U.S. patent application number 14/848825 was filed with the patent office on 2016-02-25 for monoclonal antibody capable of binding to anexelekto, and use thereof.
This patent application is currently assigned to CHUGAI SEIYAKU KABUSHIKI KAISHA. The applicant listed for this patent is Chugai Seiyaku Kabushiki Kaisha. Invention is credited to Takehisa Kitazawa, Hajime Miyamoto, Shigehisa Nagahashi, Tsukasa Suzuki.
Application Number | 20160053019 14/848825 |
Document ID | / |
Family ID | 40638811 |
Filed Date | 2016-02-25 |
United States Patent
Application |
20160053019 |
Kind Code |
A1 |
Kitazawa; Takehisa ; et
al. |
February 25, 2016 |
MONOCLONAL ANTIBODY CAPABLE OF BINDING TO ANEXELEKTO, AND USE
THEREOF
Abstract
The present inventors have succeeded in producing anti-AXL
antibodies with specific functions. The present inventors also
discovered that the antibodies have an angiogenesis-suppressive
effect and an antitumor effect, and thereby completed the present
invention. The anti-AXL antibodies of the present invention are
useful as angiogenesis inhibitors and agents for inducing or
inhibiting phosphorylation activity.
Inventors: |
Kitazawa; Takehisa;
(Shizuoka, JP) ; Suzuki; Tsukasa; (Shizuoka,
JP) ; Nagahashi; Shigehisa; (Kanagawa, JP) ;
Miyamoto; Hajime; (Kanagawa, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chugai Seiyaku Kabushiki Kaisha |
Tokyo |
|
JP |
|
|
Assignee: |
CHUGAI SEIYAKU KABUSHIKI
KAISHA
Tokyo
JP
|
Family ID: |
40638811 |
Appl. No.: |
14/848825 |
Filed: |
September 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12742947 |
Oct 1, 2010 |
9175091 |
|
|
PCT/JP2008/070739 |
Nov 14, 2008 |
|
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14848825 |
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Current U.S.
Class: |
424/138.1 ;
435/375; 530/387.7 |
Current CPC
Class: |
C07K 2317/73 20130101;
A61P 35/00 20180101; C07K 16/40 20130101; A61K 2039/505 20130101;
A61P 9/00 20180101; C07K 2317/76 20130101; C07K 2317/56 20130101;
A61P 43/00 20180101; A01K 2267/0331 20130101; C07K 2317/75
20130101; C07K 2317/565 20130101; C07K 16/32 20130101; C07K 2319/30
20130101 |
International
Class: |
C07K 16/32 20060101
C07K016/32 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 15, 2007 |
JP |
2007-297168 |
Claims
1.-46. (canceled)
47. A monoclonal antibody that binds to fibronectin type III domain
1 (FND1) of human anexelekto (AXL), has cell-growth-suppressive
activity, and does not inhibit phosphorylation of AXL.
48. The antibody according to claim 47, wherein the antibody
suppresses cancer cell growth.
49. The antibody according to claim 47, wherein the antibody has an
activity that reduces AXL expression level.
50. The antibody according to claim 47, wherein the antibody has
angiogenesis inhibitory activity.
51. The antibody of claim 47, wherein phosphorylation of AXL is
induced by the binding of an AXL ligand to AXL.
52. The antibody of claim 48, wherein phosphorylation of AXL is
induced by the binding of an AXL ligand to AXL.
53. A monoclonal antibody prepared using as an antigen a peptide
consisting of the entire amino acid sequence of FND1 of human AXL
or an amino acid sequence comprising five or more consecutive amino
acids thereof, wherein the antibody has cell-growth-suppressive
activity and does not inhibit phosphorylation of AXL.
54. The antibody according to claim 53, wherein the antibody has an
activity that reduces AXL expression level.
55. The antibody according to claim 53, wherein the antibody has
angiogenesis inhibitory activity.
56. The antibody of claim 53, wherein phosphorylation of AXL is
induced by the binding of an AXL ligand to AXL.
57. A monoclonal anti-AXL antibody that competes for binding to the
same epitope bound by either of (a) a monoclonal antibody (Ax284)
produced from a hybridoma deposited with the IPOD, AIST, and
assigned Accession No. FERM BP-10857; or (b) a monoclonal antibody
(Ax225) produced from a hybridoma deposited with the IPOD, AIST,
and assigned Accession No. FERM BP-10854, wherein the anti-AXL
antibody blocks the binding of either Ax284 or Ax225 to AXL protein
by at least 50% as compared to a control level of binding of either
Ax284 or Ax225 to AXL protein in the absence of the anti-AXL
antibody.
58. A method of inhibiting angiogenesis in a subject, the method
comprising administering to a subject in need thereof the antibody
according to claim 47.
59. A method of suppressing cell-growth in a subject, the method
comprising administering to a subject in need thereof the antibody
according to claim 47.
60. The method of claim 59, wherein the antibody suppresses growth
of a cancer cell.
61. A method of treating cancer in a subject, the method comprising
administering to a subject in need thereof the antibody according
to claim 47.
62. The method of claim 61, wherein the cancer is glioma, gastric
cancer, endometrial cancer, or non-small-cell lung cancer.
63. The method of claim 61, wherein the cancer is pancreatic
cancer, gastric cancer, lung cancer, osteosarcoma, colon cancer,
prostate cancer, melanoma, endometrial cancer, ovarian cancer,
uterine leiomyoma, thyroid cancer, cancer stem cell, breast cancer,
bladder cancer, renal cancer, glioma, neuroblastoma, or esophageal
cancer.
64. The method of claim 63, wherein the cancer is pancreatic
adenocarcinoma or breast cancer.
65. A method for reducing a level of AXL protein expression in a
cell, the method comprising contacting a cell that expresses AXL
protein with the antibody according to claim 47.
66. The method of claim 65, wherein the cell is contacted with the
antibody in vitro.
67. The method of claim 65, wherein the cell is contacted with the
antibody in vivo.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 12/742,947, having a 371(c) date of Oct. 1,
2010, which is the National Stage of International Application
Serial No. PCT/JP2008/070739, filed on Nov. 14, 2008, which claims
the benefit of Japanese Application Serial No. 2007-297168, filed
on Nov. 15, 2007. The contents of the foregoing applications are
incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to monoclonal antibodies that
bind to anexelekto, agents containing the antibodies as an active
ingredient, and methods for using the antibodies.
BACKGROUND
[0003] Anexelekto (also referred to as "AXL", "UFO", "ARK", or
"TYRO7"; hereinafter referred to as "AXL"), which has been cloned
from patients with chronic myeloid leukemia, is an oncogene capable
of transforming mouse NIH3T3 cells when highly expressed
(Non-patent Documents 1 and 2). AXL protein is a 140-kDa receptor
tyrosine kinase (Non-patent Document 3), and is said to be
responsible for signal transduction to downstream molecules through
its autophosphorylation, which occurs after it binds to the ligand
Gas6 (growth arrest specific gene 6) (Non-patent Document 4).
Receptor tyrosine kinases, such as Sky, Mer, and AXL, are known as
receptor tyrosine kinases with Gas6 as a ligand (Non-patent
Document 5).
[0004] AXL is presumed to have molecular functions involved in cell
growth enhancement, suppression of apoptosis, cell migration, and
cell adhesion. Experimentally observed phenomena in cells treated
with Gas6 protein support this presumption. Reported experimental
results include its suppression of cell death and its enhancement
of cell growth in rat vascular smooth muscle (Non-patent Documents
6 and 7), the acceleration of cell growth and the suppression of
cell death after serum starvation in mouse NIH3T3 cells (Non-patent
Documents 8 and 9), the promotion of cell growth in mouse cardiac
fibroblasts (Non-patent Document 10), the enhancement of cell
growth in human prostate cancer cells (Non-patent Document 11), the
enhancement of cell growth and infiltration and the suppression of
cell death in human gastric carcinoma cells (Non-patent Document
12), the enhancement of the migration ability of human and rat
vascular smooth muscle cells (Non-patent Document 13), the
enhancement of the cell migration ability of mouse neurons
(Non-patent Document 14), and the aggregation of cells highly
expressing mouse AXL (Non-patent Document 15).
[0005] Similarly, PI3K-Akt pathway and MAPK pathway are said to
function as downstream pathways of the signal transduction mediated
by AXL based on molecular analyses of intracellular signals after
treatment with Gas6 (Non-patent Document 5). An analysis with a
yeast two-hybrid method using an AXL intracellular region as the
bait confirmed the direct molecular interactions with these
downstream pathways. As a result,
GrbB2/PI3K/p55.gamma./SOCS-1/NcK2/RanBP2/C1-TEN were identified
(Non-patent Document 16). The interactions of these molecules
suggest the presence of intracellular signal transduction pathways
as downstream from the AXL signal. Furthermore, the observed
interactions support the presumption that AXL functions in cell
growth enhancement, the suppression of apoptosis, cell migration,
and cell adhesion. AXL has also been identified as a gene highly
expressed when TNF.alpha.-induced cell death of mouse fibroblasts
is suppressed by IL-15. The suppression of TNF.alpha.-induced cell
death was abolished by suppressing AXL expression, and the
phosphorylation of IL-15 receptors and AXL was enhanced by
treatment with IL-15. These experimental findings also suggest that
signal transduction is mediated by the complex of AXL and IL-15
receptor (Non-patent Document 17).
[0006] Tumorigenicity of nude mice has been reported to dissipate
as a result of inhibiting Gas6-dependent phosphorylation of AXL in
human glioma lines overexpressing the AXL dominant negative form
(Non-patent Document 18). However, there have been no reports or
suggestions and remain unclear whether any anti-AXL antibody which
inhibits phosphorylation exists.
[0007] AXL is a single-pass transmembrane receptor tyrosine kinase,
and the extracellular region is composed of two immunoglobulin-like
domains (referred to as IgD1 and IgD2) and two fibronectin type III
domains (referred to as FND1 and FND2) from the N-terminal side
(Non-patent Document 3). Although FND is known to be expressed in
molecules such as neural cell adhesion molecules and nephrins
involved in intercellular adhesion, detailed functions of FND in
AXL are unclear (Non-patent Document 19).
[0008] AXL has been identified as an oncogene that retains an
inherent ability to transform cells, and has been studied as a
carcinogenesis-related molecule. Many analyses of AXL expression
have been reported on the protein and mRNA. The high expression of
AXL protein has been reported in human tumor tissues and tumor
cells, including lung cancer (Non-patent Document 20), breast
cancer (Non-patent Document 21), ovarian cancer (Non-patent
Document 22), thyroid cancer (Non-patent Document 23), melanoma
(Non-patent Document 23), renal cancer (Non-patent Document 24),
gastric cancer (Non-patent Document 12), and glioma (Non-patent
Document 25). Furthermore, the high expression of AXL protein is
suggested by high levels of AXL mRNA in esophageal cancer
(Non-patent Document 26), colon cancer (Non-patent Document 27),
and acute myeloid leukemia (Non-patent Document 28). There are also
reports of the inhibition of lumen formation via the suppression of
AXL by RNAi in HUVEC (Non-patent Document 29), the reduced
tumor-forming ability of human breast cancer cells in mice
resulting from the constitutive suppression of AXL (Non-patent
Document 29), and the reduced tumor-forming ability of human glioma
cells in mice resulting from the constitutive high expression of
dominant negative mutants (Non-patent Document 25). The involvement
of AXL molecular functions in tumor growth is strongly
suggested.
[0009] Polyclonal antibodies to the extracellular domain of AXL
have been reported to have a migration inhibitory action on highly
invasive breast cancer cell lines (Patent Document 1). However,
non-clinical studies showed that drugs demonstrating
cancer-cell-migration-inhibitory action do not necessarily
demonstrate antitumor activity. For example, matrix
metalloproteinase (hereinafter abbreviated to "MMP") has been known
to promote tumor infiltration and migration. Thus, attention has
been focused on various matrix metalloproteinase inhibitors that
inhibit the enzyme activity of MMP, and clinical studies have been
conducted on various pharmaceutical agents such as Batimastat,
Marimastat, and Prinomastat. However, antitumor effects have not
been observed in the clinical trials (Non-patent Document 30).
[0010] Accordingly, there have been no reports or suggestions and
it remains unknown whether anti-AXL antibodies have antitumor
effects particularly in vivo, whether they can suppress
angiogenesis, and whether they can suppress cancer. [0011] Patent
Document 1: WO 2004/008147 [0012] Non-patent Document 1: Liu, et
al., Proc. Natl Acad. Sci. U.S.A (1988) 85, 1952-6 [0013]
Non-patent Document 2: Janssen, et al., Oncogene (1991) 6, 2113-20
[0014] Non-patent Document 3: O'Bryan, et al., Mol. Cell. Biol.
(1991) 11, 5016-31 [0015] Non-patent Document 4: Varnum, et al.,
Nature (1995) 373, 623-626 [0016] Non-patent Document 5: Hafizi, et
al., FEBS J. (2006) 273, 5231-5244 [0017] Non-patent Document 6:
Nakano, et al., FEBS Lett. (1996) 387, 78-80 [0018] Non-patent
Document 7: Nakano, et al., J. Biol. Chem. (1995) 270, 5702-5
[0019] Non-patent Document 8: Goruppi, et al., Mol. Cell. Biol.
(1997) 17, 4442-53 [0020] Non-patent Document 9: Bellosta, et al.,
Oncogene (1997) 15, 2387-97 [0021] Non-patent Document 10:
Stenhoff, et al., Biochem. Biophys. Res. Commun. (2004) 319, 871-8
[0022] Non-patent Document 11: Sainaghi, et al., J. Cell. Physiol.
(2005) 204, 36-44 [0023] Non-patent Document 12: Sawabu, et al.,
Mol. Carcinog. (2007) 46, 155-164 [0024] Non-patent Document 13:
Fridell, et al., J. Biol. Chem. (1998) 273, 7123-6 [0025]
Non-patent Document 14: Allen, et al., Mol. Cell. Biol. (2002) 22,
599-613 [0026] Non-patent Document 15: McCloskey, et al., J. Biol.
Chem. (1997) 272, 23285-91 [0027] Non-patent Document 16: Hafizi,
et al., Biochem. Biophys. Res. Commun. (2002) 299, 793-800 [0028]
Non-patent Document 17: Budagian et al., Embo J. (2005) 24, 4260-70
[0029] Non-patent Document 18: Vajkoczy P et al., Proc. Natl Acad.
Sci. U.S.A (2006) 103, 5799-804 [0030] Non-patent Document 19:
Yamagata et al., Curr. Opin. Cell. Biol. (2003) 15, 621-632 [0031]
Non-patent Document 20: Shieh, et al., Neoplasia (2005) 7,
1058-1064 [0032] Non-patent Document 21: Meric, et al., Clin.
Cancer Res. (2002) 8, 361-367 [0033] Non-patent Document 22: Sun,
et al., Oncology (2004) 66, 450-457 [0034] Non-patent Document 23:
Ito, et al., Thyroid (2002) 12, 971-975 [0035] Non-patent Document
24: Chung, et al., DNA Cell Biol. (2003) 22, 533-540 [0036]
Non-patent Document 25: Vajkoczy, et al., Proc. Natl. Acad. Sci.
U.S.A (2006) 103, 5799-804 [0037] Non-patent Document 26: Nemoto,
et al., Pathobiology (1997) 65, 195-203 [0038] Non-patent Document
27: Craven, et al., Int. J. Cancer (1995) 60, 791-797 [0039]
Non-patent Document 28: Neubauer, et al., Blood (1994) 84,
1931-1941 [0040] Non-patent Document 29: Holland, et al., Cancer
Res. (2005) 65, 9294-9303 [0041] Non-patent Document 30: Pavlaki et
al., Cancer Metastasis Rev. (2003) 22, 177-203
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0042] The objectives of the present invention are to provide
anti-AXL antibodies and uses thereof. More specifically, the
objectives of the present invention are to provide methods for
inhibiting angiogenesis using anti-AXL antibodies, methods for
suppressing cell growth, methods for inhibiting AXL function,
methods for accelerating AXL function, and methods for reducing the
AXL expression level. A further objective of the present invention
is to provide anti-AXL antibodies with novel effects.
