Note: Descriptions are shown in the official language in which they were submitted.
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Description
Title of Invention: ANTI-RAMP2 ANTIBODY
Technical Field
[0001]
The present invention relates to an antibody or
immunoreactive fragment thereof against receptor
activity-modifying protein 2 (RAMP2), having an
antiangiogenic effect, and an angiogenesis inhibitor
containing the antibody or immunoreactive fragment
thereof against RAMP2 as an active ingredient.
Background Art
[0002]
Adrenomedullin (hereinafter, referred to as "AM") is
a peptide identified from human phaeochromocytoma and
consisting of 52 amino acids, and known as a vasoactive
substance that is primarily secreted from blood vessels
and functions to vasodilate. In addition, the
adrenomedullin has been reported to have various effects
including an organ protection effect, a tissue
regeneration effect, and an anti-inflammatory effect.
Receptor activity-modifying protein 2 (RAMP2) is a single
transmembrane protein, and forms complex with a
calcitonin receptor-like receptor (CRLR), a seven-
transmembrane protein, to exert function as coreceptor
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for AM. Expression of RAMP2 is found in vascular
endothelial cells, where RAMP2 transmit a signal by
elevating the intracellular cAMP level on receiving AM
through autocrine or paracrine and maintain the vascular
homeostasis.
[0003]
Inhibition of the AM-RAMP2/CRLR signal is expected
to enable development of pharmaceuticals for various
diseases including: cardiovascular diseases by
controlling blood pressure; inflammation and edema by
controlling vasopermeability; cancer and retinal diseases
involving angiogenesis; and immunorejection after organ
transplantation. As such an inhibitor, AM37-52, an AM
partial sequence peptide (antagonist), has been reported
(Patent Literature 1). The weak antagonist activity and
poor blood kinetics due to a low molecular weight of 5
kDa or lowermake it difficult todevelop AM37-52 as a
pharmaceutical.
[0004]
In addition, polyclonal antibodies against RAMP2 and
mixtures thereof have been previously reported (Patent
Literatures 2, 3). However, effective prevention of
angiogenesis has not yet been achieved with an RAMP2
antibody (in particular, a monoclonal antibody) alone.
Citation List
Patent Literature
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[0005]
Patent Literature 1: International Publication No. WO
2016/183479
Patent Literature 2: International Publication No. WO
2010/012911
Patent Literature 3: International Publication No. WO
2012/029023
Summary of Invention
[0006]
The inventors tried to obtain an anti-RAMP2 antibody
that inhibits activation of an AM-dependent RAMP2/CRLR
complex, in particular, an anti-RAMP2 antibody being able
to inhibit the growth of HUVECs (human umbilical vein
endothelial cells), by generating polyclonal antibodies
by immunizing a peptide fragment encoding a human RAMP2
protein. However, the serum-derived polyclonal
antibodies did not have cAMP inhibitory activity. Then,
an anti-RAMP2 antibody was generated by immunizing a
protein produced through cell-free synthesis of the
extracellular domain of RAMP2. Using a single RAMP2
protein and a complex of the coreceptor CRLR with the
RAMP2 protein as an antigen, anti-RAMP2 antibodies with
superior cAMP inhibitory activity were obtained.
However, even these antibodies did not prevent the growth
of HUVECs.
[0007]
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Then, the present inventors performed immunization
with transformed overexpressing cells as an antigen.
Considering that human RAMP2 alone does not function as a
receptor for an AM peptide, CRLR gene was coexpressed
with RAMP2 gene in fibroblast cell line NIH-3T3.
[0008]
However, no high-quality antibody was successfully
obtained through general immunization of a rat, a
hamster, or a mouse with the overexpressing cells. The
reason may be due to the difficulty in successful
immunization of these animals with the functional domain
of human RAMP2, because the RAMP2 protein is a small
molecule consisting of 175 amino acids in total length
(20 kDa), and in particular, the extracellular domain of
RAMP2 consists of only 102 amino acids (12 kDa), and in
addition, human RAMP2 is highly homologous with mouse
RAMP2 and 62% of their extracellular domains encode the
same amino acids. In view of these results, an MRL/lpr
mouse which is able to generate self-immunoreaction
(autoantibody) upon hyperimmunization was immunized with
the overexpressing cells, which successfully provided
anti-RAMP2 antibodies with superior functionality.
[0009]
To screen for anti-RAMP2 antibodies with superior
functionality from the obtained hybridomas, Cell ELISA
was performed using transformed overexpressing cells. In
using the same mouse NIH-3T3 cells as an antigen for Cell
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Elisa, antibodies binding to the cell itself but not
transfected RAMP2 protein could be detected as pseudo-
positiveness. Hence hamster ovary cells (CHO-K1) were
transfected with human RAMP2-CRLR to obtain stable cells
(hCR2-CHOK1), which were used for Cell ELISA.
[0010]
Candidate antibodies selected through Cell ELISA
were further screened for signal-inhibiting activity
according to inhibitory effect on the intracellular cAMP
level elevated by addition of AM in hCR2-CHOK1 cells.
Further, the selected signal-inhibiting antibodies were
subjected to a HUVECs growth inhibition test upon AM
supplementation as screening for bioactivity inhibiting
test, and finally cAMP inhibitory anti-RAMP2 antibodies
having an activity and a HUVEC growth preventing activity
were successfully obtained.
[0011]
Accordingly, the present invention is directed to an
anti-RAMP2 antibody having the cAMP inhibitory activity
and HUVEC growth preventing activity, as well as to an
angiogenesis inhibitor containing said antibody as an
active ingredient.
(1) An antibody or an immunoreactive fragment
thereof that specifically binds to receptor activity-
modifying protein 2 (RAMP2) and has an inhibitory
activity on a vascular endothelial cell growth in an in
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vitro test for vascular endothelial cell growth induced
by adrenomedullin supplementation.
(2) The antibody or immunoreactive fragment thereof
of (1), which has the inhibitory activity on vascular
endothelial cell growth with 1050 value of 0.5 to 5 nM.
(3) The antibody or immunoreactive fragment thereof
of (1) or (2), wherein the antibody or immunoreactive
fragment thereof has an inhibitory activity on cAMP
production induced by adrenomedullin supplementation in
animal cells.
(4) The antibody or immunoreactive fragment thereof
of (3), which has the inhibitory activity on cAMP
production in an in vitro test for cAMP production
induced by adrenomedullin supplementation in animal cells
with 1050 value of 0.03 to 0.5 nM.
(5) An antibody or an immunoreactive fragment
thereof that competes for binding to RAMP2 with any one
antibody selected from an antibody comprising a heavy
chain variable region (VH) having the amino acid sequence
of SEQ ID NO: 2 and a light chain variable region (VL)
having the amino acid sequence of SEQ ID NO: 4, an
antibody comprising VH having the amino acid sequence of
SEQ ID NO: 12 and VL having the amino acid sequence of
SEQ ID NO: 14, and an antibody comprising VH having the
amino acid sequence of SEQ ID NO: 19 and VL having the
amino acid sequence of SEQ ID NO: 21; or
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an antibody or an immunoreactive fragment thereof
that binds to the same epitope as any one antibody
selected from an antibody comprising VH having the amino
acid sequence of SEQ ID NO: 2 and VL having the amino
acid sequence of SEQ ID NO: 4, an antibody comprising VH
having the amino acid sequence of SEQ ID NO: 12 and VL
having the amino acid sequence of SEQ ID NO: 14, and an
antibody comprising VH having the amino acid sequence of
SEQ ID NO: 19 and VL having the amino acid sequence of
SEQ ID NO: 21.
(6) An antibody or an immunoreactive fragment
thereof, comprising a heavy chain having heavy chain
complementarity-determining region (CDRH) 3 consisting of
the amino acid sequence of LX1IX2DX3YX4YX5MX6X7 (SEQ ID NO:
24), wherein X1 is G or M, X2 is F or Y, X3 is A or V, X4
is absent or H, X5 is A or V, X6 is D or E, and X7 is Y or
N.
(7) The antibody or immunoreactive fragment thereof
of (6), comprising a heavy chain having CDRH3 consisting
of the amino acid sequence of LMIFDAYYAMDY (SEQ ID NO: 7)
or LGIYDVYHYVMEN (SEQ ID NO: 16).
(8) The antibody or immunoreactive fragment thereof
of (6), comprising a heavy chain having CDRH1 consisting
of the amino acid sequence of GYFMN (SEQ ID NO: 5), CDRH2
consisting of the amino acid sequence of
RX8NPYNGDX9X1oYNQKFKG (SEQ ID NO: 23), and CDRH3
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consisting of the amino acid sequence of
LX1IX2DX3YX4YX5MX6X7 (SEQ ID NO: 24), wherein
X1 is G or M, X2 is F or Y, X3 is A or V. X4 is
absent or H, X5 is A or V, X6 is D or E, X7 is Y or N, X8
is N or I, X9 is S or T, and Xlo is I, F, or L.
(9) The antibody or immunoreactive fragment thereof
of (8), further comprising a light chain having light
chain complementarity-determining region (CDRL) 1
consisting of the amino acid sequence of RASQDIRNYLN (SEQ
ID NO: 8), CDRL2 consisting of the amino acid sequence of
YTSRLHS (SEQ ID NO: 9), and CDRL3 consisting of the amino
acid sequence of QQDSKXIIPWT (SEQ ID NO: 25), wherein
XII is H or N.
(10) The antibody or immunoreactive fragment thereof
of (6), comprising:
(i) CDRH1 consisting of the amino acid sequence of
GYFMN (SEQ ID NO: 5),
CDRH2 consisting of the amino acid sequence of
RNNPYNGDSIYNQKFKG (SEQ ID NO: 6), and
CDRH3 consisting of the amino acid sequence of
LMIFDAYYAMDY (SEQ ID NO: 7);
(ii) CDRH1 consisting of the amino acid sequence of
GYFMN (SEQ ID NO: 5),
CDRH2 consisting of the amino acid sequence of
RINPYNGDTFYNQKFKG (SEQ ID NO: 15), and
CDRH3 consisting of the amino acid sequence of
LGIYDVYHYVMEN (SEQ ID NO: 16); or
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(iii) CDRH1 consisting of the amino acid sequence of
GYFMN (SEQ ID NO: 5),
CDRH2 consisting of the amino acid sequence of
RINPYNGDTLYNQKFKG (SEQ ID NO: 22), and
CDRH3 consisting of the amino acid sequence of
LGIYDVYHYVMEN (SEQ ID NO: 16).
(11) The antibody or immunoreactive fragment thereof
of (10), further comprising:
for (i), CDRL1 consisting of the amino acid sequence
of RASQDIRNYLN (SEQ ID NO: 8),
CDRL2 consisting of the amino acid sequence of
YTSRLHS (SEQ ID NO: 9), and
CDRL3 consisting of the amino acid sequence of
QQDSKHPWT (SEQ ID NO: 10);
for (ii), CDRL1 consisting of the amino acid
sequence of RASQDIRNYLN (SEQ ID NO: 8),
CDRL2 consisting of the amino acid sequence of
YTSRLHS (SEQ ID NO: 9), and
CDRL3 consisting of the amino acid sequence of
QQDSKNPWT (SEQ ID NO: 17);
for (iii), CDRL1 consisting of the amino acid
sequence of RASQDIRNYLN (SEQ ID NO: 8),
CDRL2 consisting of the amino acid sequence of
YTSRLHS (SEQ ID NO: 9), and
CDRL3 consisting of the amino acid sequence of
QQDSKNPWT (SEQ ID NO: 17).
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(12) The antibody of any one of (1) to (11), which
is any one antibody or immunoreactive fragment thereof
selected from the group of:
an antibody or an immunoreactive fragment thereof,
wherein VH has an amino acid sequence encoded by a
nucleic acid sequence that can hybridize with a nucleic
acid sequence encoding the amino acid sequence of SEQ ID
NO: 2 under stringent conditions, and VL has an amino
acid sequence encoded by a nucleic acid sequence that can
hybridize with a nucleic acid sequence encoding the amino
acid sequence of SEQ ID NO: 4 under stringent conditions;
an antibody or an immunoreactive fragment thereof,
wherein VH has an amino acid sequence encoded by a
nucleic acid sequence that can hybridize with a nucleic
acid sequence encoding the amino acid sequence of SEQ ID
NO: 12 under stringent conditions, and VL has an amino
acid sequence encoded by a nucleic acid sequence that can
hybridize with a nucleic acid sequence encoding the amino
acid sequence of SEQ ID NO: 14 under stringent
conditions;
an antibody or an immunoreactive fragment thereof,
wherein VH has an amino acid sequence encoded by a
nucleic acid sequence that can hybridize with a nucleic
acid sequence encoding the amino acid sequence of SEQ ID
NO: 19 under stringent conditions, and VL has an amino
acid sequence encoded by a nucleic acid sequence that can
hybridize with a nucleic acid sequence encoding the amino
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acid sequence of SEQ ID NO: 21 under stringent
conditions;
an antibody or an immunoreactive fragment thereof,
wherein VH has an amino acid sequence with identity of
80% or more to the amino acid sequence of SEQ ID NO: 2,
and VL has an amino acid sequence with identity of 80% or
more to the amino acid sequence of SEQ ID NO: 4;
an antibody or an immunoreactive fragment thereof,
wherein VH has an amino acid sequence with identity of
80% or more to the amino acid sequence of SEQ ID NO: 12,
and VL has an amino acid sequence with identity of 80% or
more to the amino acid sequence of SEQ ID NO: 14; and
an antibody or an immunoreactive fragment thereof,
wherein VH has an amino acid sequence with identity of
80% or more to the amino acid sequence of SEQ ID NO: 19,
and VL has an amino acid sequence with identity of 80% or
more to the amino acid sequence of SEQ ID NO: 21.
(13) The antibody or immunoreactive fragment thereof
of (11), which is any one antibody or immunoreactive
fragment thereof selected from the group of:
an antibody or an immunoreactive fragment thereof,
comprising VH having the amino acid sequence of SEQ ID
NO: 2 and VL having the amino acid sequence of SEQ ID NO:
4;
an antibody or an immunoreactive fragment thereof,
comprising VH having the amino acid sequence of SEQ ID
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NO: 12 and VL having the amino acid sequence of SEQ ID
NO: 14; and
an antibody or an immunoreactive fragment thereof,
comprising VH having the amino acid sequence of SEQ ID
NO: 19 and VL having the amino acid sequence of SEQ ID
NO: 21.
(14) A nucleic acid molecule encoding an amino acid
sequence of the antibody or immunoreactive fragment
thereof of any one of (1) to (13).
(15) The nucleic acid molecule of (14), comprising:
a polynucleotide encoding VH having the nucleotide
sequence of SEQ ID NO: 1, and a polynucleotide encoding
VL having the nucleotide sequence of SEQ ID NO: 3;
a polynucleotide encoding VH having the nucleotide
sequence of SEQ ID NO: 11, and a polynucleotide encoding
VL having the nucleotide sequence of SEQ ID NO: 13; or
a polynucleotide encoding VH having the nucleotide
sequence of SEQ ID NO: 18, and a polynucleotide encoding
VL having the nucleotide sequence of SEQ ID NO: 20.
