti-ITM2A antibodies to ITM2A by Western blot. Lane A
represents a whole cell lysate of ITM2A-expressing CHO cells. Lane
B represents a whole cell lysate of ITM2A-furin-expressing CHO
cells. Lane C represents a whole cell lysate of CHO cells. FIG.
5(A) shows the binding activity of the antibody BE5-1. FIG. 5(B)
shows the binding activity of the antibody BE6-1. FIG. 5(C) shows
the binding activity of the antibody BE7-1-1. FIG. 5(D) shows the
binding activity of the antibody BE13-1. FIG. 5(E) shows the
binding activity of the anti-HA antibody.
[0023] FIG. 6 shows the expression of ITM2A in human cancer cell
lines examined using FACS. FIG. 6(A) shows the expression of ITM2A
in the Ewing's sarcoma cell line A-673. FIG. 6(B) shows the
expression of ITM2A in the Ewing's sarcoma cell line RD-ES. FIG.
6(C) shows the expression of ITM2A in the Ewing's sarcoma cell line
SK-ES-1. FIG. 6(D) shows the expression of ITM2A in the Ewing's
sarcoma cell line SK-N-MC. FIG. 6(E) shows the expression of ITM2A
in the T cell acute lymphocytic leukemia cell line CCRF-CEM. FIG.
6(F) shows the expression of ITM2A in the T cell acute lymphocytic
leukemia cell line Jurkat. FIG. 6(G) shows the expression of ITM2A
in the T cell acute lymphocytic leukemia cell line MOLT4. FIG. 6(H)
shows the expression of ITM2A in the T cell lymphoma cell line
HuT78. FIG. 6(I) shows the expression of ITM2A in the acute myeloid
leukemia cell line KG-1a. FIG. 6(J) shows the expression of ITM2A
in the acute myeloid leukemia cell line TF-1a.
[0024] FIG. 7 shows an ADCC activity exerted by an isolated
anti-ITM2A antibody against various cancer cell lines. FIG. 7(A)
shows the ADCC activity against the Ewing's sarcoma cell line
A-673. FIG. 7(B) shows the ADCC activity against the Ewing's
sarcoma cell line SK-N-MC. FIG. 7(C) shows the ADCC activity
against the T cell acute lymphocytic leukemia cell line CCRF-CEM.
FIG. 7(D) shows the ADCC activity against the acute myeloid
leukemia cell line KG-1a.
[0025] FIG. 8 shows a cytotoxic activity exerted by an isolated
anti-ITM2A antibody against various cancer cell lines in the
presence of a toxin-conjugated secondary antibody. FIG. 8(A) shows
the cytotoxic activity against the Ewing's sarcoma cell line A-673.
FIG. 8(B) shows the cytotoxic activity against the T cell acute
lymphocytic leukemia cell line CCRF-CEM. FIG. 8(C) shows the
cytotoxic activity against the T cell lymphoma cell line HuT78.
[0026] FIG. 9 shows results of evaluating the expression of
EWS-FLI1 fusion genes and ITM2A in clinical Ewing's sarcoma samples
by PCR. FIG. 9(A) shows the expression of EWS-FLI1 fusion genes in
clinical Ewing's sarcoma samples. FIG. 9(B) shows the expression of
ITM2A in clinical Ewing's sarcoma samples.
DESCRIPTION OF EMBODIMENTS
ITM2A
[0027] In the present invention, ITM2A is a type II membrane
protein. The amino acid sequence of human ITM2A and a gene sequence
encoding this amino acid sequence are disclosed in NCBI Accession
Nos. NP.sub.--004858.1 (SEQ ID NO: 1) and NM.sub.--004867.4 (SEQ ID
NO: 2), respectively. An ITM2A used in the present invention may be
a splicing variant or a variant (or a mutant). In the present
invention, the ITM2A protein is meant to include both of the
full-length protein and its fragment. The fragment refers to a
polypeptide comprising an arbitrary region of the ITM2A protein and
may not have the functions of the natural ITM2A protein. Examples
of the fragment include a fragment comprising the extracellular
region of the ITM2A protein. The extracellular region of the ITM2A
protein corresponds to positions 75 to 263 in the amino acid
sequence of SEQ ID NO: 1. In another aspect, examples of the
fragment preferably include a polypeptide consisting of amino acids
75 to 227 in the ITM2A protein represented by SEQ ID NO: 1.
Preparation of Anti-ITM2A Antibody
[0028] The anti-ITM2A antibody used in the present invention needs
only to bind to the ITM2A protein and is not limited by its origin,
type, shape, etc. Specifically, an antibody known in the art can be
used, such as a non-human animal antibody (e.g., a mouse, rat, or
camel antibody), a human antibody, a chimeric antibody, or a
humanized antibody. In the present invention, a monoclonal or
polyclonal antibody can be used. A monoclonal antibody can be
preferably used. The binding of the antibody to the ITM2A protein
is preferably specific binding. Also, the anti-ITM2A antibody used
in the present invention may be an antibody that recognizes human
ITM2A. In such a case, an antibody that specifically recognizes
human ITM2A can be used. Alternatively, an antibody that
simultaneously recognizes human ITM2A and non-human animal-derived
ITM2A (e.g., mouse ITM2A) can also be preferably used.
[0029] The anti-ITM2A antibody used in the present invention can be
obtained as a polyclonal or monoclonal antibody using means known
in the art. The anti-ITM2A antibody used in the present invention
is particularly preferably a mammal-derived monoclonal antibody.
The mammal-derived monoclonal antibody encompasses, for example,
those produced by hybridomas and those produced by hosts
transformed with expression vectors containing an antibody gene by
a genetic engineering approach.
[0030] Basically, monoclonal antibody-producing hybridomas can be
prepared according to a technique known in the art as follows:
first, animals are immunized with an ITM2A protein used as a
sensitizing antigen according to a usual immunization method.
Immunocytes obtained from the immunized animals can be fused with
parental cells known in the art by a usual cell fusion method to
obtain hybridomas. These hybridomas can be further screened for
cells producing the antibody of interest by a usual screening
method to select hybridomas producing anti-ITM2A antibodies.
[0031] Specifically, a monoclonal antibody is prepared, for
example, as follows: first, ITM2A gene can be expressed to obtain
ITM2A protein used as a sensitizing antigen to obtain antibodies.
The nucleotide sequence of the ITM2A gene is disclosed in, for
example, NCBI Accession No. NM.sub.--004867.4 (SEQ ID NO: 2).
Specifically, an ITM2A-encoding gene sequence is inserted into an
expression vector known in the art, with which appropriate host
cells are then transformed. Then, the human ITM2A protein of
interest can be purified from the host cells or from a culture
supernatant thereof by a method known in the art. Also, purified
natural ITM2A protein can be used similarly. Alternatively, fusion
proteins comprising the desired partial polypeptide of the ITM2A
protein fused with a different polypeptide may be used as
immunogens, as used in the present invention. For example, antibody
Fc fragments, peptide tags, and the like can be used for producing
the fusion proteins serving as immunogens. Two or more genes
respectively encoding the desired polypeptide fragments are fused
in frame, and the fusion gene can be inserted into expression
vectors to prepare expression vectors for the fusion proteins. The
method for preparing the fusion proteins is described in Molecular
Cloning 2nd ed. (Sambrook, J. et al., Molecular Cloning 2nd ed.,
9.47-9.58, Cold Spring Harbor Lab. Press, 1989).
[0032] An ITM2A protein thus purified can be used as a sensitizing
antigen for the immunization of a mammal. A partial peptide of
ITM2A may also be used as a sensitizing antigen. For example, the
following peptides can be used as a sensitizing antigen:
a peptide obtained by chemical synthesis on the basis of the amino
acid sequence of human ITM2A, a peptide obtained by the
incorporation of a portion of the ITM2A gene into an expression
vector, followed by gene expression, and a peptide obtained by the
degradation of the ITM2A protein with a proteolytic enzyme.
[0033] The region and size of the partial peptide of ITM2A used are
not limited. A preferable region can be selected from the amino
acid sequence constituting the extracellular domain of ITM2A
(positions 75 to 263 in the amino acid sequence of SEQ ID NO: 2).
The number of amino acids constituting the peptide serving as a
sensitizing antigen is preferably at least 3 or more, for example,
5 or more or 6 or more. More specifically, peptides of 6 to 263
residues, preferably 7 to 200 residues, more preferably 8 to 100, 8
to 50, or 10 to 30 residues, can be used as sensitizing
antigens.
[0034] The mammal to be immunized with the sensitizing antigen is
not particularly limited. For obtaining the monoclonal antibody by
the cell fusion method, the animal to be immunized is preferably
selected in consideration of compatibility with the parental cells
used in cell fusion. In general, a rodent is preferable as the
animal to be immunized. Specifically, mouse, rat, hamster, or
rabbit can be used as the animal to be immunized. In addition,
monkey or the like may be used as the animal to be immunized.
[0035] The animal can be immunized with the sensitizing antigen
according to a method known in the art. For example, a general
method can involve immunizing the mammal with the sensitizing
antigen by intraperitoneal or subcutaneous injection. Specifically,
the sensitizing antigen is administered to the mammal several times
at 4- to 21-day intervals. The sensitizing antigen is diluted with
PBS (phosphate-buffered saline), saline, or the like at an
appropriate dilution ratio and used in the immunization. The
sensitizing antigen may be administered together with an adjuvant.
For example, the antigen is mixed with a Freund's complete adjuvant
and emulsified, and the resulting emulsion can be used as the
sensitizing antigens. Also, an appropriate carrier can be used in
the immunization with the sensitizing antigens. Particularly, in
the case of using partial peptides having a small molecular weight
as the sensitizing antigen, the sensitizing antigen peptide bound
with a carrier protein such as albumin or keyhole limpet hemocyanin
can be preferably used in the immunization.
[0036] On the other hand, the monoclonal antibody can also be
obtained by DNA immunization. The DNA immunization is an
immunostimulation method involving: immunizing an animal by the
administration of vector DNA that has been constructed in a form
capable of expressing an antigenic protein-encoding gene (e.g., the
gene represented by SEQ ID NO: 2) in the immunized animal; and
allowing the immunized animal to express the immunizing antigen in
vivo. The DNA immunization can be expected to be superior to
general immunization methods using the administration of a protein
antigen as follows:
[0037] the DNA immunization can provide immunostimulation with
membrane protein (e.g., ITM2A) with its structure maintained;
and
[0038] the DNA immunization eliminates the need of purifying an
immunizing antigen.
[0039] The DNA immunization, however, is difficult to combine with
immunostimulation means such as an adjuvant. The amino acid
sequence of ITM2A is highly homologous among species. The amino
acid sequence of human-derived ITM2A represented by SEQ ID NO: 1
has identity of 99%, 98%, 96%, 94%, 94%, and 90% to, for example,
the amino acid sequences of rabbit (Oryctolagus cuniculus)-,
horse-, mouse-, giant panda-, rat-, and pig-derived ITM2A proteins,
respectively. In light of such structural identity, it is an
unexpected consequence that the monoclonal antibody binding to
ITM2A was obtained by the DNA immunization and the administration
of protein antigens involving immunostimulation means such as
adjuvant.
[0040] In order to obtain the monoclonal antibody of the present
invention by the DNA immunization, an animal is first immunized by
the administration of a DNA expressing an ITM2A protein. An
ITM2A-encoding DNA can be synthesized by a method known in the art
such as PCR. The obtained DNA is inserted into an appropriate
expression vector, which is then administered to an animal. For
example, a commercially available expression vector such as
pcDNA3.1 can be used as the expression vectors. Also, a method
generally used can be used for administering the vectors to the
animals. For example, gold particles with the expression vector
adsorbed thereon can be inserted into cells using a gene gun to
perform the DNA immunization.
[0041] A rise in the amount of the desired antibody is confirmed in
the serum of the mammals thus immunized. Then, immunocytes are
collected from the mammal and subjected to cell fusion.
Particularly, spleen cells can be used as preferable
immunocytes.
[0042] Mammalian myeloma cells are used in the cell fusion with the
immunocytes. The myeloma cells preferably have an appropriate
selection marker for screening. The selection marker refers to a
character that can survive (or cannot survive) under particular
culture conditions. For example, hypoxanthine-guanine
phosphoribosyltransferase deficiency (hereinafter, abbreviated to
HGPRT deficiency) or thymidine kinase deficiency (hereinafter,
abbreviated to TK deficiency) is known in the art as the selection
marker. Cells having the HGPRT or TK deficiency is sensitive to
hypoxanthine-aminopterin-thymidine (hereinafter, abbreviated to
HAT-sensitive). The HAT-sensitive cells are killed in a HAT
selective medium because the cells fail to synthesize DNA. By
contrast, these cells, when fused with normal cells, grow even in
the HAT selective medium because the fused cells can continue DNA
synthesis by use of the salvage pathway of the normal cells.
[0043] The cells having the HGPRT or TK deficiency can be selected
in a medium containing 6-thioguanine or 8-azaguanine (hereinafter,
abbreviated to 8AG) for the HGPRT deficiency or
5'-bromodeoxyuridine for the TK deficiency. The normal cells are
killed by incorporating these pyrimidine analogs into their DNAs.
By contrast, the cells deficient in these enzymes can survive in
the selective medium because the cells cannot incorporate the
pyrimidine analogs therein. A selection marker based on an index of
2-deoxystreptamine antibiotic (gentamicin analog) resistance
brought about by a neomycin resistance gene is called G418
resistance. Various myeloma cells suitable for the cell fusion are
known in the art. For example, the following myeloma cells can be
used in the production of the monoclonal antibody according to the
present invention:
P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U.1
(Current Topics in Microbiology and Immunology (1978) 81, 1-7),
NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6,
511-519), MPC-11 (Margulies. D. H. et al., Cell (1976) 8,
405-415),
SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270),
[0044] FO (de St. Groth, S. F. et al., J. Immunol. Methods (1980)
35, 1-21), S194 (Trowbridge, I. S. J. Exp. Med. (1978) 148,
313-323),
R210 (Galfre, G. et al., Nature (1979) 277, 131-133), etc.
[0045] Basically, the cell fusion of the immunocytes with the
myeloma cells is performed according to a method known in the art,
for example, the method of Kohler and Milstein et al. (Kohler. G.
and Milstein, C., Methods Enzymol. (1981) 73, 3-46).
[0046] More specifically, the cell fusion can be carried out, for
example, in a usual nutrient culture medium in the presence of a
cell fusion promoter. For example, polyethylene glycol (PEG) or
hemagglutinating virus of Japan (HVJ) can be used as the fusion
promoter. In addition, an auxiliary such as dimethyl sulfoxide is
preferably added thereto, if desired, for enhancing fusion
efficiency.
[0047] The ratio between the immunocytes and the myeloma cells used
can be arbitrarily set. For example, the amount of the immunocytes
is preferably set to 1 to 10 times that of the myeloma cells. For
example, an RPMI1640 or MEM culture medium suitable for the growth
of the myeloma cell line as well as a usual culture medium used in
this kind of cell culture can be used as the culture medium in the
cell fusion. A solution supplemented with serum (e.g., fetal calf
serum (FCS)) can be further added to the culture medium.
[0048] In the procedures of the cell fusion, the immunocytes and
the myeloma cells are well mixed in the predetermined amounts in
the culture medium and mixed with a PEG solution preheated to
approximately 37.degree. C. to form the fusion cells (hybridomas)
of interest. In the procedures of the cell fusion, for example, PEG
having an average molecular weight on the order of 1000 to 6000 can
usually be added at a concentration of 30 to 60% (w/v) to the cell
suspension containing the immunocytes and the myeloma cells.
Subsequently, the appropriate culture medium exemplified above is
sequentially added to the cell suspension, and its supernatant is
removed by centrifugation. This removal procedure is repeated to
remove the cell fusion agents or the like unfavorable for hybridoma
growth.
[0049] The hybridomas thus obtained can be grown in a selective
medium appropriate for the selection marker of the myeloma cells
used in the cell fusion. For example, the cells having the HGPRT or
TK deficiency can be selected by culture in a HAT medium (culture
medium containing hypoxanthine, aminopterin, and thymidine).
Specifically, when HAT-sensitive myeloma cells are used in the cell
fusion, only cells successfully fused with normal cells can be
grown selectively in the HAT culture medium. The culture using the
HAT culture medium is continued for a time long enough to kill
cells (non-fused cells) other than the hybridomas of interest.
Specifically, the culture can generally be performed for a few days
to a few weeks to select the hybridomas of interest. Subsequently,
hybridomas producing the antibody of interest are screened for and
cloned as single clones by a usual limiting dilution method.
Alternatively, the antibody that recognizes ITM2A may be prepared
by a method described in International Publication No.
WO2003104453.
[0050] The screening for the antibody of interest and the cloning
as single clones thereof can be preferably carried out by a
screening method based on antigen-antibody reaction known in the
art. For example, a carrier (e.g., beads made of polystyrene or the
like) or a commercially available 96-well microtiter plate bound
with antigens are reacted with the culture supernatant of the
hybridomas. Subsequently, the carrier is washed and then reacted
with enzyme-labeled secondary antibodies or the like. When the
culture supernatant contains the antibody of interest reactive with
the sensitizing antigen, the secondary antibodies bind to the
carrier via this antibody. The secondary antibodies bound with the
carrier can be finally detected to determine the presence of the
antibody of interest in the culture supernatant. As described
above, the hybridomas producing the desired antibody capable of
binding to the antigen can be cloned by a limiting dilution method
or the like. In this screening and cloning as single clones, in
addition to the ITM2A protein used in the immunization, an ITM2A
protein substantially identical thereto can be preferably used as
an antigen. As an example of the ITM2A protein substantially
identical thereto, cell lines expressing ITM2A, the extracellular
domain of ITM2A, or oligopeptides consisting of a partial amino
acid sequence constituting the domain can be used as an
antigen.
[0051] In addition to the method for obtaining the hybridomas by
immunizing non-human animal with the antigen, human lymphocytes may
be sensitized with the antigen to obtain the desired antibody.
Specifically, the human lymphocytes are first sensitized with an
ITM2A protein in vitro. Subsequently, the sensitized lymphocytes
are fused with appropriate fusion partners. For example,
human-derived myeloma cells capable of dividing throughout their
lives can be used as the fusion partners (Japanese Patent
Publication No. 1-59878). For example, human myeloma cells such as
U266 can be used as the fusion partners. The anti-ITM2A antibody
obtained by this method is a human antibody having a binding
activity to the ITM2A protein.
[0052] The anti-ITM2A human antibody can also be obtained by
administering the antigen ITM2A protein to transgenic animals
having all repertoires of human antibody genes or by immunizing the
animals with a DNA that has been constructed so as to express ITM2A
in the animals. Antibody-producing cells from the immunized animals
can acquire immortalizing characters by cell fusion with
appropriate fusion partners or infection with Epstein-Barr virus.
From the immortalized cells thus obtained, human antibodies against
the ITM2A protein can be isolated (WO1994025585, WO1993012227,
WO1992003918, and WO1994002602). The immortalized
antibody-producing cells can be further cloned as cells producing
antibodies having the reaction specificity of interest. In the case
of immunizing the transgenic animals, the immune systems of the
animals recognize human ITM2A as foreigners. Thus, the human
antibodies against human ITM2A can be easily obtained.
[0053] The monoclonal antibody-producing hybridomas thus prepared
can be subcultured in a usual medium. The hybridomas can also be
stored over a long period in liquid nitrogen.
[0054] The hybridomas can be cultured according to a usual method.
The monoclonal antibody of interest can be obtained from the
culture supernatant thereof. Alternatively, the hybridomas may be
administered to a mammal compatible therewith and grown, and the
monoclonal antibody can be obtained from the ascitic fluids
thereof. The former method is suitable for obtaining highly pure
antibodies.
[0055] In the present invention, an antibody encoded by an antibody
gene cloned from an antibody-producing cell may also be used. The
cloned antibody gene incorporated in an appropriate vector is
expressed as an antibody encoded thereby in a host by the
introduction of the vector into the host. Methods for the antibody
gene isolation, the introduction into vector, and the
transformation of host cells have already been established (e.g.,
Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-775).
[0056] For example, cDNAs encoding the variable regions (V regions)
of the anti-ITM2A antibody can be obtained from the anti-ITM2A
antibody-producing hybridoma cells. In order to obtain the cDNAs,
usually, the total RNA is first extracted from the hybridoma. For
example, the following methods can be used for the total RNA
extraction from the cells:
guanidine ultracentrifugation method (Chirgwin, J. M. et al.,
Biochemistry (1979) 18, 5294-5299), and AGPC method (Chomczynski,
P. et al., Anal. Biochem. (1987) 162, 156-159).
[0057] From the extracted total RNA, mRNA can be purified using
mRNA Purification Kit (manufactured by GE Healthcare Bio-Sciences
Corp.) or the like. Alternatively, a kit for directly extracting
total mRNA from cells is also commercially available, such as
QuickPrep mRNA Purification Kit (manufactured by GE Healthcare
Bio-Sciences Corp.). The total mRNA may be obtained from the
hybridoma using such a kit. From the mRNA thus obtained, antibody V
region-encoding cDNAs can be synthesized using reverse
transcriptase. Arbitrary 15- to 30-base sequences selected from
sequences common to mouse antibody genes can be used as primers for
cDNA synthesis. Specifically, the antibody V region-encoding cDNAs
can be obtained using primers having a DNA sequence represented by
any of SEQ ID NOs: 97 to 100. The cDNAs can be synthesized using
reverse transcriptase such as AMV Reverse Transcriptase
First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corp.).
Alternatively, 5'-Ampli FINDER RACE Kit (manufactured by Clontech
Laboratories, Inc.) and 5'-RACE PCR (Frohman, M. A. et al., Proc.
Natl. Acad. Sci. USA (1988) 85, 8998-9002; and Belyavsky, A. et
al., Nucleic Acids Res. (1989) 17, 2919-2932) may be appropriately
used for the cDNA synthesis and amplification. In the course of
such cDNA synthesis, appropriate restriction sites as described
later can be introduced into both ends of the cDNAs.
[0058] Also, a cDNA library may be appropriately used for obtaining
the antibody variable region-encoding genes. First, cDNAs are
synthesized with mRNA extracted from the antibody-producing cells
as template to obtain a cDNA library. A commercially available kit
can be appropriately used in the cDNA library synthesis. In
actuality, mRNA from only a small number of cells are obtained in a
very small amount. Therefore, the direct purification of the mRNA
generally results in low mRNA yields. Thus, the mRNA are usually
purified from a mRNA preparation supplemented with carrier RNA
shown to be free from the antibody genes. Alternatively, RNA may be
extracted in a given amount. In such a case, antibody variable
region-encoding mRNAs can be efficiently extracted from RNA
obtained only from the antibody-producing cells without the
addition of carrier RNA. The addition of the carrier RNA may be
unnecessary for RNA extraction from, for example, 10 or more or 30
or more, preferably 50 or more antibody-producing cells.
[0059] The antibody genes can be amplified by PCR using the
obtained cDNA library as template. A plurality of primers for the
PCR amplification of the antibody genes are known in the art. For
example, primers for human antibody gene amplification can be
preferably designed on the basis of the disclosure of the paper (J.
Mol. Biol. (1991) 222, 581-597) or the like. These primers have
their respective nucleotide sequences differing on an
immunoglobulin subclass basis. Thus, when cDNA library whose
subclass is unknown is used as template, PCR is carried out using
primers in consideration of every possibility.
[0060] From the PCR products thus obtained, the desired cDNA
fragments are purified. Subsequently, the purified cDNA fragments
are ligated with a vector DNA. The recombinant vectors thus
prepared are introduced into E. coli or the like. A colony of E.
coli transformed with the recombinant vectors is selected. A
recombinant vector can be isolated from the E. coli that has formed
the colony. Then, the cDNA inserted in the isolated recombinant
vector can be sequenced by a method known in the art, for example,
a dideoxynucleotide chain termination method.