Means for Solving the Problems
[0043] As a result of conducting dedicated studies, the present
inventors succeeded in producing anti-AXL antibodies with specific
functions and discovered that these antibodies have an
angiogenesis-suppressive effect and an antitumor effect, and they
therefore completed the present invention. More specifically, the
present invention includes:
[1] a monoclonal antibody that binds to AXL; [2] the antibody
according to [1], which has cell-growth-suppressive activity; [3]
the antibody according to [1], which suppresses cancer cell growth;
[4] the antibody according to any of [1] to [3], which binds to
FND1; [5] an antibody prepared using as an antigen a peptide
comprising entire FND1 or a sequence comprising at least five or
more consecutive amino acids thereof; [6] the antibody according to
any of [1] to [5], which has agonistic activity for AXL; [7] the
antibody according to any of [1] to [5], which has antagonistic
activity for AXL; [8] the antibody according to [7], which is
obtained by selecting an antibody in which phosphorylated tyrosine
is not detected in AXL when contacting it to an AXL-expressing cell
together with an AXL ligand; [9] the antibody according to any of
[1] to [8], which has an activity that reduces AXL expression
level; [10] the antibody according to any of [1] to [9], which has
angiogenesis inhibitory activity; [11] an antibody according to any
of the following (a) to (j): (a) an antibody (Ax285) produced from
a hybridoma deposited under Accession No. FERM BP-10858; (b) an
antibody (Ax292) produced from a hybridoma deposited under
Accession No. FERM BP-10859; (c) an antibody (Ax223) produced from
a hybridoma deposited under Accession No. FERM BP-10853; (d) an
antibody (Ax96) produced from a hybridoma deposited under Accession
No. FERM BP-10852; (e) an antibody (Ax258) produced from a
hybridoma deposited under Accession No. FERM BP-10856; (f) an
antibody (Ax284) produced from a hybridoma deposited under
Accession No. FERM BP-10857; (g) an antibody (Ax7) produced from a
hybridoma deposited under Accession No. FERM BP-10850; (h) an
antibody (Ax51) produced from a hybridoma deposited under Accession
No. FERM BP-10851; (i) an antibody (Ax225) produced from a
hybridoma deposited under Accession No. FERM BP-10854; and (j) an
antibody (Ax232) produced from a hybridoma deposited under
Accession No. FERM BP-10855; [12] an antibody that binds to the
same epitope as an epitope bound by any of the antibodies according
to [11]; [13] an antibody that comprises a CDR sequence identical
to a CDR sequence comprised in any of the antibodies according to
[11]; [14] an antibody in which sequences of heavy chain CDR1, 2,
and 3 are SEQ ID NOs: 4, 5, and 6, respectively; [15] an antibody
that comprises a heavy chain CDR comprising an amino acid sequence
of the heavy chain CDR of the antibody according to [14] with a
substitution, deletion, insertion, and/or addition of one or more
amino acids, and is functionally equivalent with the antibody
according to [14]; [16] an antibody in which sequences of light
chain CDR1, 2, and 3 are SEQ ID NOs: 8, 9, and 10, respectively;
[17] an antibody that comprises a light chain CDR comprising an
amino acid sequence of the light chain CDR of the antibody
according to [16] with a substitution, deletion, insertion, and/or
addition of one or more amino acids, and is functionally equivalent
with the antibody according to [16]; [18] the antibody according to
any of [13] to [17] that is a chimeric antibody; [19] the antibody
according to any of [13] to [17] that is a humanized antibody; [20]
a hybridoma according to any of the following (a) to (j): (a) a
hybridoma deposited under Accession No. FERM BP-10858 (Ax285); (b)
a hybridoma deposited under Accession No. FERM BP-10859 (Ax292);
(c) a hybridoma deposited under Accession No. FERM BP-10853
(Ax223); (d) a hybridoma deposited under Accession No. FERM
BP-10852 (Ax96); (e) a hybridoma deposited under Accession No. FERM
BP-10856 (Ax258); (f) a hybridoma deposited under Accession No.
FERM BP-10857 (Ax284); (g) a hybridoma deposited under Accession
No. FERM BP-10850 (Ax7); (h) a hybridoma deposited under Accession
No. FERM BP-10851 (Ax51); (i) a hybridoma deposited under Accession
No. FERM BP-10854 (Ax225); and (j) a hybridoma deposited under
Accession No. FERM BP-10855 (Ax232); [21] an angiogenesis inhibitor
that comprises an anti-AXL antibody as an active ingredient; [22]
the angiogenesis inhibitor according to [21], wherein the antibody
is an antibody according to any of [1] to [19]; [23] a cell-growth
suppressant that comprises an anti-AXL antibody as an active
ingredient; [24] the suppressant according to [23], wherein the
cells are cancer cells; [25] the suppressant according to [23],
wherein the antibody is an antibody according to any of [1] to
[19]; [26] the suppressant according to [23], wherein the anti-AXL
antibody is an antibody that binds to FND1; [27] the suppressant
according to [23], which comprises as an active ingredient an
antibody that binds to IgD2 and has a phosphorylation-inhibition
activity; [28] an AXL phosphorylation activity inducer, which
comprises an anti-AXL antibody as an active ingredient; [29] the
inducer according to [28], wherein the anti-AXL antibody is an
antibody that binds to IgD; [30] the inducer according to [28],
wherein the antibody is an antibody according to [6]; [31] an AXL
phosphorylation activity inhibitor, which comprises an anti-AXL
antibody as an active ingredient; [32] the inhibitor according to
[31], wherein the anti-AXL antibody is an antibody that binds to
IgD2; [33] the inhibitor according to [31], wherein the antibody is
an antibody according to [7] or [8]; [34] an agent that reduces an
AXL expression level, which comprises an anti-AXL antibody as an
active ingredient; [35] the agent for reducing an expression level
according to [34], wherein the anti-AXL antibody is an antibody
that binds to FND1; [36] the agent that reduces the expression
level according to [34], wherein the antibody is an antibody
according to [9]; [37] a method for inducing phosphorylation of AXL
using an anti-AXL antibody; [38] a method for reducing an AXL
expression level using an anti-AXL antibody; [39] a method for
inhibiting the phosphorylation of AXL using an anti-AXL antibody;
[40] an anti-cancer agent that comprises an anti-AXL antibody as an
active ingredient; [41] the anti-cancer agent according to [40],
wherein the antibody is an antibody according to any of [1] to
[19]; [42] The anti-cancer agent according to [40], which comprises
as an active ingredient an antibody that binds to IgD2 and has a
phosphorylation-inhibition activity; [43] the anti-cancer agent
according to [40], wherein the cancer is pancreatic cancer, gastric
cancer, lung cancer, osteosarcoma, colon cancer, prostate cancer,
melanoma, endometrial cancer, ovarian cancer, uterine leiomyoma,
thyroid cancer, cancer stem cell, breast cancer, bladder cancer,
renal cancer, glioma, neuroblastoma, or esophageal cancer; [44] the
anti-cancer agent according to [42], wherein the cancer is glioma,
gastric cancer, endometrial cancer, non-small-cell lung cancer,
pancreatic adenocarcinoma, or breast cancer; [45] the anti-cancer
agent according to [43], wherein the cancer is pancreatic
adenocarcinoma or breast cancer; [46] the antibody according to
[1], which has an AXL phosphorylation-inhibition activity; [47] a
method for inhibiting angiogenesis using an anti-AXL antibody; [48]
a method for using an anti-AXL antibody in manufacturing an
angiogenesis inhibitor; [49] a method for suppressing cell growth
using an anti-AXL antibody; [50] a method for treating and/or
preventing cancer using an anti-AXL antibody; [51] a method for
using an anti-AXL antibody in manufacturing a cell-growth
suppressant; [52] a method for using an anti-AXL antibody in
manufacturing an anti-cancer agent; [53] a method for using an
anti-AXL antibody in manufacturing a phosphorylation inducer; [54]
a method for using an anti-AXL antibody in manufacturing a
phosphorylation inhibitor; [55] a method for using an anti-AXL
antibody in manufacturing an agent for lowering the AXL expression
level; and [56] a method for producing an anti-AXL specific
antibody comprising: (a) immunizing a non-human animal with a
peptide comprising entire FND1 or a sequence comprising at least
five or more consecutive amino acids thereof; and (b) collecting an
antibody from the non-human animal of (a) or collecting an
antibody-producing cell to collect an antibody produced by the
antibody-producing cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1A is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax292) of the present invention, in inducing
AXL phosphorylation in cancer cells. The antibody was shown to
induce the phosphorylation of a kinase domain of AXL.
[0045] FIG. 1B is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax258) of the present invention, in inducing
AXL phosphorylation in cancer cells. The antibody was shown to
induce the phosphorylation of a kinase domain of AXL.
[0046] FIG. 1C is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax285) of the present invention, in inducing
AXL phosphorylation in cancer cells. The antibody was shown to
induce the phosphorylation of a kinase domain of AXL.
[0047] FIG. 1D is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax223) of the present invention, in inducing
AXL phosphorylation in cancer cells. The antibody was shown to
induce the phosphorylation of a kinase domain of AXL.
[0048] FIG. 1E is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax96) of the present invention, in inducing
AXL phosphorylation in cancer cells. The antibody was shown to
induce the phosphorylation of a kinase domain of AXL.
[0049] FIG. 2A is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax51) of the present invention, in inhibiting
ligand-dependent phosphorylation of AXL in a cell. The antibody was
shown to inhibit the ligand-dependent phosphorylation of a kinase
domain of AXL.
[0050] FIG. 2B is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax7) of the present invention, in inhibiting
ligand-dependent phosphorylation of AXL in a cell. The antibody was
shown to inhibit the ligand-dependent phosphorylation of a kinase
domain of AXL.
[0051] FIG. 3A is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax292) of the present invention, in inducing
AXL downmodulation in cancer cells. The antibody was shown to
induce the downmodulation of AXL protein.
[0052] FIG. 3B is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax284) of the present invention, in inducing
AXL downmodulation in cancer cells. The antibody was shown to
induce the downmodulation of AXL protein.
[0053] FIG. 3C is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax285) of the present invention, in inducing
AXL downmodulation in cancer cells. The antibody was shown to
induce the downmodulation of AXL protein.
[0054] FIG. 3D is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax223) of the present invention, in inducing
AXL downmodulation in cancer cells. The antibody was shown to
induce the downmodulation of AXL protein.
[0055] FIG. 3E is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax7) of the present invention, in inducing AXL
downmodulation in cancer cells. The antibody was shown to induce
the downmodulation of AXL protein.
[0056] FIG. 3F is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax225) of the present invention, in inducing
AXL downmodulation in cancer cells. The antibody was shown to
induce the downmodulation of AXL protein.
[0057] FIG. 3G is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax96) of the present invention, in inducing
AXL downmodulation in cancer cells. The antibody was shown to
induce the downmodulation of AXL protein.
[0058] FIG. 3H is a photograph showing the activity of an anti-AXL
monoclonal antibody (Ax258) of the present invention, in inducing
AXL downmodulation in cancer cells. The antibody was shown to
induce the downmodulation of AXL protein.
[0059] FIG. 4 is a drawing and photograph showing the activity of
the anti-AXL monoclonal antibodies of the present invention (Ax232,
Ax292, Ax285, and Ax284) in inhibiting in vitro angiogenesis. The
antibodies were shown to have an inhibitory activity of in vitro
angiogenesis.
[0060] FIG. 5 is a drawing showing the antitumor effects of the
anti-AXL monoclonal antibodies of the present invention (Ax223,
Ax285, Ax96, Ax292, Ax258, Ax7, Ax51, Ax284, and Ax225) in a mouse
xenograft model of human pancreatic adenocarcinoma.
[0061] FIG. 6 is a table showing the antitumor effects of anti-AXL
monoclonal antibodies of the present invention (Ax223, Ax285, Ax96,
Ax292, Ax258, Ax7, Ax51, Ax284, and Ax225) (2) in a mouse xenograft
model of human pancreatic adenocarcinoma, and summarizing the
binding domain and phosphorylation-inhibiting activity of each
antibody.
[0062] FIG. 7 is a drawing showing the antitumor effects of an
anti-AXL monoclonal antibody (Ax225) of the present invention in a
mouse xenograft model of human breast cancer.
DETAILED DESCRIPTION
[0063] A novel anti-AXL antibody is provided by the present
invention. Moreover, a novel use of the anti-AXL antibody is
provided by the present invention.
[0064] There are no particular limitations on the anti-AXL antibody
of the present invention so long as it binds to AXL, and there are
also no particular limitations on its origin (such as human, mouse,
rat, rabbit, or chicken), type (polyclonal antibody or monoclonal
antibody), form (such as an altered antibody, modified antibody,
antibody fragment, or minibody [low-molecular-weight antibody]), or
such. Although there are no particular limitations on the anti-AXL
antibody of the present invention, the antibody preferably
specifically binds to anexelekto and is preferably a monoclonal
antibody.
[0065] The anti-AXL antibody of the present invention also
preferably has a cell-growth-suppressive activity.
[0066] A preferable embodiment of the anti-AXL antibodies of the
present invention is anti-AXL antibodies binding to FND1.
[0067] As is clear from the Examples described later, antibodies
that bind to FND1 in particular have significantly high in vivo
antitumor activity compared to those of other antibodies.
[0068] Binding activity of anti-AXL antibodies to FND1 can be
evaluated by a method known to those skilled in the art, for
example, the methods described below. Binding activity of anti-AXL
antibody to FND1 is confirmed by electrophoresing FND1 and western
blotting with anti-AXL antibody.
[0069] An anti-AXL antibody with agonistic activity for AXL is an
example of the preferable embodiment of the anti-AXL antibody of
the present invention. An anti-AXL antibody with agonistic activity
for AXL refers to the induction of phosphorylation mediated by AXL,
and particularly to the induction of the phosphorylation reaction
of tyrosine, when the anti-AXL antibody binds to AXL. Although
there are no particular limitations on the target of the
phosphorylation reaction that is induced by the anti-AXL antibody
with agonistic activity, an example includes the
autophosphorylation of AXL.
[0070] Whether or not an anti-AXL antibody has agonistic activity
can be determined with a method known by those skilled in the art,
for example, by the method described below. A test anti-AXL
antibody is contacted with cells expressing AXL (such as Calu-1,
MDA-MB-231, or DU-145 cells), and AXL is subsequently extracted
from the cells. The tyrosine in the extracted AXL is confirmed to
be phosphorylated using an anti-phosphotyrosine antibody. More
specifically, an anti-AXL antibody can be confirmed as having an
agonistic activity with the methods described in the Examples.
[0071] Examples of anti-AXL antibodies with agonistic activity
include the antibodies (a) to (g) below:
(a) an antibody produced from a hybridoma deposited under Accession
No. FERM BP-10858 (Ax285); (b) an antibody produced from a
hybridoma deposited under Accession No. FERM BP-10852 (Ax96); (c)
an antibody produced from a hybridoma deposited under Accession No.
FERM BP-10856 (Ax258); (d) an antibody produced from a hybridoma
deposited under Accession No. FERM BP-10859 (Ax292); (e) an
antibody produced from a hybridoma deposited under Accession No.
FERM BP-10853 (Ax223); (f) an antibody recognizing the same epitope
as the epitope recognized by an antibody of any one of (a) to (e);
and (g) an antibody with a CDR sequence identical to the CDR
sequence of an antibody of any one of (a) to (e).
[0072] An antibody recognizing the same epitope as an antibody
described above can be obtained according to, for example, the
method described below.
[0073] Whether a test antibody shares an epitope with a certain
antibody can be confirmed by the competition of the two antibodies
for the same epitope. Competition between antibodies is detected
with a cross-blocking assay or the like. A competitive ELISA, for
example, is a preferable cross-blocking assay. Specifically, in a
cross-blocking assay, AXL protein coated onto the wells of a
microtiter plate is preincubated in the presence or absence of the
candidate competitive antibody, then an anti-AXL antibody, as
indicated above, is added. The amount of the aforementioned
anti-AXL antibody bound to the AXL protein in the wells is
indirectly correlated to the binding ability of the candidate
competitive antibody (test antibody) competing for binding to the
same epitope. Thus, the greater the affinity of the test antibody
for the same epitope, the greater is the reduction in the amount of
the aforementioned anti-AXL antibody bound to the wells coated with
AXL protein and the greater the increase in the amount of test
antibody bound to the wells coated with AXL protein.
[0074] The amount of antibody bound to the wells can be measured
easily by labeling the antibody in advance. For example, a
biotin-labeled antibody can be measured using an avidin-peroxidase
conjugate and a suitable substrate. A cross-blocking assay that
uses an enzyme label such as peroxidase is specifically referred to
as a competitive ELISA. The antibody can be labeled with other
labeling substances that can be detected or measured. Specifically,
radioactive labels and fluorescent labels are known.
[0075] When the test antibody has a constant region derived from a
species differing from that of the anti-AXL antibody indicated
above, the amount of antibody bound to the wells can also be
measured with a labeled antibody that recognizes the constant
region of that antibody. Alternatively, when an antibody is derived
from the same species but is of a different class, the amount of
antibody bound to the wells can be measured with antibodies that
recognize each class.
[0076] If a candidate competitive antibody can block the binding of
the anti-AXL antibody by at least 20%, preferably by at least
20%-50%, and more preferably by at least 50% compared with the
binding activity achieved in a control test performed in the
absence of the candidate competitive antibody, then the candidate
competitive antibody is an antibody that substantially binds to the
same epitope or that competes for binding to the same epitope as
the aforementioned anti-AXL antibody.
[0077] The determination of a CDR sequence to obtain an antibody
with a CDR sequence identical to that of a certain antibody can be
performed by one skilled in the art according to known methods. For
example, a CDR sequence can be determined by determining the
full-length amino acid sequence of an antibody or the amino acid
sequence of a variable region, and investigating its homology by
applying the determined amino acid sequence to the database of
antibody amino acid sequences developed by Kabat et al. ("Sequence
of Proteins of Immunological Interest", US Dept. of Health and
Human Services, 1983). The numbers in the framework and the numbers
in the CDR sequence can be determined according to the definition
of Kabat (Kabat, A. E. et al., US Dept. of Health and Human
Services, US Government Printing Offices, 1991).