(16) A vector comprising the nucleic acid molecule
of (14) or (15).
(17) A host cell comprising the vector of (16).
(18) A method for producing the antibody or
immunoreactive fragment thereof of any one of (1) to
(13), comprising culturing the host cell of (17).
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(19) A pharmaceutical composition comprising the
antibody or immunoreactive fragment thereof of any one of
(1) to (13) as an active ingredient.
(20) The pharmaceutical composition of (19), which
is an angiogenesis inhibitor.
(21) The pharmaceutical composition of (19), which
is a therapeutic drug or prophylactic drug for cancer,
cancer metastasis, diabetic retinopathy, retinopathy of
prematurity, age-related macular degeneration,
retinopathy of diabetes mellitus, retinal vein occlusion,
neovascular glaucoma, inflammatory skin disease,
rheumatoid arthritis, or osteoarthritis.
Advantageous Effects of Invention
[0012]
The single use of the antibody or immunoreactive
fragment thereof of the present invention can inhibit the
growth of vascular endothelial cells through binding to
RAMP2 expressed on cell surfaces, and thus the present
invention enables provision of angiogenesis inhibitors
based on a novel mechanism as well as therapeutic drugs
and prophylactic drugs for diseases or disorders in which
angiogenesis contributes to the onset or exacerbation.
Brief Description of Drawings
[0013]
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[Figure 1] Figure 1 shows a graph showing an inhibitory
activity of each anti-RAMP2 antibody (10 nM) on cAMP
production by hCR2-CHOK1 cells on addition of AM. The
ordinate indicates ratios (%) of amounts of cAMP for each
group to that of control group (100%) to which AM was
added without any antibody. The abscissa indicates the
addition or no-addition of AM and the antibody, and the
name of the antibody added.
[Figure 2] Figure 2 shows a graph showing the results of
evaluating the ability of various concentrations of 25H4-
4F9, 85H7-2B11, and 33H4-1G3 antibodies to inhibit cAMP
production induced by supplementation with AM by using
hCR2-CHOK1 cells. The ordinate indicates ratios (%) of
amounts of cAMP with supplementation with each antibody
to that without supplementation with any antibody (100%).
The abscissa indicates antibody concentrations (nM).
[Figure 3] Figure 3 shows a graph showing a preventing
activity of each anti-RAMP2 antibody (10 nM) on HUVEC
growth induced by supplementation with AM. The ordinate
indicates ratios (%) of HUVEC counts for each group to
that of control group (100%) to which AM was added
without any antibody. The abscissa indicates addition or
no-addition of AM and the antibody, and the name of the
antibody added.
[Figure 4] Figure 4 shows a graph showing the results of
evaluating the ability of various concentrations of 25H4-
4F9, 85H7-2B11, and 33H4-1G3 antibodies to prevent HUVEC
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growth induced by supplementation with AM. The ordinate
indicates ratios (%) of HUVEC counts with supplementation
witheach antibody to that without supplementation with an
antibody (100%). The abscissa indicates antibody
concentrations (nM).
[Figure 5] Figure 5 shows amino acid sequences of VH (SEQ
ID NO: 2) and the light chain variable region (SEQ ID NO:
4) of a 25H4-4F9 antibody, VH (SEQ ID NO: 12) and the
light chain variable region (SEQ ID NO: 14) of an 85H7-
2B11 antibody, and VH (SEQ ID NO: 19) and the light chain
variable region (SEQ ID NO: 21) of a 33H4-1G3 antibody,
and CDR parts included therein.
[Figure 6] Figure 6 shows a graph confirming that
obtained antibodies are anti-RAMP2-specific by Cell
ELISA. The abscissa indicates antibodies used. The
dotted open bars represent results with human CRLR single
expression cells, and black solid bars represent those
with human CRLR and human RAMP2 coexpression cells (hCR2-
CHOK1). The ordinate indicates binding amount (0D450) of
antibodies to these expression cells.
[Figure 7] Figure 7 shows a graph showing the analyzed
results of binding affinity to cell membrane surface
protein by Cell ELISA using 25H4-4F9 (black solid
rectangle), 85H7-2B11 (black solid circle), and 33H4-1G3
(black solid triangle) obtained in Example 1, and
commercially available anti-RAMP2 antibodies 4E5 (open
circle), 2F5 (open rectangle), and B5 (open triangle).
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The ordinate indicates binding rate (%), and the abscissa
indicates log values of antibody concentrations (nM). An
antibody concentration of each antibody with 50% binding
rate was determined from the graph as an EC50 value.
[Figure 8] Figure 8 shows graphs showing the results of
FACS analysis of binding to an antigen on cell membrane
surfaces for test antibodies of 25H4-4F9, 85H7-2B11, and
33H4-1G3 obtained in Example 1, and the commercially
available anti-RAMP2 antibodies 4E5, 2F5, and B5. The
name of each test antibody used is shown in the top of
the corresponding graph. In each graph, the ordinate
indicates cell counts (x 103 cells), and the abscissa
indicates fluorescence intensity of an R-phycoerythrin-
labeled secondary antibody binding to cells. In the
graphs, peaks in the left side indicate the results for a
control (without a test antibody), and peaks in the right
side indicate the results with the test antibody.
[Figure 9] Figure 9 shows a graph showing the results of
binding competition to an antigen on cell membrane
surfaces for 25H4-4F9 (black solid triangle), 85H7-2B11
(black solid circle), or 33H4-1G3 (black solid rectangle)
obtained in Example 1, or the commercially available
antibody 4E5 (open circle) with a biotinylated 85H7-2B11
antibody analyzed by FACS. The ordinate indicates mean
fluorescence intensity (MFI) of a fluorescent label
conjugated to the biotinylated 85H7-2B11 antibody that
bound to the cells. The abscissa indicates
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concentrations ( g/mL) of antibodies used. The result
for 85H7-2B11 (black solid circle), which was allowed to
compete with the same antibody as the labeled antibody,
indicates that the binding is competed.
[Figure 10] Figure 10 shows the sequences of variable
regions of the heavy chain and light chain of a humanized
antibody designed from the sequence of a 25H4-4F9
antibody. From the sequence of VH of the 25H4-4F9
antibody, h25H4-4F9vh (SEQ ID NO: 2), antibodies of
h25H4-4F9vh1 (SEQ ID NO: 27), h25H4-4F9vh2 (SEQ ID NO:
29), h25H4-4F9vh3 (SEQ ID NO: 31), h25H4-4F9vh4 (SEQ ID
NO: 33), and h25H4-4F9vh5 (SEQ ID NO: 35) were designed.
From the sequence of VL of the 25H4-4F9 antibody,
antibodies of h25H4-4F9vk (SEQ ID NO: 4), were h25H4-
4F9vk1 (SEQ ID NO: 37), h25H4-4F9vk2 (SEQ ID NO: 39),
h25H4-4F9vk3 (SEQ ID NO: 41), and h25H4-4F9vk4 (SEQ ID
NO: 43) were designed. Each underline indicates a CDR
sequence.
[Figure 11] Figure 11 shows the sequences of variable
regions of the heavy chain and light chain of a humanized
antibody designed from the sequence of an 85H7-2B11
antibody. From the sequence of VH of the 85H7-2B11
antibody, antibodies of h85H7-2B11vh (SEQ ID NO: 12),
were h85H7-2B11vh1 (SEQ ID NO: 45), h85H7-2B11vh2 (SEQ ID
NO: 47), h85H7-2B11vh3 (SEQ ID NO: 49), h85H7-2B11vh4
(SEQ ID NO: 51), and h85H7-2B11vh5 (SEQ ID NO: 53) were
designed. From the sequence of VL of the 85H7-2B11
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antibody, antibodies of h85H7-2B1lvk (SEQ ID NO: 14),
were h85H7-2B1lvkl (SEQ ID NO: 55), h85H7-2B1lvk2 (SEQ ID
NO: 57), h85H7-2B11vk3 (SEQ ID NO: 59), and h85H7-2B11vk4
(SEQ ID NO: 61) were designed. Each underline indicates
a CDR sequence.
[Figure 12] Figure 12 shows a graph showing the results
of evaluating the ability of 16.7 nM humanized 25H4-4F9
antibodies (25H4-4F9v1, 25H4-4F9v2, 25H4-4F9v3, 25H4-
4F9v4, 25H4-4F9v5, 25H4-4F9v6, and 25H4-4F9v8) to inhibit
cAMP production induced by supplementation with AM by
using hCR2-CHOK1 cells. The ordinate indicates ratios
(%) of amounts of cAMP with supplementation with each
antibody to that without supplementation with an antibody
(100%). The abscissa indicates addition or no-addition
of AM, and the name of the antibody added.
[Figure 13] Figure 13 shows a graph showing the results
of evaluating the ability of 16.7 nM humanized 85H7-2B11
antibodies (85H7-2B11v1, 85H7-2B11v2, 85H7-2B11v3, 85H7-
2B11v4, 85H7-2B11v5, 85H7-2B11v6, 85H7-2B11v7, and 85H7-
2B11v8) to inhibit cAMP production induced by
supplementation with AM by using hCR2-CHOK1 cells. The
ordinate indicates ratios (%) of amounts of cAMP with
supplementation with each antibody to that without
supplementation with any antibody (100%). The abscissa
indicates addition or no-addition of AM, and the name of
the antibody added.
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[Figure 14] Figure 14 shows graphs showing the results of
evaluating the ability of different concentrations of
humanized 25H4-4F9 antibodies (25H4-4F9v1, 25H4-4F9v2,
25H4-4F9v3, 25H4-4F9v4, 25H4-4F9v5, 25H4-4F9v6, and 25H4-
4F9v8) (top graph) and humanized 85H7-2B11 antibodies
(85H7-2B11v1, 85H7-2B11v2, 85H7-2B11v3, 85H7-2B11v4,
85H7-2B11v5, 85H7-2B11v6, 85H7-2B11v7, and 85H7-2B11v8)
(bottom graph) to inhibit cAMP production induced by
supplementation with AM by using hCR2-CHOK1 cells. In
each graph, the ordinate indicates ratios (%) of amounts
of cAMP with supplementation with each antibody to that
without supplementation with an antibody (100%). The
abscissa indicates log values of antibody concentrations
(nM). The IC50 values (concentrations for 50% inhibition
of cAMP in the graphs) of the antibodies are shown
together with legends.
[Figure 15] Figure 15 shows graphs showing the results of
binding affinity analysis to cell membrane surface
protein by Cell ELISA using 6.7 nM humanized 25H4-4F9
antibodies (25H4-4F9v1, 25H4-4F9v2, 25H4-4F9v3, 25H4-
4F9v4, 25H4-4F9v5, 25H4-4F9v6, and 25H4-4F9v8) (top
graph), and 6.7 nM humanized 85H7-2B11 antibodies (85H7-
2B11v1, 85H7-2B11v2, 85H7-2B11v3, 85H7-2311v4, 85H7-
2B11v5, 85H7-2311v6, 85H7-2B11v7, and 85H7-2B11v8)
(bottom graph). The ordinate indicates binding rate
(0D450%) and the abscissa indicates antibodies used.
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[Figure 16] Figure 16 shows graphs showing the results of
binding affinity analysis to cell membrane surface
protein by Cell ELISA using humanized 25H4-4F9 antibodies
(25H4-4F9v1, 25H4-4F9v2, 25H4-4F9v3, 25H4-4F9v4, 25H4-
4F9v5, 25H4-4F9v6, and 25H4-4F9v8) (top graph), and
humanized 85H7-2B11 antibodies (85H7-2B11v1, 85H7-2B11v2,
85H7-2B11v3, 85H7-2B11v4, 85H7-2B11v5, 85H7-2B11v6, 85H7-
2B11v7, and 85H7-2B11v8) (bottom graph). The ordinate
indicates binding rate (%), and the abscissa indicates
log values of antibody concentrations (nM). An antibody
concentration of each antibody with 50% binding rate was
determined from the graph as an EC50 value.
[Figure 17] Figure 17 shows a graph showing the results
of epitope analysis for an antibody. The ordinate
indicates amounts of a bound antibody as 0D450 (coloring
intensity of a secondary antibody). The abscissa
indicates peptides derived from the extracellular region
of RAMP2 used for binding analysis.
Description of Embodiments
[0014]
(Antibody or Immunoreactive Fragment Thereof)
The antibody of the present invention specifically
binds to RAMP2. In particular, the antibody of the
present invention specifically binds to RAMP2 expressed
on cell membranes or cell surfaces. In the context of an
antibody or immunoreactive fragment thereof,
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"specifically" recognizes (binds) herein means that the
antibody or immunoreactive fragment thereof binds to
RAMP2 with substantially higher affinity than to other
proteins and peptides. The phrase "bind with
substantially higher affinity" means high affinity to
such a degree that a specific protein or peptide of
interest can be detected apart from other proteins and
peptides with a desired measurement apparatus or method.
Substantially higher affinity may mean, for example, to
have 3 or more times, 4 or more times, 5 or more times, 6
or more times, 7 or more times, 8 or more times, 9 or
more times, 10 or more times, 20 or more times, 30 or
more times, 40 or more times, 50 or more times, or 100 or
more times intensity detected by ELISA or EIA (e.g.,
fluorescence intensity).
[0015]
The association rate constant (Kal) for binding
between the antibody of the present invention and RAMP2
is, for example, 1 x 104 Ms-1 or more, 1 x 103 Ms-1 or
more, or 5 x 103 Ms-1 or more. The dissociation rate
constant (Kdl) for binding between the antibody of the
present invention and RAMP2 is, for example, 1 x 10-3 or
less, or 1 x 10-4 or less. The association constant (KD)
for binding between the antibody of the present invention
and RAMP2 can be, for example, 1 x 10-8 (M) or less, 5 x
10-8 (M) or less, 1 x 10-9 (M) or less, or 5 x 10-9 (M) or
less. The association rate constant (Kal), dissociation
CA 03075867 2020-03-13
- 22 -
rate constant (Kdl), and association constant (KD) of an
antibody herein can be determined as follows:
biotinylated RAMP2 is fixed on an SA chip by using a
BIACORE (GE Healthcare Bio-Sciences Corp., BIACORE-X100)
in accordance with a manual provided by the manufacturer
and a test antibody is then flowed therein to measure the
association rate constant Kal, and dissociation rate
constant Kdl; and the association constant value KD can
be determined by using bivalent fitting.