[0061] Specifically, primers capable of amplifying genes
respectively encoding .gamma.1 to .gamma.5 heavy chains and .kappa.
and .lamda. light chains can be used, for example, for the purpose
of obtaining human IgG-encoding genes. Primers annealing to a
portion corresponding to the hinge region are generally used as 3'
primers for amplifying IgG variable region genes. On the other
hand, primers appropriate for each subclass are used as 5'
primers.
[0062] The PCR products obtained by amplification using the primers
for gene amplification corresponding to these heavy and light chain
subclasses are synthesized as their respective independent gene
library. The library thus synthesized can be combined to reshape
immunoglobulins comprising the heavy and light chains in
combination. The immunoglobulins thus reshaped can be screened for
the antibody of interest with their binding activities to ITM2A as
an index.
[0063] For example, for obtaining the antibody against ITM2A, more
preferably, the antibody specifically binds to ITM2A. The antibody
binding to ITM2A can be screened for, for example, by the following
steps:
(1) contacting antibodies comprising V regions encoded by the
obtained cDNAs, with ITM2A; (2) detecting a complex of an
ITM2A-bound antibody; and (3) selecting the antibody binding to
ITM2A.
[0064] The complex of an ITM2A-bound antibody (antigen-antibody
complex) is detected by a method known in the art. Specifically, a
test antibody is contacted with ITM2A immobilized on a carrier.
Next, a labeled antibody that recognizes the antibody is contacted
therewith. When the labeled antibody remaining on the carrier after
washing of the carrier is detected, the binding of the test
antibody to ITM2A can be demonstrated. An enzymatically active
protein such as peroxidase or .beta.-galactosidase, or a
fluorescent material such as FITC can be appropriately used in the
antibody labeling. Also, fixed preparations of ITM2A-expressing
cells can be appropriately used for evaluating the binding activity
of the antibody.
[0065] Panning using phage vectors may be used as a method for
screening for the antibody with its binding activity as an index.
When the antibody genes are obtained as libraries of heavy and
light chain subclasses as described above, a screening method using
phage vectors is advantageous. Genes respectively encoding heavy
and light chain variable regions can be linked via an appropriate
linker sequence to prepare genes encoding single chain Fv (scFv)
molecules in which the heavy and light variable regions of the
antibody are arranged on one chain. The scFv-encoding genes can be
inserted to phage vectors to obtain phages expressing scFvs on
their surface. The phages thus obtained are contacted with the
antigen of interest. Then, antigen-bound phages are recovered. In
this way, DNAs encoding scFvs having a binding activity to the
desired antigen can be recovered. This procedure can be repeated,
if necessary, to concentrate scFvs having the desired binding
activity to the antigen.
[0066] In the present invention, the polynucleotide encoding the
antibody may be a polynucleotide encoding the full-length antibody
or may be a polynucleotide encoding a portion of the antibody. The
term "a portion of the antibody" refers to an arbitrary portion of
the antibody molecule. Hereinafter, the term "antibody fragment" is
also used to represent a portion of the antibody. The antibody
fragment according to the present invention is preferably an
antibody fragment comprising a complementarity determining region
(CDR) of the antibody. An antibody fragment comprising heavy and
light chain complementarity determining regions (CDRs) is also
preferable. More preferably, the antibody fragment of the present
invention is an antibody fragment comprising three CDRs of a heavy
chain variable region or/and a light chain variable region.
[0067] After obtainment of each cDNA encoding the V region of the
desired anti-ITM2A antibody, the cDNA is digested with restriction
enzymes that recognize the restriction sites inserted in both ends
of the cDNA. The restriction enzymes are preferably restriction
enzymes that can recognize and digest a nucleotide sequence
unlikely to appear in the nucleotide sequences constituting the
antibody genes. For inserting one copy of the digested fragment in
the correct direction in an expression vector, restriction sites
that provide cohesive ends are preferably inserted in the ends of
the cDNA. The anti-ITM2A antibody V region-encoding cDNAs thus
digested can be inserted to appropriate expression vectors to
obtain antibody expression vectors. In this case, antibody constant
region (C region)-encoding genes and the V region-encoding genes
can be fused in frame to obtain chimeric antibodies. In this
context, the chimeric antibodies refer to antibodies comprising
constant and variable regions of different origins. Thus,
heterogeneous (e.g., mouse-human) chimeric antibodies as well as
human-human homogeneous chimeric antibodies are also encompassed by
the chimeric antibody according to the present invention. The V
region genes may be inserted in frame to expression vectors
preliminarily having constant region-encoding DNA inserts to
construct chimeric antibody expression vectors.
[0068] Specifically, for example, recognition sequences for
restriction enzymes that digest the ends of the restriction sites
inserted in both ends of the V region gene can be located on the 5'
side of each expression vector carrying the DNA encoding the
desired antibody constant region (C region). This expression vector
and a vector having an insert of the V region gene are digested
with the same combination of restriction enzymes. The resulting
expression vector and V region gene are fused in frame to construct
a chimeric antibody expression vector.
[0069] In order to produce the anti-ITM2A antibody used in the
present invention, the antibody genes can be inserted into an
expression vector such that these genes are expressed under the
control of expression control regions. The expression control
regions for antibody expression encompass, for example, enhancers
and promoters. Subsequently, appropriate host cells can be
transformed with these expression vectors to obtain recombinant
cells expressing anti-ITM2A antibody-encoding DNAs.
[0070] For the antibody gene expression, the antibody heavy chain
(H chain)- and light chain (L chain)-encoding DNAs can be
incorporated separately in different expression vectors. The same
host cell can be co-transfected with the H chain- and L
chain-incorporated vectors and thereby allowed to express antibody
molecules comprising the H and L chains. Alternatively, the H
chain- and L chain-encoding DNAs may be inserted to a single
expression vector, with which host cells can also be transformed to
express antibody molecules comprising the H and L chains
(WO1994011523).
[0071] Expression systems having many combinations of hosts and
expression vectors used for preparing the antibody by introducing
the isolated antibody genes into appropriate hosts are known in the
art. All of these expression systems can be applied to the present
invention. In the case of using eukaryotic cells as the hosts,
animal, plant, or fungus cells can be used. Specifically, examples
of the animal cells that can be used in the present invention can
include the following cells:
(1) mammalian cells such as CHO, COS, myeloma, BHK (baby hamster
kidney), Hela, Vero, HEK293, Ba/F3, HL-60, Jurkat, and SK-HEP1
cells; (2) amphibian cells such as Xenopus oocytes; and (3) insect
cells such as sf9, sf21, and Tn5 cells.
[0072] Alternatively, antibody gene expression systems using cells
derived from the genus Nicotiana (e.g., Nicotiana tabacum) as the
plant cells that can be used in the present invention are known in
the art. Cultured callus cells can be appropriately used for the
plant cell transformation.
[0073] The following cells can be used as the fungus cells
according to the present invention:
cells derived from yeasts of the genus Saccharomyces (e.g.,
Saccharomyces cerevisiae) and the genus Pichia (e.g., Pichia
pastoris), and cells derived from filamentous fungi of the genus
Aspergillus (e.g., Aspergillus niger).
[0074] Alternatively, antibody gene expression systems using
prokaryotic cells as hosts are also known in the art as the
expression systems that can be used in the present invention. In
the case of using, for example, bacterial cells, cells of bacteria
such as E. coli and Bacillus subtilis can be preferably used in the
present invention.
[0075] In the case of using mammalian cells in the present
invention, a useful promoter routinely used (regardless of the
presence or absence of an enhancer), the antibody gene to be
expressed, and a poly A signal to be located 3'-downstream thereof
can be functionally linked so that the antibody gene is expressed.
Examples of the promoter/enhancer preferably include a human
cytomegalovirus immediate early promoter/enhancer.
[0076] In addition, for example, virus promoters/enhancers or
mammalian cell-derived promoters/enhancers (e.g., human elongation
factor 1.alpha. (HEF1.alpha.)) may be used for the antibody
expression. Specific examples of the viruses whose
promoter/enhancer can be used preferably include retrovirus,
polyomavirus, adenovirus, and simian virus 40 (SV40).
[0077] The SV40 promoter/enhancer can be preferably used according
to the method of Mulligan et al. (Nature (1979) 277, 108). Also,
the HEF1.alpha. promoter/enhancer can be easily used in the desired
gene expression by the method of Mizushima et al. (Nucleic Acids
Res. (1990) 18, 5322).
[0078] In the case of using E. coli as hosts, a useful promoter
routinely used, a signal sequence for antibody secretion, and the
antibody gene to be expressed can be functionally linked so that
the gene is expressed. Examples of the promoter used preferably
include lacZ and araB promoters. The lacZ promoter can be used
according to the method of Ward et al. (Nature (1989) 341, 544-546;
and FASEBJ. (1992) 6, 2422-2427). Alternatively, the araB promoter
can be preferably used in the gene expression of interest according
to the method of Better et al. (Science (1988) 240, 1041-1043).
[0079] In the case of antibody production in E. coli periplasm, a
pelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169,
4379) can be used for antibody secretion. The antibody thus
produced in the periplasm is isolated and then refolded by use of a
protein denaturant such as guanidine hydrochloride of urea such
that the resulting antibody has the desired binding activity.
[0080] In the case of antibody production using animal cells, the
signal sequence of the heavy or light chain gene of the antibody is
preferably used as a signal sequence required for the extracellular
secretion of the antibody. Alternatively, the signal sequence of a
secretory protein such as IL-3 or IL-6 is also preferably used.
[0081] A replication origin derived from SV40, polyomavirus,
adenovirus, bovine papillomavirus (BPV), or the like can be
inserted in the expression vectors. A selection marker may be
further inserted in the expression vectors for increasing the copy
numbers of the inserted genes in the host cells. Specifically, the
following selection markers can be used:
aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK)
gene, E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt)
gene, dihydrofolate reductase (dhfr) gene, etc.
[0082] Subsequently, the host cells transformed by the introduction
of these expression vectors are cultured in vitro or in vivo. In
this way, the antibody of interest is produced in, for example, the
cultures of the host cells. The host cells are cultured according
to a method known in the art. For example, a DMEM, MEM, RPMI1640,
or IMDM medium can be used and may be used in combination with a
solution supplemented with serum such as fetal calf serum
(FCS).
[0083] The antibody thus expressed and produced can be purified by
using, alone or in appropriate combination, usual protein
purification methods known in the art. For example, affinity or
chromatography columns (e.g., protein A columns), filters,
ultrafiltration, salting-out, and dialysis can be selected and
combined appropriately to separate and purify the antibody
(Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring
Harbor Laboratory, 1988).
[0084] In addition to the host cells, transgenic animals may be
used for the recombinant antibody production. Specifically, the
antibody of interest can be obtained from animals transfected with
the genes encoding this antibody of interest. For example, the
antibody genes can be inserted in frame into genes encoding
proteins specifically produced in milk to construct fusion genes
encoding the protein and the antibody. For example, goat .beta.
casein can be used as the proteins secreted into milk. DNA
fragments comprising the fusion genes having the antibody gene
insert are injected into goat embryos, which are in turn introduced
into female goats. From milk produced by transgenic goats (or
progeny thereof) brought forth by the goats that have received the
embryos, the desired antibody can be obtained as a fusion protein
with the milk protein. In addition, hormone is appropriately
administered to the transgenic goats for increasing the amount of
milk containing the desired antibody produced from the transgenic
goats (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).
[0085] Non-human animal antibody-derived C regions can be used as
the C regions in the recombinant antibody of the present invention.
For example, C.gamma.1, C.gamma.2a, C.gamma.2b, C.gamma.3, C.mu.,
C.delta., C.alpha.1, C.alpha.2, and C.epsilon. can be used as mouse
antibody H chain C regions, and C.kappa. and C.gamma. can be used
as mouse antibody L chain C regions. Alternatively, an antibody of
a rat, a rabbit, a goat, sheep, a camel, a monkey, or the like may
be used as an antibody of an animal other than the mouse. Their
sequences are known in the art. The C regions may be appropriately
modified for improving the stability of the antibody itself or of
its production.
[0086] In the case of administering the antibody according to the
present invention to humans, a genetically recombinant antibody
that has been engineered artificially can be administered, for
example, for the purpose of reducing heteroantigenicity in humans.
The genetically recombinant antibody encompasses, for example,
chimeric antibodies and humanized antibodies. These engineered
antibodies can be produced using a method known in the art.
[0087] The chimeric antibodies refer to antibodies comprising
variable and constant regions of different origins linked to each
other. For example, mouse-human heterogeneous chimeric antibodies
are antibodies consisting of the heavy and light chain variable
regions of a mouse antibody and the heavy and light chain constant
regions of a human antibody. Mouse antibody variable
region-encoding DNAs ligated in frame with human antibody constant
region-encoding DNAs can be incorporated into expression vectors to
prepare chimeric antibody-expressing recombinant vectors. Cells
transformed with these vectors (recombinant cells) can be cultured
to express the DNA inserts. The chimeric antibodies produced during
the culture can be obtained from the cultures of the recombinant
cells. Human antibody C regions are preferably used as the C
regions of the chimeric antibodies and humanized antibodies.
[0088] For example, C.gamma.1, C.gamma.2, C.gamma.3, C.gamma.4,
C.mu., C.delta., C.alpha.1, C.alpha.2, and C.epsilon. can be used
as H chain C regions. Also, C.kappa. and C.lamda. can be used as L
chain C regions. The amino acid sequences of these C regions and
nucleotide sequences encoding these amino acid sequences are known
in the art. The human antibody C regions may be appropriately
modified for improving the stability of the antibody itself or of
its production.
[0089] In general, the chimeric antibodies are composed of
non-human animal-derived antibody V regions and human
antibody-derived C regions. By contrast, the humanized antibodies
are composed of non-human animal-derived antibody complementarity
determining regions (CDRs), human antibody-derived framework
regions (FRs), and human antibody-derived C regions. The humanized
antibodies are useful as active ingredients for a therapeutic agent
of the present invention, owing to their reduced immunogenicity in
the human body.
[0090] Each antibody variable region is typically composed of three
complementarity determining regions (CDRs) flanked by four
framework regions (FRs). The CDR regions substantially determine
the binding specificity of the antibody for its antigen. The CDRs
have diverse amino acid sequences. On the other hand, amino acid
sequences constituting the FRs are often highly analogous even
among antibodies having binding specificity for different antigens.
Therefore, in general, the binding specificity of a certain
antibody can allegedly be transplanted to other antibodies through
CDR grafting to FRs.
[0091] The humanized antibodies are also called reshaped human
antibodies. Specifically, for example, humanized antibodies
comprising non-human animal (e.g., mouse) antibody CDRs grafted in
human antibodies are known in the art. General gene recombination
approaches are also known for obtaining the humanized
antibodies.
[0092] Specifically, for example, Overlap Extension PCR is known in
the art as a method for grafting mouse antibody CDRs to human FRs.
In the Overlap Extension PCR, a nucleotide sequence encoding each
mouse antibody CDR to be grafted is added to the sequences of
primers used for human antibody FR synthesis. The primers used are
prepared for each of the four FRs. For grafting the mouse CDRs to
the human FRs, in general, it is allegedly advantageous to select
human FRs highly homologous to the FRs of the mouse antibody from
which the mouse CDRs are derived, in order to maintain the CDR
functions. Specifically, in general, the mouse CDRs are preferably
grafted to human FRs consisting of amino acid sequences highly
homologous to those of the mouse FRs adjacent to the mouse CDRs to
be grafted.
[0093] As described above, the nucleotide sequences to be ligated
are designed such that the sequences are ligated in frame. The
human FR-encoding nucleotide sequences are individually synthesized
by PCR using their respective primers. The resulting PCR products
contain the mouse CDR-encoding DNA added to each human FR-encoding
sequence. The mouse CDR-encoding nucleotide sequences are designed
such that the nucleotide sequence in each product overlaps with
another. Subsequently, the overlapping CDR portions in the PCR
products synthesized with human antibody genes as templates are
annealed to each other for complementary strand synthesis reaction.
Through this reaction, the human FR sequences are ligated via the
mouse CDR sequences.
[0094] Finally, the full-length gene of the V region comprising
three CDRs and four FRs ligated is amplified by PCR using primers
that respectively anneal to the 5' and 3' ends thereof and have an
added recognition sequence for an appropriate restriction enzyme.
The V region gene DNA thus obtained and a human antibody C
region-encoding DNA can be inserted into expression vectors such
that these DNAs are fused in frame to prepare vectors for
human-type antibody expression. These expression vectors are
introduced into hosts to establish recombinant cells. The
recombinant cells are cultured for the expression of the humanized
antibody-encoding DNA to produce the humanized antibodies into the
cultures of the cultured cells (EP239400 and WO1996002576).
[0095] The humanized antibodies thus prepared can be evaluated for
their binding activities to the antigen by qualitative or
quantitative assay to thereby preferably select human antibody FRs
such that these FRs allow CDRs to form a favorable antigen-binding
site when ligated via the CDRs. If necessary, human antibody FR
amino acid residue(s) may be substituted such that the CDRs of the
resulting reshaped human antibody form an appropriate
antigen-binding site. For example, the desired mutation can be
introduced in the amino acid sequence of human FR by the
application of the PCR method used in the mouse CDR grafting to the
human FRs. Specifically, a mutation of a partial nucleotide
sequence can be introduced to the primers annealing to a human FR
nucleotide sequence. The human FR nucleotide sequence synthesized
using such primers contains the mutation thus introduced so as to
bring about the desired amino acid substitution. The variant
antibodies having the substituted amino acid(s) can be evaluated
for their binding activities to the antigen by the same assay as
above to select variant FR sequences having the desired properties
(Sato, K. et al., Cancer Res, 1993, 53, 851-856).
[0096] As described above, the method for obtaining human
antibodies is also known in the art. In addition, a technique of
obtaining human antibodies by panning using human antibody
libraries is also known. For example, human antibody V regions are
expressed as a single chain antibody (scFv) on the surface of
phages by a phage display method. The gene of a phage selected with
its binding activity to the antigen as an index can be analyzed to
determine DNA sequences encoding the V regions of the human
antibody binding to the antigen. After the determination of the DNA
sequence of the antigen-binding scFv, the V region sequences fused
in frame with the sequences of the desired human antibody C regions
are inserted to appropriate expression vectors to prepare human
antibody expression vectors. The expression vectors are introduced
into the preferable expression cells as exemplified above. The
expression cells are cultured for the expression of the human
antibody-encoding genes to obtain the human antibodies. These
methods are already known in the art (WO1992001047, WO19992020791,
WO1993006213, WO1993011236, WO1993019172, WO1995001438, and
WO1995015388).
[0097] In a preferable aspect, examples of the antibody used in the
present invention also include an antibody having a human constant
region, as described above.
[0098] The antibody of the present invention encompasses not only
bivalent antibodies typified by IgG but also monovalent antibodies
or polyvalent antibodies typified by IgM as long as these
antibodies bind to the ITM2A protein. The polyvalent antibody of
the present invention encompasses polyvalent antibodies having
antigen-binding sites, all of which are the same as each other or
some or all of which are different from each other. The antibody of
the present invention is not limited to whole antibody molecules,
and a low-molecular antibody or a modified form thereof can be
preferably used as long as the antibody binds to the ITM2A
protein.
[0099] The low-molecular antibody encompasses an antibody fragment
deficient in a portion of the whole antibody (e.g., whole IgG).
Such partial deficiency of the antibody molecule is accepted as
long as the resulting antibody fragment is capable of binding to
the ITM2A antigen. The antibody fragment according to the present
invention preferably comprises one or both of heavy chain variable
(VH) and light chain variable (VL) regions. Also, the antibody
fragment according to the present invention preferably contains
CDRs. The amino acid sequence of VH or VL may have substitution,
deletion, addition, and/or insertion. The antibody fragment of the
present invention may be deficient in a portion of one or both of
VH and VL as long as the resulting antibody fragment is capable of
binding to the ITM2A antigen. Alternatively, a chimerized or
humanized variable region may be used. Specific examples of the
antibody fragment preferably include Fab, Fab', F(ab')2, and Fv.
Specific examples of the low-molecular antibody preferably include
Fab, Fab', F(ab')2, Fv, scFv (single chain Fv), Diabody, sc(Fv)2
(single chain (Fv)2), and scFv-Fc. Multimers (e.g., dimmers,
trimers, tetramers, and polymers) of these antibodies are also
encompassed by the low-molecular antibody of the present
invention.
[0100] The antibody fragment can be obtained by the enzymatic
treatment of the whole antibody. For example, papain, pepsin, or
plasmin is known in the art as an enzyme for forming the antibody
fragment. Alternatively, genes encoding such antibody fragments may
be constructed, and these genes can be introduced into expression
vectors so that the antibody fragments are expressed in appropriate
host cells (e.g., Co, M. S. et al., J. Immunol. (1994) 152,
2968-2976; Better, M. & Horwitz, A. H. Methods in Enzymology
(1989) 178, 476-496; Plueckthun, A. & Skerra, A. Methods in
Enzymology (1989) 178, 476-496; Lamoyi, E., Methods in Enzymology
(1989) 121, 652-663; Rousseaux, J. et al., Methods in Enzymology
(1989) 121, 663-669; and Bird, R. E. et al., TIBTECH (1991) 9,
132-137).
[0101] Each digestive enzyme recognizes a particular amino acid
sequence in the whole antibody and cleaves the whole antibody into
the following antibody fragment having a particular structure:
papain digestion: F(ab).sub.2 or Fab, pepsin digestion: F(ab')2 or
Fab', and plasmin digestion: Facb.
[0102] The use of a genetic engineering approach for the antibody
fragments thus obtained enzymatically can delete an arbitrary
portion of the antibody.
[0103] Thus, the low-molecular antibody according to the present
invention encompasses antibody fragments that lack an arbitrary
region as long as these antibody fragments have binding affinity
for ITM2A.
[0104] The Diabody refers to a bivalent antibody fragment
constructed by gene fusion (e.g., Holliger P et al., Proc. Natl.
Acad. Sci. USA (1993) 90, 6444-6448, EP404097, and WO1993011161).
The Diabody is a dimer composed of two polypeptide chains. Usually,
each of the polypeptide chains constituting the dimer comprises VL
and VH linked in frame via a linker. The linker in the Diabody is
generally too short to form a single chain variable region fragment
having an antigen-binding site in which VL and VH on the same
polypeptide chain are associated with each other. Specifically, the
number of amino acid residues constituting the linker is, for
example, approximately 5 residues. Therefore, VL and VH encoded on
the same polypeptide chain form a dimer by association with VH and
VL, respectively, on another polypeptide chain. As a result, the
Diabody has two antigen-binding sites.
[0105] The scFv is obtained by linking VH and VL of the antibody.
In the scFv, VH and VL are linked via a linker, preferably, a
peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. USA,
(1988), 85, 5879-5883). VH and VL in the scFv may be derived from
any of the antibodies described herein. The structure of the
peptide linker that links VH and VL is not particularly limited.
For example, an arbitrary single chain peptide of approximately 3
to 25 residues can be used as the linker. Specifically, for
example, a peptide linker described later can be used.
[0106] VL and VH can be linked, for example, by the PCR method
described above. First, of the following DNA sequences, DNAs
encoding the whole or desired partial amino acid sequence are used
as templates for linking VL and VH by PCR:
DNA sequences encoding the H chain or VH of the antibody, and DNA
sequences encoding the L chain or VL of the antibody.