[0078] The full-length amino acid sequence of an antibody or the
amino acid sequence of a variable region can be determined by one
skilled in the art in accordance with known methods.
[0079] An antibody with a CDR sequence identical to that of a
certain antibody may have an identical sequence in at least one CDR
of the six CDRs that are present in the antibody. However, the
antibody preferably has an identical sequence in all three CDRs
present in the heavy chain or an identical sequence in all three
CDRs present in the light chain, and even more preferably, the
antibody has an identical sequence in all six CDRs present in the
antibody.
[0080] Antibodies with a CDR sequence that is identical to a CDR
sequence of a certain antibody include chimeric antibodies and
humanized antibodies. Chimeric antibodies and humanized antibodies
will be described below.
[0081] An example of another preferable embodiment of the anti-AXL
antibody of the present invention is an anti-AXL antibody with
antagonistic activity against AXL. An anti-AXL antibody with
antagonistic activity against AXL refers to an antibody with
activity that inhibits the phosphorylation reaction mediated by AXL
induced by the binding of an AXL ligand (such as Gash) to AXL, and
particularly the tyrosine phosphorylation reaction. The inhibition
of the phosphorylation reaction can be carried out by inhibiting
the binding between the AXL ligand and AXL, or by another method.
Although there are no particular limitations on the subjects of
phosphorylation inhibition reaction induced by an anti-AXL antibody
with antagonistic activity, examples include the
autophosphorylation of AXL.
[0082] Whether an anti-AXL antibody has antagonistic activity can
be determined by a method known to those skilled in the art, and
for example, by the method described below. A test antibody is
contacted with cells expressing AXL (such as Calu-1, MDA-MB-231, or
DU-145 cells) together with an AXL ligand, and AXL is subsequently
extracted from the cells. Phosphorylated tyrosine is confirmed not
to be detected in the extracted AXL using an anti-phosphotyrosine
antibody. More specifically, an anti-AXL antibody can be confirmed
as having antagonistic activity using the methods described in the
Examples.
[0083] Examples of anti-AXL antibodies with antagonistic activity
include antibodies (a) to (d) below:
(a) an antibody produced from a hybridoma deposited under Accession
No. FERM BP-10850 (Ax7); (b) an antibody produced from a hybridoma
deposited under Accession No. FERM BP-10851 (Ax51); (c) an antibody
recognizing the same epitope as the epitope recognized by an
antibody of any one of (a) to (b); and (d) an antibody having a CDR
sequence identical to the CDR sequence of an antibody of any one of
(a) to (c).
[0084] An antibody recognizing the same epitope and an antibody
with an identical CDR sequence can be obtained with the methods
previously described.
[0085] An antibody with antagonistic activity is useful for
inhibiting angiogenesis, suppressing cell growth, and the like.
[0086] An example of another preferable embodiment of the antibody
of the present invention is an antibody with activity that reduces
the AXL expression level. In the present invention, reducing the
expression level of AXL can indicate a reduction in the amount of
AXL already present through the degradation of AXL or such, or can
indicate a reduction in the amount of newly expressed AXL by
suppressing the expression of AXL. Whether the AXL expression level
has decreased can be confirmed by a method known to those skilled
in the art, and for example, by the method described below. A test
anti-AXL antibody is contacted with cells expressing AXL (such as
Calu-1, MDA-MB-231, or DU-145 cells), and the amount of AXL present
in the cells is subsequently detected by immunoblotting or such. A
comparison is then made between the amount of AXL detected when the
test antibody is contacted and the amount of AXL detected when the
test antibody is not contacted. More specifically, this can be
confirmed according to methods described in the Examples.
[0087] Examples of anti-AXL antibodies with activity that reduces
AXL expression levels include antibodies (a) to (j) below:
(a) an antibody (Ax285) produced from a hybridoma deposited under
Accession No. FERM BP-10858; (b) an antibody (Ax96) produced from a
hybridoma deposited under Accession No. FERM BP-10852; (c) an
antibody (Ax258) produced from a hybridoma deposited under
Accession No. FERM BP-10856; (d) an antibody (Ax7) produced from a
hybridoma deposited under Accession No. FERM BP-10850; (e) an
antibody (Ax292) produced from a hybridoma deposited under
Accession No. FERM BP-10859; (f) an antibody (Ax223) produced from
a hybridoma deposited under Accession No. FERM BP-10853; (g) an
antibody (Ax225) produced from a hybridoma deposited under
Accession No. FERM BP-10854; (h) an antibody (Ax284) produced from
a hybridoma deposited under Accession No. FERM BP-10857; (i) an
antibody recognizing the same epitope as the epitope recognized by
an antibody of any one of (a) to (h); and (j) an antibody having a
CDR sequence identical to the CDR sequence of an antibody of any
one of (a) to (i).
[0088] An antibody recognizing the same epitope and an antibody
having an identical CDR sequence can be obtained with the methods
previously described.
[0089] An antibody with an activity that reduces the AXL expression
level is useful for inhibiting angiogenesis, suppressing cell
growth, and the like.
[0090] An example of another preferable embodiment of the antibody
of the present invention is an antibody with an
angiogenesis-inhibiting effect. Although there are no particular
limitations on the angiogenesis-inhibiting effect of the present
invention, so long as the new formation of blood vessels is
inhibited, examples include an inhibitory effect on the migration
activity of vascular endothelial cells, an apoptosis-inducing
effect on vascular endothelial cells, and an inhibitory effect on
the vascular morphogenesis of vascular endothelial cells. A
preferred example of an antibody with an angiogenesis-inhibiting
effect is an antibody with an angiogenesis-inhibiting effect on
tumor tissues. There are no particular limitations on the tumor
tissues, and examples include pancreatic cancer tissue (such as
pancreatic adenocarcinoma tissue), gastric cancer tissue, lung
cancer tissue (tissues of small-cell lung cancer, non-small-cell
lung cancer, and such), osteosarcoma tissue, colon cancer tissue,
prostate cancer tissue, melanoma tissue, endometrial cancer tissue,
ovarian cancer tissue, uterine leiomyoma tissue, thyroid cancer
tissue, cancer stem cell tissue, breast cancer tissue, bladder
cancer tissue, renal cancer tissue, glioma tissue, neuroblastoma
tissue, and esophageal cancer tissue. More preferable tissues are
glioma tissue, gastric cancer tissue, endometrial cancer tissue,
non-small-cell lung cancer tissue, pancreatic adenocarcinoma
tissue, and breast cancer tissue, particularly pancreatic
adenocarcinoma tissue and breast cancer tissue.
[0091] Whether or not an antibody has an angiogenesis-inhibiting
effect can be confirmed by a method known to those skilled in the
art, and for example, this can be confirmed using a commercially
available angiogenesis kit. More specifically, this can be
confirmed with the methods described in the Examples.
[0092] Specific examples of antibodies with angiogenesis-inhibiting
effects include the previously described antibodies.
[0093] An example of another preferable embodiment of the antibody
of the present invention is an antibody with
cell-growth-suppressive activity.
[0094] Although there are no particular limitations on the cells
whose growth is suppressed by the anti-AXL antibody, they are
preferably cells related to a disease, and more preferably cancer
cells. When the cells are cancer cells, there are no particular
limitations on the type of cancer, and examples include pancreatic
cancer (such as pancreatic adenocarcinoma), gastric cancer, lung
cancer (small-cell lung cancer, non-small-cell lung cancer, and
such), osteosarcoma, colon cancer, prostate cancer, melanoma,
endometrial cancer, ovarian cancer, uterine leiomyoma, thyroid
cancer, cancer stem cell, breast cancer, bladder cancer, renal
cancer, glioma, neuroblastoma, and esophageal cancer. More
preferable cancers are glioma, gastric cancer, endometrial cancer,
non-small-cell lung cancer, pancreatic adenocarcinoma, and breast
cancer, particularly pancreatic adenocarcinoma and breast
cancer.
[0095] There are no particular limitations on the mechanism for the
suppression of cell growth by the antibody of the present
invention, and cell growth can be suppressed by any mechanism, such
as the inhibition of angiogenesis, the inhibition of
phosphorylation, the induction of phosphorylation, or the reduction
of the AXL expression level.
[0096] The following methods are preferably used to evaluate or
measure the cell-growth-suppressive effects based on the anti-AXL
antibody.
[0097] As a method of evaluating or measuring
cell-growth-suppressive activity in vitro, a method is used in
which the uptake by viable cells of [.sup.3H]-labeled thymidine
added to their medium is measured as an indicator of DNA
replication ability. As a simpler method, a dye expulsion method is
used, in which the ability to exclude a dye such as Trypan Blue
outside from the cells is measured under a microscope, or an MTT
method is used. The latter uses the ability of living cells to
convert MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide), a tetrazolium salt, to a blue formazan product.
Specifically, a test antibody is added to the culture solution of
the test cells together with a ligand, and after a predetermined
period of time, MTT solution is added to the culture solution and
this is left to stand for a predetermined period to allow the MTT
to be incorporated into the cells. The MTT, which is a yellow
compound, is converted to a blue compound by succinate
dehydrogenase in the mitochondria of the cells. After the
dissolution of this blue product and coloration, the absorbance is
measured and used as an indicator of the number of viable cells.
Besides MTT, reagents such as MTS, XTT, WST-1, and WST-8 are
commercially available (Nacalai Tesque, and such) and can be
appropriately used. A control antibody can also be used when
measuring this activity.
[0098] A tumor-bearing mouse model can be used to evaluate or
measure the cell-growth-suppressive activity in vivo. For example,
after cancer cells expressing AXL are transplanted into a non-human
test animal, either intradermally or subcutaneously, a test
antibody is administered intravenously or intraperitoneally
starting from the day of transplantation or from the following day,
either daily or at intervals of a few days. The
cell-growth-suppressive activity can be evaluated by measuring the
tumor size over time. In a similar manner to the evaluation in
vitro, cell-growth-suppressive activity can be determined by
administering a control antibody and observing whether the tumor
size in the anti-AXL antibody-administered group is significantly
smaller than the tumor size in the control antibody-administered
group. When a mouse is used as the non-human test animal, nude
(nu/nu) mice can be suitably used, in which T-lymphocyte function
has been lost due to a genetic deficiency in the thymus. The use of
these mice makes it possible to eliminate the involvement of T
lymphocytes in the test animal during the evaluation and
measurement of the cell-growth-suppressive activity of the
administered antibody.
[0099] Examples of anti-AXL antibodies with cell-growth-suppressive
effects include the previously described antibodies.
[0100] The anti-AXL monoclonal antibody of the present invention
can be acquired using known methods. A monoclonal antibody derived
from a mammal is particularly preferable as the anti-AXL antibody
of the present invention. Monoclonal antibodies derived from
mammals include those produced from hybridomas as well as those
produced by a host transformed with an expression vector containing
the antibody genes, using genetic engineering techniques.
[0101] A monoclonal-antibody-producing hybridoma can be generated
using known technology, such as that described below. First, AXL
protein is used as the sensitizing antigen for immunization
according to ordinary immunization methods. Immune cells obtained
from an immunized animal are then fused with known parent cells,
according to ordinary cell fusion methods, to obtain hybridomas. A
hybridoma that produces the anti-AXL antibody can be selected from
these hybridomas by screening for cells that produce the target
antibody using ordinary screening methods.
[0102] More specifically, monoclonal antibodies can be generated
out according to, for example, the method described below. First,
the AXL protein that is used as the sensitizing antigen for
obtaining the antibodies can be obtained by expressing the AXL
gene. The nucleotide sequence of the human AXL gene is already
known (GenBank Accession No. M76125). After inserting the gene
sequence encoding AXL into a known expression vector with which to
transform suitable host cells, the human AXL protein of interest
can be purified from the host cells or from the culture supernatant
using known methods. Purified naturally occurring AXL protein can
also be used in the same manner. Purification can be carried out by
using several chromatographies, such as the usual ion
chromatography and affinity chromatography, performed once or
multiple times, either in combination or alone. A fusion protein,
in which the desired partial polypeptide of the AXL protein is
fused to a different polypeptide, can also be used as an immunogen.
An antibody Fc fragment or peptide tag, or the like, can be used to
produce a fusion protein for use as an immunogen. A vector that
expresses a fusion protein can be produced by fusing two or more
types of desired genes encoding polypeptide fragments in frame and
inserting the fused genes into an expression vector, as previously
described. Methods of preparing fusion proteins are described in
Molecular Cloning, 2nd edition (Sambrook, J. et al., Molecular
Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Laboratory Press,
1989). AXL protein purified in this manner can be used as a
sensitizing antigen for the immunization of a mammal.
[0103] A partial peptide of AXL can also be used as a sensitizing
antigen. Examples of partial peptides of AXL include a peptide
obtained by chemical synthesis from an amino acid sequence of human
AXL, a peptide obtained by incorporating a portion of the human AXL
gene into an expression vector and expressing it, and a peptide
obtained by degrading human AXL protein using a protease. There are
no particular limitations on the region used as the partial
peptide, and an extracellular region of A.times.L, for example, can
be used.
[0104] Moreover, a peptide having the sequence of entire FND1 or
containing at least its five consecutive amino acids can be
preferably used as a partial peptide. Sequences containing at least
five consecutive amino acids refer to those preferably containing
six or more and more preferably eight or more consecutive amino
acids. In addition, sequences containing at least five or more
consecutive amino acids refer to amino acid sequences having
antigenicity.
[0105] There are no particular limitations on the mammal immunized
with the sensitizing antigen. To obtain a monoclonal antibody by
cell fusion, it is preferable to select an animal to be immunized
after consideration of its compatibility with the parent cells used
for the cell fusion. In general, rodents are preferred as the
immunized animal. More specifically, mice, rats, hamsters, or
rabbits can be used for as the immunized animal. Monkeys and the
like can also be used as the immunized animal.
[0106] The animal described above can be immunized with a
sensitizing antigen using known methods. For example, in a typical
method, the mammal is immunized by injecting the sensitizing
antigen intraperitoneally or subcutaneously. Specifically, the
sensitizing antigen is administered to a mammal several times every
four to 21 days. The sensitizing antigen is used for immunization
after dilution to a suitable dilution ratio with phosphate-buffered
saline (PBS), physiological saline, or the like. The sensitizing
antigen can also be administered with an adjuvant. For example, it
can be mixed with Freund's complete adjuvant and emulsified for use
as the sensitizing antigen. A suitable carrier can also be used
when immunizing with the sensitizing antigen. In particular, when a
partial peptide with a low molecular weight is used as the
sensitizing antigen, it is desirable to bind the sensitizing
antigen to a carrier protein, such as albumin, keyhole limpet
hemocyanin, and the like, for immunization.
[0107] After the mammal has been immunized in this manner and it
has been confirmed that the level of the desired antibody in the
serum has increased, the immune cells are harvested from the mammal
and used for cell fusion. In particular, spleen cells can be used
preferably as the immune cells.
[0108] Mammalian myeloma cells are used as the cells to be fused
with the immune cells. The myeloma cells preferably have a suitable
selection marker for screening. A selection marker refers to a
trait that allows cells to live (or not) under certain culture
conditions. Known selection markers include hypoxanthine-guanine
phosphoribosyl transferase deficiency (hereinafter abbreviated to
"HGPRT deficiency") and thymidine kinase deficiency (hereinafter
abbreviated to "TK deficiency"). Cells deficient in HGPRT or TK are
hypoxanthine-aminoptering-thymidine sensitive (hereinafter
abbreviated to "HAT sensitivity"). HAT-sensitive cells are unable
to synthesize DNA and die in HAT selection medium. However, when
fused with normal cells, they can continue to synthesize DNA using
the salvage pathway of normal cells and therefore they begin to
grow in HAT selection medium.
[0109] HGPRT-deficient cells and TK-deficient cells can both be
selected with a medium containing 6-thioguanine, 8-azaguanine
(hereinafter abbreviated to "8AG") or 5'-bromodeoxyuridine.
Although normal cells die as a result of incorporating these
pyrimidine analogs into their DNA, cells deficient in these enzymes
are unable to incorporate these pyrimidine analogs and can thus
survive in the selection medium. A selection marker referred to as
G418 resistance also imparts resistance to 2-deoxystreptamine-type
antibiotics (gentamycin analogs) because it is a
neomycin-resistance gene. Various myeloma cells that are suitable
for cell fusion are known, and examples of myeloma cells that can
be used include 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), FO (de St. Groth, S. F. et al., J. Immunol. Methods
(1980) 35, 1-21), 5194 (Trowbridge, I. S., J. Exp. Med. (1978) 148,
313-323), and 8210 cells (Galfre, G. et al., Nature (1979) 277,
131-133).
[0110] The fusion of the aforementioned immune cells and myeloma
cells can be carried out according to known methods, such as the
method of Kohler and Milstein (Kohler, G and Milstein, C., Methods
Enzymol. (1981) 73, 3-46).
[0111] More specifically, the aforementioned cell fusion can be
carried out in an ordinary nutritive culture medium in the presence
of a cell fusion promoter. Examples of cell fusion promoters that
can be used include polyethylene glycol (PEG) and Sendai virus
(HVJ). An auxiliary agent, such as dimethylsulfoxide, can also be
added as desired to further enhance the fusion efficiency.