[0016]
The antibody of the present invention may be a
polyclonal antibody or a monoclonal antibody, and is
preferably a monoclonal antibody. The term "monoclonal
antibody" in the present invention refers to an antibody
that reacts with a single antigen determinant and has an
almost homogeneous structure. The antibody of the
present invention further encompasses non-human animal
antibodies, antibodies having an amino acid sequence
derived from a non-human animal antibody and an amino
acid sequence derived from a human-derived antibody, and
human antibodies. Examples of non-human animal
antibodies include antibodies of a mouse, a rat, a
hamster, a guinea pig, a rabbit, a dog, a monkey, a
sheep, a goat, a camel, a chicken, and a duck, in which
an antibody of an animal whose hybridomas can be produced
is preferable, and more preferably an antibody of a
mouse, a rat, or a rabbit. Examples of antibodies having
CA 03075867 2020-03-13
A - 23 -
an amino acid sequence derived from a non-human animal
antibody and an amino acid sequence derived from a human
antibody include human chimeric antibodies and humanized
antibodies. The "chimeric antibody" in this context
refers to an antibody derived from non-human animal and
specifically binds to RAMP2 in which the constant region
of the antibody has been modified to have the same
constant region as a human antibody by gene engineering,
and is preferably a human/mouse chimeric antibody (see
European Patent Publication No. EP0125023). The
"humanized antibody" refers to an antibody derived from a
non-human animal and specifically binds to RA4P2 in which
the primary structure excluding complementarity-
determining regions (CDRs) of H chains and L chains has
been modified to have the primary structure corresponding
to that of a human antibody by gene engineering. CDRs
may be defined by Kabat et al. ("Sequences of Proteins of
Immunological Interest", Kabat, E. et al., U. S.
Department of Health and Human Services, 1983) or by
Chothia et al. (Chothia & Lesk (1987) J. Mol. Biol., 196:
901-917). The "human antibody" refers to a human
antibody which is an expression product of a completely
human origin antibody gene, for example, monoclonal
antibodies produced by a transgenic animal introduced
with human genes involved in antibody production (see
European Patent Publication No. EP0546073). In using the
antibody of the present invention for therapy,
CA 03075867 2020-03-13
- 24 -
prevention, or diagnosis to be administered into the
body, the antibody of the present invention is preferably
a human/non-human animal chimeric antibody, a humanized
antibody, or a human antibody. Preferably, the antibody
of the present invention is a non-naturally occurring
antibody.
[0017]
The immunoglobulin class of the antibody of the
present invention is not limited and may be any
immunoglobulin class (isotype) of IgG, IgM, IgA, IgE,
IgD, and IgY, and is preferably IgG. When being IgG, the
antibody of the present invention may be of any subclass
(IgGl, IgG2, IgG3, or IgG4). The antibody of the present
invention may be monospecific, bispecific (bispecific
antibody), trispecific (trispecific antibody) (e.g.,
W01991/003493), or multispecific (multispecific
antibody).
[0018]
It is known that the variable regions (in
particular, CDRs) contribute binding properties in
antibody, and it is widely known to those skilled in the
art that binding properties are available even in an
antibody fragment, which is not a complete antibody. The
term "immunoreactive fragment" herein refers to a protein
or peptide including a part of an antibody (partial
fragment) that retains the activity of the antibody on an
antigen (immunoreactivity, binding ability). Examples of
CA 03075867 2020-03-13
- 25 -
such immunoreactive fragments include F(abi)2, Fab', Fab,
Fab3, single chain Fv (hereinafter, referred to as
"scFv"), (tandem) bispecific single chain Fv (sc(Fv)2),
single chain triplebodies, nanobodies, divalent VHH,
pentavalent VHH, minibodies, (double chain) diabodies,
tandem diabodies, bispecific tribodies, bispecific
bibodies, dual-affinity retargeting molecules (DART),
triabodies (or tribodies), tetrabodies (or [sc(Fv)2]2),
or (scFv-SA)4 disulfide-stabilized Fv (hereinafter,
referred to as "dsFv"), compact IgG, heavy chain
antibodies, and polymers of them (see Nature
Biotechnology, 29(1): 5-6 (2011); Maneesh Jain et al.,
TRENDS in Biotechnology, 25(7) (2007): 307-316; and,
Christoph stein et al., Antibodies (1): 88-123 (2012)).
The immunoreactive fragment herein may be monospecific,
bispecific, trispecific, and multispecific. The term
"antibody" herein is intended to encompass immunoreactive
fragments of the antibody, except in the case that such
interpretation leads to inconsistency.
[0019]
In one aspect, the present invention relates to a
25H4-4F9 antibody, a 25H4-4F9v1 antibody, a 25H4-4F9v2
antibody, a 25H4-4F9v3 antibody, a 25H4-4F9v4 antibody, a
25H4-4F9v5 antibody, a 25H4-4F9v6 antibody, a 25H4-4F9v8
antibody, an 85H7-2B11 antibody, an 85H7-2B11v1 antibody,
an 85H7-2B11v2 antibody, an 85H7-2B11v3 antibody, an
85H7-2B11v4 antibody, an 85H7-2B11v5 antibody, an 85H7-
CA 03075867 2020-03-13
- 26 -
2B11v6 antibody, an 85H7-2B11v7 antibody, an 85H7-2B11v8
antibody, or a 33H4-1G3 antibody. These antibodies each
have a heavy chain (HC) and light chain (LC) consisting
of amino acid sequences of sequence numbers listed in
Table 2.
[0020]
In one aspect, the antibody of the present invention
specifically recognizes the conformation of the
extracellular region of cell surface RAMP2. For example,
the antibody of the present invention specifically
recognizes an possible epitope in cell surface RAMP2 to
which the 25H4-4F9 antibody, the 25H4-4F9v1 antibody, the
25H4-4F9v2 antibody, the 25H4-4F9v3 antibody, the 25H4-
4F9v4 antibody, the 25H4-4F9v5 antibody, the 25H4-4F9v6
antibody, the 25H4-4F9v8 antibody, the 85H7-2B11
antibody, the 85H7-2B11v1 antibody, the 85H7-2B11v2
antibody, the 85H7-2B11v3 antibody, the 85H7-2B11v4
antibody, the 85H7-2B11v5 antibody, the 85H7-2B11v6
antibody, the 85H7-2B11v7 antibody, the 85H7-2B11v8
antibody, or the 33H4-1G3 antibody binds.
[0021]
The antibody of the present invention does not
recognize the primary structure of an amino acid sequence
that constitutes the extracellular region of RAMP2 as an
epitope. Here, the phrase "does not recognize the
primary structure of an amino acid sequence as an
epitope" means that the antibody of the present invention
CA 03075867 2020-03-13
- 27 -
does not bind to a linear peptide or protein of an amino
acid sequence which is not folded (not forming a
conformation). More specifically, the antibody of the
present invention does not bind to any peptide having an
amino acid sequence of SEQ ID NOs: 71 to 101 which
constitutes the extracellular region of RAMP2.
[0022]
Herein, whether a test antibody "specifically binds
to (recognizes)" an epitope (e.g., an epitope to which
any of the above specific antibodies binds), antigen, or
peptide of interest can be determined by assaying for
binding of the test antibody to not only a peptide or
protein having the epitope, the antigen, or the peptide,
but to other peptides or proteins, or other antigens. In
particular, the antibody of the present invention
specifically recognizes the conformation of the
extracellular region of cell surface RAMP2, and thus the
binding specificity of the test antibody can be
determined by using RAMP2 expressed on cell surfaces as a
protein having the epitope in the aforementioned method.
If the test antibody binds to a peptide or protein having
an epitope of interest, an antigen of interest, or a
peptide of interest, and does not bind to other peptides
or proteins, or other antigens, the test antibody is
determined to specifically bind to (recognize) the
epitope, antigen, or peptide of interest.
[0023]
CA 03075867 2020-03-13
- 28 -
Herein, a test antibody can be determined not bind
to a specific peptide or protein by assaying binding of
the antibody to the peptide or protein.
[0024]
In another aspect, the antibody of the present
invention competes for binding to RA14P2 (in particular,
cell surface RAMP2) with the 25H4-4F9 antibody, the 25H4-
4F9v1 antibody, the 25H4-4F9v2 antibody, the 25H4-4F9v3
antibody, the 25H4-4F9v4 antibody, the 25H4-4F9v5
antibody, the 25H4-4F9v6 antibody, the 25H4-4F9v8
antibody, the 85H7-2B11 antibody, the 85H7-2B11v1
antibody, the 85H7-2B11v2 antibody, the 85H7-2B11v3
antibody, the 85H7-2B11v4 antibody, the 85H7-2B11v5
antibody, the 85H7-2B11v6 antibody, the 85H7-2B11v7
antibody, the 85H7-2B11v8 antibody, or the 33H4-1G3
antibody (in the present paragraph, referred to as "25H4-
4F9 antibody or the like"). Whether the test antibody
competes with the 25H4-4F9 antibody or the like for
binding to RAMP2 can be determined by placing the test
antibody and a labeled 25H4-4F9 antibody or the like in
contact with the antigen (RAMP2). Preferably, the
labeled 25H4-4F9 antibody or the like without the test
antibody is placed in contact with the antigen (RAMP2) as
a control. Amounts of a label of the labeled 25H4-4F9
antibody or the like bound to the antigen are compared.
The test antibody can be determined as competing with the
25H4-4F9 antibody or the like, if the binding amount of
. 7 CA 03075867 2020-03-13
- 29 -
the labeled 25H4-4F9 antibody or the like to the antigen
in a sample placing the labeled 25H4-4F9 antibody or the
like and test antibody with the antigen is lower than
that in a sample placing the labeled 25H4-4F9 antibody or
the like with the antigen in the absence of the test
antibody
[0025]
A wide variety of methods for measuring binding
between an antibody and a protein or peptide are well
known in the art, such as an EIA, an ELISA, an FACS
method, a coimmunoprecipitation, a pull-down assay, a
far-Western blotting, a crosslinking using a homo-
bifunctional crosslinker or a hetero-bifunctional
crosslinker, a label transfer reaction, an interaction
mapping, a surface plasmon resonance method, an FRET
(Fluorescence resonance energy transfer) method, a
BIACORE method, and an AlphaPPI assay (PerkinElmer) such
as AlphaScreen (R) and AlphaLISA (R), and a preferred
method for detection of binding of interest can be
appropriately employed.
[0026]
The present invention relates to, in one aspect, an
antibody or an immunoreactive fragment thereof in which
VH has an amino acid sequence encoded by a nucleic acid
sequence that can hybridize with a nucleic acid sequence
encoding an amino acid sequence of any one selected from
r CA 03075867 2020-03-13
- 30 -
SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49,
51, and 53 under stringent conditions, and VL has an
amino acid sequence encoded by a nucleic acid sequence
that can hybridize with a nucleic acid sequence encoding
an amino acid sequence of any one selected from SEQ ID
NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61 under
stringent conditions. The phrase "can hybridize under
stringent conditions" herein means being able to
hybridize under hybridization conditions commonly used by
those skilled in the art. Hybridization can be achieved,
for example, by using the method described in Molecular
Cloning, A Laboratory Manual, Second Edition, Cold Spring
Harbor Laboratory Press (1989). The hybridization may be
performed under conditions, for example, at 42 C in 6 x
SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 x SSPE (3
M NaCl, 0.2 M NaH2PO4, 20 mM EDT.A2Na, pH 7.4), followed
by washing with 0.5 x SSC at 42 C. Preferably, the
present invention is an antibody or an immunoreactive
fragment thereof in which VH has an amino acid sequence
encoded by a nucleic acid sequence that can hybridize
with a nucleic acid sequence encoding an amino acid
sequence of SEQ ID NO: 2, 12, 19, 27, 29, 31, 33, 35, 45,
47, 49, 51, or 53 under stringent conditions, and VL has
an amino acid sequence encoded by a nucleic acid sequence
that can hybridize with a nucleic acid sequence encoding
an amino acid sequence of SEQ ID NO: 4, 14, 21, 37, 39,
. ) CA 03075867 2020-03-13
- 31 -
41, 43, 55, 57, 59, or 61 under stringent conditions,
correspondingly in the order presented.
[0027]
Further, the present invention relates to an
antibody or an immunoreactive fragment thereof in which
VH has an amino acid sequence with identity of 80% or
more to any one amino acid sequence selected from SEQ ID
NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and
53, and VL has an amino acid sequence with identity of
80% or more to any one amino acid sequence selected from
SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and
61. Identity of an amino acid sequence refers to the
ratio (%) of the number of identical amino acids between
two proteins in an range of an amino acid sequence to be
compared, and can be determined, for example, by using a
known program such as BLAST and FASTA. The antibody of
the present invention may be, for example, an antibody or
an immunoreactive fragment thereof in which VH has an
amino acid sequence with identity of 85% or more, 90% or
more, 95% or more, 98% or more, or 99% or more to any one
amino acid sequence selected from SEQ ID NOs: 2, 12, 19,
27, 29, 31, 33, 35, 45, 47, 49, 51, and 53, and VL has an
amino acid sequence with identity of 85% or more, 90% or
more, 95% or more, 98% or more, or 99% or more to any one
amino acid sequence selected from SEQ ID NOs: 4, 14, 21,
37, 39, 41, 43, 55, 57, 59, and 61. Preferably, the
antibody of the present invention is an antibody or an
0
. CA 03075867 2020-03-13
- 32 -
immunoreactive fragment thereof in which VH has an amino
acid sequence with identity of 80% or more, 85% or more,
90% or more, 95% or more, 98% or more, or 99% or more to
any one amino acid sequence selected from SEQ ID NOs: 2,
12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53, and
VL has an amino acid sequence with identity of 80% or
more, 85% or more, 90% or more, 95% or more, 98% or more,
or 99% or more to any one amino acid sequence selected
from SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59,
and 61, respectively.
[0028]
The present invention is preferably an antibody
having a combination of amino acid sequences of a heavy
chain (VC) and light chain (LC), or of VH and VL, or of
CDRH1 to 3 and CDRL1 to 3, comprising amino acid
sequences of SEQ ID NOs in Table 2.
[0029]
Preferably, the above described antibodies or
immunoreactive fragments thereof in which VH and VL have
amino acid sequences encoded by nucleic acid sequences
that can hybridize with nucleic acids encoding specific
amino acid sequences under stringent conditions, and in
which VH and VL have amino acid sequences with identity
of 80% or more to specific amino acid sequences have
CDRHs and CDRLs described in below.
[0030]
= CA 03075867 2020-03-13
- 33 -
The present invention relates to, in one aspect, an
antibody or immunoreactive fragment thereof comprising a
heavy chain comprising heavy chain complementarity-
determining region (CDRH) 3 consisting of the amino acid
sequence of LX1IX2DX3YX4YX5MX6X7 (SEQ ID NO: 24). In the
sequence, Xi is G or M, X2 is F or Y, X3 is A or V, X4 is
absent or H, X5 is A or V. X6 is D or E, and X7 is Y or N.