[0107] The VL-encoding DNA and the VH-encoding DNA are separately
amplified by PCR using a pair of primers respectively having both
terminal partial sequences of each DNA to be amplified.
Subsequently, a DNA encoding the peptide linker moiety is prepared.
The peptide linker-encoding DNA can also be synthesized using PCR.
Specifically, nucleotide sequences that can be linked to the
amplification products of the VL and VH nucleotide sequences
synthesized separately are respectively added in advance to the 5'
sequences of primers used. Subsequently, PCR is performed using
each DNA of [VH-encoding DNA]-[peptide linker-encoding
DNA]-[VL-encoding DNA] and primers for assembly PCR.
[0108] The primers for assembly PCR consist of the combination of a
primer annealing to the 5' sequence of the [VH-encoding DNA] and a
primer annealing to the 3' sequence of the [VL-encoding DNA].
Specifically, the primers for assembly PCR are a primer set that
allows PCR amplification of a DNA encoding the full-length sequence
of the scFv to be synthesized. By contrast, the [peptide
linker-encoding DNA] contains a preliminarily added nucleotide
sequence that can be linked to the VH- and VL-encoding DNAs. As a
result, these DNAs are linked and, further, finally prepared into a
full-length scFv gene amplification product by PCR using the
primers for assembly PCR. Once the scFv-encoding DNA is prepared,
expression vectors containing this DNA and cells transformed with
the expression vectors (recombinant cells) can be obtained
according to a routine method. In addition, the resulting
recombinant cells can be cultured for the expression of the
scFv-encoding DNA to obtain the scFv from the cultures of the
cells.
[0109] The scFv-Fc is a low-molecular antibody comprising an Fc
region fused in frame to scFv comprising antibody VH and VL
(Cellular & Molecular Immunology (2006) 3, 439-443). The origin
of the scFv used in scFv-Fc preparation is not particularly
limited, and, for example, scFv derived from IgM can be preferably
used. The origin of the Fc is not particularly limited, and, for
example, Fc derived from mouse IgG (mouse IgG2a, etc.) or human IgG
(human IgG1, etc.) can be appropriately used. Thus, in a preferable
aspect, examples of the scFv-Fc can include scFv-Fc comprising an
IgM antibody scFv fragment linked to mouse IgG2a CH2 (e.g.,
C.gamma.2) and CH3 (e.g., C.gamma.3) via the hinge region
(H.gamma.) of mouse IgG2a, and scFv-Fc comprising an IgM antibody
scFv fragment linked to human IgG1 CH2 and CH3 via the hinge region
of human IgG1.
[0110] The sc(Fv)2 is a low-molecular antibody having a single
chain polypeptide formed by two VHs and two VLs linked via linkers
or the like (Hudson et al, J. Immunol. Methods (1999) 231,
177-189). The sc(Fv)2 can be prepared, for example, by linking two
scFvs via a linker.
[0111] Examples of the sc(Fv)2 include an antibody wherein two VHs
and two VLs are aligned as VH, VL, VH, and VL (i.e.,
[VH]-linker-[VL]-linker-[VH]-linker-[VL]) in this order starting at
the N-terminus of the single chain polypeptide.
[0112] The order of two VHs and two VLs is not particularly limited
to the arrangement described above and may be any order of
arrangement. Examples thereof can also include the following
arrangements:
[VL]-linker-[VH]-linker-[VH]-linker-[VL],
[VH]-linker-[VL]-linker-[VL]-linker-[VH],
[VH]-linker-[VH]-linker-[VL]-linker-[VL],
[VL]-linker-[VL]-linker-[VH]-linker-[VH], and
[VL]-linker-[VH]-linker-[VL]-linker-[VH].
[0113] For example, an arbitrary peptide linker or a synthetic
compound linker (e.g., linkers disclosed in Protein Engineering
(1996) 9 (3), 299-305) that can be introduced by genetic
engineering can be preferably used as the linker that links the
antibody variable regions. The peptide linker can be preferably
used as the linker according to the present invention. The length
of the peptide linker is not particularly limited and may be
appropriately selected by those skilled in the art according to the
purpose. The number of amino acid residues constituting the peptide
linker is usually 1 to 100 amino acids, preferably 3 to 50 amino
acids, more preferably 5 to 30 amino acids, particularly preferably
12 to 18 amino acids (e.g., 15 amino acids).
[0114] An arbitrary sequence can be appropriately adopted as the
amino acid sequence constituting the peptide linker as long as this
sequence does not inhibit the binding effect of the scFv. For
example, the following amino acid sequences can be used for the
peptide linker:
TABLE-US-00001 Ser, Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, (SEQ ID NO:
79) Gly-Gly-Gly-Ser, (SEQ ID NO: 80) Ser-Gly-Gly-Gly, (SEQ ID NO:
81) Gly-Gly-Gly-Gly-Ser, (SEQ ID NO: 82) Ser-Gly-Gly-Gly-Gly, (SEQ
ID NO: 83) Gly-Gly-Gly-Gly-Gly-Ser, (SEQ ID NO: 84)
Ser-Gly-Gly-Gly-Gly-Gly, (SEQ ID NO: 85)
Gly-Gly-Gly-Gly-Gly-Gly-Ser, (SEQ ID NO: 86)
Ser-Gly-Gly-Gly-Gly-Gly-Gly, (Gly-Gly-Gly-Gly-Ser).sub.n, and
(Ser-Gly-Gly-Gly-Gly).sub.n
wherein n is an integer of 1 or larger.
[0115] The amino acid sequence of the peptide linker can be
appropriately selected by those skilled in the art according to the
purpose. For example, the integer n that determines the length of
the peptide linker is usually 1 to 5, preferably 1 to 3, more
preferably 1 or 2.
[0116] Accordingly, in a particularly preferable aspect, examples
of the sc(Fv)2 according to the present invention can include the
following sc(Fv)2: [VH]-peptide linker (15 amino
acids)-[VL]-peptide linker (15 amino acids)-[VH]-peptide linker (15
amino acids)-[VL].
[0117] Alternatively, the V regions may be linked using the
chemically synthesized linker (chemical cross-linking agent).
Cross-linking agents usually used in the cross-link of peptide
compounds or the like can be preferably used in the present
invention. For example, chemical cross-linking agents as shown
below are known in the art. These cross-linking agents are
commercially available:
N-hydroxysuccinimide (NHS),
[0118] disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)
suberate (BS3), dithiobis(succinimidyl propionate) (DSP),
dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol
bis(succinimidyl succinate) (EGS), ethylene glycol
bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl
tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),
bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), and
bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES),
etc.
[0119] Three linkers are usually necessary for linking four
antibody variable regions. A plurality of linkers having the same
sequences may be used, and linkers having different sequences can
also be preferably used. In the present invention, the
low-molecular antibody is preferably Diabody or sc(Fv)2. Such a
low-molecular antibody is formed by the treatment of the whole
antibody with an enzyme, for example, papain or pepsin, as
described above. Alternatively, such a low-molecular antibody is
isolated from the cultures of appropriate host cells transfected
with expression vectors having an insert of DNA encoding the
antibody fragment (e.g., 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; and
Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9,
132-137).
[0120] The antibody of the present invention also encompasses not
only monovalent antibodies but also polyvalent antibodies. The
polyvalent antibody of the present invention encompasses polyvalent
antibodies having antigen-binding sites, all of which are the same
as each other or some or all of which are different from each
other.
[0121] Antibodies conjugated with various molecules such as
polyethylene glycol (PEG) may be used as modified antibodies.
Alternatively, antibodies conjugated with cytotoxic substances such
as chemotherapeutic agents, toxic peptide, or radioactive chemicals
may also be used as modified antibodies. Such modified antibodies
(hereinafter, referred to as antibody conjugates) can be obtained
by chemically modifying the obtained antibody. A method for the
antibody modification has already been established in the art. The
toxic peptide-conjugated modified antibodies can be obtained by
allowing appropriate host cells to express fusion genes of the
antibody genes linked in frame with genes encoding the toxic
peptides, and then isolating the resulting fusion proteins from the
cultures of the cells. As described later, the modified antibody of
the present invention may be obtained in a molecular form such as a
bispecific antibody designed by a gene recombination technique so
as not only to recognize the ITM2A protein but also to recognize a
cytotoxic substance such as a chemotherapeutic agent, a toxic
peptide, or a radioactive chemical. These antibodies are also
encompassed by the "antibody" according to the present
invention.
[0122] Examples of the chemotherapeutic agent whose cytotoxic
activity functions through the conjugation to the ITM2A antibody
can include the following chemotherapeutic agents: azaribine,
anastrozole, azacytidine, bleomycin, bortezomib, bryostatin-1,
busulfan, camptothecin, 10-hydroxycamptothecin, carmustine,
Celebrex, chlorambucil, cisplatin, irinotecan, carboplatin,
cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel,
dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone,
diethylstilbestrol, doxorubicin, doxorubicin glucuronide,
epirubicin, ethinyl estradiol, estramustine, etoposide, etoposide
glucuronide, floxuridine, fludarabine, flutamide, fluorouracil,
fluoxymesterone, gemcitabine, hydroxyprogesterone caproate,
hydroxyurea, idarubicin, ifosfamide, leucovorin, lomustine,
maytansinoid, mechlorethamine, medroxyprogesterone acetate,
megestrol acetate, melphalan, mercaptopurine, methotrexate,
mitoxantrone, mithramycin, mitomycin, mitotane, phenylbutyrate,
prednisone, procarbazine, paclitaxel, pentostatin, semustine,
streptozocin, tamoxifen, taxanes, Taxol, testosterone propionate,
thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil
mustard, vinblastine, vinorelbine, and vincristine.
[0123] In the present invention, the chemotherapeutic agent is
preferably a low-molecular chemotherapeutic agent. The
low-molecular chemotherapeutic agent is unlikely to interfere with
the antibody functions even after its conjugation to the antibody.
In the present invention, the low-molecular chemotherapeutic agent
usually has a molecular weight of 100 to 2000, preferably 200 to
1000. All of the chemotherapeutic agents exemplified herein are
low-molecular chemotherapeutic agents. These chemotherapeutic
agents according to the present invention encompass prodrugs that
are converted in vivo to active chemotherapeutic agents. The
prodrug activation may be enzymatic conversion or nonenzymatic
conversion.
[0124] Alternatively, the antibody may be modified with the toxic
peptide (toxin). Examples of the toxic peptide preferably include
the followings:
diphtheria toxin A chain (Langone J. J., et al., Methods in
Enzymology (1983) 93, 307-308), Pseudomonas exotoxin (Nature
Medicine (1996) 2, 350-353), ricin A chain (Fulton R. J. et al., J.
Biol. Chem. (1986) 261, 5314-5319; Sivam G. et al., Cancer Res.
(1987) 47, 3169-3173; Cumber A. J. et al., J. Immunol. Methods
(1990) 135, 15-24; Wawrzynczak E. J. et al., Cancer Res. (1990) 50,
7519-7562; and Gheeite V. et al., J. Immunol. Methods (1991) 142,
223-230), deglycosylated ricin A chain (Thorpe P. E. et al., Cancer
Res. (1987) 47, 5924-5931), abrin A chain (Wawrzynczak E. J. et
al., Br. J. Cancer (1992) 66, 361-366; Wawrzynczak E. J., et al.
Cancer Res. (1990) 50, 7519-7562; Sivam G., et al. Cancer Res.
(1987) 47, 3169-3173; and Thorpe P. E. et al., Cancer Res. (1987)
47, 5924-5931), gelonin (Sivam G. et al., Cancer Res. (1987) 47,
3169-3173; Cumber A. J. et al., J. Immunol. Methods (1990) 135,
15-24; Wawrzynczak E. J. et al. Cancer Res., (1990) 50, 7519-7562;
and Bolognesi A. et al., Clin. exp. Immunol. (1992) 89, 341-346),
pokeweed anti-viral protein from seeds (PAP-s) (Bolognesi A. et
al., Clin. exp. Immunol. (1992) 89, 341-346), bryodin (Bolognesi A.
et al., Clin. exp. Immunol. (1992) 89, 341-346), saporin (Bolognesi
A., et al., Clin. exp. Immunol. (1992) 89, 341-346), momordin
(Cumber A. J. et al., J. Immunol. Methods (1990) 135, 15-24;
Wawrzynczak E. J. et al., Cancer Res. (1990) 50, 7519-7562; and
Bolognesi A. et al., Clin. exp. Immunol. (1992) 89, 341-346),
momorcochin (Bolognesi A. et al., Clin. exp. Immunol. (1992) 89,
341-346), dianthin 32 (Bolognesi A. et al., Clin. exp. Immunol.
(1992) 89, 341-346), dianthin 30 (Stirpe F., Barbieri L., FEBS
letter (1986) 195, 1-8), modeccin (Stirpe F., Barbieri L., FEBS
letter (1986) 195, 1-8), viscumin (Stirpe F., Barbieri L., FEBS
letter (1986) 195, 1-8), volkensin (Stirpe F., Barbieri L., FEBS
letter (1986) 195, 1-8), dodecandrin (Stirpe F., Barbieri L., FEBS
letter (1986) 195, 1-8), tritin (Stirpe F., Barbieri L., FEBS
letter (1986) 195, 1-8), luffin (Stirpe F., Barbieri L., FEBS
letter (1986) 195, 1-8), and trichokirin (Casellas P., et al., Eur.
J. Biochem. (1988) 176, 581-588; and Bolognesi A., et al., Clin.
exp. Immunol., (1992) 89, 341-346).
[0125] In the present invention, the radioactive chemical refers to
a chemical containing a radioisotope. The radioisotope used is not
particularly limited, and any radioisotope may be used. Examples
thereof can preferably include .sup.32P, .sup.14C, .sup.125I,
.sup.3H, .sup.131I, .sup.186Re, and .sup.188Re.
[0126] In another aspect, one or two or more low-molecular
chemotherapeutic agents and one or two or more toxic peptides can
be used in combination to modify the antibody. The anti-ITM2A
antibody can be conjugated to the low-molecular chemotherapeutic
agent via a covalent or noncovalent bond. Such a chemotherapeutic
agent-conjugated antibody is prepared by a method known in the
art.
[0127] A proteinous agent or toxin can be conjugated to the
antibody by a genetic engineering approach. Specifically, for
example, the anti-ITM2A antibody-encoding DNAs are fused in frame
with DNAs encoding the toxic peptides, and the resulting fused DNAs
can be incorporated into expression vectors to construct
recombinant vectors. The vectors are introduced into appropriate
host cells, and the resulting transformed cells are cultured so
that the DNA inserts are expressed. In this way, toxic
peptide-conjugated anti-ITM2A antibodies can be obtained as fusion
proteins. In the case of obtaining such antibody-fusion proteins,
the proteinous agent or toxin is generally conjugated to the
C-terminal side of each antibody. A peptide linker may be allowed
to intervene between the antibody and the proteinous agent or
toxin.
[0128] The antibody of the present invention further encompasses
bispecific antibodies. The bispecific antibodies refer to
antibodies containing, in the same antibody molecule, variable
regions that recognize different epitopes. The bispecific antibody
according to the present invention can have antigen-binding sites
that recognize different epitopes on the ITM2A molecule. When such
bispecific antibody molecules are to bind to ITM2A, two or more
molecules of the bispecific antibody can bind to one ITM2A
molecule. As a result, the bispecific antibodies, if having
cytotoxic activities, can be expected to recruit a larger number of
effector cells, resulting in stronger cytotoxic effect.
[0129] Alternatively, a bispecific antibody having antigen-binding
sites, one of which binds to ITM2A and the other of which binds to
a cytotoxic substance may be used in the present invention. The
cytotoxic substance specifically encompasses, for example,
chemotherapeutic agents, toxic peptides, and radioactive chemicals.
Such a bispecific antibody binds to ITM2A-expressing cells, while
capturing the cytotoxic substance. As a result, the cytotoxic
substance can be allowed to directly act on the ITM2A-expressing
cells. Specifically, use of the bispecific antibody that recognizes
ITM2A as well as the cytotoxic substance can specifically damage
tumor cells, resulting in the inhibited growth of the tumor
cells.
[0130] Also, a bispecific antibody that binds to ITM2A as well as
an antigen other than ITM2A expressed in tumor cells may be used in
the present invention. For example, a bispecific antibody that
binds to ITM2A and an antigen that is specifically expressed on the
surface of target cancer cells, as with ITM2A, but is different
from ITM2A, can be used.
[0131] The bispecific antibody is produced by a method known in the
art. For example, two types of antibodies differing in antigen
recognized thereby can be bound to prepare the bispecific antibody.
Each of the antibodies bound may be a 1/2 molecule having H and L
chains or may be a 1/4 molecule consisting of H chains.
Alternatively, different monoclonal antibody-producing hybridomas
may be fused to prepare fusion cells producing bispecific
antibodies. The bispecific antibody can also be prepared by a
genetic engineering approach.
[0132] The antigen binding activity of the antibody (Antibodies A
Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor
Laboratory, 1988) can be determined using means known in the art.
For example, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme
immunoassay), RIA (radioimmunoassay), flow cytometry such as FACS,
or fluoroimmunoassay can be preferably used.
[0133] The antibody of the present invention also encompasses an
antibody having a modified sugar chain of the antibody of the
present invention. The cytotoxic activities of antibodies are known
to be enhanced by the modification of their sugar chains. For
example, the following antibodies are known in the art as the
antibody having a modified sugar chain:
glycosylated antibodies (WO1999054342, etc.), antibodies deficient
in fucose added to their sugar chains (WO2000061739, WO2002031140,
WO2006067913, etc.), and antibodies having a sugar chain having
bisecting GlcNAc (WO2002079255, etc.).
[0134] The antibody of the present invention used for the
therapeutic purpose is preferably an antibody having a cytotoxic
activity. Examples of the cytotoxic activity according to the
present invention preferably include antibody-dependent
cell-mediated cytotoxicity (ADCC) and complement-dependent
cytotoxicity (CDC) activities. Another example of the antibody
having a cytotoxic activity includes an antibody having both ADCC
and CDC activities. In the present invention, the CDC activity
refers to a cytotoxic activity mediated by the complement system.
On the other hand, the ADCC activity refers to an activity of
damaging target cells by Fc.gamma. receptor-expressing cells
(immunocytes, etc.) as a result of binding of the Fc.gamma.
receptor-expressing cells (immunocytes, etc.) via the Fc.gamma.
receptors to the Fc domains of antibodies specifically attached to
the cell surface antigens of the target cells.
[0135] Whether or not the anti-ITM2A antibody has an ADCC activity
or has a CDC activity can be determined by a method known in the
art (e.g., Current protocols in Immunology, (1993) Chapter 7.
Immunologic studies in Humans, Editor, John E, Coligan et al., John
Wiley & Sons, Inc.).
[0136] Specifically, effector cells, a complement solution, and
target cells are first prepared.
[0137] (1) Preparation of Effector Cells
[0138] The spleens are excised from CBA/N mice or the like, and
spleen cells are separated therefrom in an RPMI1640 medium
(manufactured by Invitrogen Corp.). The spleen cells can be washed
with this medium containing 10% fetal bovine serum (FBS,
manufactured by HyClone Laboratories, Inc.) and then adjusted to a
cell concentration of 5.times.10.sup.6 cells/ml to prepare effector
cells.
[0139] (2) Preparation of Complement Solution
[0140] Baby Rabbit Complement (manufactured by CEDARLANE
Laboratories Ltd.) can be diluted 10-fold with a medium
(manufactured by Invitrogen Corp.) containing 10% FBS to prepare a
complement solution.
[0141] (3) Preparation of Target Cells
[0142] Cells expressing ITM2A proteins can be cultured at
37.degree. C. for 1 hour, together with 0.2 mCi 51Cr-sodium
chromate (manufactured by GE Healthcare Bio-Sciences Corp.), in a
DMEM medium containing 10% FBS to radiolabel the target cells.
Cells transformed with ITM2A protein-encoding genes, Ewing's
sarcoma cells, acute myeloid leukemia cells, T cell lymphoma cells,
T cells lymphocytic leukemia cells, or the like can be used as the
cells expressing ITM2A proteins. The cells thus radiolabeled can be
washed three times with an RPMI1640 medium containing 10% FBS and
adjusted to a cell concentration of 2.times.10.sup.5 cells/ml to
prepare the target cells.
[0143] The ADCC or CDC activity can be assayed by a method
described below. For the ADCC activity assay, a U-bottom 96-well
plate (manufactured by Becton, Dickinson and Company) supplemented
with the target cells and the anti-ITM2A antibody (each 50
.mu.l/well) is left standing for 15 minutes on ice. Then, 100 .mu.l
of the effector cells is added to each well of the plate, and the
resulting plate is incubated for 4 hours in a CO.sub.2 incubator.
The final concentration of the antibody is adjusted to 0 or 10
.mu.g/ml. After the incubation, the radioactivity of 100 .mu.l of
the supernatant recovered from each well is measured using a gamma
counter (COBRA II AUTO-GAMMA, MODEL D5005, manufactured by Packard
Instrument Company). The cytotoxic activity (%) can be calculated
on the basis of the calculation expression (A-C)/(B-C).times.100
using the following values obtained by such measurement:
A represents radioactivity (cpm) from each sample, B represents
radioactivity (cpm) from a sample supplemented with 1% NP-40
(manufactured by Nacalai Tesque, Inc.), and C represents
radioactivity (cpm) from a sample containing only the target
cells.
[0144] For the CDC activity assay, a flat-bottomed 96-well plate
(manufactured by Becton, Dickinson and Company) supplemented with
the target cells and the anti-ITM2A antibody (each 50 .mu.l/well)
is left standing for 15 minutes on ice. Then, 100 .mu.l of the
complement solution is added to each well of the plate, and the
resulting plate is incubated for 4 hours in a CO.sub.2 incubator.
The final concentration of the antibody is adjusted to 0 or 3
.mu.g/ml. After the incubation, the radioactivity of 100 .mu.l of
the supernatant recovered from each well is measured using a gamma
counter. The cytotoxic activity based on the CDC activity can be
calculated according to a calculation expression similar to that of
the ADCC activity.
[0145] In the case of assaying the cytotoxic activity of the
antibody conjugate, a flat-bottomed 96-well plate (manufactured by
Becton, Dickinson and Company) supplemented with the target cells
and the anti-ITM2A antibody conjugate (each 50 .mu.l/well) is left
standing for 15 minutes on ice. Subsequently, the plate is
incubated for 1 to 4 hours in a CO.sub.2 incubator. The final
concentration of the antibody is adjusted to 0 or 3 .mu.g/ml. After
the incubation, the radioactivity of 100 .mu.l of the supernatant
recovered from each well is measured using a gamma counter. The
cytotoxic activity of the antibody conjugate can be calculated
according to a calculation expression similar to that of the ADCC
activity assay.
[0146] In an alternative aspect, examples of the antibody used in
the present invention also preferably include an antibody having an
internalization activity. In the present invention, the "antibody
having an internalization activity" means an antibody that is
transported into a cell (cytoplasm, vesicle, any other organelle,
etc.) through its binding to ITM2A on the cell surface.
[0147] Whether or not the antibody has an internalization activity
can be confirmed by a method generally known to those skilled in
the art and can be confirmed by, for example, a method involving
contacting labeling material-bound anti-ITM2A antibodies with
ITM2A-expressing cells and confirming whether or not the labeling
material is incorporated into the cells by the contact, or a method
involving contacting cytotoxic substance-conjugated anti-ITM2A
antibodies with ITM2A-expressing cells and confirming whether or
not the death of the ITM2A-expressing cells is induced by the
contact. More specifically, whether or not the antibody has an
internalization activity can be confirmed by a method described in,
for example, Examples below.
[0148] For example, the cytotoxic substance-conjugated antibody
having an internalization activity can be used as a pharmaceutical
composition such as an anticancer agent.