[0112] The ratio in which the immune cells and myeloma cells are
used can be set arbitrarily. For example, there are preferably 1-10
times more immune cells than myeloma cells. Examples of culture
media that can be used for the cell fusion described above include
MEM and RPMI1640 culture medium, preferably used for the growth of
the aforementioned myeloma cell lines, as well as ordinary culture
medium used for this type of cell culture. A serum supplement, such
as fetal calf serum (FCS), can also be added to the culture
medium.
[0113] Cell fusion is carried out to form target fused cells
(hybridomas) by thoroughly mixing predetermined amounts of the
immune cells and myeloma cells in the culture medium and then
mixing in PEG solution, prewarmed to about 37.degree. C. During
cell fusion, PEG, with an average molecular weight of about
1000-6000, for example, can normally be added at a concentration of
30%-60% (w/v). Subsequently, the cell fusion agents and other
agents not amenable to hybridoma growth are removed by the repeated
sequential addition of a suitable culture medium, as indicated
above, centrifugation, and the removal of the supernatant.
[0114] The hybridomas thus obtained can be selected with a
selective culture medium corresponding to the selection marker
possessed by the myeloma used for cell fusion. For example, HGPRT-
or TK-deficient cells can be selected by culture in HAT culture
medium (culture medium containing hypoxanthine, aminopterin, and
thymidine). When HAT-sensitive myeloma cells are used for cell
fusion, those cells that have successfully fused with normal cells
can be selectively grown in HAT culture medium. Culture in HAT
medium is continued for an adequate amount of time for cells other
than the target hybridomas (nonfused cells) to die. Specifically,
the target hybridomas can generally be selected by culture for
several days to several weeks. Next, screening and monocloning for
a hybridoma that produces the target antibody can be performed with
an ordinary limiting dilution method. Alternatively, an antibody
that recognizes AXL can be prepared using the method described in
International Publication No. WO 03/104453.
[0115] Screening and monocloning for a target antibody is
preferably carried out with a known screening method based on an
antigen-antibody reaction. For example, an antigen is bound to a
carrier, such as polystyrene beads or a commercially available
96-well microtiter plate, and reacted with the culture supernatant
of the hybridoma. The carrier is then washed, and reacted with an
enzyme-labeled secondary antibody or the like. If a target antibody
that reacts with the sensitizing antigen is present in the culture
supernatant, the secondary antibody binds to the carrier through
this antibody. Finally, whether or not the target antibody is
present in the culture supernatant can be determined by detecting
the secondary antibody bound to the carrier. A hybridoma producing
the desired antibody, which can bind to the antigen, can be cloned
by a method such as limiting dilution. At this time, the antigen
used for immunization or a substantially equivalent AXL protein can
be used preferentially as the antigen.
[0116] In addition to the method for producing a hybridoma by
immunizing an animal other than a human with an antigen, a target
antibody can also be obtained by sensitizing human lymphocytes with
the antigen. Specifically, human lymphocytes are first sensitized
with AXL protein in vitro. The immunosensitized lymphocytes are
then fused to a suitable fusion partner. Myeloma cells of human
origin, with the ability to divide continuously, for example, can
be used as the fusion partner (see Japanese Patent Application
Kokoku Publication No. (JP-B) H1-59878 (examined, approved Japanese
patent application published for opposition)). An anti-AXL antibody
produced with this method is a human antibody with binding activity
for AXL protein.
[0117] An anti-AXL human antibody can also be obtained by
administering AXL protein as the antigen to a transgenic animal
with the entire repertoire of human antibody genes.
Antibody-producing cells of the immunized animal can be
immortalized by treatments such as fusion with a suitable fusion
partner or infection with Epstein-Barr virus. An anti-AXL antibody
can also be obtained by isolating a human antibody directed against
AXL protein from immortalized cells obtained in this manner (see
International Publication Nos WO 94/25585, WO 93/12227, WO
92/03918, and WO 94/02602). Cells producing antibodies with target
reaction specificity can also be cloned by cloning the immortalized
cells. When using a transgenic animal as the immunized animal, the
immune system of the animal recognizes human AXL as foreign. Thus,
a human antibody directed against human AXL can easily be obtained.
A hybridoma producing a monoclonal antibody prepared in this manner
can be subcultured in ordinary culture medium. The hybridoma can
also be stored for an extended period of time in liquid
nitrogen.
[0118] The hybridoma can be cultured in accordance with ordinary
methods to obtain the target monoclonal antibody from its culture
supernatant. Alternatively, the monoclonal antibody can be produced
by administering the hybridoma to a mammal compatible with it to
allow the hybridoma to grow, using the resulting ascites as the
monoclonal antibody. The former method is suitable for obtaining
highly pure antibody.
[0119] In the present invention, an antibody encoded by antibody
genes cloned from antibody-producing cells can also be used. Cloned
antibody genes can be expressed as antibody by incorporating them
in a suitable vector and introducing the vector into a host.
Methods for isolating the antibody genes, introducing them into a
vector, and transforming host cells with it have already been
established (see, for example, Vandamme, A. M. et al., Eur. J.
Biochem. (1990) 192, 767-775).
[0120] For example, a cDNA encoding a variable region (V region) of
the anti-AXL antibody can be obtained from hybridoma cells
producing the anti-AXL antibody. To accomplish this, total RNA is
usually first extracted from the hybridoma. Examples of methods for
extracting mRNA from cells include guanidine ultracentrifugation
(Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299) and the
AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162,
156-159).
[0121] The extracted mRNA can be purified using an mRNA
Purification Kit (GE Healthcare Bio-sciences) and the like.
Alternatively, kits such as the QuickPrep mRNA Purification Kit (GE
Healthcare Bio-sciences) are commercially available for the
extraction of all mRNAs directly from cells. These kits can be used
to obtain all mRNAs from a hybridoma. The cDNA encoding an antibody
V region can be synthesized from the resulting mRNAs using reverse
transcriptase. The cDNA can be synthesized with, for example, the
AMV Reverse Transcriptase First-strand cDNA Synthesis Kit
(Seikagaku Corp.). The 5'-Ampli FINDER RACE Kit (Clontech) and
5'-RACE method using PCR (Frohman, M. A. et al., Proc. Natl Acad.
Sci. U.S.A (1988) 85, 8998-9002, Belyaysky, A. et al., Nucleic
Acids Res. (1989) 17, 2919-2932) can be used to synthesize and
amplify the cDNA. Suitable restriction sites, described below, can
also be introduced at both ends of the cDNA during the course of
cDNA synthesis.
[0122] Target cDNA fragments are purified from the resulting PCR
product and linked to vector DNA. A recombinant vector is thus
prepared, and after its introduction into Escherichia coli or the
like and the selection of colonies, the desired recombinant vector
can be prepared from the E. coli that formed colonies. Whether or
not the recombinant vector has the nucleotide sequences of the
target cDNA can be confirmed by a known method, such as
dideoxynucleotide chain termination sequencing.
[0123] PCR using a primer that amplifies a variable region gene can
also be used to obtain a gene encoding a variable region. First,
cDNA is synthesized using the extracted mRNA as the template to
construct a cDNA library. It is convenient to use a commercially
available kit to synthesize the cDNA library. Because the amount of
mRNA obtained from only a small number of cells is extremely small,
its direct purification results in a low yield. Thus, mRNA is
normally purified after the addition of a carrier RNA that clearly
does not contain any antibody gene. Alternatively, when it is
possible to extract a certain amount of RNA, RNA from only
antibody-producing cells can be efficiently extracted. For example,
the addition of carrier RNA may not be necessary for the extraction
of RNA from 10 or more, 30 or more, or preferably 50 or more
antibody-producing cells.
[0124] The antibody gene is then amplified by PCR using the cDNA
library thus constructed as the template. Primers for amplifying
the antibody genes by PCR are known. For example, primers to
amplify human antibody genes can be designed based on the
literature (for example, J. Mol. Biol. (1991) 222, 581-597). These
primers have nucleotide sequences that differ for each
immunoglobulin subclass. Thus, when a cDNA library of an unknown
subclass is used as the template, PCR is performed with all
possibilities considered.
[0125] For example, when a gene encoding human IgG is to be
obtained, primers that amplify a gene encoding .gamma.1 to .gamma.5
as heavy chains and .kappa. and .lamda. chains as light chains can
be used. To amplify a variable region gene of IgG, a primer that
anneals to a sequence corresponding to the hinge region is
typically used for the primer on the 3' side. Conversely, a primer
corresponding to each subclass can be used for the primer on the 5'
side.
[0126] The PCR products amplified with primers that amplify the
genes of each subclass of heavy chains and light chains are made
into independent libraries. The use of a library synthesized in
this manner makes it possible to reconstitute immunoglobulins
comprised of combinations of heavy chains and light chains. A
target antibody can then be screened for using the binding activity
of the reconstituted immunoglobulins to AXL as an indicator.
[0127] After a cDNA encoding a V region of the target anti-AXL
antibody is obtained, the cDNA is digested with a restriction
enzyme that recognizes a restriction site inserted into both ends
of the cDNA. A preferred restriction enzyme recognizes and digests
a nucleotide sequence that is unlikely to occur in the nucleotide
sequence constituting the antibody gene. A restriction enzyme that
imparts a cohesive end is preferable when inserting a single copy
of the digested fragment into a vector in the proper direction. An
antibody expression vector can be generated by inserting the cDNA
encoding V regions of the anti-AXL antibody, digested as described
above, into a suitable expression vector. At this time, a chimeric
antibody can be produced by fusing in frame genes encoding an
antibody constant region (C region) and genes encoding the V region
described above. Herein, "chimeric antibody" refers to an antibody
containing constant and variable regions derived from different
organisms. Thus, xenogeneic chimeric antibodies, such as
mouse-human antibodies and human-human allogeneic chimeric
antibodies, are included in the chimeric antibodies of the present
invention. A chimeric antibody expression vector can also be
constructed by inserting the V region genes into an expression
vector that originally had constant regions.
[0128] Specifically, the recognition sequence of a restriction
enzyme that digests the V region gene can be arranged on the 5'
side of an expression vector retaining a DNA encoding the desired
antibody constant region (C region). A chimeric antibody expression
vector is constructed by digesting the two with the same
combination of restriction enzymes and then fusing them in
frame.
[0129] Antibody genes can be incorporated into an expression vector
for expression under the control of an expression control domain to
produce the anti-AXL antibody of the present invention. An
expression control domain for expressing antibody can include, for
example, an enhancer and a promoter. Recombinant cells expressing
DNA encoding the anti-AXL antibody can then be obtained by
transforming suitable host cells with this expression vector.
[0130] In the expression of antibody genes, DNAs encoding the
antibody heavy chain (H chain) and light chain (L chain) can each
be incorporated into different expression vectors. Vectors
incorporating either the H chain or the L chain can express an
antibody molecule with the H chain and L chain after the vectors
are simultaneously transformed (cotransfected) into the same host
cell. Alternatively, DNAs encoding H chain and L chain can be
incorporated in a single expression vector to transform host cells
(see International Publication No. WO 94/11523).
[0131] Many combinations of hosts and expression vectors are known
for the preparation of antibodies by first isolating antibody genes
and then introducing them into a suitable host. All of these
expression systems can be applied to the present invention. Animal
cells, plant cells, or fungal cells can be used when eukaryotic
cells are used as hosts. Specific examples of animal cells that can
be used in the present invention include mammalian cells (such as
CHO, COS, myeloma, BHK [baby hamster kidney], Hela, and Vero
cells), amphibian cells (such as Xenopus oocytes), and insect cells
(such as sf9, sf21, and Tn5 cells).
[0132] Known examples of plant cells used in antibody gene
expression systems are cells from the genus Nicotiana, such as
Nicotiana tabacum. Callus-cultured cells can be used for plant cell
transformation.
[0133] Examples of fungal cells that can be used include those of
yeast (the genus Saccharomyces, such as Saccharomyces cerevisiae,
and the methanol-utilizing yeast genus Pichia, such as Pichia
pastoris) and of filamentous fungi (the genus Aspergillus, such as
Aspergillus niger).
[0134] Antibody gene expression systems that use prokaryotic cells
are also known. For example, cells of bacteria such as E. coli or
Bacillus subtilis can be used in the present invention. When using
mammalian cells, an expression vector can be constructed in which a
routinely used useful promoter, the antibody genes to be expressed,
and a polyA signal at the 3' side downstream from it are operably
linked. An example of a promoter/enhancer is human cytomegalovirus
immediate early promoter/enhancer.
[0135] Other examples of promoter/enhancers that can be used to
express an antibody of the present invention include viral
promoter/enhancers or mammalian cell promoter/enhancers, such as
human elongation factor 1.alpha. (HEF1.alpha.). Specific examples
of viruses whose promoter/enhancers are useful include
retroviruses, polyomaviruses, adenoviruses, and simian virus 40
(SV40).
[0136] When using an SV40 promoter/enhancer, the method of Mulligan
et al. can be used (Nature (1979) 277, 108). An HEF1.alpha.
promoter/enhancer can also be used to easily express a target gene
with the method of Mizushima et al. (Nucleic Acids Res. (1990) 18,
5322).
[0137] With E. coli, antibody genes can be expressed by operably
linking a routinely used useful promoter, an antibody secretion
signal sequence, and the antibody genes to be expressed. Examples
of promoters include the lacZ promoter and the araB promoter. When
using the lacZ promoter, the method of Ward et al. can be used
(Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427).
Alternatively, the araB promoter can be used to express target
genes according to the method of Better et al. (Science (1988) 240,
1041-1043).
[0138] An example of the antibody secretion signal sequence that
can be used for the production into the periplasm of E. coli is the
pelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169,
4379). The antibody produced in the periplasm is separated and then
structurally refolded using a protein denaturant such as a
guanidine hydrochloride or urea so that the antibody has the
desired binding activity.
[0139] Examples of useful replication origins that can be inserted
into an expression vector include those originating in SV40,
polyomaviruses, adenoviruses, and bovine papillomavirus (BPV). A
selection marker can also be inserted into the expression vector to
amplify the number of gene copies in a host cell system. Specific
examples of selection markers that can be used include the
aminoglycoside transferase (APH) gene, the thymidine kinase (TK)
gene, the E. coli xanthine-guanine phosphoribosyl transferase
(Ecogpt) gene, and the dihydrofolate reductase (dhfr) gene.
[0140] A target antibody is produced by introducing these
expression vectors into host cells and culturing the transformed
host cells in vitro or in vivo. Culture of the host cells is
carried out in accordance with known methods. Examples of culture
media that can be used include DMEM, MEM, RPMI1640, and IMDM, and
these can be used in combination with a serum supplement such as
FCS.
[0141] An antibody expressed and produced in the manner described
above can be purified using known methods that are routinely used
for protein purification, either alone or in a suitable
combination. For example, antibodies can be separated and purified
by the suitable selection and combination of, for example, an
affinity column such as a Protein A column, a chromatography
column, a filter, ultrafiltration, salting out, or dialysis
(Antibodies--A Laboratory Manual, Ed Harlow and David Lane, Cold
Spring Harbor Laboratory, 1988).
[0142] In addition to the host cells described above, a transgenic
animal can also be used to produce the recombinant antibody. A
target antibody can be obtained from an animal into which a gene
encoding the target antibody has been introduced. For example,
antibody genes can be constructed as fused genes by inserting them
into a gene encoding a protein inherently produced in frame in
milk. Goat .beta. casein, for example, can be used as this protein
secreted in milk. A DNA fragment containing the fused genes into
which the antibody genes have been inserted is injected into a goat
embryo and the injected embryo is introduced into a female goat.
The desired antibody can be acquired in the form of a fusion
protein, fused to milk protein, from milk produced by the
transgenic goat (or offspring thereof) born from the goat that
received the embryo. Hormones can be given as appropriate to the
transgenic goat to increase the amount of milk containing the
desired antibody produced by it (Ebert, K. M. et al.,
Bio/Technology (1994) 12, 699-702). A C region originating in an
animal antibody can be used for the C region of a recombinant
antibody of the present invention. Examples of useful mouse
antibody H chain C regions include C.gamma.1, C.gamma.2a,
C.gamma.2b, C.gamma.3, C.delta., C.alpha.1, C.alpha.2, and
C.epsilon., and examples of L chain C regions include C.kappa. and
C.lamda.. Examples of useful animal antibodies other than mouse
antibodies include rat, rabbit, goat, sheep, camel, and monkey
antibodies. The sequences of these antibodies are known. The C
region can also be modified to improve the stability of the
antibody or its production. In the present invention, when
administering the antibody to a human, an artificially modified
recombinant antibody can be made in order to, for example, lower
its xenogeneic antigenicity in humans. Examples of recombinant
antibodies include chimeric antibodies and humanized
antibodies.
[0143] These modified antibodies can be produced using known
methods. Chimeric antibodies refer to antibodies in which variable
regions and constant regions of different origins are linked. For
example, an antibody with heavy chain and light chain variable
regions of a mouse antibody and heavy chain and light chain
constant regions of a human antibody is a mouse-human xenogeneic
chimeric antibody. A recombinant vector expressing a chimeric
antibody can be prepared by linking DNA encoding variable regions
of a mouse antibody with a DNA encoding a constant region of a
human antibody and then incorporating it into an expression vector.