Preferably, the present invention is an antibody or
immunoreactive fragment thereof comprising a heavy chain
comprising CDRH3 consisting of the amino acid sequence of
LMIFDAYYAMDY (SEQ ID NO: 7) or LGIYDVYHYVMEN (SEQ ID NO:
16). It is already known that CDRH3 is important for
determination of the specificity of an antibody (Xu JL et
al., Immunity. 2000 Jul; 13(1): 37-45.). CDRH3
preferably has the amino acid sequence of LMIFDAYYAMDY
(SEQ ID NO: 7) or LGIYDVYHYVMEN (SEQ ID NO: 16).
[0031]
Preferably, the antibody or immunoreactive fragment
thereof of the present invention has CDRH1 consisting of
the amino acid sequence of GYFMN (SEQ ID NO: 5).
Further, the antibody or immunoreactive fragment
thereof of the present invention preferably has CDRH2
consisting of the amino acid sequence of
RX8NPYNGDX9X10YX11QKFX12G (SEQ ID NO: 66) or
RX8NPYNGDX9X1DYNQKFKG (SEQ ID NO: 23). In the sequences,
X8 is N or I, X9 is S or T, Xn is I, F, or L, X11 is A or
N, and X12 is Q or K. CDRH2 preferably has the amino acid
= CA 03075867 2020-03-13
- 34 -
sequence of RNNPYNGDSIYNQKFKG (SEQ ID NO: 6),
RINPYNGDTFYNQKFKG (SEQ ID NO: 15), RINPYNGDTLYNQKFKG (SEQ
ID NO: 22), RNNPYNGDSIYNEKFQG (SEQ ID NO: 62),
RNNPYNGDSIYAEKFQG (SEQ ID NO: 63), RINPYNGDTFYNQKFQG (SEQ
ID NO: 64), or RINPYNGDTFYAQKFQG (SEQ ID NO: 65).
[0032]
More preferably, the antibody or immunoreactive
fragment thereof of the present invention has light chain
complementarity-determining region (CDRL) 1 consisting of
the amino acid sequence of XnASQDIRNYLN (SEQ ID NO: 69).
In the sequence, Xn is Q or R. CDRL1 preferably has the
amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8) or
QASQDIRNYLN (SEQ ID NO: 67).
[0033]
The antibody or immunoreactive fragment thereof of
the present invention has CDRL2 consisting of the amino
acid sequence of YT5RLX14X15 (SEQ ID NO: 70). In the
sequence, X1.4 is E or H and X15 is S or T. CDRL2
preferably has the amino acid sequence of YTSRLHS (SEQ ID
NO: 9) or YTSRLET (SEQ ID NO: 68).
[0034]
The antibody or immunoreactive fragment thereof of
the present invention has CDRL3 consisting of the amino
acid sequence of QQDSKX16PWT (SEQ ID NO: 25). In the
sequence, X16 is H or N. CDRL3 preferably has the amino
acid sequence of QQDSKHPWT (SEQ ID NO: 10) or QQDSKNPWT
(SEQ ID NO: 17).
` ^ CA 03075867 2020-03-13
- 35 -
[0035]
In an example, the antibody of the present invention
has any one combination of heavy chain CDRs of the
following (i) to (vii):
(i) CDRH1 consisting of the amino acid sequence of
SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence
of SEQ ID NO: 6, and CDRH3 consisting of the amino acid
sequence of SEQ ID NO: 7;
(ii) CDRH1 consisting of the amino acid sequence of
SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence
of SEQ ID NO: 62, and CDRH3 consisting of the amino acid
sequence of SEQ ID NO: 7;
(iii) CDRH1 consisting of the amino acid sequence of
SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence
of SEQ ID NO: 63, and CDRH3 consisting of the amino acid
sequence of SEQ ID NO: 7;
(iv) CDRH1 consisting of the amino acid sequence of
SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence
of SEQ ID NO: 15, and CDRH3 consisting of the amino acid
sequence of SEQ ID NO: 16;
(v) CDRH1 consisting of the amino acid sequence of
SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence
of SEQ ID NO: 64, and CDRH3 consisting of the amino acid
sequence of SEQ ID NO: 16;
(vi) CDRH1 consisting of the amino acid sequence of
SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence
CA 03075867 2020-03-13
- 36 -
of SEQ ID NO: 65, and CDRH3 consisting of the amino acid
sequence of SEQ ID NO: 16; and
(vii) CDRH1 consisting of the amino acid sequence of
SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence
of RINPYNGDTLYNQKFKG (SEQ ID NO: 22), and CDRH3
consisting of the amino acid sequence of SEQ ID NO: 16.
[0036]
The antibody of the present invention may further
have any one combination of light chain CDRs of the
following (a) to (f):
(a) CDRL1 consisting of the amino acid sequence of
SEQ ID NO: 8, CDRL2 consisting of the amino acid sequence
of SEQ ID NO: 9, and CDRL3 consisting of the amino acid
sequence of SEQ ID NO: 10;
(b) CDRL1 consisting of the amino acid sequence of
QASQDIRNYLN (SEQ ID NO: 67), CDRL2 consisting of the
amino acid sequence of SEQ ID NO: 9, and CDRL3 consisting
of the amino acid sequence of SEQ ID NO: 10;
(c) CDRL1 consisting of the amino acid sequence of
SEQ ID NO: 67, CDRL2 consisting of the amino acid
sequence of SEQ ID NO: 68, and CDRL3 consisting of the
amino acid sequence of SEQ ID NO: 10;
(d) CDRL1 consisting of the amino acid sequence of
SEQ ID NO: 8, CDRL2 consisting of the amino acid sequence
of SEQ ID NO: 9, and CDRL3 consisting of the amino acid
sequence of SEQ ID NO: 17;
. CA 03075867 2020-03-13
- 37 -
(e) CDRL1 consisting of the amino acid sequence of
SEQ ID NO: 67, CDRL2 consisting of the amino acid
sequence of SEQ ID NO: 9, and CDRL3 consisting of the
amino acid sequence of SEQ ID NO: 17; and
(f) CDRL1 consisting of the amino acid sequence of
SEQ ID NO: 67, CDRL2 consisting of the amino acid
sequence of SEQ ID NO: 68, and CDRL3 consisting of the
amino acid sequence of SEQ ID NO: 17.
[0037]
For example, the present invention includes an
antibody or immunoreactive fragment thereof having any of
the following combinations of CDRs.
[0038]
[Table 1]
1M N3IN 9>IJAON
dN>isa00
LI SH12:IS1A 6 N1AN2:11CIOSVd AAHAAGA191 .. A1109NAdN12:1 8 .. 91 ..
ZZ .. NV\HAD 9
1M Er
13-TdS1A 89 N L9 N]lAl
91 DOdAbv 99 NIALAA0 9
dN>iSGOO 1ANdIGOSVO AAHAACIAI91
A.J109NAdN18
1M N NIA) obd>ibv
LI SH1eIS1A 6 L9 9T 99 NIAIJA9 9
cINASObb lANHIGOSVb AAHAAGAI91
AJ1CIONAdNI8
,
N3IAJ 90JAON
d
t79 NIA1dA9 9
' dNNSGOO LI SH1S1A 6 lAN2 L9 :JIGOSVO
AAHAAGAI91 91 AJ109NAdN18,
,I, 1 1M NBA
90d>iON
,s, LT SH-IdS1A 6
N1AN2:11GOSV2:1 8 91 179 NIAJAAO 9
,cs'i oo c11\1>iSCIOCt
AAHAAGAI91 AA1G9NAdNld
N m
. 1M N3IAI
OXON
w
01 dNASCIOO LT
SH12:1S1A 6 N1ANdICIOSVI 8 91 91 NIAIAA9 S
N
AAHAAGAI91 AJ109NAdN18
,,,
o 1M N
AG 90dA3VA
6 dHNSGOO 01 1]-12:1S1A 89
1ANdICIOSVO L9
LAIVAAVaillAil L
ISCISNAcINNH S9 NV\IdA9 S
IAA OI SH12:1S1A 6 N
L9 AG L 90d>13VA
9 NIAJJA9 9
dHASGOO lAN2:11GOSVO AVAAVGJIIAll
ISGONAdNN2J
1M ,
L SH1eIS1A 6 N
L9 AG L SOJN3NA
Z9 NIAIJA9 9
dH>ISCIOO 0 1AN2:110OSVO
IAIVAAVGJIIAll ISCIONAdNNH
IM 01 SH12:IS1A 6 1\11ANIIGOSVI 8 AG L ObJ>13NA
Z9 NIAIAA9 9
dH>ISCIOb INVAAVGJIIAll
ISCI9NAdNNei
,
IM L OT SH1dS1A 6 NiANdIGOSV2:1 8 AG
9>LOONA
9 NVUAO 9
dH>ISGOO lAIVAAVCHItAll
ISCIDNAdNN2:1
ON 01 ON GI ON al ON 01
ON GI ON GI
eauanbas epuenbas ananbas aouanbas
nuenbas aouanbas
O3S OBS 03s 03s
OBS Ins
E18G3 Z12:IGD 112:100
H271G3 ZI-IdGO IH800
,
A
CA 03075867 2020-03-13
- 39 -
[0039]
Herein, amino acids are described by the single-
letter code. Specifically, A denotes alanine, L denotes
leucine, R denotes arginine, K denotes lysine, N denotes
asparagine, M denotes methionine, D denotes aspartic
acid, F denotes phenylalanine, C denotes cysteine, P
denotes proline, Q denotes glutamine, S denotes serine, E
denotes glutamic acid, T denotes threonine, G denotes
glycine, W denotes tryptophan, H denotes histidine, Y
denotes tyrosine, I denotes isoleucine, and V denotes
valine.
[0040]
The antibody and immunoreactive fragment thereof of
the present invention have an inhibitory activity on
vascular endothelial cell growth in an in vitro test for
vascular endothelial cell growth induced by
adrenomedullin supplementation. To determine whether a
test antibody or immunoreactive fragment thereof has an
inhibitory activity on vascular endothelial cell growth
in an in vitro test for vascular endothelial cell growth
induced by adrenomedullin supplementation, the test
antibody or immunoreactive fragment thereof is added to
vascular endothelial cells which have been cultured in an
appropriate medium for about several hours, to which AM
is added to a final concentration of 50 to 200 nM,
followed by culturing for 2 to 7 days, and then the
growth of the vascular endothelial cells is observed.
CA 03075867 2020-03-13
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Determination of the growth of vascular endothelial cells
may be performed by counting the number of cells, or by
measuring, for example, 0D450 as an indicator of the
number of cells. If the number of vascular endothelial
cells cultured in the presence of the test antibody or
immunoreactive fragment thereof is smaller than that of
vascular endothelial cells cultured without the test
antibody or immunoreactive fragment thereof, the test
antibody or immunoreactive fragment thereof is determined
to have an inhibitory activity on the cell growth in an
in vitro test for vascular endothelial cell growth
induced by adrenomedullin supplementation. In addition,
the 1050 value (M) may be determined as the concentration
of the test antibody or immunoreactive fragment thereof
that brings 50% of the number of vascular endothelial
cells cultured in the presence of the test antibody or
immunoreactive fragment thereof, wherein the number of
vascular endothelial cells cultured without the test
antibody or immunoreactive fragment thereof set to as
100%. For example, the 1050 value of the antibody of the
present invention may be 35 nM or less, 30 nM or less, 25
nM or less, 20 nM or less, 15 nM or less, 10 nM or less,
nM or less, 4 nM or less, 3 nM or less, 2 nM or less,
or 1.6 nM or less. The lower limit of the 1050 value is
not needed to be specified, but may be, for example, 0.1
nM or more, 0.2 nM or more, 0.3 nM or more, 0.4 nM or
more, 0.5 nM or more, 0.6 nM or more, 0.7 nM or more, or
* CA 03075867 2020-03-13
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0.8 nM or more. The IC50 value may be within a range of
any combination of the upper limit values and lower limit
values, and may be, for example, 0.1 to 35 nM, 0.8 to
32.7 nM, 0.2 to 30 nM, 0.3 to 25 nM, 0.4 to 20 nM, 0.5 to
15 nM, 0.5 to 10 nM, 0.5 to 5 nM, or 0.8 to 1.6 nM. If
the IC50 value is any of these, the test antibody or
immunoreactive fragment thereof may be determined to have
an inhibitory activity on vascular endothelial cell
growth in an in vitro test for vascular endothelial cell
growth induced by adrenomedullin supplementation.
[0041]
Preferably, the antibody or immunoreactive fragment
thereof of the present invention not only has an
inhibitory activity on vascular endothelial cell growth
in an in vitro test for vascular endothelial cell growth
induced by adrenomedullin supplementation, but also has
an inhibitory activity on cAMP production induced by
adrenomedullin supplementation in animal cells. To
determine whether a test antibody or immunoreactive
fragment thereof has an inhibitory activity on cAMP
production induced by adrenomedullin supplementation in
animal cells, animal cells that produce cAMP upon
supplementation with adrenomedullin (e.g., hCR2-CHOK1
cells) are cultured in an appropriate medium overnight,
the test antibody or immunoreactive fragment thereof is
added thereto, AM is added thereto to a final
concentration of 50 to 300 pM, and the cAMP level is
CA 03075867 2020-03-13
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measured, cAMP levels can be measured by using a
commercially available kit (e.g., LANCE Ultra cAMP kit
(PerkinElmer, Inc.)). If cAMP production by cells with
addition of a test antibody or immunoreactive fragment
thereof is less than cAMP production by cells without
addition of the antibody, the antibody or immunoreactive
fragment thereof is determined to have an inhibitory
activity on cAMP production induced by adrenomedullin
supplementation in animal cells. In addition, a
concentration of the test antibody or immunoreactive
fragment thereof at which the amount of cAMP produced by
cells cultured in the presence of the test antibody or
immunoreactive fragment thereof reaches 50 may be
determined as the IC50 value (M), with the amount of cAMP
produced by cells cultured without addition of the test
antibody or immunoreactive fragment thereof set to 100.
For example, the IC50 value of the antibody of the
present invention may be 2 nM or lower, 1.5 nM or lower,
1 nM or lower, 0.5 nM or lower, 0.4 nM or lower, 0.3 nM
or lower, 0.2 nM or lower, or 0.15 nM or lower. The
lower limit value of the IC50 value is not needed to be
specified, but may be, for example, 0.01 nM or higher,
0.02 nM or higher, or 0.03 nM or higher. The IC50 value
may be within a range of any combination of those upper
limit values and lower limit values, and may be, for
example, 0.01 to 2 nM, 0.01 to 1.5 nM, 0.01 to 1 nM, 0.01
to 0.5 nM, 0.03 to 1.35 nM, or 0.03 to 0.15 nM. When the
. 0
CA 03075867 2020-03-13
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I050 value is any of them, the test antibody or
immunoreactive fragment thereof may be determined to have
an inhibitory activity on cAMP production induced by
adrenomedullin supplementation in animal cells.