[0149] An arbitrary antibody binding to ITM2A can be used as the
antibody of the present invention. Preferable examples of the
antibody can include antibodies (1) to (25) shown below. These
antibodies may be, for example, whole antibodies, low-molecular
antibodies, animal antibodies, chimeric antibodies, humanized
antibodies, or human antibodies:
(1) an antibody comprising an H chain having the amino acid
sequence represented by SEQ ID NO: 3 as CDR1, the amino acid
sequence represented by SEQ ID NO: 4 as CDR2, and the amino acid
sequence represented by SEQ ID NO: 5 as CDR3; (2) an antibody
comprising an L chain having the amino acid sequence represented by
SEQ ID NO: 6 as CDR1, the amino acid sequence represented by SEQ ID
NO: 7 as CDR2, and the amino acid sequence represented by SEQ ID
NO: 8 as CDR3; (3) an antibody comprising the H chain described in
(1) and the L chain described in (2); (4) an antibody comprising an
H chain having the amino acid sequence represented by SEQ ID NO: 9
as CDR1, the amino acid sequence represented by SEQ ID NO: 10 as
CDR2, and the amino acid sequence represented by SEQ ID NO: 11 as
CDR3; (5) an antibody comprising an L chain having the amino acid
sequence represented by SEQ ID NO: 12 as CDR1, the amino acid
sequence represented by SEQ ID NO: 13 as CDR2, and the amino acid
sequence represented by SEQ ID NO: 14 as CDR3; (6) an antibody
comprising the H chain described in (4) and the L chain described
in (5); (7) an antibody comprising an H chain having the amino acid
sequence represented by SEQ ID NO: 15 as CDR1, the amino acid
sequence represented by SEQ ID NO: 16 as CDR2, and the amino acid
sequence represented by SEQ ID NO: 17 as CDR3; (8) an antibody
comprising an L chain having the amino acid sequence represented by
SEQ ID NO: 18 as CDR1, the amino acid sequence represented by SEQ
ID NO: 19 as CDR2, and the amino acid sequence represented by SEQ
ID NO: 20 as CDR3; (9) an antibody comprising the H chain described
in (7) and the L chain described in (8); (10) an antibody
comprising an H chain having the amino acid sequence represented by
SEQ ID NO: 21 as CDR1, the amino acid sequence represented by SEQ
ID NO: 22 as CDR2, and the amino acid sequence represented by SEQ
ID NO: 23 as CDR3; (11) an antibody comprising an L chain having
the amino acid sequence represented by SEQ ID NO: 24 as CDR1, the
amino acid sequence represented by SEQ ID NO: 25 as CDR2, and the
amino acid sequence represented by SEQ ID NO: 26 as CDR3; (12) an
antibody comprising the H chain described in (10) and the L chain
described in (11); (13) the antibody described in any of (1) to
(12) which is a chimeric antibody; (14) the antibody described in
any of (1) to (12) which is a humanized antibody; (15) the antibody
described in (1) or (3), comprising the amino acid sequence
represented by SEQ ID NO: 28; (16) the antibody described in (2) or
(3), comprising the amino acid sequence represented by SEQ ID NO:
30; (17) the antibody described in (4) or (6), comprising the amino
acid sequence represented by SEQ ID NO: 32; (18) the antibody
described in (5) or (6), comprising the amino acid sequence
represented by SEQ ID NO: 34; (19) the antibody described in (7) or
(9), comprising the amino acid sequence represented by SEQ ID NO:
36; (20) the antibody described in (8) or (9), comprising the amino
acid sequence represented by SEQ ID NO: 38; (21) the antibody
described in (10) or (12), comprising the amino acid sequence
represented by SEQ ID NO: 40; (22) the antibody described in (11)
or (12), comprising the amino acid sequence represented by SEQ ID
NO: 42; (23) the antibody described in any of (15) to (22) which is
a chimeric antibody; (24) an antibody that has an amino acid
sequence of the antibody described in any of (1) to (23) with a
substitution, deletion, addition, and/or insertion of one or more
amino acid(s) and has an activity equivalent to or a binding
activity equivalent to that of the antibody; and (25) an antibody
capable of binding to an epitope to which a second antibody binds,
wherein the second antibody is the antibody described in any of (1)
to (23).
[0150] In the present invention, the phrase "having an activity
equivalent to that of the antibody of the present invention" means
that a cytotoxic activity against ITM2A-expressing cells is
equivalent to that of the antibody of the present invention. In the
present invention, the phrase "having a binding activity equivalent
to that of the antibody of the present invention" means that an
ITM2A binding activity is equivalent to that of the antibody of the
present invention.
[0151] A method for introducing a mutation to a polypeptide is one
of methods well known to those skilled in the art for preparing a
polypeptide functionally equivalent to a certain polypeptide. For
example, those skilled in the art can appropriately introduce a
mutation in the antibody of the present invention using
site-directed mutagenesis (Hashimoto-Gotoh, T. et al. Gene (1995)
152, 271-275; Zoller, M J, and Smith, M. Methods Enzymol., (1983)
100, 468-500; Kramer, W. et al. Nucleic Acids Res., (1984) 12,
9441-9456; Kramer W, and Fritz H J Methods. Enzymol., (1987) 154,
350-367; Kunkel, T A Proc. Natl. Acad. Sci. USA., (1985) 82,
488-492; and Kunkel, Methods Enzymol., (1988) 85, 2763-2766) or the
like and thereby prepare an antibody functionally equivalent to the
antibody concerned. Amino acid mutations may occur in the natural
world. Such an antibody that has an amino acid sequence of the
antibody of the present invention with a mutation of one or more
amino acid(s) and has an activity functionally equivalent to or a
binding activity equivalent to that of the antibody concerned is
also encompassed by the antibody of the present invention.
[0152] The number of amino acids mutated in such a variant is
usually within 50 amino acids, preferably within 30 amino acids,
more preferably within 15 amino acids or within 10 amino acids
(e.g., within 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s)).
[0153] For amino acid residues to be mutated, this mutation is
preferably performed conservatively between amino acids having the
same side chain properties. For example, the following
classification based on the properties of amino acid side chains
has been established:
hydrophobic amino acids (A, I, L, M, F, P, W, Y, and V),
hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, and T),
amino acids having an aliphatic side chain (G, A, V, L, I, and P),
amino acids having a hydroxy group-containing side chain (S, T, and
Y), amino acids having a sulfur atom-containing side chain (C and
M), amino acids having a side chain containing carboxylic acid and
amide (D, N, E, and Q), amino acids having a base-containing side
chain (R, K, and H), and amino acids having an aromatic
group-containing side chain (H, F, Y, and W) (all symbols within
the parentheses represent single letter codes of amino acids).
[0154] A polypeptide having an amino acid sequence modified from a
certain amino acid sequence by the deletion and/or addition of one
or more amino acid residue(s) and/or the substitution thereof by
other amino acids is already known to maintain the biological
activity of the original polypeptide (Mark, D. F. et al., Proc.
Natl. Acad. Sci. USA (1984) 81, 5662-5666; Zoller, M. J. and Smith,
M., Nucleic Acids Research (1982) 10, 6487-6500; Wang, A. et al.,
Science (1984) 224, 1431-1433; and Dalbadie-McFarland, G. et al.,
Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413). Specifically,
when amino acids in an amino acid sequence constituting a certain
polypeptide are substituted by amino acids classified in the same
group thereas, it is generally said that the polypeptide is likely
to maintain its activity. In the present invention, the
substitution between amino acids within the same amino acid group
described above is referred to as conservative substitution.
[0155] The present invention also provides an antibody binding to
the same epitope as that to which the anti-ITM2A antibody disclosed
herein binds. Specifically, the present invention relates to an
antibody binding to the same epitope as that to which BE5-1, BE6-1,
BE7-1-1, or BE13-1 binds, and use thereof. Such an antibody can be
obtained, for example, by a method show below.
[0156] Whether a test antibody shares an epitope with a certain
antibody can be confirmed on the basis of their competition for the
same epitope. The competition between the antibodies is detected by
cross-blocking assay or the like. The cross-blocking assay is
preferably, for example, competitive ELISA assay.
[0157] Specifically, the cross-blocking assay involves
preincubating ITM2A proteins coated on the wells of a microtiter
plate in the presence or absence of a candidate competing antibody
and then adding the anti-ITM2A antibody of the present invention to
the wells. The amount of the anti-ITM2A antibody of the present
invention bound to the ITM2A protein in each well indirectly
correlates with the binding capability of the candidate competing
antibody (test antibody) that competes therewith for binding to the
same epitope. Specifically, correlation is confirmed such that the
higher affinity of the test antibody for the same epitope results
in the smaller amount of the anti-ITM2A antibody of the present
invention bound to the ITM2A protein-coated well and instead, the
larger amount of the test antibody bound to the ITM2A
protein-coated well.
[0158] The amount of each antibody bound to the well can be easily
determined by labeling the antibody in advance. For example, the
amount of a biotinylated antibody can be determined using an
avidin-peroxidase conjugate and an appropriate substrate. The
cross-blocking assay using enzyme (e.g., peroxidase) labeling is
particularly called competitive ELISA assay. The antibody may be
labeled with any of other detectable or measurable labeling
materials. Specifically, for example, radiolabeling or fluorescent
labeling is known in the art.
[0159] Alternatively, the cross-blocking assay is preferably
competitive FACS assay.
[0160] Specifically, the competitive FACS assay employs cells
containing expressed ITM2A proteins instead of ITM2A proteins
coated on the wells of a microtiter plate in the competitive ELISA
assay. The cells containing expressed ITM2A proteins are
preincubated in the presence or absence of a candidate competing
antibody. Then, the biotinylated anti-ITM2A antibody of the present
invention is added to the wells. Fluorescence can be detected using
a streptavidin-fluorescein conjugate to determine the competition
between the antibodies. The cross-blocking assay using flow
cytometry is particularly called competitive FACS assay. The
antibody can be preferably labeled with any of other detectable or
measurable fluorescent labeling materials.
[0161] When the test antibody contains constant regions derived
from an organism species different from that of the anti-ITM2A
antibody of the present invention, the antibody (derived from any
organism species) bound to the well can be assayed using a labeled
antibody that recognizes the constant region of the antibody of the
organism species. Alternatively, even in the case of detecting the
binding of antibodies derived from the same organism species but
differing in class, each antibody bound to the well can be assayed
using an antibody specifically binding to the antibody of each
class.
[0162] Provided that the candidate antibody can block the binding
of the anti-ITM2A antibody by at least 20%, preferably at least
30%, more preferably at least 40%, even more preferably 50%,
compared with the binding activity obtained as a result of the
control test conducted in the absence of the candidate competing
antibody, this candidate competing antibody is determined as an
antibody that binds to substantially the same epitope as that to
which the anti-ITM2A antibody of the present invention binds or as
an antibody that competes therewith for the binding to the same
epitope. For the epitope assay, the constant region of the antibody
of the present invention may be replaced with the same constant
region as that of the test antibody.
[0163] The epitope to which the anti-ITM2A antibody of the present
invention binds can be appropriately determined by the method
described above. Preferably, the epitope can be present in a
fragment comprising the extracellular region of the ITM2A protein.
Examples of such a fragment also preferably include a polypeptide
consisting of amino acids 75 to 263 in the ITM2A protein
represented by SEQ ID NO: 1. In another aspect, examples of the
fragment preferably include a polypeptide consisting of amino acids
75 to 227 in the ITM2A protein represented by SEQ ID NO: 1.
Pharmaceutical Composition
[0164] In an alternative aspect, the present invention provides a
pharmaceutical composition comprising the antibody binding to ITM2A
protein as an active ingredient. The present invention also relates
to a cell growth inhibitor, particularly, an anticancer agent,
comprising the antibody binding to ITM2A protein as an active
ingredient. The cell growth inhibitor and the anticancer agent of
the present invention are preferably administered to a subject
having cancer or possibly having cancer. As shown later in
Examples, ITM2A is expressed at a low level in normal cells, but is
overexpressed in cancer cells. Therefore, the administration of the
anti-ITM2A antibody probably produces cancer cell-specific
cytotoxic effect.
[0165] The anti-ITM2A antibody used in the pharmaceutical
composition (e.g., the anticancer agent) of the present invention
is not particularly limited and may be any anti-ITM2A antibody. For
example, any of the anti-ITM2A antibodies described above can be
preferably used.
[0166] In the present invention, the phrase "comprising the
antibody binding to ITM2A as an active ingredient" means containing
the anti-ITM2A antibody as a main active ingredient and is not
intended to limit the content of the anti-ITM2A antibody.
[0167] When the disease targeted by the pharmaceutical composition
of the present invention is cancer, the targeted cancer is not
particularly limited as long as the ITM2A protein is expressed in
the cancer. The cancer is preferably Ewing's sarcoma or blood
cancer such as T cell leukemia, T cell lymphoma, acute myeloid
leukemia, B cell tumor, or multiple myeloma, particularly
preferably Ewing's sarcoma. Among the Ewing's sarcomas, Ewing's
sarcoma having t(11;22)(q24;q12) chromosomal translocation may be
preferably targeted. Such cancer may be any of primary foci and
metastatic foci.
[0168] In the present invention, a method known in the art such as
FISH or PCR can be appropriately adopted as a cell damaging method
or for determining whether or not Ewing's sarcoma cells whose
growth is to be inhibited have t(11;22)(q24;q12) chromosomal
translocation.
[0169] In order to determine whether to have the t(11;22)(q24;q12)
chromosomal translocation by the FISH method, for example, a probe
for EWS gene detection and a probe for FLI-1 gene detection
separately labeled so as to emit different fluorescences are
hybridized to test tissue samples immobilized by a method known in
the art. The presence of the t(11;22)(q24;q12) chromosomal
translocation can be confirmed by determining whether or not fusion
signals of these fluorescences are detected as a result of the
hybridization.
[0170] Alternatively, FISH using two probes for respectively
detecting portions of a chromosome split by the translocation may
also be appropriately adopted for determining whether to have the
t(11;22)(q24;q12) chromosomal translocation. Specifically, these
two probes (e.g., a set of probes for detecting the EWS gene split
by the translocation or a set of probes for FLI-1 gene detection)
separately labeled so as to emit different fluorescences are
hybridized to test tissue samples immobilized by a method known in
the art. The presence of the t(11;22)(q24;q12) chromosomal
translocation can be confirmed by determining whether or not split
signals of these fluorescences are detected as a result of the
hybridization.
[0171] In order to determine whether to have the t(11;22)(q24;q12)
chromosomal translocation by the PCR method, a set of two primers
is designed such that the EWS gene and the FLI-1 gene can be
detected. The primers are designed such that a fusion gene formed
by the translocation is amplified as a result of PCR using the set
of the primers. The presence of the t(11;22)(q24;q12) chromosomal
translocation can be confirmed by detecting the PCR amplification
of the fusion gene formed by the translocation.
[0172] Alternatively, a set of two primers that allow detection of
fragments of a chromosome split by the translocation is designed
for determining whether to have the t(11;22)(q24;q12) chromosomal
translocation. For example, PCR is carried out using a set of
primers for detecting the EWS gene split by the translocation or a
set of primers for FLI-1 gene detection, and a test tissue sample
as a template. The presence of the t(11;22)(q24;q12) chromosomal
translocation can be confirmed provided that chromosomal fragments
formed by PCR using a sample free from the translocation as a
template are not detected in the PCR products obtained with the
test tissue sample as a template.
[0173] The pharmaceutical composition of the present invention can
be administered either orally or parenterally to a patient.
Parenteral administration is preferable. Specific examples of such
an administration method preferably include injection, transnasal,
pulmonary, and transdermal administrations. Examples of the
injection administration include intravenous, intramuscular,
intraperitoneal, and subcutaneous injections, through which the
pharmaceutical composition of the present invention can be
administered systemically or locally. The administration method can
be appropriately selected according to the age or symptoms of the
patient. The dose of the pharmaceutical composition of the present
invention can be selected from among the range of, for example,
0.0001 mg to 1000 mg per kg body weight per dosing. Alternatively,
the dose in each patient may be selected from among the range of,
for example, 0.001 to 100000 mg per body. However, the
pharmaceutical composition of the present invention is not limited
by these doses.
[0174] The pharmaceutical composition of the present invention can
be formulated according to a routine method (e.g., Remington's
Pharmaceutical Science, Latest edition, Mark Publishing Company,
Easton, U.S.A.). The pharmaceutical composition preferably used may
additionally contain pharmaceutically acceptable carriers or
additives. Examples of such carriers or additives include, but not
limited thereto, surfactants, excipients, coloring agents,
flavoring agents, preservatives, stabilizers, buffers, suspending
agents, tonicity agents, binders, disintegrants, lubricants, flow
promoters, and corrigents. Other carriers routinely used may be
appropriately used. Specific examples of such carriers routinely
used can include light anhydrous silicic acid, lactose, crystalline
cellulose, mannitol, starch, carmellose calcium, carmellose sodium,
hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl
acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium
chain fatty acid triglyceride, polyoxyethylene hydrogenated castor
oil 60, white sugar, carboxymethylcellulose, corn starch, and
inorganic salts.
[0175] The present invention also provides a method for damaging
ITM2A-expressing cells or inhibiting the growth of ITM2A-expressing
cells, comprising contacting the ITM2A-expressing cells with the
antibody binding to ITM2A protein.
[0176] The antibody used in the method of the present invention is
not particularly limited, and, for example, any of the antibodies
described above may be used. The cells to which the anti-ITM2A
antibody binds are not particularly limited as long as ITM2A is
expressed in the cells. The ITM2A-expressing cells according to the
present invention are preferably cancer cells, more preferably
Ewing's sarcoma cells or cells of blood cancer such as T cell
leukemia, T cell lymphoma, acute myeloid leukemia, B cell tumor, or
multiple myeloma. The method of the present invention can be
applied to any of primary foci and metastatic foci of these
cancers. The cancer cells are more preferably Ewing's sarcoma cells
or metastatic Ewing's sarcoma cells. Among the Ewing's sarcoma
cells, Ewing's sarcoma cells having t(11;22)(q24;q12) chromosomal
translocation may be preferably targeted. Such Ewing's sarcoma
cells having the chromosomal translocation may be located in any of
primary foci and metastatic foci.
[0177] In the present invention, a method known in the art such as
FISH or PCR can be appropriately adopted as a cell damaging method
or for determining whether or not Ewing's sarcoma cells whose
growth is to be inhibited have t(11;22)(q24;q12) chromosomal
translocation.
[0178] In order to determine whether to have the t(11;22)(q24;q12)
chromosomal translocation by the FISH method, for example, a probe
for EWS gene detection and a probe for FLI-1 gene detection
separately labeled so as to emit different fluorescences are
hybridized to test tissue samples immobilized by a method known in
the art. The presence of the t(11;22)(q24;q12) chromosomal
translocation can be confirmed by determining whether or not
adjacent or fusion signals of these fluorescences are detected as a
result of the hybridization.
[0179] Alternatively, FISH using two probes for respectively
detecting portions of a chromosome split by the translocation may
also be appropriately adopted for determining whether to have the
t(11;22)(q24;q12) chromosomal translocation. Specifically, these
two probes (e.g., a set of probes for detecting the EWS gene split
by the translocation or a set of probes for FLI-1 gene detection)
separately labeled so as to emit different fluorescences are
hybridized to test tissue samples immobilized by a method known in
the art. The presence of the t(11;22)(q24;q12) chromosomal
translocation can be confirmed by determining whether or not split
signals of these fluorescences are detected as a result of the
hybridization.
[0180] In order to determine whether to have the t(11;22)(q24;q12)
chromosomal translocation by the PCR method, a set of two primers
is designed such that the EWS gene and the FLI-1 gene can be
detected. The primers are designed such that a fusion gene formed
by the translocation is amplified as a result of PCR using the set
of the primers. The presence of the t(11;22)(q24;q12) chromosomal
translocation can be confirmed by detecting the PCR amplification
of the fusion gene formed by the translocation.
[0181] Alternatively, a set of two primers that allow detection of
fragments of a chromosome split by the translocation is designed
for determining whether to have the t(11;22)(q24;q12) chromosomal
translocation. For example, PCR is carried out using a set of
primers for detecting the EWS gene split by the translocation or a
set of primers for FLI-1 gene detection, and a test tissue sample
as a template. The presence of the t(11;22)(q24;q12) chromosomal
translocation can be confirmed provided that chromosomal fragments
formed by PCR using a sample free from the translocation as a
template are not detected in the PCR products obtained with the
test tissue sample as a template.
[0182] In the present invention, the "contact" is performed, for
example, by adding the antibody to cultures of ITM2A-expressing
cells cultured in vitro. In the present invention, the "contact" is
also performed by administering the antibody to non-human animals
implanted with ITM2A-expressing cells in their bodies or to animals
endogenously having ITM2A-expressing cancer cells.
[0183] Methods shown below are preferably used for evaluating or
determining cytotoxicity caused against the ITM2A-expressing cells
by the contact of the anti-ITM2A antibody. Examples of the methods
for evaluating or determining the cytotoxic activity in vitro can
include the antibody-dependent cell-mediated cytotoxicity (ADCC)
and complement-dependent cytotoxicity (CDC) activity assay methods
described above. Whether or not the anti-ITM2A antibody has an ADCC
activity or has a CDC activity can be determined by a method known
in the art (e.g., Current protocols in Immunology, Chapter 7.
Immunologic studies in Humans, Editor, John E, Coligan et al., John
Wiley & Sons, Inc., (1993)). In the activity assay, a binding
antibody that has an isotype identical to that of the anti-ITM2A
antibody and does not have the cytotoxic activity is used as a
control antibody in the same way as in the anti-ITM2A antibody.
When the anti-ITM2A antibody exhibits a stronger cytotoxic activity
than that of the control antibody, the anti-ITM2A antibody can be
determined to have the activity.
[0184] The isotype of an antibody is defined by the sequence of the
H chain constant region in the amino acid sequence of this
antibody. The antibody isotype is finally determined depending on
class switching caused by genetic recombination on the chromosome
during the maturation of antibody-producing B cells in vivo.
Difference in isotype is reflected by the difference between the
physiological/pathological functions of antibodies. Specifically,
it is known that, for example, the strength of the cytotoxic
activity is influenced not only by the expression level of the
antigen but also by the isotype of the antibody. Thus, for the
cytotoxic activity assay described above, the antibody used as a
control preferably has an isotype identical to that of the test
antibody.
[0185] In order to evaluate or determine the cytotoxic activity in
vivo, for example, ITM2A-expressing cancer cells are intradermally
or subcutaneously transplanted to non-human test animals. Then, the
test antibody is intravenously or intraperitoneally administered
thereto on a daily basis or at a few day-intervals from the
administration day or the next day. The cytotoxic activity of the
test antibody can be determined by measuring tumor sizes over time.
A control antibody having an isotype identical thereto is
administered, as in the in vitro evaluation. When the test
anti-ITM2A antibody-administered group exhibits a significantly
smaller tumor size than that of the control antibody-administered
group, the test anti-ITM2A antibody can be determined to have the
cytotoxic activity. In the case of using mice as the non-human test
animals, nude (nu/nu) mice can be preferably used, which are
genetically deficient in the thymus gland and thus lack the
functions of T lymphocytes. The use of these mice excludes the
involvement of the endogenous T lymphocytes of the test animals in
the evaluation or determination of the cytotoxic activities of
administered antibodies.
[0186] In one aspect, the method of the present invention provides
the diagnosis of cancer by detecting ITM2A protein in a test
sample. In this aspect, preferably, the extracellular region of the
ITM2A protein is detected. An antibody that recognizes the ITM2A
protein can be preferably used in the detection of the ITM2A
protein.