Recombinant cells transformed with the vector are cultured and the
incorporated DNAs are expressed to obtain the chimeric antibody
produced in a culture. C regions of a human antibody are used as
the C regions of chimeric antibodies and humanized antibodies. For
example, C.gamma.1, C.gamma.2, C.gamma.3, C.gamma.4, C.delta.,
C.alpha.1, C.alpha.2, and C.epsilon. can be used for the C region
in H chains. C.kappa. and C.lamda., can be used for the C region in
L chains. The amino acid sequences of these C regions and the
nucleotide sequences that encode them are known. A human antibody C
region can also be modified to improve the stability of the
antibody itself or the antibody production.
[0144] In general, chimeric antibodies are composed of V regions
originating from antibodies of an animal other than a human and C
regions originating from human antibodies. In contrast, humanized
antibodies are composed of complementarity determining regions
(CDRs) originating from antibodies of animals other than humans,
framework regions (FRs) originating from human antibodies, and C
regions originating from human antibodies. Because humanized
antibodies have reduced antigenicity in the human body, they are
useful as an active ingredient of a therapeutic agent of the
present invention.
[0145] Antibody variable regions are normally composed of three
CDRs flanked by four FRs. A CDR is substantially a region that
determines the binding specificity of an antibody. The amino acid
sequences of CDRs are rich in diversity. Conversely, the amino acid
sequences that constitute FRs often demonstrate high homology, even
among antibodies with different binding specificities.
Consequently, it is generally considered that the binding
specificity of a certain antibody can be grafted onto another
antibody by grafting the CDRs.
[0146] A humanized antibody is also referred to as a "reshaped"
human antibody. Specifically, humanized antibodies in which the
antibody CDRs of an animal other than a human, such as a mouse,
have been grafted onto human antibodies, are known. General genetic
recombination techniques for producing humanized antibodies are
also known.
[0147] A specific example of a known method of grafting the CDRs of
a mouse antibody to human FRs is overlap extension PCR. In the
overlap extension PCR, a nucleotide sequence encoding a CDR of the
mouse antibody to be grafted is added to primers used to synthesize
a human antibody FR. Primers are prepared for each of the four FRs.
In general, it is considered to be advantageous in terms of
maintaining the CDR function to select a human FR with high
homology to the mouse FR when grafting a mouse CDR onto a human FR.
That is, it is generally preferable to use a human FR with an amino
acid sequence with high homology to the amino acid sequence of the
FR adjacent to the mouse CDR to be grafted.
[0148] The nucleotide sequences to be linked are designed so that
they are mutually connected in frame. Human FRs are individually
synthesized by specific primer sets. As a result, products are
obtained in which a DNA that encodes a mouse CDR has been added to
each FR. The nucleotide sequences encoding mouse CDRs of the
products are designed to overlap one another. A
complementary-strand synthesis reaction is then carried out by
mutually annealing the overlapping CDR portions of the products
synthesized using the human antibody gene as the template. As a
result of this reaction, human FRs are linked through the mouse CDR
sequence.
[0149] Finally, the full length of a V region gene in which three
CDRs and four FRs have been linked is amplified by primers that
anneal to its 5' and 3' ends and which have suitable restriction
enzyme recognition sequences added. A vector for expressing the
humanized antibody can then be prepared by inserting the DNA
obtained in the manner described above and DNA encoding a human
antibody C region into an expression vector so that they are fused
in frame. The humanized antibody is then produced in a culture of
cultured cells by introducing the recombinant vector into a host to
establish recombinant cells, followed by culturing the recombinant
cells and expressing the DNA encoding the humanized antibody (see
European Patent Publication No. EP 239400 and International
Publication No. WO 96/02576).
[0150] FRs of a human antibody can be preferentially selected so
that the CDRs form a favorable antigen-binding site when linked
through the CDRs, by qualitatively or quantitatively measuring and
evaluating its binding activity to the antigen of the humanized
antibody prepared in the manner described above. Amino acid
residues of the FRs can also be substituted as necessary, so that
the CDRs of the reshaped human antibody form a suitable
antigen-binding site. For example, an amino acid sequence mutation
can be introduced into FRs by applying PCR used to graft the mouse
CDRs to the human FRs. Specifically, a mutation of a partial
nucleotide sequence can be introduced into a primer that anneals to
the FR. A mutated nucleotide sequence is introduced into the FR
synthesized with such a primer. A mutant FR sequence with a desired
property can be selected by measuring and evaluating the binding
activity of the amino-acid-substituted mutant antibody to the
antigen, using the method described above (Sato, K. et al., Cancer
Res. (1993) 53, 851-856).
[0151] Methods for acquiring human antibodies are also known. For
example, human lymphocytes are sensitized with the desired antigen
or cells expressing the desired antigen in vitro. Next, the desired
human antibody with binding activity for the antigen can be
acquired by fusing the sensitized lymphocytes to human myeloma
cells (see JP-B H1-59878). U266 cells, for example, can be used as
the human myeloma cells, to serve as the fusion partner.
[0152] A desired human antibody can also be acquired by immunizing
with the desired antigen a transgenic animal with the entire
repertoire of human antibody genes (see International Publication
Nos WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO
96/34096, and WO 96/33735). Moreover, technologies by which human
antibodies can be acquired by panning, using a human antibody
library, are also known. For example, the V region of a human
antibody can be expressed on the surface of a phage in the form of
a single-chain antibody (scFv) using the phage display method, thus
allowing the selection of a phage that binds to an antigen. By
analyzing the genes of the selected phage, it is possible to
determine the DNA sequence encoding the V region of the human
antibody that binds to the antigen. After determining the DNA
sequence of the scFv that binds to the antigen, the V region
sequence is fused in frame to the sequence of the C region of the
desired human antibody, and is then inserted into a suitable
expression vector to prepare an expression vector. The human
antibody can be acquired by introducing the expression vector into
the preferred expression cells, as described above, and expressing
the gene encoding the human antibody. These methods are already
known (International Publication Nos WO 92/01047, WO 92/20791, WO
93/06213, WO 93/11236, WO 93/19172, WO 95/01438, and WO
95/15388).
[0153] The antibodies of the present invention include not only
bivalent antibodies as represented by IgG, but also monovalent
antibodies or polyvalent antibodies as represented by IgM, as long
as they bind to the AXL protein. The polyvalent antibodies of the
present invention include those with the same antigen-binding
sites, and those in which some or all of the antigen-binding sites
are different. The antibody of the present invention is not limited
to the entire antibody molecule, but may also be a minibody or
modified antibody thereof, as long as it binds to the AXL
protein.
[0154] Minibodies include antibody fragments in which a portion of
the whole antibody (such as whole IgG) is deleted. Partial
deficiencies in antibody molecules are permitted as long as the
ability to bind to the AXL antigen is retained. The antibody
fragment of the present invention preferably comprises one or both
of the heavy chain variable regions (VH) and light chain variable
regions (VL). The amino acid sequences of VH or VL can comprise
substitutions, deletions, additions, and/or insertions. Moreover, a
portion of one or both of VH and VL can be deleted as long as the
ability to bind to the AXL antigen is retained. The variable
regions may also be chimerized or humanized. Specific examples of
antibody fragments include, for example, Fab, Fab', F(ab')2, and
Fv. Specific examples of minibodies include Fab, Fab', F(ab')2, Fv,
scFV (single-chain Fv), diabody, sc(Fv)2 (single-chain (Fv)2), etc.
Polymers of these antibodies (such as dimers, trimers, tetramers,
or polymers) are also included in the minibodies of the present
invention.
[0155] Antibody fragments can be obtained by producing an antibody
fragment by treating the antibody with an enzyme. Known examples of
enzymes used to produce antibody fragments include papain, pepsin,
plasmin, etc. Alternatively, genes encoding these antibody
fragments can be constructed, introduced into an expression vector,
and then expressed in suitable host cells (see, for example, Co, M.
S. et al., J. Immunol. (1994) 152, 2698-2976; Better, M. and
Horwitz, A. H., Methods in Enzymology (1989) 178, 476-496;
Plueckthun, A. and 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).
[0156] Digestive enzymes cleave a specific position of an antibody
fragment to yield an antibody fragment with a specific structure,
as indicated below. An arbitrary portion of an antibody can be
deleted by applying genetic engineering techniques to an antibody
fragment enzymatically obtained in this manner.
[0157] Papain digestion: F(ab)2 or Fab
[0158] Pepsin digestion: F(ab')2 or Fab'
[0159] Plasmin digestion: Facb
[0160] "Diabody" refers to bivalent antibody fragments constructed
by gene fusion (see Holliger, P. et al., Proc. Natl Acad. Sci.
U.S.A (1993) 90, 6444-6448; EP 404,097; WO 93/11161, etc.).
Diabodies are dimers composed of two polypeptide chains. Normally,
VL and VH within the same chain of the polypeptide chains that
forms a dimer are both bound by linkers. The linkers in a diabody
are typically too short to allow the VL and VH to bind to each
other. Specifically, the number of amino acid residues that
constitute a linker is, for example, about five residues. Thus, the
VL and VH encoded in the same polypeptide chain cannot form a
single-chain variable region fragment, but instead form a dimer
with a different single-chain variable region fragment. As a
result, a diabody has two antigen-binding sites.
[0161] An scFv is obtained by linking the H chain V region and the
L chain V region of an antibody. In an scFv, the H chain V region
and L chain V region are linked through a linker, preferably a
peptide linker (Huston, J. S. et al., Proc. Natl Acad. Sci. U.S.A
(1988) 85, 5879-5883). The H chain V region and L chain V region in
an scFv may be derived from any antibody described as an antibody
herein. There is no particular limitation on the peptide linkers
that link the V regions. For example, any arbitrary single-chain
peptide comprising about three to 25 residues can be used as a
linker. The V regions can be linked by, for example, the PCR method
described above. To link the V regions using the PCR method, a DNA
encoding the entire or desired partial amino acid sequence of the
DNA sequence encoding the H chain or the H chain V region of the
above antibody, and a DNA sequence encoding the L chain or the L
chain V region of the above antibody, are used as templates.
[0162] DNA encoding the V regions of the H chain and that encoding
L chain are both amplified by the PCR method using pairs of primers
with sequences corresponding to the sequences at both ends of the
DNA to be amplified. Next, DNA encoding the peptide linker portion
is prepared. The DNA encoding the peptide linker can also be
synthesized by PCR. Nucleotide sequences that can link the
amplification products of each separately synthesized V region are
added to the 5' side of the primers used at this time. Next, a PCR
reaction is carried out using the "H chain V region DNA", the
"peptide linker DNA", and the "L chain V region DNA" together with
the primers for the assembly PCR. The primers for the assembly PCR
consist of a combination of a primer that anneals to the 5' side of
the "H chain V region DNA" and a primer that anneals to the 3' side
of the "L chain V region DNA". Therefore, the primers for the
assembly PCR consist of a primer set that can amplify the DNA
encoding the entire sequence of the scFv to be synthesized.
Conversely, nucleotide sequences that can link to each V region DNA
are added to the "peptide linker DNA". As a result, these DNAs are
linked together and the full length of scFv is finally produced as
an amplification product of the primers used for the assembly PCR.
Once a DNA encoding an scFv is prepared, an expression vector
comprising the DNA and recombinant cells transformed with the
expression vector can be acquired with ordinary methods. The scFv
can also be acquired by expressing the DNA encoding the scFv in
cultures of the resulting recombinant cells.
[0163] An sc(Fv)2 is a minibody in which two VHs and two VLs are
linked by a linker or such to form a single chain (Hudson, et al.,
J. Immunol. Methods (1999) 231, 177-189). An sc(Fv)2 can be
prepared, for example, by connecting scFvs with a linker.
[0164] An sc(Fv)2 is preferably an antibody in which two VHs and
two VLs are arranged in the order VH, VL, VH, VL
(VH-linker-VL-linker-VH-linker-VL) using the N-terminal side of a
single-chain polypeptide as the starting point.
[0165] Any arbitrary peptide linker that can be introduced by
genetic engineering, a synthetic compound linker (for example,
those disclosed in Protein Engineering, (1996) 9(3), 299-305) or
such, can be used as the linker to link antibody variable regions.
Peptide linkers are preferred in the present invention. There is no
particular limitation on the length of the peptide linkers, and the
length can be suitably selected by those skilled in the art
according to the purpose of use. Normally, the number of amino acid
residues constituting a peptide linker ranges from one to 100 amino
acids, preferably from three to 50 amino acids, more preferably
from five to 30 amino acids, and particularly preferably from 12 to
18 amino acids (for example, 15 amino acids).
[0166] The amino acid sequence constituting a peptide linker can be
any arbitrary sequence as long as it does not inhibit the binding
function of the scFv.
[0167] Alternatively, V regions can be linked using a synthetic
chemical linker (chemical cross-linking agent). Cross-linking
agents ordinarily used to cross-link peptide compounds and such can
be used in the present invention. Examples of cross-linking agents
that can be used include N-hydroxysuccinimide (NHS),
disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS3),
dithiobis(succinimidylpropionate) (DSP),
dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethyleneglycol
bis(succinimidylsuccinate) (EGS), ethyleneglycol
bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl
tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),
bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and
bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone
(sulfo-BSOCOES).
[0168] Normally, three linkers are required when four antibody
variable regions are linked. The multiple linkers used may be
identical or different. A preferred minibody of the present
invention is a diabody or sc(Fv)2. To obtain these minibodies, an
antibody is treated with an enzyme such as papain or pepsin to
produce antibody fragments. Alternatively, a DNA encoding these
antibody fragments is constructed, introduced into an expression
vector, and then expressed in suitable host cells (see, for
example, Co, M. S. et al., J. Immunol. (1994) 152, 2698-2976;
Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178,
476-496; Plueckthun, 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).
[0169] The antibody of the present invention can also be used as a
modified antibody bound to various molecules such as polyethylene
glycol (PEG) or cytotoxic substances. Such modified antibodies can
be obtained by chemically modifying an antibody of the present
invention. Antibody modification methods have already been
established in the art.
[0170] The antibody of the present invention may also be a
bispecific antibody. "Bispecific antibody" refers to an antibody
that has variable regions that recognize different epitopes within
the same antibody molecule. The epitopes may be present in
different molecules or present in the same molecule. In the present
invention, a bispecific antibody can have antigen-binding sites
that recognize different epitopes on an AXL molecule.
Alternatively, a bispecific antibody can recognize AXL via one
recognition site and a cytotoxic substance by the other recognition
site. These antibodies are also included in the antibodies of the
present invention.
[0171] A bispecific antibody that recognizes an antigen other than
AXL can be combined in the present invention. For example, a
bispecific antibody that recognizes an antigen other than AXL,
which is specifically expressed on the surfaces of target cancer
cells in the same manner as AXL, can be combined.
[0172] Methods for producing bispecific antibodies are known. For
example, a bispecific antibody can be produced by linking two types
of antibodies that recognize different antigens. Each of the linked
antibodies may be a half molecule, with the H and L chains, or a
quarter molecule comprising only the H chain. Alternatively, fused
cells that produce bispecific antibodies can be prepared by fusing
hybridomas producing different monoclonal antibodies. Bispecific
antibodies can also be prepared with genetic engineering
techniques.
Binding Activity of an Antibody
[0173] Known means can be used to measure the antigen-binding
activity of an antibody (Antibodies A Laboratory Manual, Ed Harlow
and David Lane, Cold Spring Harbor Laboratory, 1988). Examples of
methods that can be used include ELISA (enzyme-linked immunosorbent
assay), EIA (enzyme immunoassay), RIA (radioimmunoassay),
fluorescent immunoassay, etc. Examples of the means for measuring
the binding activity of an antibody to an antigen expressed in
cells include the method described on pages 359-420 of "Antibodies
A Laboratory Manual" mentioned above.
[0174] Methods using a flow cytometer are particularly preferably
used to measure the binding between an antigen expressed on the
surface of cells suspended in a buffer and such, and an antibody to
that antigen. Examples of flow cytometers used include the
FACSCanto.TM. II, FACSAria.TM., FACSArray.TM., FACSVantage.TM. SE,
and FACSCalibur.TM. (all from BD Biosciences), and the EPICS ALTRA
HyPerSort, Cytomics FC 500, EPICS XL-MCL ADC, EPICS XL ADC, and
Cell Lab Quanta/Cell Lab Quanta SC (all from Beckman Coulter).
[0175] An example of a preferred method of measuring the binding
activity of a test AXL antibody to an antigen is the method of
staining with a secondary antibody labeled with FITC, which
recognizes a test antibody reacted with cells expressing AXL, and
then measuring with the FACSCalibur (BD Biosciences) and analyzing
the fluorescence intensity using CellQuest software (BD
Biosciences).
Hybridomas
[0176] The present invention also provides hybridomas deposited
under Accession Nos. FERM BP-10858 (AX285), FERM BP-10859 (AX292),
FERM BP-10853 (AX223), FERM BP-10852 (AX96), FERM BP-10856 (AX258),
FERM BP-10857 (AX284), FERM BP-10850 (Ax7), FERM BP-10851 (Ax51),
FERM BP-10854 (Ax225), and FERM BP-10855 (Ax232). These hybridomas
produce anti-AXL antibodies with agonistic activity, anti-AXL
antibodies with antagonistic activity, anti-AXL antibodies with
activity that lowers the expression level of AXL, anti-AXL
antibodies with angiogenesis inhibitory activity, and/or anti-AXL
antibodies with cell-growth-suppressing activity.