[0042]
Preferably, the antibody or immunoreactive fragment
thereof of the present invention binds to cells
coexpressing human CRLR and human RAMP2 in vitro. "Cells
coexpressing human CRLR and human RAMP2" herein are not
limited as long as it is stable expression cells obtained
by transfecting cells with human CRLR and human RAMP2 and
coexpresses human CRLR and human RAMP2, and are
preferably a stable cell line obtained by transfecting
hamster ovary cells (CHO-K1) or NIH3T3 cells with human
CRLR and human RAMP2, named herein as hCR2-CHOK1 or hCR2-
NIH3T3, respectively. Cells coexpressing human CRLR and
human RAMP2 can be obtained by transfecting cells of
interest with a transfection vector which has been
introduced tandemly arranged human CRLR gene (NM 005795)
and human RAMP2 (NM 005854). A stable expression cell
_
line can be obtained by culturing the cells in selective
culture containing a drug which corresponds to a drug
resistance gene inserted in a vector.
[0043]
For example, the binding affinity (EC50) of the
antibody of the present invention determined by a Cell
ELISA test using cells coexpressing human CRLR and human
=
CA 03075867 2020-03-13
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RA14P2 may be 3.5 nM or lower. In case that the
immunoglobulin class of a test antibody is IgG, for
example, a Cell ELISA test can be performed in the
following manner. Cells coexpressing human CRLR and
human RAMP2 suspended in an appropriate medium are added
to a 96-well culture plate at 1 x 103 to 1 x 105
cells/well and cultured overnight, to which 4%
paraformaldehyde solution is added, and then the cells
are fixed by incubating for several tens of minutes at
4 C, which are washed and blocked with 1% BSA-PBS for 1
hour or longer. Thereto, a purified test antibody
appropriately serially diluted is added, which is reacted
with a labeled anti-IgG antibody as a secondary antibody,
and then reacted with a reagent corresponding to the
label, and the luminescence intensity or coloring
intensity emitted from the label is measured to determine
the binding amount. The EC50 value can be determined as
an antibody concentration in which the antibody binds50%
of the maximum binding amount. For example, the EC50
value of the antibody of the present invention may be 4
nM or less, 3.5 nM or less, 3.19 nM or less, 3 nM or
less, 2.5 nM or less, 2 nM or less, 1.5 nM or less, 1 nM
or less, or 0.5 nM or less. The lower limit value of the
EC50 value is not needed to be specified, but may be, for
example, 0.01 nM or more, 0.03 nM or more, 0.05 nM or
more, or 0.14 nM or more. The EC50 value may be within a
range of any combination of the upper limit values and
,
CA 03075867 2020-03-13
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lower limit values, and may be, for example, 0.01 to 4
nM, 0.03 to 3 nM, 0.05 to 2 nM, or 0.14 to 3.19 nM. If
the EC50 value is any of them, the test antibody or
immunoreactive fragment thereof may be determined to bind
to cells coexpressing human CRLR and human RAMP2 in
vitro.
[0044]
(Nucleic Acid Molecule)
In another aspect, the present invention relates to
a nucleic acid molecule having a polynucleotide encoding
the above-described antibody or immunoreactive fragment
thereof of the present invention. Specifically, the
nucleic acid molecule of the present invention may be a
nucleic acid molecule having a polynucleotide encoding
any one amino acid sequence selected from SEQ ID NOs: 2,
12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53 as VH,
and a polynucleotide encoding any one amino acid sequence
selected from SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55,
57, 59, and 61 as VL. For example, the nucleic acid
molecule of the present invention may be: a nucleic acid
having polynucleotides encoding the amino acid sequence
of SEQ ID NO: 2 as VH and the amino acid sequence of SEQ
ID NO: 4 as VL; a nucleic acid having polynucleotides
encoding the amino acid sequence of SEQ ID NO: 27 as VH
and the amino acid sequence of SEQ ID NO: 37 as VL; a
nucleic acid having polynucleotides encoding the amino
acid sequence of SEQ ID NO: 29 as VH and the amino acid
CA 03075867 2020-03-13
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sequence of SEQ ID NO: 37 as VL; a nucleic acid having
polynucleotides encoding the amino acid sequence of SEQ
ID NO: 29 as VH and the amino acid sequence of SEQ ID NO:
39 as VL; a nucleic acid having polynucleotides encoding
the amino acid sequence of SEQ ID NO: 31 as VH and the
amino acid sequence of SEQ ID NO: 39 as VL; a nucleic
acid having polynucleotides encoding the amino acid
sequence of SEQ ID NO: 31 as VH and the amino acid
sequence of SEQ ID NO: 41 as VL; a nucleic acid having
polynucleotides encoding the amino acid sequence of SEQ
ID NO: 33 as VH and the amino acid sequence of SEQ ID NO:
41 as VL; a nucleic acid having polynucleotides encoding
the amino acid sequence of SEQ ID NO: 35 as VH and the
amino acid sequence of SEQ ID NO: 43 as VL; a nucleic
acid having polynucleotides encoding the amino acid
sequence of SEQ ID NO: 12 as VH and the amino acid
sequence of SEQ ID NO: 14 as VL; a nucleic acid having
polynucleotides encoding the amino acid sequence of SEQ
ID NO: 45 as VH and the amino acid sequence of SEQ ID NO:
55 as VL; a nucleic acid having polynucleotides encoding
the amino acid sequence of SEQ ID NO: 12 as VH and the
amino acid sequence of SEQ ID NO: 14 as VL; a nucleic
acid comprising polynucleotides encoding the amino acid
sequence of SEQ ID NO: 47 as VH and the amino acid
sequence of SEQ ID NO: 55 as VL; a nucleic acid having
polynucleotides encoding the amino acid sequence of SEQ
ID NO: 47 as VH and the amino acid sequence of SEQ ID NO:
CA 03075867 2020-03-13
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57 as VL; a nucleic acid having polynucleotides encoding
the amino acid sequence of SEQ ID NO: 49 as VH and the
amino acid sequence of SEQ ID NO: 57 as VL; a nucleic
acid having polynucleotides encoding the amino acid
sequence of SEQ ID NO: 49 as VH and the amino acid
sequence of SEQ ID NO: 59 as VL; a nucleic acid having
polynucleotides encoding the amino acid sequence of SEQ
ID NO: 51 as VH and the amino acid sequence of SEQ ID NO:
59 as VL; a nucleic acid having polynucleotides encoding
the amino acid sequence of SEQ ID NO: 51 as VH and the
amino acid sequence of SEQ ID NO: 61 as VL; a nucleic
acid having polynucleotides encoding the amino acid
sequence of SEQ ID NO: 53 as VH and the amino acid
sequence of SEQ ID NO: 61 as VL; or a nucleic acid having
polynucleotides encoding the amino acid sequence of SEQ
ID NO: 19 as VH and the amino acid sequence of SEQ ID NO:
21 as VL.
[0045]
For example, the nucleic acid molecule of the
present invention may include a polynucleotide having any
of the following sequences: the nucleotide sequence of
SEQ ID NO: 1, and the nucleotide sequence of SEQ ID NO:
3; the nucleotide sequence of SEQ ID NO: 26, and the
nucleotide sequence of SEQ ID NO: 36; the nucleotide
sequence of SEQ ID NO: 28, and the nucleotide sequence of
SEQ ID NO: 36; the nucleotide sequence of SEQ ID NO: 28,
and the nucleotide sequence of SEQ ID NO: 38; the
CA 03075867 2020-03-13
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nucleotide sequence of SEQ ID NO: 30, and the nucleotide
sequence of SEQ ID NO: 38; the nucleotide sequence of SEQ
ID NO: 30, and the nucleotide sequence of SEQ ID NO: 40;
the nucleotide sequence of SEQ ID NO: 32, and the
nucleotide sequence of SEQ ID NO: 40; the nucleotide
sequence of SEQ ID NO: 34, and the nucleotide sequence of
SEQ ID NO: 42; the nucleotide sequence of SEQ ID NO: 11,
and the nucleotide sequence of SEQ ID NO: 13; the
nucleotide sequence of SEQ ID NO: 44, and the nucleotide
sequence of SEQ ID NO: 54; the nucleotide sequence of SEQ
ID NO: 46, and the nucleotide sequence of SEQ ID NO: 54;
the nucleotide sequence of SEQ ID NO: 46, and the
nucleotide sequence of SEQ ID NO: 56; the nucleotide
sequence of SEQ ID NO: 48, and the nucleotide sequence of
SEQ ID NO: 56; the nucleotide sequence of SEQ ID NO: 48,
and the nucleotide sequence of SEQ ID NO: 58; the
nucleotide sequence of SEQ ID NO: 50, and the nucleotide
sequence of SEQ ID NO: 58; the nucleotide sequence of SEQ
ID NO: 50, and the nucleotide sequence of SEQ ID NO: 60;
the nucleotide sequence of SEQ ID NO: 52, and the
nucleotide sequence of SEQ ID NO: 60; and the nucleotide
sequence of SEQ ID NO: 18, and the nucleotide sequence of
SEQ ID NO: 20.
[0046]
The present invention further encompasses a vector
having the nucleic acid molecule. The vector is not
particularly limited as long as being able to use for
CA 03075867 2020-03-13
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antibody expression, and appropriately selected according
to a host to be used from such as a virus vector, plasmid
vector, or the like. In another aspect, the present
invention relates to a host cell comprising the vector.
The host cell is not particularly limited as long as
being able to be used for antibody expression, for
example, mammalian cells (e.g., mouse cells, rat cells,
rabbit cells, human cells), yeasts, and microorganisms
(e.g., Escherichia coli).
[0047]
In yet another aspect, the present invention relates
to a pharmaceutical composition containing the above
described antibody or immunoreactive fragment thereof of
the present invention as an active ingredient. Target
diseases for the pharmaceutical composition of the
present invention are diseases or disorders in which
angiogenesis contributes to the onset or exacerbation,
including cancer, cancer metastasis, diabetic
retinopathy, retinopathy of prematurity, age-related
macular degeneration, retinopathy of diabetes mellitus,
retinal vein occlusion, neovascular glaucoma,
inflammatory skin disease, rheumatoid arthritis, and
osteoarthritis. Accordingly, the pharmaceutical
composition of the present invention can be for
prevention, treatment, inhibiting progression, or
improvement of those diseases or disorders. Also, the
present invention relates to a use of the antibody or
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immunoreactive fragment thereof of the present invention
for manufacturing the above pharmaceutical composition.
[0048]
Any of oral or parenteral formulation may be
employed for the pharmaceutical composition of the
present invention as long as that can be administered to
patients. Examples of compositions for parenteral
administration include injections, nasal preparations,
suppositories, patches, and ointments. The
pharmaceutical composition of the present invention is
preferably an injection. Examples of the dosage form of
the pharmaceutical composition of the present invention
include liquids and lyophilized formulations. In using
the pharmaceutical composition of the present invention
as an injection, additives can be added, as necessary.
Examples of the additive include a solubilizer such as
propylene glycol and ethylenediamine; a buffer such as
phosphates; a tonicity agent such as sodium chloride and
glycerin; a stabilizer such as sulfites; a preservative
such as phenol; and a soothing agent such as lidocaine
(see "Iyakuhin Tenka-butsu Jiten (Encyclopedia of
Pharmaceutical Excipients)" Yakuji Nippo, Limited,
"Handbook of Pharmaceutical Excipients Fifth Edition"
APhA Publications). If the pharmaceutical composition of
the present invention is an injection, which is stored in
containers such as ampules, vials, pre-filled syringes,
pen-type injection cartridges, and drip infusion bags.
CA 03075867 2020-03-13
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[0049]
(Obtaining Antibody)
The antibody of the present invention can be made by
immunizing a non-human animal that generates
immunoreaction with cells expressing human CRLR and human
RAMP2 as an immunogen, and if necessary, with an
immunostimulant (e.g., mineral oil or an aluminum
precipitate and heat-killed cells or lipopolysaccharide;
Freund complete adjuvant; or Freund incomplete adjuvant).
The animal to be immunized is not particularly limited as
long as being able to make hybridomas, and includes a
mouse, a rat, a hamster, a guinea pig, a rabbit, a dog, a
monkey, a sheep, a goat, a chicken, and a duck, and is
preferably a mouse or a rat, more preferably a mouse, and
the most preferably an MRL/lpr mouse. Cells to be used
as an immunogen is desirably from the same species as the
animal to be immunized in order to inhibit antibody
productions against antigens other than the antigen of
interest. Administration of an immunogen to the animal
can be performed, for example, through subcutaneous
injection, intraperitoneal injection, intravenous
injection, intracutaneous injection, intramuscular
injection, or footpad injection of 1 x 106 cells in one
time or several times at appropriate intervals (typically
one immunization per 1 to 6 weeks, 2 to 10 immunizations
in total). After one to two weeks from the final
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immunization, blood is collected from the orbit or tail
vain of the immunized animal
, and the antibody titer is measured by using the serum.
The antibody of the present invention can be obtained by
purifying from the serum of an animal that exhibits a
sufficient antibody titer.
[0050]
A monoclonal antibody can be obtained from culture
of a hybridoma produced by fusing an antibody-producing
cell derived from the animal immunized by the above
method and a myeloma cell. The fusion method includes
the method proposed by Milstein et al. (Galfre, G. &
Milstein, C. (1981) Methods Enzymol., 73: 3-46).
Antibody-producing cells can be collected from the
spleen, pancreas, lymph node, or peripheral blood of a
mouse or a rat that has been immunized by the above
method and exhibits a sufficient antibody titer. For
example, the myeloma cell for use is not limited as long
as it is derived from a mammal such as a mouse, a rat, a
guinea pig, a hamster, a rabbit, and a human and can grow
in vitro. Examples of such cells include P3-X63Ag8 (X63)
(Nature, 256, 495, 1975), P3/NS1/1-Ag4-1 (NS1) (Eur. J.
Immunol., 6, 292, 1976), P3X63Ag8U1 (P3U1) (Curr. Top.
Microbiol. Immunol., 81, 1, 1978), P3X63Ag8.653 (653) (J.
Immunol., 123, 1548, 1979), Sp2/0-Ag14 (Sp2/0) (Nature,
276, 269, 1978), Sp2/0/F0-2 (F0-2) (J. Immunol. Methods,
35, 1, 1980), and SP2ab. Cells derived from the same
CA 03075867 2020-03-13
A
- 53 -
species as the antibody-producing cell are preferred, and
cells derived from the same strain as the antibody-
producing cell are more preferred.