[0187] One specific example of the diagnosis method of the present
invention can include a method for diagnosing cancer, comprising
the following steps:
(a) providing a sample collected from a test subject; and (b)
detecting ITM2A protein contained in the collected sample using an
antibody binding to the ITM2A protein.
[0188] In the present invention, the detection encompasses
quantitative or qualitative detection. Examples of the qualitative
detection can include the following assays: assay to simply
determine the presence or absence of the ITM2A protein,
assay to determine the presence or absence of more than a
predetermined amount of the ITM2A protein, and assay to compare the
amount of the ITM2A protein with that contained in another sample
(e.g., a control sample).
[0189] On the other hand, examples of the quantitative detection
can include the measurement of an ITM2A protein concentration and
the measurement of the amount of the ITM2A protein.
[0190] The test sample according to the present invention is not
particularly limited as long as the sample possibly contains the
ITM2A protein. Specifically, samples collected from living bodies
such as mammals are preferable. Samples collected from humans are
more preferable. Specific examples of the test sample can include
blood, interstitial fluid, plasma, extravascular fluid,
cerebrospinal fluid, synovial fluid, pleural fluid, serum, lymph,
saliva, urine, tissues, ascitic fluid, and intraperitoneal lavage.
The sample is preferably a sample obtained from the test sample,
such as a preparation in which tissues or cells collected from a
living body are fixed, or cell cultures.
[0191] The cancer diagnosed by the present invention is not
particularly limited and may be any cancer. Specific examples
thereof can include Ewing's sarcoma and blood cancer such as T cell
leukemia, T cell lymphoma, acute myeloid leukemia, B cell tumor,
and multiple myeloma. In the present invention, any of primary foci
and metastatic foci of these cancers can be diagnosed. Among the
Ewing's sarcoma cells, Ewing's sarcoma having t(11;22)(q24;q12)
chromosomal translocation may be diagnosed. Such Ewing's sarcoma
having the chromosomal translocation may be any of primary foci and
metastatic foci.
[0192] In the present invention, the cancer is diagnosed using, as
an index, the level of the ITM2A protein detected in the test
sample. Specifically, when the amount of the ITM2A protein detected
in the test sample is larger than that of a negative control or a
healthy individual, the test subject is shown to have cancer or be
highly likely to have cancer in the future. Specifically, the
present invention relates to a method for diagnosing cancer,
comprising the following steps:
(1) detecting the expression level of ITM2A in a biological sample
collected from a test subject, and (2) comparing the expression
level of ITM2A detected in step (1) with that of a control, wherein
when the expression level of ITM2A is higher than that of the
control, the test subject is determined to have cancer.
[0193] In the present invention, the control refers to a reference
sample for comparison and encompasses negative controls and
biological samples of healthy individuals. The negative controls
can be obtained by collecting biological samples of healthy
individuals and mixing them, if necessary. The expression level of
ITM2A in the control can be detected in parallel with the detection
of the expression level of ITM2A in the biological sample of the
test subject. Alternatively, the expression level of ITM2A in a
large number of biological samples of healthy individuals may be
detected in advance to statistically determine the standard
expression level in the healthy individuals. Such statistically
determined values are also used as control values for the
expression level of ITM2A in the test biological sample.
Specifically, for example, mean.+-.2.times. standard deviation
(S.D.) or mean.+-.3.times. standard deviation (S.D.) can be used as
the standard value. Statistically, the mean.+-.2.times. standard
deviation (S.D.) and the mean.+-.3.times. standard deviation (S.D.)
include values of 80% and 90% of the healthy individuals,
respectively.
[0194] Alternatively, the expression level of ITM2A in the control
may be set using an ROC curve. The ROC curve, or receiver operating
characteristic curve, is a graph showing detection sensitivity in
the ordinate and false positive rates (i.e., "1-specificity") in
the abscissa. In the present invention, the ROC curve can be
obtained by plotting changes in sensitivity and false positive rate
at a series of varying reference values for determining the
expression level of ITM2A in the biological sample.
[0195] The "reference value" for obtaining the ROC curve is a
numeric value temporarily used for statistical analysis. In
general, the "reference value" for obtaining the ROC curve is
serially varied within a range which can cover all selectable
reference values. For example, the reference value can be varied
between the minimal and maximal measured values of ITM2A in a
population to be analyzed.
[0196] A standard value that can be expected to offer the desired
detection sensitivity and precision can be selected on the basis of
the obtained ROC curve. The standard value statistically set on the
basis of the ROC curve or the like is also called a cut-off value.
In a method for detecting cancer on the basis of the cut-off value,
step (2) described above comprises comparing the expression level
of ITM2A detected in step (1), with the cut-off value. Then, when
the expression level of ITM2A detected in step (1) is higher than
the cut-off value, cancer is detected in the test subject.
[0197] In the present invention, the expression level of ITM2A can
be determined by an arbitrary method. Specifically, the expression
level of ITM2A can be determined by evaluating the amount of the
ITM2A protein and the biological activity of the ITM2A protein. The
amount of the ITM2A protein can be determined by the method as
described herein.
[0198] In the present invention, an arbitrary animal species
expressing the ITM2A protein can be selected as the test subject.
For example, many non-human mammalian individuals such as rhesus
macaque (Macaca mulatta) (ENSMMUG00000003564), common marmoset
(Callithrix jacchus) (ENSCJAG00000009591), Sumatran orangutan
(Pongo abelii) (LOC100431628), rabbit (Oryctolagus cuniculus)
(ENSOCUG00000008651), horse (Equus caballus) (ENSECAG00000011335),
mouse (Mus musculus) (ENSMUSG00000031239), giant panda (Ailuropoda
melanoleuca) (LOC100476516), rat (Rattus norvegicus)
(ENSRNOG00000002365), pig (Sus scrofa) (ENSSSCG00000012448), and
chicken (Gallus gallus) (ENSGALG00000004107) are known to express
the ITM2A protein. Thus, these animals are encompassed by the test
subject according to the present invention. The test subject is
particularly preferably a human. When a non-human animal is used as
the test subject, the ITM2A protein of the animal species is
detected.
[0199] The anti-ITM2A antibody may be used for detecting the ITM2A
protein of a non-human animal species. In such a case, an
anti-ITM2A antibody binding to only the ITM2A protein of the animal
species may be used. Alternatively, an anti-ITM2A antibody capable
of binding to not only the ITM2A protein derived from the animal
species but also the ITM2A protein derived from another animal
species, i.e., having so-called cross reactivity, can also be
preferably used. The anti-ITM2A antibody may be further used for
detecting the human ITM2A protein. In such a case, an anti-ITM2A
antibody binding to only human ITM2A as well as an anti-ITM2A
antibody capable of binding to both of human ITM2A and the ITM2A
protein derived from another animal species can be preferably
used.
[0200] A method for detecting the ITM2A protein contained in the
test sample is not particularly limited and is preferably an
immunological detection method using the anti-ITM2A antibody as
exemplified below:
radioimmunoassay (RIA), enzyme immunoassay (EIA), fluoroimmunoassay
(FIA), luminescent immunoassay (LIA), immunoprecipitation (IP),
turbidimetric immunoassay (TIA), Western blot (WB),
immunohistochemical (IHC) method, and single radial immunodiffusion
(SRID).
[0201] Among these approaches, the immunohistochemical (IHC) method
is a preferable immunological assay method for diagnosing cancer,
comprising the step of detecting the ITM2A protein on sections in
which tissues or cells obtained from a patient having cancer are
fixed. The immunological methods described above, such as the
immunohistochemical (IHC) method, are generally known to those
skilled in the art.
[0202] Specifically, since ITM2A is a membrane protein specifically
overexpressed in cancer cells, cancer cells or cancer tissues can
be detected using the anti-ITM2A antibody. Cancer cells contained
in cells or tissues collected from living bodies can be detected by
the immunohistological analysis.
[0203] In another preferable aspect, cancer tissues can be detected
in vivo by a noninvasive method using the anti-ITM2A antibody.
Specifically, the present invention relates to a method for
detecting cancer, comprising the steps of: (1) administering, to a
test subject, a labeling material (e.g., radioisotope)-labeled
antibody binding to ITM2A protein; and (2) detecting the
accumulation of the labeling material. The anti-ITM2A antibody can
be detectably labeled for tracing the anti-ITM2A antibody
administered into the living body. For example, the in vivo
behavior of the antibody labeled with a fluorescent or luminescent
material or a radioisotope can be traced. The fluorescent or
luminescent material-labeled anti-ITM2 antibody can be observed
using an endoscope or peritoneoscope. The localization of the
anti-ITM2A antibody can be imaged by tracing the radioactivity of
the radioisotope. In the present invention, the in vivo
localization of the anti-ITM2A antibody represents the presence of
cancer cells.
[0204] A positron-emitting nuclide can be used as the radioisotope
for labeling the anti-ITM2A antibody used for the purpose of
detecting cancer in vivo. For example, the antibody can be labeled
with a positron-emitting nuclide such as .sup.18F, .sup.55Co,
.sup.64Cu, .sup.66Ga, .sup.68Ga, .sup.76Br, .sup.89Zr, and
.sup.124I. A method known in the art (Acta Oncol. 32, 825-830,
1993) can be used in the labeling of the anti-ITM2A antibody with
these positron-emitting nuclides.
[0205] The anti-ITM2A antibody labeled with the positron-emitting
nuclide is administered to humans or animals. Then, radiation
emitted by the radionuclide is measured ex vivo using PET (positron
emission tomograph). The measurement results are converted to
images by a computed tomographic approach. The PET apparatus is
intended to noninvasively obtain data about in vivo
pharmacokinetics or the like. The PET can quantitatively image
radiation intensity indicated by signal intensity. By such use of
the PET, antigen molecules highly expressed in particular cancer
can be detected without collecting samples from patients.
Specifically, in the present invention, the ITM2A protein highly
expressed in Ewing's sarcoma or blood cancer such as T cell
leukemia, T cell lymphoma, acute myeloid leukemia, B cell tumor, or
multiple myeloma can be detected. In the present invention, the
ITM2A protein expressed in Ewing's sarcoma having t(11;22)(q24;q12)
chromosomal translocation, among the Ewing's sarcoma cells, can be
detected. The Ewing's sarcoma, the acute myeloid leukemia, the B
cell tumor, the multiple myeloma, or the Ewing's sarcoma having
t(11;22)(q24;q12) chromosomal translocation may be any of primary
foci and metastatic foci. The anti-ITM2A antibody may be
radiolabeled with a short-life nuclide using a positron-emitting
nuclide such as .sup.11C, .sup.13N, .sup.15O, .sup.18F, or
.sup.45Ti, in addition to the nuclides described above. i
[0206] Research and development have been pursued as to, for
example, techniques of producing short-life nuclides using a
medical cyclotron and the nuclides described above or producing
short-life radiolabeling compounds. The anti-ITM2A antibody can be
labeled with various radioisotopes by these techniques. The
anti-ITM2A antibody administered to patients accumulates in primary
foci and metastatic foci according to the specificity of the
anti-ITM2A antibody for pathological tissues at each site. When the
anti-ITM2A antibody is labeled with the positron-emitting nuclide,
its radioactivity can be detected to detect the presence of the
primary foci and the metastatic foci based on the localization of
the radioactivity. An active value of gamma radiation or positron
emission of 25 to 4000 keV can be appropriately used for the
diagnostic use. Moreover, therapeutic effect can also be expected
by selecting an appropriate nuclide and administering the selected
nuclide in larger amounts. A nuclide that provides a value of gamma
radiation or positron emission of 70 to 700 keV can be used for
obtaining anticancer effect attributed to radiation.
[0207] In an alternative aspect, the present invention provides a
method for selecting a test subject to receive the pharmaceutical
composition comprising the antibody binding to ITM2A protein as an
active ingredient, or a test subject applicable to a method for
damaging ITM2A-expressing cells or inhibiting the growth of
ITM2A-expressing cells by contacting the ITM2A-expressing cells
with the antibody binding to ITM2A protein. In a further
alternative aspect, the present invention provides a method for
predicting the efficacy of cancer treatment using the anti-ITM2A
antibody of the present invention.
[0208] As described above, a test subject containing the ITM2A
protein expressed in Ewing's sarcoma cells or cells of blood cancer
such as T cell leukemia, T cell lymphoma, acute myeloid leukemia, B
cell tumor, or multiple myeloma in vivo is preferably selected as a
test subject to receive the antibody binding to ITM2A protein or
the pharmaceutical composition comprising this antibody as an
active ingredient, or as a test subject for which the efficacy of
treatment by the administration thereof is predicted. The method of
the present invention is not limited by whether or not these tumors
or cancers are primary foci or metastatic foci. A test subject
having in vivo Ewing's sarcoma having t(11;22)(q24;q12) chromosomal
translocation, among the Ewing's sarcomas, may be selected as a
preferable subject. Such Ewing's sarcoma having the chromosomal
translocation may be any of primary foci and metastatic foci.
[0209] The cell damaging method or the method for determining
whether or not Ewing's sarcoma cells whose growth is to be
inhibited have t(11;22)(q24;q12) chromosomal translocation
according to the present invention is as described above.
[0210] The present invention also provides a diagnostic drug or kit
for cancer diagnosis, comprising a reagent for detecting ITM2A
protein in a test sample. The diagnostic drug of the present
invention comprises at least the anti-ITM2A antibody.
[0211] The reagent for cancer diagnosis of the present invention
can be used as a kit for cancer diagnosis, in combination with
other factors used in ITM2A detection. Specifically, the present
invention relates to a kit for cancer diagnosis which comprises: an
antibody binding to ITM2A; and a reagent for detecting the binding
of the antibody to ITM2A and may further comprise a control sample
consisting of a biological sample containing ITM2A. A manual for
instruction of assay procedures may be further included in the kit
of the present invention.
[0212] An aspect represented by the expression "comprising" used
herein encompasses an aspect represented by the expression
"essentially consisting of" and an aspect represented by the
expression "consisting of".
[0213] All prior art documents cited herein are incorporated herein
by reference.
EXAMPLES
[0214] Hereinafter, the present invention will be described further
specifically with reference to Examples. However, the technical
scope of the present invention is not limited by these
Examples.
Example 1
Expression Analysis of ITM2A mRNA
[0215] The expression of ITM2A mRNA was assayed in clinical Ewing's
sarcoma samples, Ewing's sarcoma cell lines, blood cancer cell
lines, and normal tissues using Human Exon 1.0 ST Array
(Affymetrix, Inc.). The expression analysis employed 1 .mu.g of
total RNAs from each sample shown in FIG. 1. The analysis was
conducted according to a method described in GeneChip Whole
Transcript (WT) Sense Target Labeling Assay Manual (Affymetrix,
Inc.). The data was digitized using Exon Array Computational Tool
software (Affymetrix, Inc.). The total RNAs of normal tissues used
in the analysis were normal tissues-derived total RNAs purchased
from Clontech Laboratories, Inc., Ambion, Inc., Stratagene Corp.,
Cell Applications, Inc., Panomics, Inc., CHEMICON International,
Inc., and BioChain Institute, Inc. Total RNAs were prepared from
the tumor sites and normal sites of clinical cancer tissues
(sampled after informed consent was obtained) and from cancer cell
lines using Trizol (Invitrogen Corp.) or Isogen (Nippon Gene Co.,
Ltd.) according to methods included in these products. A mean of
numeric values obtained with ITM2A core probe sets (probe set IDs:
4013550, 4013551, 4013552, 4013553, 4013554, 4013557, 4013559,
4013560, 4013561, 4013564, and 4013565) was estimated as expression
data.
[0216] As a result of the expression analysis, the expression level
of ITM2A mRNA was around 1000 counts at the maximum in normal
tissues, but was 2000 to 7000 counts in cell lines or clinical
samples of Ewing's sarcoma, demonstrating the expression of ITM2A
in Ewing's sarcoma (FIG. 1). Also, the expression of ITM2A was
confirmed at a level around 2000 in an acute myeloid leukemia cell
line KG-1 or HL60, a B cell tumor cell line IM9, and a multiple
myeloma cell line KMS-12-BM. These results showed that ITM2A was
able to serve as a therapeutic target and a diagnostic marker for
Ewing's sarcoma or blood cancer such as T cell leukemia, T cell
lymphoma, acute myeloid leukemia, B cell tumor, or multiple
myeloma.
Example 2
Preparation of Monoclonal Antibody Against ITM2A
(2-1) Cloning of ITM2A Gene
[0217] cDNAs were prepared using SuperScript III Reverse
Transcriptase (Invitrogen Corp.) with total RNAs prepared from a
cancer cell line IM9 using Trizol as a template. A nucleotide
sequence encoding ITM2A was amplified by PCR using the cDNAs as a
template, a forward primer (SEQ ID NO: 43), and a reverse primer
(SEQ ID NO: 44). This PCR employed PrimeSTAR GXL DNA Polymerase
(Takara Bio Inc.) and was performed by 30 repetitive reaction
cycles each involving 98.degree. C. for 10 seconds, 55.degree. C.
for 15 seconds, and 68.degree. C. for 1 minute. The amplification
products formed from the PCR were cloned into pCR2.1-TOPO vectors
(Invitrogen Corp.) to obtain pCR2.1_ITM2A. The inserted sequence of
pCR2.1_ITM2A was sequenced to confirm that the inserted sequence
was the same as a sequence registered under RefSeq Accession No.
NM.sub.--004867.4.
(2-2) Preparation of Expression Vector for DNA Immunization
[0218] A nucleotide sequence encoding the extracellular region of
ITM2A (predicted to be Tyr75-Glu263 as a result of analysis
according to http://www.uniprot.org/) was cloned into expression
vectors (pMCN2i) for mammal cells. The vector pMCN2i allows
induction of expression of a gene insert under the control of mouse
CMV promoter (GenBank Accession No. U68299) and contains a neomycin
resistance gene incorporated therein. The signal sequence used was
the signal sequence of mouse interleukin 3. First, the ITM2A
extracellular region-encoding nucleotide sequence was amplified by
PCR using pCR2.1_ITM2A as a template, a forward primer (SEQ ID NO:
45) having SfiI site, and a reverse primer (SEQ ID NO: 46) having
NotI site. The amplification products formed from the PCR were
cloned into pCR2.1-TOPO vectors. SfiI/NotI-digested fragments of
the plasmids obtained as a result of the cloning were cloned into
the SfiI-NotI sites of pMCN2i_mIL3ss-mIgG2aFc vectors to obtain
plasmids (pMCN2i_mIL3ss-ITM2Aoutside). The pMCN2i_mIL3ss-mIgG2aFc
vectors contained the mouse interleukin 3 signal sequence cloned in
the EcoRI-SfiI site and the mouse IgG2a antibody Fc region gene
cloned in the CpoI-NotI site. The nucleotide sequence from start
codon to stop codon in the inserted sequence comprising the ITM2A
extracellular region-encoding nucleotide sequence in
pMCN2i_mIL3ss-ITM2Aoutside is shown in SEQ ID NO: 47, and an amino
acid sequence encoded thereby is shown in SEQ ID NO: 48.
(2-3) Preparation of Protein for Immunization
[0219] A fusion protein (ITM2A-Fc) of the ITM2A extracellular
region (Tyr75-Glu263) and the Fc region of mouse IgG2a was
prepared. First, the ITM2A extracellular region-encoding nucleotide
sequence was amplified by PCR using pCR2.1_ITM2A as a template, a
forward primer (SEQ ID NO: 49) having SfiI site, and a reverse
primer (SEQ ID NO: 50) having CpoI site, and cloned into
pCR2.1-TOPO vectors. SfiI/CpoI-digested fragments of the plasmids
obtained as a result of the cloning were cloned into the SfiI-CpoI
sites of pMCN2i_mIL3ss-mIgG2aFc vectors to obtain plasmids
pMCN2i_mIL3ss-ITM2Aoutside-Fc. The nucleotide sequence from start
codon to stop codon in the inserted sequence comprising the
ITM2A-Fc-encoding nucleotide sequence in
pMCN2i_mIL3ss-ITM2Aoutside-Fc is shown in SEQ ID NO: 51, and an
amino acid sequence encoded thereby is shown in SEQ ID NO: 52.
[0220] Next, pMCN2i_mIL3ss-ITM2Aoutside-Fc digested with PvuI was
transduced into a CHO cell line DG44 (Invitrogen Corp.) by
electroporation. Transductants were screened for with Geneticin
(500 .mu.g/mL) to establish a CHO cell line constantly secreting
ITM2A-Fc. The cells were cultured using a CHO-S-SFM II medium
(Invitrogen Corp.) supplemented with Geneticin (500 .mu.g/mL), HT
Supplement (Invitrogen Corp.), and penicillin/streptomycin
(Invitrogen Corp.) as a culture medium. ITM2A-Fc proteins were
purified from the culture supernatant of the cells thus
established. First, the culture supernatant was applied to a HiTrap
rProtein A FF column (GE Healthcare Bio-Sciences Corp.). The column
was washed with a binding buffer (20 mM sodium phosphate, pH 7.0),
followed by antibody elution with an eluting buffer (0.1 M
glycine-HCl, pH 2.7). The buffer solution of the eluate neutralized
with a neutralizing buffer (1 M Tris-HCl, pH 9.0) was replaced with
PBS using a PD-10 column (GE Healthcare Bio-Sciences Corp.). The
protein concentration was measured using DC Protein Assay Kit I
(Bio-Rad Laboratories, Inc.).
(2-4) Preparation of Cell Line Forced to Express ITM2A
[0221] A nucleotide sequence encoding C-terminally HA-tagged ITM2A
was cloned into pMCN2i vectors. First, the ITM2A-encoding
nucleotide sequence was amplified by PCR using pCR2.1_ITM2A as a
template, a forward primer (SEQ ID NO: 53) having EcoRI site, and a
reverse primer (SEQ ID NO: 54) having NotI site and an HA tag
sequence, and cloned into pCR2.1-TOPO vectors. EcoRI/NotI-digested
fragments of the plasmids obtained as a result of the cloning were
cloned into the EcoRI-NotI sites of pMCN2i vectors to obtain
plasmids pMCN2i_ITM2A-HA. The nucleotide sequence from start codon
to stop codon in the inserted sequence comprising the
ITM2A-encoding nucleotide sequence in pMCN2i_ITM2A-HA is shown in
SEQ ID NO: 55, and an amino acid sequence encoded thereby is shown
in SEQ ID NO: 56. pMCN2i_ITM2A-HA digested with PvuI was transduced
into DG44 cells by electroporation. Transductants were screened for
with Geneticin (500 .mu.g/mL) to establish a CHO cell line
constantly expressing C-terminally HA-tagged ITM2A (ITM2A_CHO).
(2-5) Preparation of Anti-ITM2A Monoclonal Antibody
[0222] A Balb/c mouse (female, 8 weeks old, Charles River
Laboratories Japan Inc.) was subjected to DNA immunization seven
times (days 0, 7, 11, 14, 17, 21, and 24) using Helios Gene Gun
(Bio-Rad Laboratories, Inc.). The DNA immunization employed
pMCN2i_mIL3ss-ITM2Aoutside. Following the DNA immunization, 50
.mu.g of the ITM2A-Fc proteins mixed with a Freund's incomplete
adjuvant (BD Diagnostics) was subcutaneously injected to the mouse
(days 49, 91, 99, and 107). At day 115, 50 .mu.g of the ITM2A-Fc
proteins was administered to the tail vein without being mixed with
an adjuvant. Three days thereafter, the spleen was excised and used
as a starting material to prepare hybridomas. First, the excised
spleen cells were mixed with a mouse myeloma cell line P3-X63Ag8U1
(P3U1, ATCC) at a ratio of 2:1. PEG1500 (Roche Diagnostics K.K.)
was gradually added to the mixed solution to perform cell fusion.