Angiogenesis Inhibitors
[0177] The present invention also provides angiogenesis inhibitors
comprising an anti-AXL antibody. There is no particular limitation
on the mechanism by which angiogenesis is inhibited. Examples of
the mechanism include an inhibitory effect on the migration
activity of vascular endothelial cells, an apoptosis-inducing
action on vascular endothelial cells, and an inhibitory effect on
the vascular morphogenesis of vascular endothelial cells. The
angiogenesis inhibitors of the present invention preferably inhibit
angiogenesis in cancer tissues. There is no particular limitation
on the cancer tissues. Examples of these cancer tissues include
pancreatic cancer tissues (pancreatic adenocarcinoma tissues,
etc.), gastric cancer tissues, lung cancer tissues (tissues of
small-cell lung cancer, non-small-cell lung cancer, and such),
osteosarcoma tissues, colon cancer tissues, prostate cancer
tissues, melanoma tissues, endometrial cancer tissues, ovarian
cancer tissues, uterine leiomyosarcoma tissues, thyroid cancer
tissues, cancer stem cell tissues, breast cancer tissues, bladder
cancer tissues, renal cancer tissues, glioma tissues, neuroblastoma
tissues, and esophageal cancer tissues. More preferable tissues are
glioma tissue, gastric cancer tissue, endometrial cancer tissue,
non-small-cell lung cancer tissue, pancreatic adenocarcinoma
tissue, and breast cancer tissue, particularly pancreatic
adenocarcinoma tissue and breast cancer tissue.
[0178] There is no particular limitation on the antibodies used in
the angiogenesis inhibitors of the present invention, as long as
they have an angiogenesis inhibitory effect. For example, the
antibodies described above (antibodies with agonistic activity,
antibodies with antagonistic activity, antibodies with activity
that lowers the expression level of AXL, etc.) can be used.
[0179] The angiogenesis inhibitors comprising the anti-AXL antibody
of the present invention can be expressed as methods for inhibiting
angiogenesis using an anti-AXL antibody. The angiogenesis
inhibitors comprising the anti-AXL antibody of the present
invention can be expressed as use of an anti-AXL antibody for
producing an angiogenesis inhibitor.
Cell-Growth Suppressants
[0180] The present invention also provides cell-growth suppressants
comprising anti-AXL antibodies. There is no particular limitation
on the mechanism by which cell growth is suppressed. Examples of
the mechanisms include those based on the angiogenesis inhibitory
action, those based on the cytotoxic activity of the antibody, and
those based on a cytotoxic substance bound to the antibody, but
those based on the angiogenesis inhibitory action are
preferable.
[0181] There is no particular limitation on the cells whose growth
is suppressed by an anti-AXL antibody. The cells are preferably
those related to a disease, and more preferably cancer cells. Thus,
examples of the preferred embodiments of the cell-growth
suppressants of the present invention include an anticancer agent
comprising an anti-AXL antibody. When the cells are cancer cells,
there is no particular limitation on the type of cancer, and the
types include pancreatic cancer (pancreatic adenocarcinoma, etc.),
gastric cancer, lung cancer (small-cell lung cancer, non-small-cell
lung cancer, and such), osteosarcoma, colon cancer, prostate
cancer, melanoma, endometrial cancer, ovarian cancer, uterine
leiomyosarcoma, thyroid cancer, cancer stem cell, breast cancer,
bladder cancer, renal cancer, glioma, neuroblastoma, and esophageal
cancer. More preferable cancers are glioma, gastric cancer,
endometrial cancer, non-small-cell lung cancer, pancreatic
adenocarcinoma, and breast cancer, particularly pancreatic
adenocarcinoma and breast cancer.
[0182] There is no particular limitation on the antibodies used in
the cell-growth suppressants of the present invention, as long as
they have cell-growth-suppressing activity. For example, the
antibodies described above (antibodies with agonistic activity,
antibodies with antagonistic activity, antibodies with activity
that lowers the expression level of AXL, etc.) can be used.
[0183] The cell-growth suppressants comprising the anti-AXL
antibody of the present invention can be expressed as methods for
suppressing cell growth using an anti-AXL antibody. When the cells
whose growth is suppressed are cancer cells, the anticancer agents
comprising the anti-AXL antibody of the present invention can be
expressed as methods for treating and/or preventing cancer using an
anti-AXL antibody. The cell-growth suppressants comprising the
anti-AXL antibody of the present invention can be expressed as use
of an anti-AXL antibody to produce a cell-growth suppressant. When
the cells whose growth is suppressed are cancer cells, they can be
expressed as use of an anti-AXL antibody for producing an
anticancer agent.
Phosphorylation Inducers
[0184] The present invention also provides phosphorylation inducers
comprising an anti-AXL antibody. The phosphorylation inducers of
the present invention normally induce phosphorylation in cells
expressing AXL. Although there is no particular limitation on the
targets of the phosphorylation induction, the targets are normally
polypeptides having tyrosine and are preferably AXL.
[0185] There is no particular limitation on the antibodies used in
the phosphorylation inducers of the present invention. For example,
the antibodies with agonistic activity described above can be
used.
[0186] The phosphorylation inducers comprising the anti-AXL
antibody of the present invention can be expressed as methods for
inducing phosphorylation using an anti-AXL antibody. The
phosphorylation inducers comprising the anti-AXL antibody of the
present invention can also be expressed as use of an anti-AXL
antibody for producing a phosphorylation inducer.
Phosphorylation Inhibitors
[0187] The present invention also provides phosphorylation
inhibitors comprising an anti-AXL antibody. The phosphorylation
inhibitors of the present invention normally inhibit the
phosphorylation induced by the binding of an AXL ligand (such as
Gash) to AXL. Although there is no particular limitation on the
targets of phosphorylation inhibition, the targets are normally
polypeptides having tyrosine and are preferably AXL.
[0188] There is no particular limitation on the antibodies used in
the phosphorylation inhibitors of the present invention. For
example, the antibodies with antagonistic activity described above
can be used.
[0189] The phosphorylation inhibitors comprising the anti-AXL
antibody of the present invention can be expressed as methods for
inhibiting phosphorylation using an anti-AXL antibody. The
phosphorylation inhibitors comprising the anti-AXL antibody of the
present invention can also be expressed as use of an anti-AXL
antibody for producing a phosphorylation inhibitor.
Agents for Lowering the AXL Expression Level
[0190] The present invention also provides agents that lower the
AXL expression level comprising an anti-AXL antibody. The agent
that lowers the AXL expression level reduces AXL expression level
in cells expressing AXL. There is no particular limitation on the
cells that express AXL. Examples of these cells include cancer
cells (Calu-1, MDA-MB-231, DU-145, etc.).
[0191] The reduction in the expression level of AXL may be a
reduction in the amount of AXL already present by the degradation
of AXL, or such, or may be a reduction in the amount of newly
expressed AXL by suppressing the expression of AXL.
[0192] The agents that lower the AXL expression level comprising
the anti-AXL antibody of the present invention can be expressed as
methods for lowering the expression level of AXL using an anti-AXL
antibody. Moreover, the agents that lower the AXL expression level
comprising the anti-AXL antibody of the present invention can be
expressed as use of an anti-AXL antibody for producing an agent for
lowering the AXL expression level.
Pharmaceutical Compositions
[0193] The angiogenesis inhibitors, cell-growth suppressants,
phosphorylation inducers, phosphorylation inhibitors, or agents
that lower the AXL expression level of the present invention can be
administered by either oral administration methods or parenteral
administration methods. Parenteral administration methods are
particularly preferred. Specific examples of such administration
methods include injection administration, transnasal
administration, transpulmonary administration, and transcutaneous
administration. The pharmaceutical compositions of the present
invention can be administered systemically or locally by injection
administration, for example, by intravenous injection,
intramuscular injection, intraperitoneal injection, subcutaneous
injection, or such. Suitable methods of administration can also be
selected according to the age and symptoms of the patient. The
dosage can be selected, for example, within the range of 0.0001 mg
to 1000 mg per kilogram body weight per administration.
Alternatively, the dosage can be selected, for example, within the
range of 0.001 to 100,000 mg/body per patient. However, the dosage
of the pharmaceutical compositions of the present invention is not
limited thereto.
[0194] The angiogenesis inhibitors, cell-growth suppressants,
phosphorylation inducers, phosphorylation inhibitors, or agents for
lowering the AXL expression level of the present invention can be
formulated according to ordinary methods (for example, Remington's
Pharmaceutical Science, latest edition, Mark Publishing Company,
Easton, USA), and may comprise pharmaceutically acceptable carriers
or additives. Examples of the carriers and additives include, but
are not limited to, surfactants, vehicles, colorants, fragrances,
preservatives, stabilizers, buffers, suspension agents, isotonic
agents, binders, disintegration agents, lubricants, fluidity
promoters, and flavoring agents. Other commonly used carriers can
be used as appropriate. Specific examples of such carriers include
light silicic anhydride, lactose, crystalline cellulose, mannitol,
starch, carmellose calcium, carmellose sodium, hydroxypropyl
cellulose, hydroxypropyl methyl cellulose, polyvinylacetal
diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain
fatty-acid triglycerides, polyoxyethylene hydrogenated castor oil
60, saccharose, carboxymethyl cellulose, cornstarch, inorganic
salts, etc.
[0195] All prior art reference cited herein are incorporated by
reference in their entirety.
EXAMPLES
[0196] Although the present invention will be explained in more
detail by the following Examples, the present invention is not
limited by these Examples.
Example 1
1-1 Preparation of Antigen
[0197] Hamster ovary cells (CHO (dhfr.sup.-) cells) were
transfected with the expression vector for a fusion protein
(hAXL-ECD-mIgG2aFc), in which the extracellular domain of human AXL
and an Fc domain of mouse IgG2a were fused, and CHO cell lines that
produce hAXL-ECD-mIgG2aFc protein were cloned with G418 selection.
The culture supernatant of the hAXL-ECD-mIgG2aFc protein-producing
CHO cell lines collected using serum-free medium (CHO-S-SFM II;
Gibco) was added to a Protein G Column (HiTrap Protein G HP, GE
Healthcare) equilibrated with a binding buffer (20 mM phosphate
buffer, pH 7.0). After the unbound proteins were washed with the
binding buffer, fractions of hAXL-ECD-mIgG2aFc protein were
collected with an elution buffer (100 mM glycine-HCl, pH 2.7) into
tubes containing neutralizing buffer (1 M Tris-HCl, pH 9.0). Then
the buffer of the purified protein was replaced with
phosphate-buffered physiological saline (pH 7.35-7.65; Takara Bio)
and the purified protein was concentrated using an ultrafiltration
kit for a molecular weight fraction of 10 kDa (Centricon
(registered trademark), Millipore). The concentration of the
purified protein was calculated from the absorbance at 280 nm using
a molar absorption coefficient calculated according to the
calculation formula of Pace et al. (Prof Sci. (1995) 4,
2411-2423).
1-2 Preparation of Anti-AXL-Antibody-Producing Hybridoma
[0198] Four BALB/c mice (male, six weeks old at the start of
immunization, Charles River Laboratories Japan) and two MRL/lpr
mice (male, six weeks old at the start of immunization, Charles
River Laboratories Japan) were immunized as described below with
the antigen prepared in the previous section (hAXL-ECD-mIgG2aFc
protein). Antigen emulsified with Freund's complete adjuvant (H37
Ra, Difco Laboratories) was administered subcutaneously at 40
.mu.g/head as the initial immunization. Two weeks later, antigen
emulsified with Freund's incomplete adjuvant (Difco Laboratories)
was administered subcutaneously at 40 .mu.g/head. The animals were
subsequently immunized three times more at one week intervals.
Increases in the serum antibody titer in response to the antigen
were confirmed by ELISA as indicated in the following section,
followed by a final immunization of intravenous administration of
antigen diluted with phosphate-buffered physiological saline
(phosphate-buffered saline without calcium ions or magnesium ions,
PBS(-); Nissui Pharmaceutical) at 10 .mu.g/head. Three days after
the final immunization, mouse spleen cells and mouse myeloma cells
P3X63Ag8U.1 (referred to as P3U1, ATCC CRL-1597) were fused
according to ordinary methods using PEG 1500 (Roche Diagnostics).
The fused cells were cultured in RPMI1640 medium (Invitrogen)
containing 10% FBS (Invitrogen) (hereafter referred to as 10%
FBS/RPMI1640). On the day after fusion, the fused cells were
suspended in semifluid medium (StemCells) followed by the selective
culture and colonization of the hybridomas. Hybridoma colonies were
picked from the medium on the ninth or tenth day after fusion and
seeded into a 96-well plate containing HAT selective medium (10%
FBS/RPMI1640, 2 vol % HAT 50.times. concentrate [Dainippon
Pharmaceutical] and 5 vol % BM-Condimed H1 [Roche Diagnostics]) at
one colony per well. After culture for three to four days, the
supernatant was collected from each well and the hybridomas with
binding activity to the extracellular domain of human AXL were
selected by measuring their binding activity to the aforementioned
antigen and to a control protein fused with the Fc domain of mouse
IgG2a by ELISA, as indicated in the following section.
[0199] The binding activities of the supernatants of the selected
hybridomas are shown in Table 1.
TABLE-US-00001 TABLE 1 2nd AXL 2nd SC Abs 2nd SC Abs SC Abs 2nd SC
Abs IgG Clone No. AXL-mFc FGFR2-mFc Abs.DELTA. AXL-His Binding 7
2.053 0.057 1.996 1.118 0.66 51 1.844 0.058 1.786 0.538 0.55 232
1.353 0.061 1.292 1.204 0.575 96 2.122 0.058 2.064 1.554 0.635 119
2.208 0.063 2.145 1.527 0.668 223 2.076 0.071 2.005 1.542 0.339 225
0.629 0.055 0.574 0.642 0.859 258 2.005 0.078 1.927 1.028 0.74 284
0.619 0.064 0.555 0.124 0.857 285 1.804 0.058 1.746 0.914 0.965 292
1.877 0.069 1.808 1.234 1.052
[0200] The hybridomas selected by the present inventors were
deposited at the International Patent Organism Depositary of the
National Institute of Advanced Industrial Science and Technology.
The following section provides a description of the contents,
specifying the deposition.
(a) Name and Address of the Depositary Institution
[0201] Name: International Patent Organism Depositary, National
Institute of Advanced Industrial Science and Technology
[0202] Address: Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki,
Japan 305-8566
(b) Acceptance Date (Deposition Date): Jul. 5, 2007
(c) Accession No.
[0203] AXL No. 7 #070402 (Ax7) (Accession No. FERM BP-10850)
[0204] AXL No. 51 #070406 (Ax51) (Accession No. FERM BP-10851)
[0205] AXL No. 232 #070406 (Ax232) (Accession No. FERM
BP-10855)
[0206] AXL No. 96 #070402 (Ax96) (Accession No. FERM BP-10852)
[0207] AXL No. 223 #070402 (Ax223) (Accession No. FERM
BP-10853)
[0208] AXL No. 225 #070402 (Ax225) (Accession No. FERM
BP-10854)
[0209] AXL No. 258 #070402 (Ax258) (Accession No. FERM
BP-10856)
[0210] AXL No. 284 #070402 (Ax284) (Accession No. FERM
BP-10857)
[0211] AXL No. 285 #070402 (Ax285) (Accession No. FERM
BP-10858)
[0212] AXL No. 292 #070411 (Ax292) (Accession No. FERM
BP-10859)
1-3 Binding Activity to Human AXL
[0213] Antigen (hAXL-ECD-mIgG2aFc) diluted to 1 .mu.g/mL with
coating buffer (100 mM sodium bicarbonate [pH 9.6], 0.02% sodium
azide) or control protein fused with the Fc domain of mouse IgG2a
was dispensed into a 96-well plate (Nunc-Immuno.TM. 96
MicroWell.TM. MaxiSorp.TM. plate; Nalge Nunc International) at 80
.mu.L/well, followed by incubation at least overnight at 4.degree.
C. After it was washed three times with phosphate-buffered saline
containing 0.05 vol % Tween (registered trademark) 20 (tPBS[-]),
the plate was blocked at least overnight at 4.degree. C. with
diluent buffer (1/5 dilution of BlockingOne; Nacalai Tesque). After
the removal of the buffer, mouse antiserum or hybridoma culture
supernatant diluted with diluent buffer was added to the plate at
80 .mu.L/well, followed by incubation for one hour at room
temperature. After the plate had been washed three times with
tPBS(-), HRP-labeled anti-mouse IgG antibody (Stressgen), diluted
1/5000 with diluent buffer, was added at 80 .mu.L/well, followed by
incubation for one hour at room temperature. After the plate had
been washed five times with tPBS(-), a chromogenic substrate,
Peroxidase Substrate (Kirkegaad & Perry Laboratories), was
added at 80 .mu.L/well, followed by incubation for 20 minutes at
room temperature. After the addition of Peroxidase Stop Solution
(Kirkegaad & Perry Laboratories) at 80 .mu.L/well, the
absorbance at 405 nm was measured with a Microplate Reader Model
3550 (Bio-Rad Laboratories).