[0051]
After culturing, the culture supernatant is
collected, and clones that bind to RAMP2 are selected
through Cell ELISA using cells coexpressing human CRLR
and human RAMP2 (e.g., hCR2-CHOK1 cells). The selected
clones are separated into single cells by repeating a
limiting dilution one to five times. Candidate
antibodies are further selected from the antibodies
produced by the separated single cell clones through Cell
ELISA, which are screened for signal-inhibitory
activityaccording to whether they inhibit the increase of
the intracellular cAMP level induced by AM
supplementation in cells coexpressing human CRLR and
human RAMP2 (e.g., hCR2-CHOK1 cells). The selected
signal-inhibitory antibodies can be screened for
inhibitory activity against the growth of HUVECs induced
by AM supplementation as a bioactivity inhibition test,
to obtain an anti-RAMP2 antibody having a cAMP inhibitory
activity and a HUVEC growth inhibiting activity.
[0052]
The antibody obtained can be purified to a
homogeneous state. Separation and purification methods
commonly used for proteins can be used for the separation
and purification of the antibody. For example, the
CA 03075867 2020-03-13
A
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antibody can be separated and purified by combining
methods appropriately selected from column chromatography
such as affinity chromatography, a filter,
ultrafiltration, salting-out, dialysis, SDS-polyacrylic
amide gel electrophoresis, isoelectric focusing, and so
on (Antibodies: A Laboratory Manual. Ed Harlow and David
Lane, Cold Spring Harbor Laboratory, 1988). Examples of
columns for affinity chromatography include a protein A
column and a protein G column. Further, an RAMP2 (or
human CRLR and human RAMP2)-immobilized column, ion-
exchange chromatography, hydrophobic interaction
chromatography, and so on can be used regardless of the
class of the antibody.
[0053]
Alternatively, an antibody that binds to RAMP2 can
be obtained, for example, by using an antibody phage
library (Tomizuka et al., Nature Genet., 15, 146-156
(1997)). The antibody phage library can be conducted,
for example, by immobilizing cells expressing human CRLR
and human RAMP2 to a solid phase, which is reacted with
the phage antibody library, followed by washing out non-
binding phages, and then bound phages are collected to
obtain desired clones (panning)
[0054]
Alternatively, an antibody that binds to RAMP2 can
be obtained as follows: designing the amino acid sequence
of an antibody of interest or immunoreactive fragment
CA 03075867 2020-03-13
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- 55 -
thereof by referring to the amino acid sequences
described herein; preparing a DNA encoding the designed
amino acid sequence which is incorporated into an
expression vector; introducing the vector into
appropriate host cells to express antibodies; and
screening the antibodies obtained for high specificity to
RAMP2 by the above-described method to obtain an antibody
that binds to RA4P2.
[0055]
(Production of Human Chimeric Antibody)
In case that the antibody of the present invention
is a human chimeric antibody, the antibody of the present
invention can be obtained as follows (Morrison, S. L. et
al., Proc. Natl. Acad. Sci. USA, 81, 6851-6855, 1984):
preparing a DNA encoding VH and VL of a non-human animal
monoclonal antibody that specifically recognizes RAMP2;
linking the DNA to a human immunoglobulin constant region
cDNA, which is incorporated into an expression vector;
and introducing the vector into appropriate host cells
for expression to obtain the antibody of the present
invention.
[0056]
(Production of Humanized Antibody)
When the antibody of the present invention is a
humanized antibody, the antibody of the present invention
can be obtained as follows (see L. Rieohmann et al.,
Nature, 332, 323, 1988: Kettleborough, C. A. et al.,
CA 03075867 2020-03-13
x ,
- 56 -
Protein Eng., 4, 773-783, 1991; Clark M., Immunol.
Today., 21, 397-402, 2000): constructing DNA encoding a V
region in which amino acid sequences of CDRs of VH and VL
of a non-human animal monoclonal antibody that
specifically recognizes RAMP2 is placed between Framework
Regions (FRs) of VH and VL of a human antibody ; linking
the constructed DNA to a human-derived immunoglobulin
constant region cDNA, which is incorporated into an
expression vector; introducing the vector into
appropriate host cells for expression to obtain the
antibody of the present invention. The CDRs of a non-
human animal monoclonal antibody can be obtained by
comparing an amino acid sequence predicted from the DNA
sequence encoding VH and VL of the non-human animal
monoclonal antibody obtained in the above-described
manner with full amino acid sequences of VHs and VLs of
known antibodies. The amino acid sequences of known
antibodies can be obtained, for example, as amino acid
sequences of antibodies registered in a database such as
Protein Data Bank. The FR of a humanized antibody is not
particularly limited as long as the assembled antibody
exhibits the effects of the present invention, and is
preferably an FR of a human antibody which brings a
variable region (hereinafter, referred to as "V region")
conformation similar to that of a CDR-derived non-human
animal monoclonal antibody to the humanized antibody , or
human antibody FRs having high identity to the amino acid
CA 03075867 2020-03-13
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- 57 -
sequence of FRs of the non-human animal monoclonal
antibody to be used. A part of the amino acids
constituting the human-derived FRs in the humanized
antibody (in particular, amino acids present at positions
sterically close to CDRs) may be substituted, as
necessary, with the amino acid in FR sequence of the non-
human animal monoclonal antibody from which CDRs are
derived (see Queen et al., U.S. Patent No. 5585089). The
DNA sequence encoding the V region of a humanized
antibody to be used is designed to be corresponding to an
amino acid sequence comprising the amino acid sequence of
CDRs of the non-human animal monoclonal antibody and the
amino acid sequence of FRs of a human antibody linked
each other. The DNA encoding the V region of a humanized
antibody can be produced from the designed DNA sequence
by using a method well known by those skilled in the art.
[0057]
(Human Antibody)
A human antibody can be obtained, for example, by
using a human antibody phage library or a human antibody-
producing transgenic mouse (Tomizuka et al., Nature
Genet., 15, 146-156 (1997)). A desired clone can be
obtained using a human antibody phage library, for
example, by immobilizing cells expressing human CRLR and
human RAMP2on a solid phase, which is reacted with a
phage antibody library, followed by washing out not-
binding phages, and then collecting bound
CA 03075867 2020-03-13
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- 58 -
phages(panning). A human antibody-producing transgenic
mouse is a mouse obtained by introducing a human antibody
immunoglobulin (Ig) gene into an endogenous Ig gene-
knocked out mouse. A human antibody that specifically
recognizes RAMP2 can be obtained by immunizing the human
antibody-producing transgenic mouse with an antigen
(preferably, cells expressing human CRLR and human RAMP2)
by using the above-described method for generating the
antibody of the present invention.
[0058]
(Nucleic Acid, Vector, and Host Cell)
The nucleic acid of the present invention can be
obtained by cloning from an antibody-producing hybridoma
obtained in the above, or by appropriately designing a
nucleic acid sequence from the amino acid sequence of an
antibody or immunoreactive fragment thereof obtained in
the above. The vector of the present invention can be
obtained by appropriately incorporating the nucleic acid
obtained into a vector suitable for expression. The
vector of the present invention may comprise regions
needed for expression (a promoter, an enhancer, a
terminator, and so on) in addition to the nucleic acid of
the present invention. The host cell of the present
invention can be obtained by introducing the vector of
the present invention into a suitable cell line (e.g., an
animal cell, an insect cell, a plant cell, a yeast, a
microorganism such as Escherichia coli).
CA 03075867 2020-03-13
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[0059]
(Pharmaceutical Composition (Therapeutic Drug,
Prophylactic Drug) and Therapeutic Method)
The antibody or immunoreactive fragment thereof of
the present invention can be purified, as necessary, and
then formulated by using a conventional method to be used
as pharmaceutical composition. The present invention
includes use of the antibody or immunoreactive fragment
thereof of the present invention for manufacturing a
pharmaceutical composition. Further, the present
invention includes a use of the antibody or
immunoreactive fragment thereof of the present invention
for therapy or prevention of diseases or disorders in
which angiogenesis contributes to the onset or
exacerbation. Furthermore, the present invention relates
to a therapeutic method or prophylactic method for
diseases or disorders in which angiogenesis contributes
to the onset or exacerbation, comprising administering an
effective dose of the antibody or immunoreactive fragment
thereof of the present invention to a patient in need
thereof.
[0060]
For example, the pharmaceutical composition of the
present invention (therapeutic drug or prophylactic drug)
can be used as an injection in forms of, for example,
intravenous injections, subcutaneous injections,
intradermal injections, intramuscular injections,
CA 03075867 2020-03-13
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intravitreal injections, and infusion injections. Such
injections can be prepared, for example, by dissolving,
suspending, or emulsifying the antibody or the like in a
sterile aqueous or oily solution commonly used for
injections according to a known method. For the aqueous
solution for injections, for example, an isotonic
solution including physiological saline, glucose,
sucrose, mannitol, and an additional adjuvant can be
used, to which an appropriate solubilizer such as alcohol
(e.g., ethanol), polyalcohol (e.g., propylene glycol,
polyethylene glycol), nonionic surfactant (e.g.,
Polysorbate 80, Polysorbate 20, HCO-50 (polyoxyethylene
(50 mol) adduct of hydrogenated castor oil)) can be
added. For the oily solution, for example, sesame oil,
and soybean oil can be used, to which benzyl benzoate, or
benzyl alcohol can be added as a solubilizer. The
injection prepared is typically put in an appropriate
ampule, vial, or syringe. Alternatively, an appropriate
diluent is added to the antibody or immunoreactive
fragment thereof of the present invention to prepare a
lyophilized formulation, which can be dissolved in water
or physiological saline or the like to prepare an
injection before use. Although oral administration of
protein such as an antibody is generally considered to be
difficult because protein is decomposed in the digestive
organ, well-designed antibody fragments or modified
antibody fragments and dosage form are expected to enable
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oral administration. Examples of formulations for oral
administration include capsules, tablets, syrups, and
granules.
[0061]
It is preferred for the pharmaceutical composition
of the present invention to be formulated into a dosage
form with a dosage unit compatible with a dose of the
active ingredient. Examples of dosage forms with such a
dosage unit include injections (ampules, vials, pre-
filled syringes), and such a dosage form may contain 5 to
500 mg, 5 to 100 mg, or 10 to 250 mg of the antibody or
immunoreactive fragment thereof of the present invention
per dosage unit in typical cases.
[0062]
Administration of the antibody or pharmaceutical
composition (therapeutic drug or prophylactic drug) of
the present invention may be topical or systemic. There
is no limitation to the administration method, and the
antibody or pharmaceutical composition of the present
invention is administered parenterally or orally, as
described above. Examples of routes of parenteral
administration include intraocular, subcutaneous,
intraperitoneal, blood (intravenous or intraarterial) or
spinal fluid injection or infusion drop, and intraocular
and blood administrations are preferred. The
pharmaceutical composition (therapeutic drug or
prophylactic drug) of the present invention may be
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administered temporarily, or administered continuously or
intermittently. For example, continuous administration
for 1 minute to 2 weeks is permitted. The antibody of
the present invention may be administered singly or in
combination with an additional drug. Alternatively, the
antibody of the present invention may be an antibody-drug
conjugate (ADC), in which such an additional drug is
conjugated. For the additional drug, for example, a drug
known to have therapeutic effect on diseases or disorders
in which angiogenesis contributes to the onset or
exacerbation can be used.
[0063]
The dose of the pharmaceutical composition of the
present invention is not particularly limited as long as
being a dose that provides a desired therapeutic effect
or prophylactic effect, and can be appropriately
determined in view of symptoms, sex, and age. The dose
of the pharmaceutical composition of the present
invention can be determined, for example, as an
indicator, a therapeutic effect or prophylactic effect on
diseases or disorders in which angiogenesis contributes
to the onset or exacerbation. In use for prevention
and/or therapy for a patient with a disease or disorder
in which angiogenesis contributes to the onset or
exacerbation, for example, it is convenient to administer
the pharmaceutical composition of the present invention
through intravenous injection with a single dose of the
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active ingredient typically at about 0.01 to 20 mg/kg
body weight, preferably at about 0.1 to 10 mg/kg body
weight, more preferably at about 0.1 to 5 mg/kg body
weight, about 1 to 10 times per month, preferably one to
five times per month. A dose according to this can be
administered for other parenteral administration or oral
administration. For a severe symptom, the dose or number
of administrations may be increased according to the
symptom.
Examples
[0064]
Hereinafter, the present invention will be described
more specifically with reference to Examples. However,
the present invention is not limited to these Examples.
All of the literatures cited throughout the present
application are directly incorporated herein by
reference. The present application claims priority to
Japanese Patent Application No. 2017-175311. All of the
contents of Japanese Patent Application No. 2017-175311
to which the present application claims priority are
directly incorporated herein by reference.
[0065]
(Example 1) Production of Anti-RAMP2 Antibody
Before making a hybridoma, cells for use as an
antigen were prepared in the following manner. Human
CRLR (NM 005795) and human RAMP2 (NM 005854) were
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tandemly incorporated into a pCH01.0 vector from Thermo
Ltd., and NIH-3T3 cells (ATCC: CRL-1658) were transformed
therewith (hCR2-NIH3T3) by using Lipofectamine 2000
(Thermo Ltd.). To make a stable cell line, selective
culture was performed with a D-MEM medium (Wako Pure
Chemical Industries, Ltd., Japan) containing 10 g/mL
Puromycin and 100 nM Methotrexate.
[0066]
The hCR2-NIH3T3 cells obtained were prepared to
achieve 1 x 106 cells per MRL/lpr mouse, and
intraperitoneally administered to immunize with the
antigen once per week for 5 weeks. The serums before and
after immunization were collected, and the increase of
antibody titer specific to the antigen was confirmed
through Cell ELISA. After confirming the increase of
antibody titer, immunization was again performed with 1 x
106 hCR2-NIH3T3 cells as an antigen for final
immunization.
[0067]
Cells derived from the spleen and lymph node were
fused by using a PEG method with the cell line SP2ab,
mouse myeloma cells, for cell fusion, and seeded in a 96-
well culture plate. A HAT-containing 10% FBS-TIL medium
was added the day after the fusion, and medium exchange
was performed every 3 days to make hybridomas.
[0068]
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To screen for anti-human RAMP2 antibody-producing
cell lines, hCRLR/hRAMP2-expressing CHO-Kl cells (hCR2-
CHOK1) made in the same manner as for NIH-3T3 cells were
seeded in a 96-well culture plate. After the cells were
fixed with 4% PFA and blocked with 1% BSA-PBS, the
culture supernatant was added thereto, and Goat Anti-
mouse-IgG-HRP (SouthernBiotech, Inc.) as a secondary
antibody was reacted. Positive wells were picked up by
coloring with TMB solution (eBioscience, Inc.), and used
as candidate hybridomas.
[0069]
Among the candidate hybridomas, three cell lines
were conditioned with an ASF104 medium (Ajinomoto Co.,
Inc., Japan), and each supernatant was then collected,
and the IgG antibody was purified with the Protein A
derivative Ab-Capcher ExTra (ProteNova Co., Ltd., Japan)
to afford a 25H4-4F9 antibody, 85H7-2B11 antibody, and
33H4-1G3 antibody as anti-RAMP2 antibodies.