An RPMI1640 medium (Invitrogen Corp.) supplemented with
penicillin/streptomycin was added to the mixed solution, and the
mixture was centrifuged, followed by the removal of the supernatant
to remove PEG1500 from the mixed solution. Next, the cells were
suspended in a HAT medium (RPMI1640 medium supplemented with 10%
fetal bovine serum (FBS), penicillin-streptomycin, 1.times.HAT
Media Supplement (Sigma-Aldrich Corp.), and 0.5.times.BM-Condimed
H1 Hybridoma Cloning Supplement (Roche Diagnostics K.K.)), and the
resulting cell suspension was inoculated at a concentration of
1.times.10.sup.5 P3U1 cells/well to eight 96-well plates. The
plates were incubated at 37.degree. C. for 8 days in a 5% CO2
incubator, followed by screening using the culture supernatant in
each well. The screening was performed by assaying binding to the
ITM2A_CHO cells and the parent CHO cells using a flow cytometer
(FACSCalibur, Becton, Dickinson and Company). Clones producing
antibodies specifically binding to the ITM2A_CHO cells were
selected and cloned as single clones by the limiting dilution
method to isolate hybridomas producing antibodies binding to ITM2A.
From these experiments, anti-ITM2A monoclonal antibodies BE5-1,
BE6-1, BE7-1-1, and BE13-1 were established. These antibodies were
isotyped using Isostrip (Roche Diagnostics K.K.) and consequently,
all determined to be mouse IgG1.kappa..
[0223] The established hybridomas of BE5-1, BE6-1, BE7-1-1, and
BE13-1 were each cultured in a HAT medium supplemented with Ultra
Low IgG FBS (Invitrogen Corp.) instead of FBS. From each culture
supernatant, each anti-ITM2A antibody (BE5-1, BE6-1, BE7-1-1, and
BE13-1) was purified using a HiTrap Protein G HP column (GE
Healthcare Bio-Sciences Corp.). The concentration of the purified
antibody was measured using DC Protein Assay Kit I.
Example 3
Analysis on Epitope for Anti-ITM2A Monoclonal Antibody by ELISA
(3-1) Preparation of Partial ITM2A Protein
[0224] The ITM2A extracellular region (Tyr75-Glu263) or a portion
thereof (Tyr75-Lys182) was expressed as a GST-fusion protein in E.
coli (Tyr75-Glu263: GST-ITM2A-L, and Tyr75-Lys182: GST-ITM2A-S).
The fusion protein was C-terminally His-tagged. First, a nucleotide
sequence encoding ITM2A (Tyr75-Glu263) or ITM2A (Tyr75-Lys182) was
amplified by PCR using pCR2.1_ITM2A as a template, a forward primer
(SEQ ID NO: 57) having EcoRI site, and a reverse primer (SEQ ID NO:
58 or 59) having SalI site and a His tag sequence, and cloned into
pCR2.1-TOPO vectors. EcoRI/SalI-digested fragments of the plasmids
obtained as a result of the cloning were cloned into the EcoRI-SalI
sites of pGEX6P-1 vectors (GE Healthcare Bio-Sciences Corp.) to
obtain plasmids pGEX_GST-ITM2A-L and pGEX_GST-ITM2A-S,
respectively. The nucleotide sequence from start codon to stop
codon in pGEX_GST-ITM2A-L is shown in SEQ ID NO: 60, and an amino
acid sequence encoded thereby is shown in SEQ ID NO: 61. The
nucleotide sequence from start codon to stop codon in the inserted
sequence comprising the ITM2A-encoding nucleotide sequence in
pGEX_GST-ITM2A-S is shown in SEQ ID NO: 62, and an amino acid
sequence encoded thereby is shown in SEQ ID NO: 63. BL21 (DE3)
Competent Cells (Takara Bio Inc.) were transformed with
pGEX_GST-ITM2A-L or pGEX_GST-ITM2A-S, and each transformant was
induced to express GST-ITM2A-L or GST-ITM2A-S using
isopropyl-thiogalactopyranoside. After washing with B-PER (Thermo
Fisher Scientific K.K.), cell pellets were solubilized with a
solubilizing buffer (8 M urea, 50 mM Tris-HCl (pH 8.0), and 300 mM
NaCl). Cell extracts prepared with a solubilizing buffer
supplemented with 10 mM imidazole were applied to a HisTrap HP
column (GE Healthcare Bio-Sciences Corp.). The column was washed
with a solubilizing buffer supplemented with 40 mM imidazole,
followed by the elution of GST-ITM2A-L and GST-ITM2A-S using a
solubilizing buffer supplemented with 500 mM imidazole. The protein
concentrations of GST-ITM2A-L and GST-ITM2A-S were calculated on
the basis of absorbance at 280 nm.
(3-2) Analysis on Epitope for Anti-ITM2A Monoclonal Antibody by
ELISA
[0225] Each anti-ITM2A monoclonal antibody prepared in Example 2
was evaluated for its binding to GST-ITM2A-L and GST-ITM2A-S by
ELISA. First, 100 .mu.L each of GST-ITM2A-L and GST-ITM2A-S
solutions having a concentration of 3 .mu.g/mL was added to each
well of a 96-well plate for ELISA (Nunc-Immuno Plate, Thermo Fisher
Scientific K.K.) to coat the well with GST-ITM2A-L or GST-ITM2A-S.
The coated well was blocked with a buffer containing 1% bovine
serum albumin. Then, 100 .mu.L each of solutions of the antibodies
BE5-1, BE6-1, BE7-1-1, and BE13-1 diluted with the same buffer as
above was added to each well. The plate was incubated at room
temperature for 1 hour. The positive control used was an anti-His
antibody (mouse IgG1, Santa Cruz Biotechnology, Inc.). The negative
control used was mouse IgG1 (BD Pharmingen). Each antibody was
diluted into 8 dilutions at a common ratio of 3.16 from a
concentration of 1 .mu.g/mL. After reaction with a secondary
antibody (alkaline phosphatase-goat anti-mouse IgG (Gamma),
Invitrogen Corp.), a substrate (phosphatase substrate,
Sigma-Aldrich Corp.) was added to each well. Color developed by the
reaction solution in each well was determined by the measurement of
absorbance at 405 nm to 655 nm.
[0226] As a result, all the antibodies BE5-1, BE6-1, BE7-1-1, and
BE13-1 bound to GST-ITM2A-L, whereas only the antibodies BE5-1 and
BE6-1 bound to GST-ITM2A-S (FIG. 2). This suggested that the
antibodies BE5-1 and BE6-1 recognized ITM2A Tyr75-Lys182 and the
antibodies BE7-1-1 and BE13-1 recognized ITM2A Leu183-Glu263.
Example 4
Analysis on Epitope for Anti-ITM2A Monoclonal Antibody by FACS
(4-1) Preparation of Cell Line Forced to Express C-Terminally
Truncated ITM2A
[0227] The extracellular region of ITM2A contains a consensus
sequence (Arg226-Leu227-Arg228-Arg229) that is cleaved by furin.
Thus, a cell line expressing a protein (ITM2A-furin) comprising the
sequence from Met1 to Leu227 of ITM2A and an HA tag added
downstream thereof was prepared. First, a nucleotide sequence
encoding ITM2A (Met1-Leu227) was amplified by PCR using
pCR2.1_ITM2A as a template, a forward primer (SEQ ID NO: 53) having
EcoRI site, and a reverse primer (SEQ ID NO: 64) having NotI site
and an HA tag sequence, and cloned into pCR2.1-TOPO vectors.
EcoRI/NotI-digested fragments of the plasmids obtained as a result
of the cloning were cloned into the EcoRI-NotI sites of pMCN2i
vectors to obtain plasmids pMCN2i_ITM2A-furin-HA. The nucleotide
sequence from start codon to stop codon in the inserted sequence
comprising the ITM2A-furin-encoding nucleotide sequence in
pMCN2i_ITM2A-furin-HA is shown in SEQ ID NO: 65, and an amino acid
sequence encoded thereby is shown in SEQ ID NO: 66.
pMCN2i_ITM2A-furin-HA digested with PvuI was transduced into DG44
cells by electroporation. Transductants were screened for with
Geneticin (500 .mu.g/mL) to establish a CHO cell line
(ITM2A-furin_CHO) constantly expressing C-terminally HA-tagged
ITM2A-furin.
(4-2) Analysis on Epitope for Anti-ITM2A Monoclonal Antibody by
FACS
[0228] Each anti-ITM2A monoclonal antibody prepared in Example 2
was assayed for its binding to ITM2A-furin by flow cytometry
(FACS). The cells used were ITM2A-furin_CHO prepared in the
paragraph (4-1), ITM2A_CHO prepared in the paragraph (2-4), and
host cells CHO. These cells were separately suspended in PBS
supplemented with 0.2% bovine serum albumin and 0.1% NaN3 (FACS
buffer). To each cell suspension, the antibody BE5-1, BE6-1,
BE7-1-1, or BE13-1, mouse IgG1, or an anti-HA antibody (clone HA-7,
mouse IgG1, Sigma-Aldrich Corp.) was added, and the resulting cell
suspension was incubated for 1 hour on ice. Each antibody was
diluted into 6 dilutions at a common ratio of 5 from a
concentration of 10 .mu.g/mL. After washing of the cells with a
FACS buffer, an FITC-labeled anti-mouse IgG antibody (Goat F(ab')2
Fragment Anti-mouse IgG (Fc.gamma.)-FITC, Beckman Coulter, Inc.)
was added thereto as a secondary antibody. The resulting cell
suspension was incubated for 1 hour on ice. The cells were washed
with a FACS buffer, then suspended in a FACS buffer supplemented
with 10 .mu.g/mL propidium iodide (PI, Sigma-Aldrich Corp.), and
subjected to assay using a flow cytometer. The assay data was
analyzed using CELLQuest software (Becton, Dickinson and Company)
to evaluate a PI-negative live cell population.
[0229] All the antibodies BE5-1, BE6-1, BE7-1-1, and BE13-1 bound
to ITM2A_CHO, whereas only the antibodies BE5-1 and BE6-1 bound to
ITM2A-furin_CHO (FIGS. 3A and 3B). This suggested that the
antibodies BE5-1 and BE6-1 recognized ITM2A Tyr75-Leu227 and the
antibodies BE7-1-1 and BE13-1 recognized Arg228-Glu263. None of the
antibodies BE5-1, BE6-1, BE7-1-1, and BE13-1 (each 10 .mu.g/mL)
bound to host CHO cells (FIG. 3C). The anti-HA antibody (10
.mu.g/mL) was used to confirm the expression of ITM2A or
ITM2A-furin in each cell line (FIG. 3D).
Example 5
Analysis on Binding of Anti-ITM2A Monoclonal Antibody to Mouse
ITM2A
(5-1) Cloning of Mouse ITM2A Gene
[0230] A nucleotide sequence encoding mouse ITM2A was amplified by
PCR using mouse brain cDNA (Mouse MTC Panel I, Clontech
Laboratories, Inc.) as a template, a forward primer (SEQ ID NO:
67), and a reverse primer (SEQ ID NO: 68), and cloned into
pCR2.1-TOPO vectors to obtain pCR2.1_mITM2A. This PCR employed KOD
Plus (Toyobo Co., Ltd.) and was performed by denaturation at
94.degree. C. for 2 minutes followed by 30 repetitive reaction
cycles each involving 98.degree. C. for 10 seconds, 59.degree. C.
for 30 seconds, and 68.degree. C. for 1 minute. The inserted
sequence in pCR2.1_mITM2A was sequenced to confirm that the
inserted sequence was the same as a sequence registered under
RefSeq Accession No. NM.sub.--008409.
(5-2) Preparation of Cell Line Forced to Express Mouse ITM2A
[0231] A nucleotide sequence encoding C-terminally HA-tagged mouse
ITM2A was cloned into pMCN2i vectors. First, the mouse
ITM2A-encoding nucleotide sequence was amplified by PCR using
pCR2.1_mITM2A as a template, a forward primer (SEQ ID NO: 69)
having EcoRI site, and a reverse primer (SEQ ID NO: 70) having NotI
site and an HA tag sequence. The amplified fragments were digested
with EcoRI and NotI and cloned into the EcoRI-NotI sites of pMCN2i
vectors to obtain plasmids pMCN2i_mITM2A-HA. The nucleotide
sequence from start codon to stop codon in the inserted sequence
comprising the mouse ITM2A-encoding nucleotide sequence in
pMCN2i_mITM2A-HA is shown in SEQ ID NO: 71, and an amino acid
sequence encoded thereby is shown in SEQ ID NO: 72.
pMCN2i_mITM2A-HA digested with PvuI was transduced into DG44 cells
by electroporation. Transductants were screened for with Geneticin
(500 .mu.g/mL) to establish a CHO cell line (mITM2A_CHO) constantly
expressing C-terminally HA-tagged mouse ITM2A.
(5-3) Analysis on Binding of Anti-ITM2A Monoclonal Antibody to
Mouse ITM2A
[0232] Each anti-ITM2A monoclonal antibody prepared in Example 2
was evaluated for its binding to mouse ITM2A by FACS. The cells
used were ITM2A_CHO prepared in the paragraph (2-4), and mITM2A_CHO
prepared in the paragraph (5-2). The binding was detected using
FACS in the same way as the procedures described in the paragraph
4-2.
[0233] The antibodies BE5-1, BE7-1-1, and BE13-1 bound to
mITM2A_CHO, whereas the antibody BE6-1 did not bound thereto (FIG.
4). This suggested that the antibodies BE5-1, BE7-1-1, and BE13-1
cross-reacted with mouse ITM2A whereas the antibody BE6-1 did not
cross-react therewith.
Example 6
Evaluation of Anti-ITM2A Monoclonal Antibody for its Binding
Activity to ITM2A Using Western Blot
[0234] Each anti-ITM2A monoclonal antibody prepared in Example 2
was evaluated for whether its binding to ITM2A was detectable using
Western blot. First, 1.times.10.sup.7 cells each of ITM2A_CHO
prepared in the paragraph (2-4), ITM2A-furin_CHO prepared in the
paragraph (4-1), and host CHO cells were washed with PBS and then
lysed using 1 mL of a lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM
NaCl, 1 mM EDTA, 1% Triton X-100, and Protease Inhibitor Cocktail
(Sigma-Aldrich Corp.)) to obtain whole cell lysates. Each lysate
and 2.times. Sample Buffer (Sigma-Aldrich Corp.) mixed in equal
amounts were heat-treated. Then, 5 .mu.L of each lysate was
subjected to SDS-PAGE. After the electrophoresis, proteins, etc.
contained in SDS-PAGE gel were transferred to a PVDF membrane
(Immobilon-P, Millipore Corp.), which was then incubated at room
temperature for 1 hour with 10 .mu.g/mL of the antibody BE5-1,
BE6-1, BE7-1-1, or BE13-1, or an anti-HA antibody (clone F-7, Santa
Cruz Biotechnology, Inc.). The membrane was incubated at room
temperature for 1 hour with an HRP-labeled anti-mouse IgG antibody
(GE Healthcare Bio-Sciences Corp.) used as a secondary antibody.
Finally, light emitted using ECL Western Blotting Detection
Reagents (GE Healthcare Bio-Sciences Corp.) was exposed onto an
X-ray film to detect a band representing an antigen-antibody
complex.
[0235] The Western blot under these conditions showed that only the
antibody BE6-1 was able to form an antigen-antibody reaction
complex with the antigen ITM2A (FIG. 5).
Example 7
Determination of Gene Sequence Encoding Variable Region of
Anti-ITM2A Monoclonal Antibody
[0236] The gene sequences of variable regions of each anti-ITM2A
monoclonal antibody prepared in Example 2 were determined. Total
RNAs were prepared from hybridomas (1.times.10.sup.6 cells)
producing each antibody using RNeasy Mini Kit (Qiagen N.V.). Next,
cDNAs were synthesized using Smarter Race cDNA Amplification Kit
(Clontech Laboratories, Inc.) with the RNAs as a template. The
primers used were a primer (SEQ ID NO: 73) complementary to a
nucleotide sequence encoding the heavy chain constant region of a
mouse IgG1.kappa. antibody and a primer (SEQ ID NO: 74)
complementary to a nucleotide sequence encoding the light chain
constant region thereof. The amplification products cloned into
pCR2.1-TOPO vectors were sequenced. The variable region sequences
of each antibody are summarized in Table 1, and the variable region
CDR sequences thereof are summarized in Table 2.
TABLE-US-00002 TABLE 1 Sequence of variable region of ITM2A
antibody SEQ ID NO (nucleotide SEQ ID NO (amino Antibody sequence)
acid sequence) BE5-1 Heavy chain 27 28 variable region Light chain
29 30 variable region BE6-1 Heavy chain 31 32 variable region Light
chain 33 34 variable region BE7-1-1 Heavy chain 35 36 variable
region Light chain 37 38 variable region BE13-1 Heavy chain 39 40
variable region Light chain 41 42 variable region
TABLE-US-00003 TABLE 2 Amino acid sequence of variable region CDR
of ITM2A antibody SEQ ID NO (amino acid Antibody sequence) BE5-1
Heavy chain CDR1 3 CDR2 4 CDR3 5 Light chain CDR1 6 CDR2 7 CDR3 8
BE6-1 Heavy chain CDR1 9 CDR2 10 CDR3 11 Light chain CDR1 12 CDR2
13 CDR3 14 BE7-1-1 Heavy chain CDR1 15 CDR2 16 CDR3 17 Light chain
CDR1 18 CDR2 19 CDR3 20 BE13-1 Heavy chain CDR1 21 CDR2 22 CDR3 23
Light chain CDR1 24 CDR2 25 CDR3 26
Example 8
Analysis on Expression of ITM2A in Human Cancer Cell Line and
Evaluation of Anti-ITM2A Monoclonal Antibody for its ADCC Activity
and Cell Growth Inhibitory Activity
(8-1) Analysis on Expression of ITM2A in Human Cancer Cell Line
[0237] The expression of ITM2A was assayed in human cancer cell
lines by FACS. The antibody used was the antibody BE6-1 having a
concentration of 10 .mu.g/mL. The expression of ITM2A was assayed
in the same way as the procedures described in Example 4. The
secondary antibody used was an FITC-labeled anti-mouse Ig antibody
(goat F(ab')2) included in Qifi-Kit (Dako). The negative control
used was mouse IgG1 having a concentration of 10 .mu.g/mL. The
assay results demonstrated that ITM2A was expressed on the cells of
Ewing's sarcoma cell lines (A-673, RD-ES, SK-ES-1, and SK-N-MC), T
cell acute lymphocytic leukemia cell lines (CCRF-CEM, Jurkat, and
MOLT-4), a T cell lymphoma cell line (HuT78), and acute myeloid
leukemia cell lines (KG-1a and TF-1a) (FIG. 6).
(8-2) Study on ADCC Activity of Anti-ITM2A Monoclonal Antibody
[0238] The antibody-dependent cell-mediated cytotoxicity (ADCC)
activity of the antibody BE6-1 was assayed. Human cancer cell lines
CCRF-CEM and KG-1a were separately cultured for 1 hour in the
presence of chromium-51 (GE Healthcare Bio-Sciences Corp.) and then
washed three times with an RPMI1640 medium supplemented with 10%
fetal bovine serum and penicillin/streptomycin (hereinafter,
referred to as an RPMI medium). Each cell suspension of
1.times.10.sup.5 cells/mL was prepared using the RPMI medium. The
cell suspension was added at a concentration of 100 .mu.L/well to a
96-well plate. Next, the antibody BE6-1 or mouse IgG1 was diluted
with the RPMI medium and added thereto at a concentration of 50
.mu.L/well. The final concentration of the antibody BE6-1 was
adjusted to 10, 2, 0.4, 0.08, and 0.016 .mu.g/mL. The final
concentration of the mouse IgG1 was adjusted to 10 .mu.g/mL. The
plate was left standing at room temperature for 15 minutes. Then, a
cell suspension of effector cells adjusted to 1.times.10.sup.6
cells/mL with the RPMI medium was added thereto at a concentration
of 50 .mu.L/well. The effector cells used were NK-92 cells (ATCC)
constantly expressing chimeric proteins comprising the
extracellular region of mouse Fc.gamma. receptor III (RefSeq
Accession No. NM.sub.--010188) fused in frame with the
transmembrane and intracellular regions of human Fc.epsilon.
receptor I-gamma (RefSeq Accession No. NM.sub.--004106)
(WO2008093688). The plate was incubated at 37.degree. C. for 4
hours in a 5% CO2 incubator. Then, 100 .mu.L/well of the culture
supernatant was recovered, and the radioactivity (cpm) of the
culture supernatant was measured using a gamma counter (1480 WIZARD
3'', Wallac). The measurement value was applied to the following
expression to calculate the rate (%) of specific chromium
release:
Rate(%) of specific chromium release=(A-C).times.100/(B-C).
[0239] In the expression, A represents the radioactivity in each
well; B represents a mean of radioactivity values of wells
containing cells lysed with 1% (final concentration) Nonidet P-40;
and C represents a mean of radioactivity values of wells
supplemented with only target cells. The experiment was triplicated
to calculate a mean of the rates of specific chromium release and
standard deviation.
[0240] Human cancer cell lines A-673 and SK-N-MC were inoculated at
each concentration of 5.times.10.sup.3 cells/well to a plate
(Cellbind surface 96-well cell culture plate (Corning Inc.)). The
plate was incubated at 37.degree. C. for 4 days in a 5% CO2
incubator. After addition of chromium-51 to each well, the plate
was further incubated for 1 hour. Each well was carefully washed
with a medium so as not to dissociate the cells. Then, a medium was
added thereto at a concentration of 50 .mu.L/well. Next, the
antibody BE6-1 or mouse IgG1 was added thereto at a concentration
of 50 .mu.L/well. The final concentration of the antibody BE6-1 was
adjusted to 10, 2, 0.4, 0.08, and 0.016 .mu.g/mL. The final
concentration of the mouse IgG1 was adjusted to 10 .mu.g/mL. The
plate was left standing at room temperature for 15 minutes. Then,
effector cells adjusted to 8.times.10.sup.5 cells/mL with a medium
were added thereto at a concentration of 100 .mu.L/well. The rate
of specific chromium release was calculated according to the
expression described above. All the media used were a DMEM medium
(Invitrogen Corp.) supplemented with 10% fetal bovine serum and
penicillin/streptomycin.
[0241] The antibody BE6-1 induced an ADCC activity against the
cells A-673, SK-N-MC, CCRF-CEM, and KG-1a in a
concentration-dependent manner (FIG. 7).
(8-3) Study on Cell Growth Inhibitory Activity of Anti-ITM2A
Monoclonal Antibody
[0242] The cell growth inhibitory activity of the antibody BE6-1
was assayed in the presence of a toxin-conjugated secondary
antibody. The toxin-conjugated secondary antibody used was a
saporin-labeled anti-mouse IgG antibody (Mab-Zap, Advanced
Targeting Systems). A human cancer cell line CCRF-CEM was
inoculated at a concentration of 6.times.10.sup.3 cells/well to a
96-well plate. The antibody BE6-1 (500, 100, 20, and 4 ng/mL) or
mouse IgG1 (500 ng/mL) was added to each well. Mab-Zap was added at
a concentration of 500 ng/mL to each well. The plate was incubated
for 3 days. Then, cell growth in each well was assayed using WST-8
(Cell Count Reagent SF, Nacalai Tesque, Inc.). The experiment was
triplicated to calculate a mean and standard deviation with cell
grown in a well supplemented with only a medium as 0% and cell
growth in a well supplemented with only cells as 100%. The medium
used was an RPMI1640 medium supplemented with 10% fetal bovine
serum and penicillin/streptomycin.