1-4 Purification of Antibody from Hybridoma Culture Supernatant
[0214] The resulting hybridomas described above were cultured in
HAT selective medium using low-IgG FBS (Invitrogen) as the FBS.
Protein G beads (Pharmacia), in which the solvent was replaced with
wash buffer (20 mM sodium acetate buffer, pH 5.0), were added to
20-50 mL of the culture supernatant at 50 .mu.L per 10 mL of
culture supernatant, followed by mixing by inversion overnight at
4.degree. C. After the Protein G beads had been retrieved and
washed with wash buffer, the antibody was eluted with elution
buffer (50 mM sodium acetate buffer, pH 3.3), followed immediately
by neutralization with neutralizing buffer (Tris-HCl buffer, pH
7.8). The buffer was replaced with phosphate-buffered physiological
saline (pH 7.35-7.65; Nissui Pharmaceutical) and the purified
antibody was concentrated using an ultrafiltration kit for a
molecular weight fraction of 10 kDa (Amicon (registered trademark),
Millipore), followed by sterilization with a 0.22 .mu.m
sterilization filter (Millipore GV, Millipore).
Example 2
Assay of Antibody-Induced Phosphorylation
[0215] The ability of the anti-AXL monoclonal antibody obtained in
Example 1 to induce the phosphorylation of AXL in cancer cells was
tested. Cells (human non-small-cell lung cancer cell line Calu-1,
human breast cancer cell line MDA-MB-231, and human prostate cancer
cell line DU-145) were seeded into six-well plates at a density of
4.times.10.sup.5 cells/well and 24 hours later, the medium was
replaced with medium from which the serum had been removed
(serum-starved medium) and the cells were cultured overnight. Next,
the above-prepared anti-AXL monoclonal antibody was added at 2
.mu.g/mL, and recombinant GAS6 (R&D) was added at 200 ng/mL to
act as the positive control, followed by incubation for 30 minutes
at 37.degree. C. Next, the cells were washed with PBS(-) and lysed
on ice for 30 minutes with cell lysis buffer (137 mM NaCl, 20 mM
Tris-HCl [pH 8.0], 10% glycerol, 2 mM EDTA, 1 mM sodium vanadate, 1
vol % NP-40, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 .mu.g/mL
aprotinin, 10 .mu.g/mL leupeptin, 10 .mu.g/mL pepstatin). The cell
solution mixture was homogenized with an ultrasonic homogenizer
(Tomy Seiko) followed by centrifugation (20,000.times.g) for 10
minutes at 4.degree. C. The supernatant of the cell solution
mixture was mixed for 30 minutes with 0.05 volumes of Protein G
Agarose (Roche Diagnostics). After centrifugation (2,300.times.g)
for one minute at 4.degree. C., 1.2 .mu.g of anti-AXL monoclonal
antibody (R&D) was added to the supernatant, which was shaken
for one hour at 4.degree. C. Then, 10 .mu.L of Protein G Agarose
was added and the solution was shaken for a further one hour at
4.degree. C. After centrifugation (2,300.times.g) for one minute at
4.degree. C., the immunoprecipitate was washed and suspended in
NuPAGE-LDS sample buffer (Invitrogen), and then heated for 10
minutes at 70.degree. C. The immunoprecipitate was electrophoresed
for one hour at 150 V using 7% NuPAGE (Invitrogen).
[0216] After immunoprecipitation and electrophoresis on 7% NuPAGE,
the protein was electrophoretically transferred to a 0.45 .mu.m
polyvinylidene difluoride filter (Immobilon-FL, Millipore) over the
course of one hour at 30 mA with NuPAGE transfer buffer
(Invitrogen) and the buffer containing 20 vol % methanol. The
filter was washed with TBS (50 mM Tris-HCl [pH 7.6], 150 mM NaCl)
and then blocked by incubation overnight in Odyssey blocking buffer
(Li-COR). The filter was washed four times for five minutes each
with TBST (TBS containing 0.05 vol % Tween (registered trademark)
20) and then incubated for two hours at room temperature with
biotinylated 4G10 anti-phosphotyrosine antibody (diluted 1:1,000
with TBST; Upstate) and anti-AXL antibody (diluted 1:15,000 with
TBST; Santa Cruz). After the filter had been washed four times for
five minutes each with TBST, the filter was incubated for one hour
with Alexa 680-labeled streptavidin (Invitrogen) diluted 1:10,000
with TBST and IRDye 800-labeled anti-goat secondary antibody
(Rockland) diluted 1:10,000 with TBST. After the filter had been
washed three times for five minutes each with TBST, it was washed
again once for five minutes with TBS, and then scanned with the
Odyssey infrared imaging system (Li-COR).
[0217] A band obtained by immunoblotting the immunoprecipitated
intracellular AXL with anti-AXL antibody and a band obtained by
immunoblotting it with anti-phosphotyrosine antibody overlapped,
and the intensification of the band for tyrosine-phosphorylated AXL
was observed after the addition of the anti-AXL monoclonal
antibodies Ax285, Ax292, Ax223, Ax96, and Ax258, and after the
addition of the recombinant GAS6 used as the positive control
(FIGS. 1a, b, c, d, and e). Thus, intensified tyrosine
phosphorylation of AXL was observed as a result of the addition of
the anti-AXL monoclonal antibody acquired by the present inventors.
Thus, these anti-AXL monoclonal antibodies can induce the
phosphorylation of the kinase domain of AXL.
Example 3
Assay of the Inhibition of Ligand-Dependent Phosphorylation by the
Antibody
[0218] The ability of the anti-AXL monoclonal antibody to inhibit
ligand-dependent phosphorylation within cancer cells was tested.
Cells (human non-small-cell lung cancer cell line Calu-1, human
breast cancer cell line MDA-MB-231, or human prostate cancer cell
line DU-145) were seeded into six-well plates at a density of
4.times.10.sup.5 cells/well and 24 hours later, the medium was
replaced with medium from which the serum had been removed
(serum-starved medium) and then the cells were cultured overnight.
Next, the anti-AXL monoclonal antibody prepared in Example 1 was
added at 2 .mu.g/mL, and then recombinant GAS6 (R&D) was added
simultaneously at 200 ng/mL and incubated for 30 minutes at
37.degree. C. Next, the cells were washed with PBS(-) and the
protein was extracted from the cells with the previously described
cell lysis buffer. The cell lysis products, immunoprecipitated with
commercially available anti-AXL antibody (Santa Cruz.TM.), were
separated on 7% NuPAGE (Invitrogen), immunoblotted by western
blotting, and tyrosine phosphorylation assay, as previously
described. The immunoprecipitated intracellular AXL was blotted
with anti-phosphotyrosine antibody by treatment with GAS6, which is
its ligand. However, the blot of the anti-phosphotyrosine antibody
was weakened by the anti-AXL monoclonal antibodies Ax7 and Ax51
(FIGS. 2a and b). Thus, the ligand-dependent tyrosine
phosphorylation of AXL was confirmed to be inhibited by exposing to
the anti-AXL monoclonal antibodies acquired by the present
inventors. These anti-AXL monoclonal antibodies can inhibit
ligand-dependent phosphorylation of the kinase domain of AXL.
Example 4
Assay of the Induction of AXL Protein Downmodulation by the
Antibody
[0219] The ability of the anti-AXL monoclonal antibody to induce
the downmodulation of AXL within cancer cells was tested. Cells
(human non-small-cell lung cancer cell line Calu-1, human breast
cancer cell line MDA-MB-231, or human prostate cancer cell line
DU-145) were seeded into six-well plates at a density of
4.times.10.sup.5 cells/well and 24 hours later, the medium was
replaced with medium from which the serum had been removed
(serum-starved medium) and then the cells were cultured overnight.
Next, the anti-AXL monoclonal antibody prepared as described above
was added at 2 .mu.g/mL, and recombinant GAS6 (R&D) was added
at 200 ng/mL to act as the positive control, followed by incubation
for 24 hours at 37.degree. C. Next, the cells were washed with
PBS(-) and the protein was extracted from the cells with the
previously described cell lysis buffer. The cell lysis products,
immunoprecipitated with a commercially available anti-AXL antibody
(Santa Cruz.TM.), were separated on 7% NuPAGE (Invitrogen),
immunoblotted by western blotting, and tyrosine phosphorylation
assay, as previously described.
[0220] 25 .mu.g of each protein solution was suspended in
NuPAGE-LDS sample buffer (Invitrogen), heated for 10 minutes at
70.degree. C., and electrophoresed for one hour at 150 V on 7%
NuPAGE (Invitrogen). The gels separated by electrophoresis were
electrophoretically transferred to a 0.45 .mu.m polyvinylidene
difluoride filter (Immobilon-FL, Millipore) over the course of one
hour at 30 mA in NuPAGE transfer buffer (Invitrogen) and the buffer
containing 20 vol % methanol. The filter was washed with TBS (50 mM
Tris-HCl [pH 7.6], 150 mM NaCl) and then blocked by incubation
overnight in Odyssey blocking buffer (Li-COR). The filter was
washed four times for five minutes each with TBST and then
incubated for two hours at room temperature with anti-AXL antibody
(diluted 1:15,000 with TBST; Santa Cruz) and anti-actin antibody
(diluted 1:5,000 with TBST). After the filter had been washed four
times for five minutes each with TBST, it was incubated for one
hour with Alexa 680-labeled anti-rabbit secondary antibody
(Invitrogen) diluted 1:10,000 with TBST and IRDye 800-labeled
anti-goat secondary antibody (Rockland) diluted 1:10,000 with TBST.
After it had been washed three times for five minutes each with
TBST, the filter was washed again once for five minutes with TBS,
and then scanned with the Odyssey infrared imaging system
(Li-COR).
[0221] The AXL blots were observed to weaken following exposure to
the anti-AXL monoclonal antibodies Ax285, Ax292, Ax223, Ax96,
Ax258, Ax284, Ax7, and Ax225 (FIGS. 3a, b, c, d, e, f, g, and h).
Therefore, these anti-AXL monoclonal antibodies can induce the
downmodulation of AXL protein.
Example 5
In Vitro Angiogenesis Inhibitory Activity of Anti-AXL Antibody
[0222] The activity of anti-AXL antibody to inhibit the lumen
formation of human umbilical vein endothelial cells (HUVEC) was
measured using an angiogenesis kit available from Kurabo
Industries. The experimental procedure was in accordance with the
protocol provided with the kit and is summarized below. HUVEC and
fibroblasts were cocultured, and a 24-well plate (provided with the
kit) containing cells in the growth state of early lumen formation
was placed in an incubator for three hours at 37.degree. C. under
5% CO.sub.2 and humidified air. The caps of three containers
containing 25 mL of special-purpose medium (provided with the kit)
were loosened and placed in the incubator for about 30 minutes at
37.degree. C. under 5% CO.sub.2 and humidified air. The plate was
then removed from the incubator and the well cap sheet was peeled
off. The plate cover was then replaced with a new one (provided
with the kit). The cells were confirmed to be normal by observation
under a microscope. Culture medium (>12 mL/plate), warmed to
37.degree. C., was dispensed into Falcon tubes and VEGF-A (2
.mu.g/mL) was added to the medium to a final concentration of 10
ng/mL by 200-fold dilution. The anti-AXL antibody prepared as
described above was added to the medium dispensed into the tubes to
a final concentration of 10 .mu.g/mL. PBS(-) was used in place of
antibody for the negative control. The medium in the wells of the
24-well plate was gently removed by aspiration and 500 .mu.L of
drug-containing medium was then gently added. The condition of the
cells was observed microscopically and they were then returned to
the incubator. The medium was replaced using the same procedure on
days 4, 7, and 9, counting the day on which the antibody was added
as day 1.
[0223] The cell layer was fixed and stained using a lumen staining
kit (Kurabo) on the 11th day after the addition of the antibody.
The procedure was carried out according to the protocol provided
with the kit and is summarized below. After the cells were observed
under a microscope, the medium was removed by aspiration and the
well was washed by the addition of 1 mL of wash buffer (PBS(-) pH
7.4; Sigma) to each well, and then the wash buffer was removed by
aspiration. 1 mL of ice-cold fixing solution (70% ethanol) was
added to each well and allowed to stand for 30 minutes at room
temperature. The fixing solution was then removed, 1 mL of blocking
solution was added to each well, the well was washed, and the
blocking solution was removed by aspiration. 0.5 mL of the primary
antibody provided with the kit was diluted according to the
protocol and added to each well, followed by incubation for one
hour at 37.degree. C. The primary antibody was removed by
aspiration and each well was washed three times with 1 mL of
blocking solution (PBS(-) containing 1% BSA, pH 7.4; Sigma). 0.5 mL
of the secondary antibody provided with the kit and diluted in
accordance with the protocol was added to each well, followed by
incubation for one hour at 37.degree. C. The secondary antibody was
removed by aspiration and each well was washed three times with 1
mL of distilled water. 0.5 mL of the substrate solution provided
with the kit was added to each well, followed by incubation for
10-30 minutes at 37.degree. C. until the lumen became dark purple.
The substrate solution was then removed by aspiration and each well
was washed three times with 1 mL of distilled water and allowed to
air dry. Microscopic images of each fixed well were captured at
five locations with a CCD camera (Nikon Digital Camera, dxm1200),
and the vessel areas were calculated using angiogenesis
quantification software (Ver. 1.0, Kurabo).
[0224] The rate of the reduction in the area of the vessels that
formed in a lumen in the wells to which was added anti-AXL antibody
relative to the area of the vessels that formed in a lumen in the
wells to which was added the negative control PBS(-) was used as
the index of the inhibitory activities of the antibodies, and
Ax232, Ax292, Ax285, and Ax284 displayed inhibitory activity (FIG.
4).
Example 6
Binding Activity of the Anti-AXL Antibody to Mouse AXL
[0225] After the extracellular domain of mouse AXL (hereinafter
referred to as mAXL-ECD; R&D) was diluted with coating buffer
(100 mM sodium bicarbonate buffer, pH 9.6) to 2 .mu.g/mL, 100 .mu.L
was dispensed into a 96-well plate (Nunc-Immuno.TM. 96
MicroWell.TM. MaxiSorp.TM. plates; Nalge Nunc International). After
the plate was placed in a refrigerator overnight, the antibody
solution in the plate was removed, 200 .mu.L/well of diluent buffer
(BlockingOne; Nacalai Tesque) was dispensed, and then blocked for
two hours at room temperature. After the removal of the diluent
buffer, the anti-AXL antibody prepared above diluted to 3 .mu.g/mL
with diluent buffer was dispensed at 100 .mu.L/well, and allowed to
stand for 1.5 hours at room temperature. After the removal of the
antibody solution, the wells were washed three times with tPBS(-).
A labeled antibody cocktail containing alkaline-phosphatase-labeled
goat anti-mouse IgG1 antibody, alkaline-phosphatase-labeled goat
anti-mouse IgG2a antibody, and alkaline-phosphatase-labeled goat
anti-mouse IgG2b antibody (SouthernBiotech) was prepared with final
dilutions of each antibody of 1/2250:1/4000:1/4000, and was
dispensed at 100 .mu.L/well, and allowed to stand for one hour at
room temperature. After the removal of the antibody solution, the
wells were washed three times with tPBS(-). 100 .mu.L/well of
alkaline phosphatase chromogenic substrate solution (BluePhos
Microwell Phosphatase Substrate System, Kirkegaad & Perry
Laboratories) was dispensed, followed by color development at room
temperature. The absorbance at 650 nm was then measured with a
microplate reader (Emax, Molecular Devices).
[0226] Binding to mouse AXL was confirmed for Ax96, Ax119, Ax223,
Ax225, and Ax284.
Example 7
In Vitro Cancer Cell Growth Inhibitory Activity of the Anti-AXL
Antibody
[0227] Evaluation was performed using HCT-116 (CCL-247), Calu-1
(HTB-54), DU-145 (HTB-81), and T-47D (HTB-133) purchased from ATCC,
and AsPC-1, MDA-MB-231, and PANC-1 purchased from Dainippon
Sumitomo Pharma. The cells were maintained under the conditions
recommended by the supplier of each cell. A dilution series was
prepared of the anti-AXL antibody produced as described above with
10% FBS/RPMI1640, and 20 .mu.L was dispensed into a 96-well plate
(flat bottom). Each of the suspensions of HCT-116, Calu-1, DU145,
T-47D, AsPC-1, MDA-MB-231, and PANC-1 cells were prepared at 2000,
3000, 2000, 5000, 3000, 5000, and 3,000 cells per well,
respectively, and 180 .mu.L of cell suspension was added to each
well and then cultured in an incubator at 37.degree. C. in 5%
CO.sub.2. Four days later, 10 .mu.L of WST-8 (Cell Counting Kit-8,
Dojindo Laboratories) was added to each well and the absorbance at
450 nm was measured with a microplate reader (Model 3550-UV,
Bio-Rad), according to the protocol provided with the kit. The cell
inhibitory activity (%) of the anti-AXL antibodies was calculated
by assigning a value of 0% inhibition to the value measured when no
test substance was included, and assigning a value of 100%
inhibition to a value measured when no test substance or cells were
included.
[0228] Ax51 demonstrated CGI activity of 30% or more against HCT116
cells.