[0070]
(Example 2) cAMP Inhibition Test
For the anti-RAMP2 antibodies obtained, a cAMP
Inhibition test was conducted with hCR2-CHOK1 cells.
Known commercially available antibodies (Novus
Biologicals, 4E5, Cat No. NBP2-01853; Abnova Corporation,
2F5, Cat No. H00010266-M05; and Santa Cruz Biotechnology,
Inc., B5, Cat No. sc-365240) were used as control
antibodies. A Half area 96-well plate was used for
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measurement. In 10% FBS-containing D-MEM/Ham's F-12
(Wako Pure Chemical Industries, Ltd., Japan), 2500 cells
of hCR2-CHOK1 cells were cultured overnight, and each
antibody (25H4-4F9, 85H7-2B11, 33H4-1G3, 4E5, 2F5, and
B5) was added to reach a final concentration of 0.001 nM,
0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, or 1000 nM, and
human AM (PEPTIDE INSTITUTE, INC., Japan) was added to
reach a final concentration of 150 pM. By using a LANCE
Ultra cAMP kit (PerkinElmer, Inc.), the cAMP level was
measured in accordance with a manual provided by the
manufacturer. As controls, cAMP productions by hCR2-
CHOK1 cells without addition of an antibody/with addition
of AM and without addition of an antibody/without
addition of AM were measured in the same manner.
[0071]
Figure 1 shows the results representing cAMP
inhibition when 10 nM of an antibody was added. Figure 1
shows relative cAMP concentration for each group with
addition of an antibody, with the cAMP concentration
without addition of the antibody set to 100. In contrast
to the control antibodies, which hardly inhibited cAMP
production, the 25H4-4F9 antibody, 85H7-2B11 antibody,
and 33H4-1G3 antibody inhibited cAMP production to a
degree comparable to the group without addition of AM.
Figure 2 shows the relation between concentration and
cAMP inhibition rates for the antibodies. The IC50
values of the 25H4-4F9 antibody, 85H7-2B11 antibody, and
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=
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33H4-1G3 antibody for cAMP inhibition were 0.15 nM, 0.03
nM, and 0.045 nM, respectively.
[0072]
(Example 3) HUVEC Growth prevention Test
HUVECs (KURABO INDUSTRIES LTD.) in a growth factor-
free 2% FBS-containing Humedia-EG2 medium were seeded in
a 96-well culture plate at 2500 cells/well, and allowed
to adhere through preculture for 3 hours. Each antibody
(25H4-4F9, 85H7-2B11, 33H4-1G3, 4E5, 2F5, and B5) was
added to reach a final concentration of 0.0001 nM, 0.001
nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, or 1000 nM, AM
was added thereto to reach a final concentration of 100
nM, and the growth of HUVECs after 96 hours was observed
as a HUVEC growth prevention test. In counting the
number of cells, 100 L of a solution diluted by 10-fold
with a growth factor-free 2% FBS-containing Humedia-EG2
medium was added, coloring was caused in a CO2 incubator
for 4 hours, and 0D450 was measured as a cell count by
using a Cell Counting Kit-8 (DOJINDO LABORATORIES).
[0073]
Figure 3 shows the results of growth prevention when
nM of an antibody was added. Figure 3 shows ratios of
0D450 (the number of HUVECs) for groups with addition of
an antibody with 0D450 (the number of HUVECs) without
addition of an antibody set to 100. In contrast to the
control antibodies, which hardly prevented the growth of
HUVECs, the 25H4-4F9 antibody, 85H7-2B11 antibody, and
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33H4-1G3 antibody prevented the growth to about 30%.
Figure 4 shows the relation between concentration and
HUVEC growth prevention rates for the antibodies. The
IC50 values of the 25H4-4F9 antibody, 85H7-2B11 antibody,
and 33H4-1G3 antibody for HUVEC growth prevention were
1.2 nM, 0.8 nM, and 1.6 nM, respectively.
[0074]
(Example 4) Sequence Analysis
An RNA was extracted from a cell pellet for each
antibody-producing hybridoma by using an SV-Total RNA
Isolation kit (Promega Corporation), and cDNA was
synthesized by using a Transcriptor First Strand cDNA
Synthesis Kit (F. Hoffmann-La Roche Ltd.), and the
antibody sequence of each hybridoma was analyzed. A
wobble primer (degenerate primer) was designed from the
CDR sequence of an existing mouse, and the sequences for
the heavy chain and light chain were each amplified by
using Ex Taq polymerase from Takara Bio Inc. The amino
acid sequences were determined from the nucleotide
sequences obtained, and the CDR region was specified with
a KABAT method.
[0075]
Figure 5 shows the determined sequences of VH (SEQ
ID NO: 2) and VL (SEQ ID NO: 4) of the 25H4-4F9 antibody,
those of VH (SEQ ID NO: 12) and VL (SEQ ID NO: 14) of the
85H7-2B11 antibody, and those of VH (SEQ ID NO: 19) and
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VL (SEQ ID NO: 21) of the 33H4-1G3 antibody. The
determined CDR sequences are shown in the following.
Heavy chain CDR sequences of 25H4-4F9 antibody:
CDRH1: GYFMN (SEQ ID NO: 5)
CDRH2: RNNPYNGDSIYNQKFKG (SEQ ID NO: 6)
CDRH3: LMIFDAYYAMDY (SEQ ID NO: 7)
light chain CDR sequences of 25H4-4F9 antibody:
CDRL1: RASQDIRNYLN (SEQ ID NO: 8)
CDRL2: YTSRLHS (SEQ ID NO: 9)
CDRL3: QQDSKHPWT (SEQ ID NO: 10)
Heavy chain CDR sequences of 85H7-2B11 antibody:
CDRH1: GYFMN (SEQ ID NO: 5)
CDRH2: RINPYNGDTFYNQKFKG (SEQ ID NO: 15)
CDRH3: LGIYDVYHYVMEN (SEQ ID NO: 16)
light chain CDR sequences of 85H7-2B11 antibody:
CDRL1: RASQDIRNYLN (SEQ ID NO: 8)
CDRL2: YTSRLHS (SEQ ID NO: 9)
CDRL3: QQDSKNPWT (SEQ ID NO: 17)
Heavy chain CDR sequences of 33H4-1G3 antibody:
CDRH1: GYFMN (SEQ ID NO: 5)
CDRH2: RINPYNGDTLYNQKFKG (SEQ ID NO: 22)
CDRH3: LGIYDVYHYVMEN (SEQ ID NO: 16)
light chain CDR sequences of 33H4-1G3 antibody:
CDRL1: RASQDIRNYLN (SEQ ID NO: 8)
CDRL2: YTSRLHS (SEQ ID NO: 9)
CDRL3: QQDSKNPWT (SEQ ID NO: 17)
[0076]
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(Example 5) Confirmation of Anti-RAMP2-Specific
Antibodies
Cell ELISA confirmed that the antibodies obtained
were anti-RAMP2-specific. Specifically, hamster ovary
cells (CHO-K1) were transformed with human CRLR and human
RAMP2 as in Example 1. Comparison on antibody binding
ability was made between cells transformed with human
CRLR and human RAMP2 and cells transformed only with
human CRLR.
[0077]
Figure 6 shows the results. The 25H4-4F9 antibody,
85H7-2B11 antibody, and 33H4-1G3 antibody hardly bound to
CHO-K1 cells expressing only human CRLR, which indicates
that these antibodies specifically bind to CHO-Kl cells
coexpressing human CRLR and human RAMP2.
[0078]
(Example 6) Binding Affinity Analysis with Cell Membrane
Surface Protein by Cell ELISA
To a 96-well culture plate (BD Falcon), hCR2-CHOK1
cells in a 10% FCS-containing DMEM/F-12 medium were added
at 1 x 104 cells/well, and cultured overnight. An
equivalent amount of 4% paraformaldehyde solution (MUTO
PURE CHEMICALS CO., LTD.) was added thereto, and the
cells were left to stand at 4 C for 20 minutes to fix the
cells, washed with PBST, and then subjected to blocking
treatment with 1% BSA-PBS for 1 hour or longer. The
antibodies used were 25H4-4F9, 85H7-2B11, and 33H4-1G3
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obtained in Example 1, and the commercially available
anti-RAMP2 antibodies 4E5, 2F5, and E5. Each antibody
purified was serially diluted by 1/2 from 67.5 nM, and
Goat Anti-mouse-IgG-HRP (SouthernBiotech, Inc.) as a
secondary antibody was reacted. TMB solution
(eBioscience, Inc.) was added to cause coloring, and the
reaction was then terminated with addition of 2 N H2SO4
solution. 0D450 was measured by using an iMark (Bio-Rad
Laboratories, Inc.). From the binding data acquired,
binding affinity (EC50) was calculated by using GraphPad
Prism 7.
[0079]
As demonstrated in Figure 7, 25H4-4F9, 85H7-2B11,
and 33H4-1G3 obtained in Example 1 each exhibited binding
activity clearly superior to those of the commercially
available antibodies 4E5, 2F5, and B5. The ordinate
indicates ratios to the maximum binding amount calculated
with the maximum binding amount (OD value) of an antibody
obtained in the same test set to 100%. EC50, which is
indicative of binding affinity of an antibody, was 0.40
nM for 25H4-4F9, 0.14 nM for 85H7-2B11, and 0.30 nM for
33H4-1G3. The commercially available antibodies each had
low binding ability and thus failed to provide a normal
binding curve, and the EC50 value was 37.7 nM or higher
for 4E5, and 66.7 nM or higher for 2F5 and B5.
[0080]
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(Example 7) Analysis of Binding to Cell Membrane Surface
Protein by FACS
The binding ability of each of the antibodies (25H4-
4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1, and
the commercially available anti-RAMP2 antibodies 4E5,
2F5, and B5) to membrane surface RAMP2 protein on hCR2-
CHOK1 cells was measured by using a flow cytometer (ACEA
Biosciences, Inc.). To a 96-well V-bottom plate (Thermo
Fisher Scientific), 3 x 105 peeled hCR2-CHOK1 cells and
50 L of 3 g/mL antibody solution were added, and the
plate was left to stand on ice for 1 hour to bind the
antibody. The antibody was washed three times with FACS
buffer (1% BSA/0.05% NaN3/PBS), and R-Phycoerythrin-
conjugated Affinipure F(ab')2 Fragment Goat Anti-Mouse
IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc.)
prepared at 2.5 g/mL was added thereto, and the plate
was left to stand on ice under shading for 30 minutes to
bind the secondary antibody. After one washing, the
cells were suspended in 500 L of FACS buffer, and
fluorescence was measured by FACS in the same manner as
in Example 7.
[0081]
As demonstrated in Figure 8, 25H4-4F9, 85H7-2B11,
and 33H4-1G3 obtained in Example 1 all bound to the cell
membrane surface protein. Among the commercially
available antibodies, 4E5 was found to bind to the cell
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membrane surface protein, but no binding was found for
the commercially available antibodies 2F5 and B5.
[0082]
(Example 8) Analysis of Epitope Competition by FACS
A test on competition for an antigen was conducted
by simultaneously reacting biotinylated 85H7-2B11 and
each of the antibodies (25H4-4F9, 85H7-2B11, and 33H4-1G3
obtained in Example 1, and the commercially available
antibody 4E5), not biotinylated, with hCR2-CHOK1. The
85H7-2B11 antibody was biotinylated by using a Biotin
Labeling kit-NH2 (DOJINDO LABORATORIES) in accordance
with a manual provided by the manufacturer. To hCR2-
CHOK1 cells, 3 g/mL of the biotinylated 85H7-2B11
antibody and 0, 0.1, 0.3, 1, 3, 10, or 30 g/mL of each
antibody not biotinylated were added, and the cells were
left to stand on ice for 1 hour to bind the antibody
under competition conditions. After three washings,
Streptavidin-Fluorescein Isothiocyanate (BioLegend, Inc.)
prepared at 2.5 g/mL was added thereto, and the cells
were left to stand on ice under shading for 30 minutes to
bind the secondary antibody. After one washing, the
cells were suspended in 500 L of FACS buffer, and
fluorescence was measured by FACS in the same manner as
in Example 7.
[0083]
As demonstrated in Figure 9, the 25H4-4F9, 85H7-
2B11, and 33H4-1G3 competed with 85H7-2B11 for binding to
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the antigen, but the commercially available antibody 4E5
did not compete with 85H7-2B11.
[0084]
(Example 9) Design of Humanized Antibodies
Humanized antibodies (humanized 25H4-4F9 and
humanized 85H7-2B11 antibodies) were designed for the
25H4-4F9 and 85H7-2B11 antibodies. In design of
humanized sequences, amino acid sequences were designed
with a method devised by Xoma Corporation (U.S. Patent
No. 5,766, 886; MODIFIED ANTIBODY VARIABLE DOMAINS, an
expired patent). The amino acid sequences of the heavy
chain and VL of a 25H4-4F9 mouse antibody or those of the
heavy chain and VL of an 85H7-2B11 mouse antibody were
used to perform homology search for a human
immunoglobulin GERMLINE database (NCBI: IGBLAST), and an
amino acid sequence with the highest homology was used as
a framework of a humanized antibody (25H4-4F9 heavy
chain: IGHV1-69-2*01, IGHV1-18*04, and IGHJ6*01; 25H4-4F9
light chain: IGKV1-33*01 and IGKJ2*01; 85H7-2B11 heavy
chain: IGHV1-46*01, IGHV1-18*04, and IGHJ6*01; 85H7-2B11
light chain: IGKV1-33*01 and IGKJ2*01; an identical
sequence was selected as a framework for VL of 25H4-4F9
and VL of 85H7-2B11 because their amino acid sequences
are homologous except at one portion). Each of the amino
acid residues in these frameworks was subjected to three-
grade evaluation (HIGH, MODERATE, LOW) for the risk of
losing the three-dimensional structure of an antibody or
,
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the binding ability of the antibody to an antigen when
the mouse sequence was changed to a human sequence.
Amino acid residues of LOW risk were first converted into
a human amino acid sequence and then conversion of amino
acid residues of MODERATE and HIGH risk was performed,
and thus five sequences (vhl to vh5) for the heavy chain
of each antibody and four sequences (vkl to vk4) for the
light chain were designed. Further, the heavy chains and
light chains were combined to make eight humanized
antibodies in total for each antibody.
[0085]
Figure 10 shows the designed sequences of VH and VL
of 25H4-4F9, and Figure 11 shows the designed sequences
of VH and VL of 85H7-2B11. Table 2 shows the
combinations of the sequences of a heavy chain and a
light chain, and VH, VL, and CDRs in the humanized
antibodies made.