[0243] A human cancer cell line HuT78 was inoculated at a
concentration of 1.times.10.sup.4 cells/well to a 96-well plate.
The antibody BE6-1 (2500, 500, 100, and 20 ng/mL) or mouse IgG1
(2500 ng/mL) was added to each well. Mab-Zap was added at a
concentration of 2500 ng/mL to each well. The plate was incubated
for 4 days. Then, cell growth in each well was assayed using WST-8.
The medium used was an IMDM medium (Invitrogen Corp.) supplemented
with 20% fetal bovine serum and penicillin/streptomycin.
[0244] A human cancer cell line A-673 was inoculated at a
concentration of 3.times.10.sup.3 cells/well to a 96-well plate.
The plate was incubated for 1 day. Then, the antibody BE6-1 (1000,
200, 40, and 8 ng/mL) or mouse IgG1 (1000 ng/mL) was added to each
well of the plate. Mab-Zap was added at a concentration of 1000
ng/mL to each well. The plate was incubated for 3 days. Then, cell
growth in each well was assayed using WST-8. The medium used was a
DMEM medium supplemented with 10% fetal bovine serum and
penicillin/streptomycin.
[0245] The antibody BE6-1 inhibited cell growth of each cell line
in a concentration-dependent manner in the presence of the
toxin-conjugated secondary antibody (FIG. 8). This suggested that
the anti-ITM2A monoclonal antibody directly conjugated with toxin
was also able to inhibit the growth of cancer cells.
Example 9
Correlation Between Expression of EWS-FLI1 Fusion Gene and
ITM2A
(9-1) Expression Analysis of EWS-FLI1 Fusion Gene in Clinical
Ewing's Sarcoma Sample
[0246] 85% of Ewing's sarcoma cases are known to have observable
t(11;22)(q24;q12) chromosomal translocation and the expression of a
fusion gene (EWS-FLI1) comprising the 5' end of the EWS gene fused
with the 3' end of the FLI-1 gene (Cancer Lett (2007) 254, 1-10).
Thus, 13 clinical Ewing's sarcoma samples including the samples
used in the expression analysis of Example 1 (ews.sub.--2,
ews.sub.--3, ews.sub.--4, ews.sub.--5, ews.sub.--6, ews.sub.--7,
ews.sub.--8, ews.sub.--9, ews.sub.--10, ews.sub.--11, ews.sub.--12,
ews.sub.--13, and ews.sub.--15) were analyzed for the expression of
the EWS-FLI1 fusion gene by PCR. First, cDNAs were synthesized from
the RNAs of each clinical Ewing's sarcoma sample using SuperScript
III First-Strand Synthesis System for RT-PCR (Invitrogen Corp.).
Next, the EWS-FLI1 fusion gene was amplified by PCR using the cDNAs
as a template, a forward primer (SEQ ID NO: 75), and a reverse
primer (SEQ ID NO: 76). This amplification employed KOD Plus
Version 2 (Toyobo Co., Ltd.) and was performed by thermal
denaturation at 94.degree. C. for 2 minutes followed by 30
repetitive cycles each involving 98.degree. C. for 10 seconds and
68.degree. C. for 1.5 minutes. The primer sequences used were the
same as those described in the literature (N Engl J Med (1994) 331,
294-9). A nucleotide sequence encoding .beta.-actin was amplified
as a control using primers included in the kit (SuperScript III
First-Strand Synthesis System for RT-PCR). The amplification
reaction using PCR employed KOD Plus Version 2 and was performed by
thermal denaturation at 94.degree. C. for 2 minutes followed by 25
repetitive cycles each involving 98.degree. C. for 10 seconds,
58.degree. C. for 30 seconds, and 68.degree. C. for 30 seconds. The
positive control used was a Ewing's sarcoma cell line SK-ES-1
expressing the EWS-FLI1 fusion gene. The negative control used was
a lymphoma cell line NK-92.
[0247] The PCR results demonstrated the expression of the EWS-FLI1
fusion gene in 9 (ews.sub.--4, ews.sub.--5, ews.sub.--7,
ews.sub.--8, ews.sub.--9, ews.sub.--11, ews.sub.--12, ews.sub.--13,
and ews.sub.--15) out of the 13 clinical Ewing's sarcoma samples
(FIG. 9A).
(9-2) Analysis on Expression of ITM2A in Clinical Ewing's Sarcoma
Sample
[0248] The expression of the ITM2A gene was analyzed in clinical
Ewing's sarcoma samples by PCR. The ITM2A gene was amplified by PCR
using the cDNAs synthesized in the paragraph (9-1) as a template, a
forward primer (SEQ ID NO: 77), and a reverse primer (SEQ ID NO:
78). This amplification employed KOD Plus Version 2 and was
performed by thermal denaturation at 94.degree. C. for 2 minutes
followed by 25 repetitive cycles each involving 98.degree. C. for
10 seconds, 65.degree. C. for 30 seconds, and 68.degree. C. for 30
seconds. The positive control used was pCR2.1_ITM2A prepared in the
paragraph (2-1). The negative control used was a lymphoma cell line
NK-92.
[0249] As a result of the PCR, ITM2A was expressed in the 9 cases
expressing the EWS-FLI1 fusion gene, demonstrating high correlation
between their expression (FIG. 9B).
Sequence CWU 1
1
861263PRThomo sapiens 1Met Val Lys Ile Ala Phe Asn Thr Pro Thr Ala
Val Gln Lys Glu Glu 1 5 10 15 Ala Arg Gln Asp Val Glu Ala Leu Leu
Ser Arg Thr Val Arg Thr Gln 20 25 30 Ile Leu Thr Gly Lys Glu Leu
Arg Val Ala Thr Gln Glu Lys Glu Gly 35 40 45 Ser Ser Gly Arg Cys
Met Leu Thr Leu Leu Gly Leu Ser Phe Ile Leu 50 55 60 Ala Gly Leu
Ile Val Gly Gly Ala Cys Ile Tyr Lys Tyr Phe Met Pro 65 70 75 80 Lys
Ser Thr Ile Tyr Arg Gly Glu Met Cys Phe Phe Asp Ser Glu Asp 85 90
95 Pro Ala Asn Ser Leu Arg Gly Gly Glu Pro Asn Phe Leu Pro Val Thr
100 105 110 Glu Glu Ala Asp Ile Arg Glu Asp Asp Asn Ile Ala Ile Ile
Asp Val 115 120 125 Pro Val Pro Ser Phe Ser Asp Ser Asp Pro Ala Ala
Ile Ile His Asp 130 135 140 Phe Glu Lys Gly Met Thr Ala Tyr Leu Asp
Leu Leu Leu Gly Asn Cys 145 150 155 160 Tyr Leu Met Pro Leu Asn Thr
Ser Ile Val Met Pro Pro Lys Asn Leu 165 170 175 Val Glu Leu Phe Gly
Lys Leu Ala Ser Gly Arg Tyr Leu Pro Gln Thr 180 185 190 Tyr Val Val
Arg Glu Asp Leu Val Ala Val Glu Glu Ile Arg Asp Val 195 200 205 Ser
Asn Leu Gly Ile Phe Ile Tyr Gln Leu Cys Asn Asn Arg Lys Ser 210 215
220 Phe Arg Leu Arg Arg Arg Asp Leu Leu Leu Gly Phe Asn Lys Arg Ala
225 230 235 240 Ile Asp Lys Cys Trp Lys Ile Arg His Phe Pro Asn Glu
Phe Ile Val 245 250 255 Glu Thr Lys Ile Cys Gln Glu 260 2792DNAhomo
sapiens 2atggtgaaaa tcgccttcaa tacccctacc gccgtgcaaa aggaggaggc
gcggcaagac 60gtggaggccc tcctgagccg cacggtcaga actcagatac tgaccggcaa
ggagctccga 120gttgccaccc aggaaaaaga gggctcctct gggagatgta
tgcttactct cttaggcctt 180tcattcatct tggcaggact tattgttggt
ggagcctgca tttacaagta cttcatgccc 240aagagcacca tttaccgtgg
agagatgtgc ttttttgatt ctgaggatcc tgcaaattcc 300cttcgtggag
gagagcctaa cttcctgcct gtgactgagg aggctgacat tcgtgaggat
360gacaacattg caatcattga tgtgcctgtc cccagtttct ctgatagtga
ccctgcagca 420attattcatg actttgaaaa gggaatgact gcttacctgg
acttgttgct ggggaactgc 480tatctgatgc ccctcaatac ttctattgtt
atgcctccaa aaaatctggt agagctcttt 540ggcaaactgg cgagtggcag
atatctgcct caaacttatg tggttcgaga agacctagtt 600gctgtggagg
aaattcgtga tgttagtaac cttggcatct ttatttacca actttgcaat
660aacagaaagt ccttccgcct tcgtcgcaga gacctcttgc tgggtttcaa
caaacgtgcc 720attgataaat gctggaagat tagacacttc cccaacgaat
ttattgttga gaccaagatc 780tgtcaagagt aa 79235PRThomo sapiens 3Asp
Tyr Arg Met His 1 5 419PRTMus musculus 4Val Ile Thr Gly Lys Ser Asp
Asn Tyr Gly Ala Ser Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly 53PRTMus
musculus 5Arg Asp Tyr 1 616PRTMus musculus 6Arg Ser Ser Lys Ser Leu
Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr 1 5 10 15 77PRTMus musculus
7Arg Met Ser Asn Leu Ala Pro 1 5 89PRTMus musculus 8Met Gln His Leu
Glu Tyr Pro Phe Thr 1 5 95PRTMus musculus 9Glu Tyr Thr Met His 1 5
1017PRTMus musculus 10Gly Ile Asn Pro Asn Asn Gly Asp Thr Ser Tyr
Asn Gln Lys Phe Lys 1 5 10 15 Gly 115PRTMus musculus 11Gly Pro Phe
Ala Tyr 1 5 1216PRTMus musculus 12Arg Ser Ser Gln Ser Leu Val His
Ser Asn Gly Asn Thr Tyr Phe His 1 5 10 15 137PRTMus musculus 13Lys
Val Ser Asn Arg Phe Phe 1 5 149PRTMus musculus 14Ser Gln Thr Thr
His Phe Pro Phe Thr 1 5 155PRTMus musculus 15Ser Tyr Trp Met His 1
5 1617PRTMus musculus 16Glu Ile Asp Pro Ser Asp Ser Tyr Asn Asp Tyr
Asn Gln Lys Phe Lys 1 5 10 15 Gly 175PRTMus musculus 17Trp Gly Glu
Asp Tyr 1 5 1816PRTMus musculus 18Lys Ser Ser Gln Ser Leu Leu Asp
Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 197PRTMus musculus 19Leu
Val Ser Lys Leu Asp Ser 1 5 209PRTMus musculus 20Trp Gln Gly Thr
His Phe Pro Arg Thr 1 5 215PRTMus musculus 21Asp Tyr Tyr Met Lys 1
5 2217PRTMus musculus 22Asp Ile Asn Pro Asn Asn Gly Gly Leu Ser Tyr
Asn Gln Lys Phe Lys 1 5 10 15 Gly 235PRTMus musculus 23Trp Ser Gly
Ala Tyr 1 5 2416PRTMus musculus 24Lys Ser Ser Gln Ser Leu Leu Asp
Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 257PRTMus musculus 25Leu
Val Ser Lys Leu Asp Ser 1 5 269PRTMus musculus 26Trp Gln Gly Thr
His Phe Pro Trp Thr 1 5 27342DNAMus musculus 27caggtgcagc
ttgtagagac cgggggaggc ttggtgaggc ctggaaattc tctgaaactc 60tcctgtgtta
cctcgggatt cactttcagt gactaccgga tgcactggct tcgccagtct
120ccagggaaga ggctggagtg gattgctgta attacaggca aatctgataa
ttatggagca 180agttatgcag agtctgtgaa aggcagattc actatttcaa
gagatgattc aaaaagcagt 240gtctacctgc agatgaacag attaagagag
gaagacactg ccacttatta ttgtagtaga 300agggactact ggggtcaagg
aacctcagtc accgtctcct ca 34228114PRTMus musculus 28Gln Val Gln Leu
Val Glu Thr Gly Gly Gly Leu Val Arg Pro Gly Asn 1 5 10 15 Ser Leu
Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30
Arg Met His Trp Leu Arg Gln Ser Pro Gly Lys Arg Leu Glu Trp Ile 35
40 45 Ala Val Ile Thr Gly Lys Ser Asp Asn Tyr Gly Ala Ser Tyr Ala
Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Arg Leu Arg Glu Glu
Asp Thr Ala Thr Tyr 85 90 95 Tyr Cys Ser Arg Arg Asp Tyr Trp Gly
Gln Gly Thr Ser Val Thr Val 100 105 110 Ser Ser 29336DNAMus
musculus 29gatattgtga tgactcaggc tgcaccctct gtacctgtca ctcctggaga
gtcagtatcc 60atctcctgca ggtctagtaa gagtctcctg catagtaatg gcaacactta
cttgtattgg 120ttcctgcaga ggccaggcca gtctcctcag ctcctgatat
atcggatgtc caaccttgcc 180ccaggagtcc cagacaggtt cagtggcagt
gggtcaggaa ctgctttcac actgagaatc 240agtagagtgg aggctgagga
tgtgggtgtt tattactgta tgcaacatct cgaatatcct 300ttcacgttcg
gtgctgggac caagctggag ctgaaa 33630112PRTMus musculus 30Asp Ile Val
Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly 1 5 10 15 Glu
Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25
30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser
35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Pro Gly
Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe
Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Phe Thr Phe Gly
Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 31342DNAMus musculus
31gaggtccagt tgcaacagtc tggacctgaa ctggtgaagc ctggggcttc agtgaagata
60tcctgcaaga cttctggata cacattcact gaatacacca tgcactgggt gaagcagagc
120catggaaaga gccttgagtg gattggaggt attaatccta acaatggtga
tactagctac 180aaccagaagt tcaagggcaa ggccacattg actgtagaca
agtcctccat cacagcctac 240atggagctcc gcagcctgac atctgaggat
tctgcagtct attactgtgc aagaggcccg 300tttgcttact ggggccaggg
gactctggtc actgtctcta ca 34232114PRTMus musculus 32Glu Val Gln Leu
Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30
Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35
40 45 Gly Gly Ile Asn Pro Asn Asn Gly Asp Thr Ser Tyr Asn Gln Lys
Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ile
Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Pro Phe Ala Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Thr 33336DNAMus
musculus 33gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga
tcaagcctcc 60atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta
cttccattgg 120tacctgcaga agccaggcca gtctccaaag ttcctgatct
acaaagtttc caaccgattt 180tttggggtcc cagacaggtt cagtggcagt
ggatcaggga cagatttcac actcaagatc 240agcagagtgg aggctgagga
tctgggagtt tatttctgct ctcaaactac acattttcca 300ttcacgttcg
gctcggggac aaagttggaa ataaaa 33634112PRTMus musculus 34Asp Val Val
Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp
Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25
30 Asn Gly Asn Thr Tyr Phe His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45 Pro Lys Phe Leu Ile Tyr Lys Val Ser Asn Arg Phe Phe Gly
Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val
Tyr Phe Cys Ser Gln Thr 85 90 95 Thr His Phe Pro Phe Thr Phe Gly
Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 35342DNAMus musculus
35caggtccaac tgcagcagcc tggggctgaa cttgtgaaac ctggggcttc agtgaagctg
60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg
120cctggacaag gccttgagtg gatcggagag attgatcctt ctgatagcta
taatgactac 180aatcaaaagt tcaagggcaa ggccacattg actgtagaca
aatcctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac
tctgcggtct attactgtgc aagatggggg 300gaggactact ggggccaagg
caccactctc acagtctcct ca 34236114PRTMus musculus 36Gln Val Gln Leu
Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Asn Asp Tyr Asn Gln Lys
Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Glu Asp Tyr Trp Gly
Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser 37336DNAMus
musculus 37gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca
accagcctcc 60atctcttgca agtcaagtca gagcctctta gatagtgatg gaaagacata
tttgaattgg 120ttgttacaga ggccaggcca gtctccaaag cgcctaatct
atctggtgtc taaactggac 180tctggagtcc ctgacaggtt cactggcagt
ggatcaggga cagatttcac actgaaaatc 240agcagagtgg aggctgagga
tttgggagtt tattattgct ggcaaggtac acattttcct 300cggacgttcg
gtggaggcac caagctggaa atcaaa 33638112PRTMus musculus 38Asp Val Val
Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln
Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25
30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly
Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val
Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 39342DNAMus musculus
39gaggtccagc tgcaacaatc tggacctgag ctggtgaagc ctggggcttc agtgaggatg
60tcctgtaagg cttctggata cacattcact gactactaca tgaagtgggt gaagcagagt
120catggaaaga gccttgagtg gattggagat attaatccta acaatggtgg
tcttagttac 180aaccagaagt tcaagggcaa ggccacattg actgtagaca
aatcctccag cacagcctac 240atgcagctcg ccagcctgac atctgaggac
tctgcagtct attactgtgc aatatggtcc 300ggggcttact ggggccaagg
gactctggtc actgtctctg ca 34240114PRTMus musculus 40Glu Val Gln Leu
Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val
Arg Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30
Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35
40 45 Gly Asp Ile Asn Pro Asn Asn Gly Gly Leu Ser Tyr Asn Gln Lys
Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Met Gln Leu Ala Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Trp Ser Gly Ala Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ala 41336DNAMus
musculus 41gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca
accagcctcc 60atctcttgca agtcaagtca gagcctctta gatagtgatg gaaagacata
tttgaattgg 120ttgttacaga ggccaggcca gtctccaaag cgcctaatct
atctggtgtc taaactggac 180tctggagtcc ctgacaggtt cactggcagt
ggatcaggga cagatttcac actgaaaatc 240agcagagtgg aggctgagga
tttgggagtt tattattgct ggcaaggtac acattttccg 300tggacgttcg
gtggaggcac caagctggaa atcaaa 33642112PRTMus musculus 42Asp Val Val
Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln
Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25
30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly
Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val
Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Trp Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 4326DNAArtificial
SequencePCR primer 43atggtgaaaa tcgccttcaa tacccc
264427DNAArtificial SequencePCR primer 44ttactcttga cagatcttgg
tctcaac 274543DNAArtificial SequencePCR primer 45ggcccagccg
gccatggcgt acaagtactt catgcccaag agc 434630DNAArtificial
SequencePCR primer 46gcggccgctt actcttgaca gatcttggtc
3047648DNAArtificial SequencepMCN2i_mIL3ss-ITM2Aoutside insert
47atggttcttg ccagctctac caccagcatc cacaccatgc tgctcctgct cctgatgctg
60gcccagccgg ccatggcgta caagtacttc atgcccaaga gcaccattta ccgtggagag
120atgtgctttt ttgattctga ggatcctgca aattcccttc gtggaggaga
gcctaacttc 180ctgcctgtga ctgaggaggc tgacattcgt gaggatgaca
acattgcaat cattgatgtg 240cctgtcccca gtttctctga tagtgaccct
gcagcaatta ttcatgactt tgaaaaggga 300atgactgctt acctggactt
gttgctgggg aactgctatc tgatgcccct caatacttct 360attgttatgc
ctccaaaaaa tctggtagag ctctttggca aactggcgag tggcagatat
420ctgcctcaaa cttatgtggt tcgagaagac ctagttgctg tggaggaaat
tcgtgatgtt 480agtaaccttg gcatctttat ttaccaactt tgcaataaca
gaaagtcctt ccgccttcgt 540cgcagagacc tcttgctggg tttcaacaaa
cgtgccattg ataaatgctg gaagattaga 600cacttcccca acgaatttat
tgttgagacc aagatctgtc aagagtaa 64848215PRTArtificial
SequencepMCN2i_mIL3ss-ITM2Aoutside insert 48Met Val Leu Ala Ser Ser
Thr Thr Ser Ile His Thr Met Leu Leu Leu 1 5 10 15 Leu Leu Met Leu
Ala Gln Pro Ala Met Ala Tyr Lys Tyr Phe Met Pro 20 25 30 Lys Ser
Thr Ile Tyr Arg Gly Glu Met Cys Phe Phe
Asp Ser Glu Asp 35 40 45 Pro Ala Asn Ser Leu Arg Gly Gly Glu Pro
Asn Phe Leu Pro Val Thr 50 55 60 Glu Glu Ala Asp Ile Arg Glu Asp
Asp Asn Ile Ala Ile Ile Asp Val 65 70 75 80 Pro Val Pro Ser Phe Ser
Asp Ser Asp Pro Ala Ala Ile Ile His Asp 85 90 95 Phe Glu Lys Gly
Met Thr Ala Tyr Leu Asp Leu Leu Leu Gly Asn Cys 100 105 110 Tyr Leu
Met Pro Leu Asn Thr Ser Ile Val Met Pro Pro Lys Asn Leu 115 120 125
Val Glu Leu Phe Gly Lys Leu Ala Ser Gly Arg Tyr Leu Pro Gln Thr 130
135 140 Tyr Val Val Arg Glu Asp Leu Val Ala Val Glu Glu Ile Arg Asp
Val 145 150 155 160 Ser Asn Leu Gly Ile Phe Ile Tyr Gln Leu Cys Asn
Asn Arg Lys Ser 165 170 175 Phe Arg Leu Arg Arg Arg Asp Leu Leu Leu
Gly Phe Asn Lys Arg Ala 180 185 190 Ile Asp Lys Cys Trp Lys Ile Arg
His Phe Pro Asn Glu Phe Ile Val 195 200 205 Glu Thr Lys Ile Cys Gln
Glu 210 215 4942DNAArtificial SequencePCR primer 49ctggcccagc
cggccatggc gtacaagtac ttcatgccca ag 425038DNAArtificial SequencePCR
primer 50gtcggtccgc gaggttcctc ttgacagatc ttggtctc
38511347DNAArtificial SequencepMCN2i_mIL3ss-ITM2Aoutside-Fc insert
51atggttcttg ccagctctac caccagcatc cacaccatgc tgctcctgct cctgatgctg
60gcccagccgg ccatggcgta caagtacttc atgcccaaga gcaccattta ccgtggagag
120atgtgctttt ttgattctga ggatcctgca aattcccttc gtggaggaga
gcctaacttc 180ctgcctgtga ctgaggaggc tgacattcgt gaggatgaca
acattgcaat cattgatgtg 240cctgtcccca gtttctctga tagtgaccct
gcagcaatta ttcatgactt tgaaaaggga 300atgactgctt acctggactt
gttgctgggg aactgctatc tgatgcccct caatacttct 360attgttatgc
ctccaaaaaa tctggtagag ctctttggca aactggcgag tggcagatat
420ctgcctcaaa cttatgtggt tcgagaagac ctagttgctg tggaggaaat