TABLE-US-00002 TABLE 2 HCT116 1st 2nd TOP 1/3 1/9 TOP 1/10 Ax51 31
11 10 12 15
Example 8
Measurement of Antitumor Effects of the Anti-AXL Antibody in a
Mouse Model Grafted with Human Pancreatic Adenocarcinoma
1. Preparation of a Mouse Model Grafted With Human Pancreatic
Adenocarcinoma
[0229] The human pancreatic adenocarcinoma cell line PANC-1,
purchased from Dainippon Pharmaceutical (currently Dainippon
Sumitomo Pharma), was prepared at 5.times.10.sup.7 cells/mL with
HBSS. 200 .mu.L of the cell suspension (1.times.10.sup.7
cells/mouse) was subcutaneously grafted into the inguinal region of
a CAnN.Cg-Foxn1<nu>/CrlCrlj nu/nu (BALB-nu/nu) mouse
purchased from Charles River Laboratories, Japan. The mouse was
subjected to the experiment when the tumor volume had reached about
210 mm.sup.3.
2. Antibody Preparation and Administration
[0230] The antibodies of Table 1 were prepared at 2 mg/mL with PBS
and administered twice a week for two weeks at 20 mg/kg into the
peritoneal cavity of the mouse grafted with human pancreatic
adenocarcinoma. As the negative control, PBS was administered in
the same manner. Gemzar (Eli Lilly Japan) was prepared at 12 mg/mL
with physiological saline as the positive control and administered
intraperitoneally at 120 mg/kg twice a week for two weeks.
3. Evaluation of Antitumor Effects
[0231] The antitumor effects in a mouse model grafted with human
pancreatic adenocarcinoma were calculated as
tumor-growth-suppressive effects by comparing the tumor growth in
the antibody-treated group with the tumor growth in the negative
control group four days after the final administration (FIG.
5).
Tumor-growth-suppressive effect (%)=(1-amount of tumor growth in
the antibody-treated group/amount of tumor growth in the control
group).times.100
4. Statistical Processing
[0232] Tumor volume was expressed as the mean.+-.standard
deviation. Statistical analysis consisted of a comparison between
the control group and the treated group by the LSD method using the
SAS Preclinical Package Ver. 5.0. Reliability of 95% (*: p<0.05)
was determined to constitute significance.
5. Results
[0233] All of the antibodies inhibited tumor growth and
demonstrated antitumor effects (FIG. 5).
Example 9
Measurement of Antitumor Effects of Anti-AXL Antibody on Mouse
Model Transplanted with Human Pancreatic Adenocarcinoma (2)
[0234] 1. Preparation of Mouse Model Grafted with Human Pancreatic
Adenocarcinoma
[0235] Human pancreatic adenocarcinoma cell line PANC-1 purchased
from Dainippon Pharmaceutical (currently Dainippon Sumitomo Pharma)
was prepared to 5.times.10.sup.7 cells/mL with HBSS. 200 .mu.L of
the cell suspension (1.times.10.sup.7 cells/mouse) were
subcutaneously grafted to the inguinal regions of
CAnN.Cg-Foxn1<nu>/CrlCrlj nu/nu (BALB-nu/nu) mice purchased
from Japan Charles River. The mice were used in the experiment when
the mean tumor volume reached about 270 mm.sup.3.
2. Antibody Preparation and Administration
[0236] Anti-AXL antibody was prepared to 2 mg/mL with PBS and
administered into the peritoneal cavity of the mice grafted with
human pancreatic adenocarcinoma twice a week for two weeks at 20
mg/kg. PBS was administered in the same manner for use as a
negative control. Gemzar (Eli Lilly Japan) was prepared to 12 mg/mL
with physiological saline for use as a positive control and
administered intraperitoneally twice a week for two weeks at 120
mg/kg.
3. Evaluation of Antitumor Effects
[0237] Antitumor effects in a mouse model grafted with human
pancreatic adenocarcinoma were calculated as tumor growth
suppressive effects by comparing with the amount of tumor growth of
a negative control group four days after final administration.
Tumor growth suppressive effect (%)=(1-amount of tumor growth of
the antibody-treated group/amount of tumor growth of the control
group).times.100
4. Results
[0238] The results for suppression of tumor growth are shown in
FIG. 6. A tumor growth suppressive effect (%) of lower than 30% is
indicated as "-", that of 30% or more is indicated as "+", and that
of 60% or more is indicated as "++". The results for the assay of
inhibition of ligand-dependent phosphorylation by antibody of
Example 3 are also shown in FIG. 6.
[0239] Antibodies that bind to FND-1 demonstrated 60% or more of
TGI activity even if administration was begun at the time when mean
tumor volumes had reached about 270 mm.sup.3. This finding that
anti-AXL antibodies that bind to FND1 have such significant
antitumor effects in vivo was determined for the first time in this
study and was completely unexpected.
[0240] In addition, the existence of anti-AXL antibodies that bind
to IgD2 that demonstrate phosphorylation inhibitory effect and in
vivo antitumor effects as indicated in Examples 3, 8, and 9 was
also discovered for the first time in this study and was also
completely unexpected.
Example 10
Binding Activity to Human AXL-FND1 and Human AXL-IgD2
1. Binding Activity to Human AXL-FND1 and Human AXL-IgD2
[0241] The binding abilities of anti-AXL monoclonal antibody to
AXL-fibronectin type 3 domain 1 (AXL-FND1) and AXL immunoglobulin
family domain 2 (AXL-IgD2) were tested.
2. Preparation of Human Recombinant AXL-FND1 and Human Recombinant
AXL-IgD2 Expression Vectors
[0242] Human recombinant AXL-FND1 was prepared by amplifying by PCR
a region equivalent to the 225th to 331st amino acids from
full-length human AXL cDNA (O'Bryan, et al., Mol. Cell. Biol.
(1991) 11, 5016-5031) (GenBank No. NM 021913), cloning the
amplified products to pET-41a(+) (Novagen) to express fusion
proteins with GST-tag, and constructing pET-AXL-FND1. Other domains
were prepared by amplifying by PCR a region equivalent to the 137th
to 224th amino acids, and cloning the amplified products to
pET-41a(+) to express fusion proteins with GST tag. Each of the
prepared vectors (5 .mu.l) was transformed to DHSa (Toyobo Co.,
Ltd., Cat. No. DNA-903) by a heat shock method and then cultured in
SOC medium. Colonies were selected after culturing overnight at
37.degree. C. on an LB plate containing kanamycin.
3. Purification of Human Recombinant AXL-FND1 and Human Recombinant
AXL-IgD2
[0243] Each of the produced colonies were precultured overnight at
37.degree. C. in 20 mL of LB medium containing kanamycin and then
transferred to 500 mL of medium. The each colony was cultured to an
A.sub.600 of 0.5.+-.0.05 and IPTG was added to be a concentration
of 0.5 mM. After culturing for one hour at 37.degree. C., the
bacterial cells were collected and suspended in Buffer A (50 mM
Tris-HCl (pH 8.0), 1 mM EDTA, 0.5 mM PMSF, and 1 mM DTT). Freezing
and thawing was repeated twice using liquid nitrogen. NP-40 was
then added to 0.5% and the cells were homogenized with an
ultrasonic homogenizer (30 seconds.times.5) and centrifuged for 30
minutes at 204,000.times.G, and then the supernatant was
recovered.
[0244] Human recombinant AXL-FND1 was purified in the manner
described below using the resulting supernatant. Solubilized E.
coli supernatant was mixed with Glutathione Sepharose.TM. 4 Fast
Flow (GE Healthcare) and stirred for one hour at 4.degree. C. with
a rotator. After centrifugation for five minutes at 500.times.G,
the supernatant was discarded and the Glutathione Sepharose.TM. 4B
was washed by adding Buffer A. This washing procedure was repeated
three times. After transferring the human recombinant AXL-FND1 from
the washed Glutathione Sepharose.TM. 4 Fast Flow to a mini-column,
it was separated and eluted from the Glutathione Sepharose.TM. 4
Fast Flow with 50 mM Tris-HCl (pH 7.5) and 25 mM glutathione. Each
of other AXL domains was expressed, separated, and eluted in the
same manner.
4. Evaluation of Binding Activity of Anti-AXL Antibody to AXL-FND1
by Western Blotting
[0245] The recombinant AXL-FND1 separated and eluted from the
Glutathione Sepharose.TM. 4 Fast Flow, as well as AXL-IgD1,
AXL-IgD2, AXL-FND2, AXL-IgD1+IgD2, AXL-IgD2+FND1, and AXL-FND1+FND2
were quantified using the BIO-RAD Dc Protein Assay. 1 .mu.g each
was mixed with NuPAGE.RTM. Sample Buffer (Invitrogen), and
electrophoresed with NuPAGE.RTM. 10% Bis-TrisGel. The
electrophoresed gel was transferred to an Immobilon.TM.-FL
(Millipore) PVDF membrane. The PVDF membrane containing the
transferred protein was blocked with Odyssey.RTM. Blocking Buffer
(LI-COR) and immersed in a primary antibody solution in which
anti-AXL antibody was diluted to 5 .mu.g/mL, and incubated
overnight at 4.degree. C. The PVDF membrane containing the
transferred protein and immersed in the primary antibody solution
was washed four times for five minutes each with 0.1% TBS-T (TBS
(Tris-Buffered Saline (Takara)) containing 0.1% Tween-20). The PVDF
membrane immersed in anti-AXL antibody was immersed in Alexa
Fluor.RTM. 680 Goat Anti-mouse IgG (H+L) (Invitrogen) secondary
antibody solution diluted to 80 ng/mL and incubated for one hour at
room temperature. After washing the PVDF membrane immersed in the
secondary antibody solution three times for five minutes each with
0.1% TBS-T, the membrane was washed for five minutes with TBS-T
containing 0.01% SDS and then washed for five minutes with TBS. The
binding of the washed PVDF membrane was then evaluated by scanning
with the Odyssey.RTM. far infrared imaging system.
5. Results
[0246] The evaluation results are shown in FIG. 6.
[0247] Anti-AXL antibody produced by a hybridoma deposited under
Accession No. FERM BP-10854 (Ax225) was demonstrated to recognize
FND1 of AXL (FIG. 6). Anti-AXL antibody produced by a hybridoma
deposited under Accession No. FERM BP-10857 (Ax284) was considered
to recognize FND1 and IgD2 of AXL (FIG. 6). Anti-AXL antibody
produced by a hybridoma deposited under Accession No. FERM BP-10850
(Ax7) and anti-AXL antibody produced by a hybridoma deposited under
Accession No. FERM BP-10851 (Ax51) were demonstrated to recognize
IgD2 of AXL (FIG. 6).
Example 11
Measurement of Antitumor Effects of Anti-AXL Antibody on Mouse
Model Grafted with Human Breast Cancer
[0248] 1. Preparation of Mouse Model Grafted with Human Breast
Cancer
[0249] Human breast cancer cell line MDA-MB-435S obtained from ATCC
was prepared to 5.times.10.sup.7 cells/mL with HBSS. 200 .mu.L of
the cell suspension (1.times.10.sup.7 cells/mouse) was
subcutaneously grafted to the inguinal regions of
CAnN.Cg-Foxn1<nu>/CrlCrlj nu/nu (BALB-nu/nu) mice purchased
from Japan Charles River. The mice were used in the experiment when
the tumor volume reached about 200 mm.sup.3.
2. Antibody Preparation and Administration
[0250] Anti-AXL antibody was prepared to 2 mg/mL with PBS and
administered into the peritoneal cavity of the mice grafted with
human breast cancer twice a week for two weeks at 20 mg/kg. PBS was
administered in the same manner for use as a negative control.
3. Evaluation of Antitumor Effects
[0251] Antitumor effects in a mouse model grafted with human breast
cancer were calculated as tumor growth suppressive effects by
comparing with the amount of tumor growth of a negative control
group four days after final administration.
Tumor growth suppressive effect (%)=(1-amount of tumor growth of
the antibody-treated group/amount of tumor growth of the control
group).times.100
4. Statistical Processing
[0252] Tumor volume was expressed as the mean.+-.standard
deviation. Statistical analysis consisted of a comparison between
the control group and the treated group by the LSD method using the
SAS Preclinical Package Ver. 5.0. Reliability of 95% (*: p<0.05)
was determined to constitute significance.
5. Results
[0253] The used anti-AXL antibodies suppressed tumor growth and
demonstrated antitumor effects (FIG. 7). Therefore, anti-AXL
antibodies that bind to FND1 are expected to have antitumor effects
against various tumors.
Example 12
Sequence Analysis of Antibody cDNA
[0254] 1. Preparation of chimeric antibody-expression vectors
[0255] Total RNA was extracted from the cells of a hybridoma
deposited under Accession No. FERM BP-10854 (Ax225) using the
RNeasy Mini Kit (Qiagen), and cDNA was synthesized using the SMART
RACE cDNA Amplification Kit (BD Biosciences). Antibody variable
region gene was isolated by carrying out PCR with PrimeSTAR HS DNA
Polymerase (Takara) using the following primers (H chain, MHCg1; L
chain, MLCk) which were set for respective constant regions of
antibody and 10.times. Universal Primer A Mix, provided with the
SMART RACE cDNA Amplification Kit (BD Biosciences).
TABLE-US-00003 MHCg1: 5'-GGGCCAGTGGATAGACAGATG-3' (SEQ ID NO. 1)
MLCk: 5'-GCTCACTGGATGGTGGGAAGATG-3' (SEQ ID NO. 2)
[0256] The nucleotide sequence of each isolated DNA fragment was
determined using the BigDye Terminator Cycle Sequencing Kit
(Applied Biosystems) with the ABI PRISM 3730xL DNA Sequencer or ABI
PRISM 3700 DNA Sequencer (Applied Biosystems) in accordance with
the method described in the instructions provided.
2. Results
[0257] The heavy chain variable region of the amino acid sequence
of the resulting AXL225 mouse antibody is shown in SEQ ID NO. 3,
the CDR1 of that region is shown in SEQ ID NO. 4, CDR2 is shown in
SEQ ID NO. 5, and CDR3 is shown in SEQ ID NO. 6. The light chain
variable region of the amino acid sequence of the resulting AXL225
mouse antibody is shown in SEQ ID NO. 7, the CDR1 of that region is
shown in SEQ ID NO. 8, CDR2 is shown in SEQ ID NO. 9, and CDR3 is
shown in SEQ ID NO. 10.
INDUSTRIAL APPLICABILITY
[0258] The present inventors discovered for the first time that
anti-AXL antibodies have an angiogenesis-suppressing effect and a
cancer-suppressing effect. The anti-AXL antibody of the present
invention is useful as an angiogenesis inhibitor and as a
cell-growth suppressant. Using an antibody of the present
invention, the phosphorylation of AXL can also be induced or
inhibited. Moreover, using an antibody of the present invention,
the expression level of AXL can be reduced.
Sequence CWU 1
1
10121DNAArtificialAn artificially synthesized primer sequence
1gggccagtgg atagacagat g 21223DNAArtificialAn artificially
synthesized primer sequence 2gctcactgga tggtgggaag atg
233138PRTArtificialAn artificially synthesized peptide sequence
3Met Ala Val Leu Val Leu Leu Phe Cys Leu Val Thr Phe Pro Ser Cys 1
5 10 15 Ile Leu Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val
Ala 20 25 30 Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly
Leu Ser Leu 35 40 45 Thr Ser Phe Gly Val Asp Trp Val Arg Gln Ser
Pro Gly Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Gly
Gly Ser Thr Asn Tyr Asn Ser 65 70 75 80 Ala Leu Lys Ser Arg Leu Ser
Ile Ser Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val Phe Leu Lys Met
Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr 100 105 110 Tyr Cys Ala
Gly Glu Gly Ser Lys Tyr Gly Ala Trp Phe Ala Tyr Trp 115 120 125 Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 45PRTArtificialAn
artificially synthesized peptide sequence 4Ser Phe Gly Val Asp 1 5
516PRTArtificialAn artificially synthesized peptide sequence 5Val
Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Lys Ser 1 5 10
15 611PRTArtificialAn artificially synthesized peptide sequence
6Glu Gly Ser Lys Tyr Gly Ala Trp Phe Ala Tyr 1 5 10
7131PRTArtificialAn artificially synthesized peptide sequence 7Met
Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10
15 Ser Ser Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val
20 25 30 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln
Asn Ile 35 40 45 Val His Thr Asn Gly Asn Thr Tyr Leu Glu Trp Tyr
Leu Gln Lys Pro 50 55 60 Gly Gln Ser Pro Glu Leu Leu Ile Tyr Lys
Val Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val
Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys 100 105 110 Phe Gln Gly Ser
His Ile Pro Phe Thr Phe Gly Thr Gly Thr Lys Leu 115 120 125 Glu Ile
Lys 130 816PRTArtificialAn artificially synthesized peptide
sequence 8Arg Ser Ser Gln Asn Ile Val His Thr Asn Gly Asn Thr Tyr
Leu Glu 1 5 10 15 97PRTArtificialAn artificially synthesized
peptide sequence 9Lys Val Ser Asn Arg Phe Ser 1 5
109PRTArtificialAn artificially synthesized peptide sequence 10Phe
Gln Gly Ser His Ile Pro Phe Thr 1 5
* * * * *