[0086]
[Table 2]
_
Heavy chain Light chain
Antibody SEQ ID NO
SEQ ID
name
HC VH CDRH1 CDRH2 CDRH3 name LC VL
CDRL1 CDRL2 CDRL3
25H4-4F9 25H4-4F9vh ¨ 2 5 6 7
25H4-4F9vk , ¨ 4 8 9 10
25H4-4F9v1 h25H4-4F9vh1 103 27
5 6 7 h25H4-4F9vk1 113 37 8 9 10
25H4-4F9v2 h25H4-4F9vh2 105 29
5 6 7 h25H4-4F9yk1 113 37 8 9 10
25H4-4F9v3 h25H4-4F9vh2 105 29
5 6 7 h25H4-4F9yk2 115 39 8 9 10
25H4-4F9v4 h25H4-4F9vh3 107 31
5 62 7 h25H4-4F9yk2 115 39 8 9 10
P
25H4-4F9v5 h25H4-4F9vh3 107 31
5 62 7 h25H4-4F9yk3 117 41 67 9 10 .
25H4-4F9v6 h25H4-4F9vh4 109 33
5 63 7 h25H4-4F9yk3 117 41 67 9 10
.3
25H4-4F9v8 h25H4-4F9vh5 111 35
5 63 7 h25H4-4F9yk4 119 43 67 68 10 ¨i
r.,
r.,
85H7-2B11 85H7-21311vh ¨ 12 5 15 16 85H7-2B11vk ¨ 14 8 9 17
,
,
85H7-2B11v1 85H7-2B11vh1 121 45 5 15 16
85H7-2811vid 131 55 8 9 17 ,
85H7-2B11v2 85H7-2B11vh2 123 47 5 15 16 85H7-21311vk1
131 55 8 9 17
85H7-2B11v3 85H7-2B11vh2 123 47 5 15 16 85H7-2B11yk2
133 57 8 9 17
85H7-2B11v4 85H7-2B11vh3 125 49
5 64 16 85H7-2B11yk2 133 57 8 9 17 ,
85H7-2B11v5 85H7-2B11vh3 125 49 5 64 16 85H7-2811vk3
135 59 67 9 17
85H7-2611v6 85H7-2B11vh4 127 51 5 65 16 85H7-2B11vk3
135 59 67 9 17
85H7-21311v7 85H7-2611vh4 127 51 5 65 16 85H7-2811vk4
137 61 67 68 17
85H7-2B11v8 85H7-2B11vh5 129 53 5 65 16 85H7-2B11vk4
137 61 67 68 17
33H4-1G3 33H4-1G3vh ¨ 19 5 _ 22 16 33H4-1G3vk ¨ 21 8 9
17
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[0087]
(Example 10) Making of Recombinant Humanized Antibodies
To make recombinant antibodies, a gene sequence
encoding a heavy chain (SEQ ID NO: 102, 104, 106, 108,
110, 120, 122, 124, 126, or 128) and a gene sequence
encoding a light chain (SEQ ID NO: 112, 114, 116, 118,
130, 132, 134, or 136) were each cloned into the
mammalian expression vector pcDNA3.4 (Thermo Fisher
Scientific). The vector into which a heavy chain had
been cloned and the vector into which a light chain had
been cloned were mixed together to reach a weight ratio
of 1:1, and the mammalian cells CHO-S (Thermo Fisher
Scientific) were transfected with 10 g to 30 g of the
vector DNA. An ExpiFectamine (TM) CHO Transfection Kit
(Thermo Fisher Scientific) was used for the transfection,
and 10 mL to 30 mL of cell suspension was subjected to
rotating culture in a flask for 14 days. Cells were
removed through centrifugation from the culture solution
after culturing for 14 days, and the culture supernatant
obtained by passing through a 0.45 Rm-filter was purified
with Protein A Resin (Amsphere A3; JSR Life Sciences
Corporation). The Protein A Resin and culture
supernatant were mixed together at 4 C overnight to bind
the recombinant antibody, and then washed with 20 mM
phosphate buffer solution at pH 7Ø The recombinant
antibody bound to the resin was eluted with 0.1 M
glycine-HC1 Elution buffer at pH 3.2, and the eluate was
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- 78 -
neutralized with 1/50 volume of 1 M Tris-HCl at pH 9Ø
After the neutralization, the buffer was exchanged with
phosphate buffer solution by using an ultrafiltration
membrane of 30 kDa (Amicon Ultra: Millipore Corporation),
and the absorbance at 280 nm was measured to determine
the concentration of the recombinant antibody.
[0088]
(Example 11) cAMP Inhibition Test for Humanized
Antibodies
A cAMP inhibition test was conducted for humanized
antibodies by using hCR2-CHOK1 cells. As in activity
measurement for mouse antibodies, a Half area 96-well
plate manufactured by PerkinElmer was used in
measurement, and 2500 cells of hCR2-CHOK1 cells were
cultured in la% FBS-containing D-MEM/Ham's F-12 (Wako
Pure Chemical Industries, Ltd., Japan) overnight, to
which an antibody (humanized 25H4-4F9, humanized 85H7-
2B11, 4E5, 2F5, or B5) was added and human AM (PEPTIDE
INSTITUTE, INC., Japan) was added thereto to reach a
final concentration of 150 pM. By using a LANCE Ultra
cAMP kit (PerkinElmer, Inc.), the cAMP level was measured
in accordance with a manual provided by the manufacturer.
As controls, cAMP productions by hCR2-CHOK1 cells without
addition of an antibody/with addition of AM and without
addition of an antibody/without addition of AM were
measured in the same manner.
[0089]
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Figures 12 and 13 show cAMP inhibition rates when
16.7 nM of each humanized 25H4-4F9 antibody was added and
those when 16.7 nM of each humanized 85H7-2B11 antibody
was added, respectively, cAMP inhibition rates were
shown as ratios of cAMP concentration for each group with
addition of an antibody with cAMP concentration without
addition of an antibody set to 100. In contrast to the
commercially available antibodies (4E5, 2F5, and B5) used
as controls, which hardly inhibited cAMP production, the
humanized 25H4-4F9 antibodies and humanized 85H7-2B11
antibodies inhibited cAMP production to a degree
comparable to the group without addition of AM.
[0090]
Figure 14 shows the relation between concentration
and cAMP inhibition rates for the antibodies. The 1050
values of the humanized 25H4-4F9 antibodies and humanized
85H7-2B11 antibodies for cAMP inhibition were 0.17 nM for
h25H4-4F9v1, 0.14 nM for h25H4-4F9v2, 0.26 nM for h25H4-
4F9v3, 0.17 nM for h25H4-4F9v4, 0.50 nM for h25H4-4F9v5,
1.35 nM for h25H4-4F9v6, 1.07 nM for h25H4-4F9v8, and,
0.07 nM for h85H7-2B11v1, 0.11 nM for h85H7-2B11v2, 0.08
nM for h85H7-2B11v3, 0.13 nM for h85H7-2B11v4, 0.14 nM
for h85H7-2B11v5, 0.18 nM for h85H7-2B11v6, 0.12 nM for
h85H7-2B11v7, and 0.18 nM for h85H7-2B11v8.
[0091]
(Example 12) Binding Affinity Analysis for Humanized
Antibodies by Cell ELISA
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To a 96-well plate (BD Falcon, Ltd.), hCR2-CHOK1
cells in a 10% FCS-containing DMEM/F-12 medium (Wako Pure
Chemical Industries, Ltd.) were added at 1 x 104
cells/well, and cultured overnight. An equivalent amount
of 4% paraformaldehyde solution (MUTO PURE CHEMICALS CO.,
LTD.) was added thereto, and the cells were left to stand
at 4 C for 20 minutes to fix the cells, washed with PBST,
and then subjected to blocking treatment with 1% BSA-PBS
for 1 hour or longer. Each antibody purified was
serially diluted by 1/10 from 67 nM, and 100 L of 40.0
' ng/mL Goat-Anti-Human-IgG-Fc-Fragment-HRP-conjugated
(Bethyl Laboratories, Inc.) as a secondary antibody was
added and reacted. TMB solution (eBioscience, Inc.) was
used to cause coloring, the reaction was then terminated
with addition of 2 N H2SO4 solution, and 0D450 was
measured by using an iMark (Bio-Rad Laboratories, Inc.).
From the binding data acquired, binding affinity (EC50)
was calculated by using GraphPad Prism 7.
[0092]
As demonstrated in Figure 15, binding when 6.7 nM of
an antibody was added was clearly superior for humanized
25H4-4F9 and humanized 85H7-2B11 to the commercially
available antibodies 4E5, 2F5, and B5.
[0093]
Figure 16 shows binding at different concentrations.
The binding activity (EC50) of an antibody was 0.73 nM
for h25H4-4F9v1, 1.05 nM for h25H4-4F9v2, 0.83 nM for
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h25H4-4F9v3, 1.83 nM for h25H4-4F9v4, 2.32 nM for h25H4-
4F9v5, 3.19 nM for h25H4-4F9v6, 3.03 nM for h25H4-4F9v8,
and, 0.18 nM for h85H7-2B11v1, 0.24 nM for h85H7-2B11v2,
0.23 nM for h85H7-2B11v3, 0.22 nM for h85H7-2B11v4, 0.25
nM for h85H7-2B11v5, 0.24 nM for h85H7-2B11v6, 0.23 nM
for h85H7-2B11v7, and 0.20 nM for h85H7-2B11v8.
[0094]
(Example 13) HUVEC Growth Prevention Test for Humanized
Antibodies
The ability of the humanized antibodies to prevent
HUVEC growth was examined in the same manner as in
Example 3. HUVECs were seeded in a 96-well culture plate
at 2,500 cells/well, and allowed to adhere through
preculture for 3 hours. Thereafter, each of the
humanized 25H4-4F9 and humanized 85H7-2B11 at different
concentrations was added thereto, AM was added thereto to
reach a final concentration of 100 nM, and the growth of
HUVECs after 96 hours was observed to check the ability
to prevent HUVEC growth. In counting the number of
cells, a Cell Counting Kit-8 (DOJINDO LABORATORIES) was
used. Added was 100 L of a coloring solution diluted by
10-fold with a growth factor-free 2% FBS-containing
Humedia-EG2 medium, coloring was caused in a CO2
incubator for 4 hours, and 0D450 was measured as a cell
count.
[0095]
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HUVEC growth prevention rates (I050) were calculated
from the ratios of concentration of each antibody added,
with 0D450 (the number of HUVECs) without addition of an
antibody set to 100. The results found that the IC50
values of the humanized 25H4-4F9 antibodies and humanized
85H7-2B11 antibodies were 16.0 nM for h25H4-4F9v1, 10.9
nM for h25H4-4F9v2, and, 5.1 nM for h85H7-2B11v1, 9.5 nM
for h85H7-2B11v2, 1.5 nM for h85H7-2B11v3, 1.4 nM for
h85H7-2B11v4, 21.4 nM for h85H7-2B11v5, 14.8 nM for
h85H7-2B11v6, 32.7 nM for h85H7-2B11v7, and 22.6 nM for
h85H7-2B11v8.
[0096]
(Example 14) Epitope Analysis for Antibodies
To determine whether there exists a specific epitope
primary sequence that the 85H7-2B11 antibody recognizes,
31 peptide fragments (SEQ ID NOs: 71 to 101), consisting
of 13 to 15 amino acids, were made by offsetting three
amino acids of the extracellular region of human RAMP2
protein to overlap 12 amino acids, and the binding
ability of the antibody to these peptides was examined
through an ELISA method. A sufficient amount of 1.0
g/mL was used for the solid phase for the peptides and
protein, and the 85H7-2B11 antibody at a concentration of
20 ng/mL was reacted for binding. As a secondary
antibody, 50.0 ng/mL Goat Anti-mouse-IgG-HRP was reacted,
the antibody bound was allowed to color with TMB
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solution, the reaction was terminated with 2 N H2SO4
solution, and then 0D450 was measured.
QPLPTTGTPGSEGGT (SEQ ID NO: 71)
PTTGTPGSEGGTVKN (SEQ ID NO: 72)
GTPGSEGGTVKNYET (SEQ ID NO: 73)
GSEGGTVKNYETAVQ (SEQ ID NO: 74)
GGTVKNYETAVQFSW (SEQ ID NO: 75)
VKNYETAVQFSWNHY (SEQ ID NO: 76)
YETAVQFSWNHYKDQ (SEQ ID NO: 77)
AVQFSWNHYKDQMDP (SEQ ID NO: 78)
FSWNHYKDQMDPIEK (SEQ ID NO: 79)
NHYKDQMDPIEKDWS (SEQ ID NO: 80)
KDQMDPIEKDWSDWA (SEQ ID NO: 81)
MDPIEKDWSDWAMIS (SEQ ID NO: 82)
IEKDWSDWAMISRPY (SEQ ID NO: 83)
DWSDWAMISRPYSTL (SEQ ID NO: 84)
DWAMISRPYSTLRDS (SEQ ID NO: 85)
MISRPYSTLRDSLEH (SEQ ID NO: 86)
RPYSTLRDSLEHFAE (SEQ ID NO: 87)
STLRDSLEHFAELFD (SEQ ID NO: 88)
RDSLEHFAELFDLGF (SEQ ID NO: 89)
LEHFAELFDLGFPNP (SEQ ID NO: 90)
FAELFDLGFPNPLAE (SEQ ID NO: 91)
LFDLGFPNPLAERII (SEQ ID NO: 92)
LGFPNPLAERIIFET (SEQ ID NO: 93)
PNPLAERIIFETHQI (SEQ ID NO: 94)
LAERIIFETHQIHFA (SEQ ID NO: 95)
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RIIFETHQIHFANSS (SEQ ID NO: 96)
FETHQIHFANSSLVQ (SEQ ID NO: 97)
HQIHFANSSLVQPTF (SEQ ID NO: 98)
HFANSSLVQPTFSDP (SEQ ID NO: 99)
NSSLVQPTFSDPPED (SEQ ID NO: 100)
LVQPTFSDPPEDV (SEQ ID NO: 101)
[0097]
As demonstrated in Figure 17, the sufficient amount
of the 85H7-2B11 antibody bound to the equivalent amount
of human RA14P2 protein. On the other hand, the 85H7-2B11
antibody did not bind to any of the human RAMP2 peptide
fragments. These results reveal that the 85H7-2B11
antibody recognizes not a specific primary structure of
RAMP2 protein, but an epitope composed of a higher-order
structure such as conformation. From the results of
Example 8 that the antibodies of the present invention
compete with each other for binding to the antigen, the
antibodies of the present invention are all inferred to
recognize not a specific primary structure of RAMP2
protein, but an epitope composed of a higher-order
structure such as conformation. These results reveal
that recognition of not a specific primary structure of
RA14P2 protein but an epitope composed of the
extracellular conformation is important to prevent the
growth of HUVECs.
Industrial Applicability
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- 85 -
[0098]
The present antibody can specifically inhibit the
function of human RAMP2, a human adrenomedullin receptor,
and hence is expected to play an important role in
providing a method for preventing, diagnosing, or
treating an RAMP2-associated disease, and developing a
prophylactic drug, diagnostic drug, or therapeutic drug
for the disease.