tcgtgatgtt 480agtaaccttg gcatctttat ttaccaactt tgcaataaca
gaaagtcctt ccgccttcgt 540cgcagagacc tcttgctggg tttcaacaaa
cgtgccattg ataaatgctg gaagattaga 600cacttcccca acgaatttat
tgttgagacc aagatctgtc aagaggaacc tcgcggaccg 660acaatcaagc
cctgtcctcc atgcaaatgc ccagcaccta acctcttggg tggaccatcc
720gtcttcatct tccctccaaa gatcaaggat gtactcatga tctccctgag
ccccatagtc 780acatgtgtgg tggtggatgt gagcgaggat gacccagatg
tccagatcag ctggtttgtg 840aacaacgtgg aagtacacac agctcagaca
caaacccata gagaggatta caacagtact 900ctccgggtgg tcagtgccct
ccccatccag caccaggact ggatgagtgg caaggagttc 960aaatgcaagg
tcaacaacaa agacctgcca gcgcccatcg agagaaccat ctcaaaaccc
1020aaagggtcag taagagctcc acaggtatat gtcttgcctc caccagaaga
agagatgact 1080aagaaacagg tcactctgac ctgcatggtc acagacttca
tgcctgaaga catttacgtg 1140gagtggacca acaacgggaa aacagagcta
aactacaaga acactgaacc agtcctggac 1200tctgatggtt cttacttcat
gtacagcaag ctgagagtgg aaaagaagaa ctgggtggaa 1260agaaatagct
actcctgttc agtggtccac gagggtctgc acaatcacca cacgactaag
1320agcttctccc ggactccggg taaatga 134752448PRTArtificial
SequencepMCN2i_mIL3ss-ITM2Aoutside-Fc insert 52Met Val Leu Ala Ser
Ser Thr Thr Ser Ile His Thr Met Leu Leu Leu 1 5 10 15 Leu Leu Met
Leu Ala Gln Pro Ala Met Ala Tyr Lys Tyr Phe Met Pro 20 25 30 Lys
Ser Thr Ile Tyr Arg Gly Glu Met Cys Phe Phe Asp Ser Glu Asp 35 40
45 Pro Ala Asn Ser Leu Arg Gly Gly Glu Pro Asn Phe Leu Pro Val Thr
50 55 60 Glu Glu Ala Asp Ile Arg Glu Asp Asp Asn Ile Ala Ile Ile
Asp Val 65 70 75 80 Pro Val Pro Ser Phe Ser Asp Ser Asp Pro Ala Ala
Ile Ile His Asp 85 90 95 Phe Glu Lys Gly Met Thr Ala Tyr Leu Asp
Leu Leu Leu Gly Asn Cys 100 105 110 Tyr Leu Met Pro Leu Asn Thr Ser
Ile Val Met Pro Pro Lys Asn Leu 115 120 125 Val Glu Leu Phe Gly Lys
Leu Ala Ser Gly Arg Tyr Leu Pro Gln Thr 130 135 140 Tyr Val Val Arg
Glu Asp Leu Val Ala Val Glu Glu Ile Arg Asp Val 145 150 155 160 Ser
Asn Leu Gly Ile Phe Ile Tyr Gln Leu Cys Asn Asn Arg Lys Ser 165 170
175 Phe Arg Leu Arg Arg Arg Asp Leu Leu Leu Gly Phe Asn Lys Arg Ala
180 185 190 Ile Asp Lys Cys Trp Lys Ile Arg His Phe Pro Asn Glu Phe
Ile Val 195 200 205 Glu Thr Lys Ile Cys Gln Glu Glu Pro Arg Gly Pro
Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn
Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Lys
Ile Lys Asp Val Leu Met Ile Ser Leu 245 250 255 Ser Pro Ile Val Thr
Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 260 265 270 Asp Val Gln
Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 275 280 285 Gln
Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 290 295
300 Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe
305 310 315 320 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile
Glu Arg Thr 325 330 335 Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro
Gln Val Tyr Val Leu 340 345 350 Pro Pro Pro Glu Glu Glu Met Thr Lys
Lys Gln Val Thr Leu Thr Cys 355 360 365 Met Val Thr Asp Phe Met Pro
Glu Asp Ile Tyr Val Glu Trp Thr Asn 370 375 380 Asn Gly Lys Thr Glu
Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp 385 390 395 400 Ser Asp
Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 405 410 415
Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly 420
425 430 Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly
Lys 435 440 445 5335DNAArtificial SequencePCR primer 53gaattcacca
tggtgaaaat cgccttcaat acccc 355462DNAArtificial SequencePCR primer
54gcggccgctt aagcgtaatc tggaacatcg tatgggtact cttgacagat cttggtctca
60ac 6255819DNAArtificial SequencepMCN2i_ITM2A-HA insert
55atggtgaaaa tcgccttcaa tacccctacc gccgtgcaaa aggaggaggc gcggcaagac
60gtggaggccc tcctgagccg cacggtcaga actcagatac tgaccggcaa ggagctccga
120gttgccaccc aggaaaaaga gggctcctct gggagatgta tgcttactct
cttaggcctt 180tcattcatct tggcaggact tattgttggt ggagcctgca
tttacaagta cttcatgccc 240aagagcacca tttaccgtgg agagatgtgc
ttttttgatt ctgaggatcc tgcaaattcc 300cttcgtggag gagagcctaa
cttcctgcct gtgactgagg aggctgacat tcgtgaggat 360gacaacattg
caatcattga tgtgcctgtc cccagtttct ctgatagtga ccctgcagca
420attattcatg actttgaaaa gggaatgact gcttacctgg acttgttgct
ggggaactgc 480tatctgatgc ccctcaatac ttctattgtt atgcctccaa
aaaatctggt agagctcttt 540ggcaaactgg cgagtggcag atatctgcct
caaacttatg tggttcgaga agacctagtt 600gctgtggagg aaattcgtga
tgttagtaac cttggcatct ttatttacca actttgcaat 660aacagaaagt
ccttccgcct tcgtcgcaga gacctcttgc tgggtttcaa caaacgtgcc
720attgataaat gctggaagat tagacacttc cccaacgaat ttattgttga
gaccaagatc 780tgtcaagagt acccatacga tgttccagat tacgcttaa
81956272PRTArtificial SequencepMCN2i_ITM2A-HA insert 56Met Val Lys
Ile Ala Phe Asn Thr Pro Thr Ala Val Gln Lys Glu Glu 1 5 10 15 Ala
Arg Gln Asp Val Glu Ala Leu Leu Ser Arg Thr Val Arg Thr Gln 20 25
30 Ile Leu Thr Gly Lys Glu Leu Arg Val Ala Thr Gln Glu Lys Glu Gly
35 40 45 Ser Ser Gly Arg Cys Met Leu Thr Leu Leu Gly Leu Ser Phe
Ile Leu 50 55 60 Ala Gly Leu Ile Val Gly Gly Ala Cys Ile Tyr Lys
Tyr Phe Met Pro 65 70 75 80 Lys Ser Thr Ile Tyr Arg Gly Glu Met Cys
Phe Phe Asp Ser Glu Asp 85 90 95 Pro Ala Asn Ser Leu Arg Gly Gly
Glu Pro Asn Phe Leu Pro Val Thr 100 105 110 Glu Glu Ala Asp Ile Arg
Glu Asp Asp Asn Ile Ala Ile Ile Asp Val 115 120 125 Pro Val Pro Ser
Phe Ser Asp Ser Asp Pro Ala Ala Ile Ile His Asp 130 135 140 Phe Glu
Lys Gly Met Thr Ala Tyr Leu Asp Leu Leu Leu Gly Asn Cys 145 150 155
160 Tyr Leu Met Pro Leu Asn Thr Ser Ile Val Met Pro Pro Lys Asn Leu
165 170 175 Val Glu Leu Phe Gly Lys Leu Ala Ser Gly Arg Tyr Leu Pro
Gln Thr 180 185 190 Tyr Val Val Arg Glu Asp Leu Val Ala Val Glu Glu
Ile Arg Asp Val 195 200 205 Ser Asn Leu Gly Ile Phe Ile Tyr Gln Leu
Cys Asn Asn Arg Lys Ser 210 215 220 Phe Arg Leu Arg Arg Arg Asp Leu
Leu Leu Gly Phe Asn Lys Arg Ala 225 230 235 240 Ile Asp Lys Cys Trp
Lys Ile Arg His Phe Pro Asn Glu Phe Ile Val 245 250 255 Glu Thr Lys
Ile Cys Gln Glu Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 260 265 270
5727DNAArtificial SequencePCR primer 57gaattctaca agtacttcat
gcccaag 275848DNAArtificial SequencePCR primer 58gtcgactcag
tggtggtggt ggtggtgctc ttgacagatc ttggtctc 485948DNAArtificial
SequencePCR primer 59gtcgactcag tggtggtggt ggtggtgttt gccaaagagc
tctaccag 48601290DNAArtificial SequencepGEX_GST-ITM2A-L insert
60atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt
60ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa
120tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta
ttatattgat 180ggtgatgtta aattaacaca gtctatggcc atcatacgtt
atatagctga caagcacaac 240atgttgggtg gttgtccaaa agagcgtgca
gagatttcaa tgcttgaagg agcggttttg 300gatattagat acggtgtttc
gagaattgca tatagtaaag actttgaaac tctcaaagtt 360gattttctta
gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa
420acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga
cgctcttgat 480gttgttttat acatggaccc aatgtgcctg gatgcgttcc
caaaattagt ttgttttaaa 540aaacgtattg aagctatccc acaaattgat
aagtacttga aatccagcaa gtatatagca 600tggcctttgc agggctggca
agccacgttt ggtggtggcg accatcctcc aaaatcggat 660ctggaagttc
tgttccaggg gcccctggga tccccggaat tctacaagta cttcatgccc
720aagagcacca tttaccgtgg agagatgtgc ttttttgatt ctgaggatcc
tgcaaattcc 780cttcgtggag gagagcctaa cttcctgcct gtgactgagg
aggctgacat tcgtgaggat 840gacaacattg caatcattga tgtgcctgtc
cccagtttct ctgatagtga ccctgcagca 900attattcatg actttgaaaa
gggaatgact gcttacctgg acttgttgct ggggaactgc 960tatctgatgc
ccctcaatac ttctattgtt atgcctccaa aaaatctggt agagctcttt
1020ggcaaactgg cgagtggcag atatctgcct caaacttatg tggttcgaga
agacctagtt 1080gctgtggagg aaattcgtga tgttagtaac cttggcatct
ttatttacca actttgcaat 1140aacagaaagt ccttccgcct tcgtcgcaga
gacctcttgc tgggtttcaa caaacgtgcc 1200attgataaat gctggaagat
tagacacttc cccaacgaat ttattgttga gaccaagatc 1260tgtcaagagc
accaccacca ccaccactga 129061429PRTArtificial
SequencepGEX_GST-ITM2A-L insert 61Met Ser Pro Ile Leu Gly Tyr Trp
Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu
Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp
Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu
Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65
70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met
Leu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg
Ile Ala Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe
Leu Ser Lys Leu Pro Glu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg
Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His
Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu
Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val
Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185
190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu
Val Leu 210 215 220 Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe Tyr Lys
Tyr Phe Met Pro 225 230 235 240 Lys Ser Thr Ile Tyr Arg Gly Glu Met
Cys Phe Phe Asp Ser Glu Asp 245 250 255 Pro Ala Asn Ser Leu Arg Gly
Gly Glu Pro Asn Phe Leu Pro Val Thr 260 265 270 Glu Glu Ala Asp Ile
Arg Glu Asp Asp Asn Ile Ala Ile Ile Asp Val 275 280 285 Pro Val Pro
Ser Phe Ser Asp Ser Asp Pro Ala Ala Ile Ile His Asp 290 295 300 Phe
Glu Lys Gly Met Thr Ala Tyr Leu Asp Leu Leu Leu Gly Asn Cys 305 310
315 320 Tyr Leu Met Pro Leu Asn Thr Ser Ile Val Met Pro Pro Lys Asn
Leu 325 330 335 Val Glu Leu Phe Gly Lys Leu Ala Ser Gly Arg Tyr Leu
Pro Gln Thr 340 345 350 Tyr Val Val Arg Glu Asp Leu Val Ala Val Glu
Glu Ile Arg Asp Val 355 360 365 Ser Asn Leu Gly Ile Phe Ile Tyr Gln
Leu Cys Asn Asn Arg Lys Ser 370 375 380 Phe Arg Leu Arg Arg Arg Asp
Leu Leu Leu Gly Phe Asn Lys Arg Ala 385 390 395 400 Ile Asp Lys Cys
Trp Lys Ile Arg His Phe Pro Asn Glu Phe Ile Val 405 410 415 Glu Thr
Lys Ile Cys Gln Glu His His His His His His 420 425
621047DNAArtificial SequencepGEX_GST-ITM2A-S insert 62atgtccccta
tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60ttggaatatc
ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa
120tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta
ttatattgat 180ggtgatgtta aattaacaca gtctatggcc atcatacgtt
atatagctga caagcacaac 240atgttgggtg gttgtccaaa agagcgtgca
gagatttcaa tgcttgaagg agcggttttg 300gatattagat acggtgtttc
gagaattgca tatagtaaag actttgaaac tctcaaagtt 360gattttctta
gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa
420acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga
cgctcttgat 480gttgttttat acatggaccc aatgtgcctg gatgcgttcc
caaaattagt ttgttttaaa 540aaacgtattg aagctatccc acaaattgat
aagtacttga aatccagcaa gtatatagca 600tggcctttgc agggctggca
agccacgttt ggtggtggcg accatcctcc aaaatcggat 660ctggaagttc
tgttccaggg gcccctggga tccccggaat tctacaagta cttcatgccc
720aagagcacca tttaccgtgg agagatgtgc ttttttgatt ctgaggatcc
tgcaaattcc 780cttcgtggag gagagcctaa cttcctgcct gtgactgagg
aggctgacat tcgtgaggat 840gacaacattg caatcattga tgtgcctgtc
cccagtttct ctgatagtga ccctgcagca 900attattcatg actttgaaaa
gggaatgact gcttacctgg acttgttgct ggggaactgc 960tatctgatgc
ccctcaatac ttctattgtt atgcctccaa aaaatctggt agagctcttt
1020ggcaaacacc accaccacca ccactga 104763348PRTArtificial
SequencepGEX_GST-ITM2A-S insert 63Met Ser Pro Ile Leu Gly Tyr Trp
Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu
Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp
Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu
Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65
70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met
Leu Glu 85 90
95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu
Pro Glu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys
Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met
Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro
Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe Lys Lys
Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190 Leu Lys Ser
Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr
Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu 210 215
220 Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe Tyr Lys Tyr Phe Met Pro
225 230 235 240 Lys Ser Thr Ile Tyr Arg Gly Glu Met Cys Phe Phe Asp
Ser Glu Asp 245 250 255 Pro Ala Asn Ser Leu Arg Gly Gly Glu Pro Asn
Phe Leu Pro Val Thr 260 265 270 Glu Glu Ala Asp Ile Arg Glu Asp Asp
Asn Ile Ala Ile Ile Asp Val 275 280 285 Pro Val Pro Ser Phe Ser Asp
Ser Asp Pro Ala Ala Ile Ile His Asp 290 295 300 Phe Glu Lys Gly Met
Thr Ala Tyr Leu Asp Leu Leu Leu Gly Asn Cys 305 310 315 320 Tyr Leu
Met Pro Leu Asn Thr Ser Ile Val Met Pro Pro Lys Asn Leu 325 330 335
Val Glu Leu Phe Gly Lys His His His His His His 340 345
6464DNAArtificial SequencePCR primer 64gcggccgctt aagcgtaatc
tggaacatcg tatgggtaaa ggcggaagga ctttctgtta 60ttgc
6465711DNAArtificial SequencepMCN2i_ITM2A-furin-HA insert
65atggtgaaaa tcgccttcaa tacccctacc gccgtgcaaa aggaggaggc gcggcaagac
60gtggaggccc tcctgagccg cacggtcaga actcagatac tgaccggcaa ggagctccga
120gttgccaccc aggaaaaaga gggctcctct gggagatgta tgcttactct
cttaggcctt 180tcattcatct tggcaggact tattgttggt ggagcctgca
tttacaagta cttcatgccc 240aagagcacca tttaccgtgg agagatgtgc
ttttttgatt ctgaggatcc tgcaaattcc 300cttcgtggag gagagcctaa
cttcctgcct gtgactgagg aggctgacat tcgtgaggat 360gacaacattg
caatcattga tgtgcctgtc cccagtttct ctgatagtga ccctgcagca
420attattcatg actttgaaaa gggaatgact gcttacctgg acttgttgct
ggggaactgc 480tatctgatgc ccctcaatac ttctattgtt atgcctccaa
aaaatctggt agagctcttt 540ggcaaactgg cgagtggcag atatctgcct
caaacttatg tggttcgaga agacctagtt 600gctgtggagg aaattcgtga
tgttagtaac cttggcatct ttatttacca actttgcaat 660aacagaaagt
ccttccgcct ttacccatac gatgttccag attacgctta a 71166236PRTArtificial
SequencepMCN2i_ITM2A-furin-HA insert 66Met Val Lys Ile Ala Phe Asn
Thr Pro Thr Ala Val Gln Lys Glu Glu 1 5 10 15 Ala Arg Gln Asp Val
Glu Ala Leu Leu Ser Arg Thr Val Arg Thr Gln 20 25 30 Ile Leu Thr
Gly Lys Glu Leu Arg Val Ala Thr Gln Glu Lys Glu Gly 35 40 45 Ser
Ser Gly Arg Cys Met Leu Thr Leu Leu Gly Leu Ser Phe Ile Leu 50 55
60 Ala Gly Leu Ile Val Gly Gly Ala Cys Ile Tyr Lys Tyr Phe Met Pro
65 70 75 80 Lys Ser Thr Ile Tyr Arg Gly Glu Met Cys Phe Phe Asp Ser
Glu Asp 85 90 95 Pro Ala Asn Ser Leu Arg Gly Gly Glu Pro Asn Phe
Leu Pro Val Thr 100 105 110 Glu Glu Ala Asp Ile Arg Glu Asp Asp Asn
Ile Ala Ile Ile Asp Val 115 120 125 Pro Val Pro Ser Phe Ser Asp Ser
Asp Pro Ala Ala Ile Ile His Asp 130 135 140 Phe Glu Lys Gly Met Thr
Ala Tyr Leu Asp Leu Leu Leu Gly Asn Cys 145 150 155 160 Tyr Leu Met
Pro Leu Asn Thr Ser Ile Val Met Pro Pro Lys Asn Leu 165 170 175 Val
Glu Leu Phe Gly Lys Leu Ala Ser Gly Arg Tyr Leu Pro Gln Thr 180 185
190 Tyr Val Val Arg Glu Asp Leu Val Ala Val Glu Glu Ile Arg Asp Val
195 200 205 Ser Asn Leu Gly Ile Phe Ile Tyr Gln Leu Cys Asn Asn Arg
Lys Ser 210 215 220 Phe Arg Leu Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
225 230 235 6729DNAArtificial SequencePCR primer 67atggtgaaga
tcgccttcaa cacccctac 296828DNAArtificial SequencePCR primer
68tcactcctga cagatcttgg tttcaacg 286937DNAArtificial SequencePCR
primer 69taagaattcc accatggtga agatcgcctt caacacc
377059DNAArtificial SequencePCR primer 70gcggccgctt aagcgtaatc
tggaacatcg tatgggtact cctgacagat cttggtttc 5971819DNAArtificial
SequencepMCN2i_mITM2A-HA insert 71atggtgaaga tcgccttcaa cacccctacg
gcggtgcaaa aggaggaggc gcggcaagat 60gtagaggcgc tcgtcagtcg cactgtccga
gctcaaatcc tgactggcaa ggagctcaga 120gttgtcccgc aggagaaaga
tggctcatct gggagatgca tgcttactct cctaggcctc 180tcattcatct
tggcaggact gattgttggt ggagcctgca tttacaagta cttcatgccc
240aagagcacca tttaccatgg tgagatgtgc ttctttgatt ctgaggatcc
tgtcaattcc 300attcctggag gagagccata ctttctgcct gtgactgagg
aggctgatat ccgtgaggat 360gacaacattg ccatcattga tgtgcctgtg
cccagtttct ctgatagcga tccggcggca 420attattcacg actttgagaa
gggaatgact gcttacctgg acttgctttt gggaaactgt 480tatctgatgc
ccctcaatac ttccattgtt atgactccaa agaatctggt ggaacttttt
540ggaaaactgg caagtggcaa gtatttgcct catacttatg tggttcgtga
agacctggtt 600gctgtggaag aaattcgtga tgttagtaac cttggtattt
ttatttacca actttgcaac 660aaccgaaaat ccttccgcct tagacgcaga
gaccttctgc tgggtttcaa caagcgtgcc 720attgacaaat gctggaagat
tagacacttc cccaatgaat ttatcgttga aaccaagatc 780tgtcaggagt
acccatacga tgttccagat tacgcttaa 81972272PRTArtificial
SequencepMCN2i_mITM2A-HA insert 72Met Val Lys Ile Ala Phe Asn Thr
Pro Thr Ala Val Gln Lys Glu Glu 1 5 10 15 Ala Arg Gln Asp Val Glu
Ala Leu Val Ser Arg Thr Val Arg Ala Gln 20 25 30 Ile Leu Thr Gly
Lys Glu Leu Arg Val Val Pro Gln Glu Lys Asp Gly 35 40 45 Ser Ser
Gly Arg Cys Met Leu Thr Leu Leu Gly Leu Ser Phe Ile Leu 50 55 60
Ala Gly Leu Ile Val Gly Gly Ala Cys Ile Tyr Lys Tyr Phe Met Pro 65
70 75 80 Lys Ser Thr Ile Tyr His Gly Glu Met Cys Phe Phe Asp Ser
Glu Asp 85 90 95 Pro Val Asn Ser Ile Pro Gly Gly Glu Pro Tyr Phe
Leu Pro Val Thr 100 105 110 Glu Glu Ala Asp Ile Arg Glu Asp Asp Asn
Ile Ala Ile Ile Asp Val 115 120 125 Pro Val Pro Ser Phe Ser Asp Ser
Asp Pro Ala Ala Ile Ile His Asp 130 135 140 Phe Glu Lys Gly Met Thr
Ala Tyr Leu Asp Leu Leu Leu Gly Asn Cys 145 150 155 160 Tyr Leu Met
Pro Leu Asn Thr Ser Ile Val Met Thr Pro Lys Asn Leu 165 170 175 Val
Glu Leu Phe Gly Lys Leu Ala Ser Gly Lys Tyr Leu Pro His Thr 180 185
190 Tyr Val Val Arg Glu Asp Leu Val Ala Val Glu Glu Ile Arg Asp Val
195 200 205 Ser Asn Leu Gly Ile Phe Ile Tyr Gln Leu Cys Asn Asn Arg
Lys Ser 210 215 220 Phe Arg Leu Arg Arg Arg Asp Leu Leu Leu Gly Phe
Asn Lys Arg Ala 225 230 235 240 Ile Asp Lys Cys Trp Lys Ile Arg His
Phe Pro Asn Glu Phe Ile Val 245 250 255 Glu Thr Lys Ile Cys Gln Glu
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 260 265 270 7321DNAArtificial
SequencePCR primer 73gggccagtgg atagacagat g 217423DNAArtificial
SequencePCR primer 74gctcactgga tggtgggaag atg 237522DNAArtificial
SequencePCR primer 75cccactagtt acccacccca aa 227622DNAArtificial
SequencePCR primer 76tgttgggctt gcttttccgc tc 227722DNAArtificial
SequencePCR primer 77ggctcctctg ggagatgtat gc 227822DNAArtificial
SequencePCR primer 78atcctcacga atgtcagcct cc 22794PRTArtificial
Sequencepeptide linker 79Gly Gly Gly Ser 1 804PRTArtificial
Sequencepeptide linker 80Ser Gly Gly Gly 1 815PRTArtificial
Sequencepeptide linker 81Gly Gly Gly Gly Ser 1 5 825PRTArtificial
Sequencepeptide linker 82Ser Gly Gly Gly Gly 1 5 836PRTArtificial
Sequencepeptide linker 83Gly Gly Gly Gly Gly Ser 1 5
846PRTArtificial Sequencepeptide linker 84Ser Gly Gly Gly Gly Gly 1
5 857PRTArtificial Sequencepeptide linker 85Gly Gly Gly Gly Gly Gly
Ser 1 5 867PRTArtificial Sequencepeptide linker 86Ser Gly Gly Gly
Gly Gly Gly 1 5
* * * * *
References