CA3075867A1

CA3075867A1 - Anti-ramp2 antibody

Info

Publication number: CA3075867A1
Application number: CA3075867A
Authority: CA
Inventors: Takayuki Shindo; Tetsuro Nakamura; Ryu Maeda; Yosuke Nakayama
Original assignee: Gene Techno Science Co Ltd
Current assignee: Kidswell Bio Corp
Priority date: 2017-09-13
Filing date: 2018-09-13
Publication date: 2019-03-21
Also published as: JP7202011B2; JPWO2019054460A1; WO2019054460A1; JP2022169737A

Abstract

With regard to an anti-RAMP2 antibody having a cAMP inhibitory effect and an HUVEC antiproliferative effect, the present invention provides an angiogenesis inhibitor containing said antibody as an active ingredient. Specifically, the present invention relates to, in an in vitro proliferation test: an antibody against a receptor activity modifying protein2 (RAMP2) having an antiproliferative effect of vascular endothelial cells, or immunoreactive fragments thereof; or an antibody having an effect of inhibiting cAMP production caused by animal cells through the addition of adrenomedullin, or immunoreactive fragments thereof.

Description

4 it t 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 4 a 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 a 1

[0005]
Patent Literature 1: International Publication No. WO

Patent Literature 2: International Publication No. WO

Patent Literature 3: International Publication No. WO

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]

i A

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 A

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 , A

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 A A

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 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 ) ) (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).

4 ,

(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 A A

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

= =

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).

A

(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]

[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

, , 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).

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 = - 17 -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 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.

[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.

=

[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, "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

- 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 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, 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 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 85H7-2B11v6 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 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]

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 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 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 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 . CA 03075867 2020-03-13 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 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 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

- 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 , 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

dHASGOO lAN2:11GOSVO AVAAVGJIIAll ISGONAdNN2J
1M , L SH1eIS1A 6 N

dH>ISCIOO 0 1AN2:110OSVO
IAIVAAVGJIIAll ISCIONAdNNH
IM 01 SH12:IS1A 6 1\11ANIIGOSVI 8 AG L ObJ>13NA

dH>ISCIOb INVAAVGJIIAll ISCI9NAdNNei , IM L OT SH1dS1A 6 NiANdIGOSV2:1 8 AG
9>LOONA

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

- 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.

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

- 41 -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

- 42 -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

- 43 -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 =

- 44 -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 ,

- 45 -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

- 46 -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:

- 47 -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

- 48 -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

- 49 -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

- 50 -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.

- 51 -[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

- 52 -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 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 A

- 54 -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 A At

- 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., x , 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 ) ,

- 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 , ,

- 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).

- 59 -[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, / , 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 m

- 61 -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 o - 62 -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 A

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 A , 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]

A

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 A

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 =
=

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 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 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]

A
#

(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 A
A

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]

(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 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 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 , r - 75 -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

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 85H7-2B11v3 85H7-2B11vh2 123 47 5 15 16 85H7-2B11yk2 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 85H7-2611v6 85H7-2B11vh4 127 51 5 65 16 85H7-2B11vk3 85H7-21311v7 85H7-2611vh4 127 51 5 65 16 85H7-2811vk4 85H7-2B11v8 85H7-2B11vh5 129 53 5 65 16 85H7-2B11vk4 33H4-1G3 33H4-1G3vh ¨ 19 5 _ 22 16 33H4-1G3vk ¨ 21 8 9 r =

[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 , , 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]

a 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

o =

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 CA 03075867 2020-03-13 .

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]

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

6 .

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) , 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 ,

[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.

Claims

[Claim 1]
An antibody or an immunoreactive fragment thereof that specifically binds to receptor activity-modifying protein 2 (RAMP2) and has an inhibitory activity on vascular endothelial cell growth in an in vitro test for vascular endothelial cell growth induced by adrenomedullin supplementation.
[Claim 2]
The antibody or immunoreactive fragment thereof of claim 1, which has the inhibitory activity on vascular endothelial cell growth with 1050 value of 35 nM or lower.
[Claim 3]
The antibody or immunoreactive fragment thereof of claim 1 or 2, wherein the antibody or immunoreactive fragment thereof has an inhibitory activity on cAMP
production induced by adrenomedullin supplementation in animal cells.
[Claim 4]
The antibody or immunoreactive fragment thereof of claim 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 2 nM or lower.
[Claim 5]

The antibody or immunoreactive fragment thereof of any one of claims 1 to 4, wherein the antibody or immunoreactive fragment thereof binds to cells coexpressing human CRLR and human RAMP2.
[Claim 6]
The antibody or immunoreactive fragment thereof of claim 5, wherein binding affinity (EC50) in a Cell ELISA
test using the cells coexpressing human CRLR and human RA4P2 is 3.5 nM or lower.
[Claim 7]
The antibody or immunoreactive fragment thereof of any one of claims 1 to 6, which competes for binding to RA4P2 with an antibody comprising a heavy chain variable region (VH) having an amino acid sequence of any one selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53, and a light chain variable region (VL) having an amino acid sequence of any one selected from SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61; or an antibody or an immunoreactive fragment thereof that binds to the same epitope as an antibody comprising VH having an amino acid sequence of any one selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53, and VL having an amino acid sequence of any one selected from SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61.
[Claim 8]

The antibody or immunoreactive fragment thereof of any one of claims 1 to 7, wherein the antibody or immunoreactive fragment thereof does not recognize a primary structure of an amino acid sequence constituting an extracellular region of RAMP2.
[Claim 9]
The antibody or immunoreactive fragment thereof of claim 8, wherein the antibody or immunoreactive fragment thereof does not bind to a peptide consisting of any of amino acid sequences of SEQ ID NOs: 71 to 101.
[Claim 10]
The antibody or immunoreactive fragment thereof of any one of claims 1 to 9, comprising a heavy chain having heavy chain complementarity-determining region (CDRH) 3 consisting of the amino acid sequence of LX1 IX2 DX3 YX4 YX5 MX6 X7 (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.
[Claim 11]
The antibody or immunoreactive fragment thereof of claim 10, comprising a heavy chain having CDRH3 consisting of the amino acid sequence of LMIFDAYYAMDY
(SEQ ID NO: 7) or LGIYDVYHYVMEN (SEQ ID NO: 16).
[Claim 12]
The antibody or immunoreactive fragment thereof of claim 10 or 11, wherein the heavy chain further has CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID

NO: 5), and CDRH2 consisting of the amino acid sequence of RXBNPYNGDX9 X10YX11 QKFX12 G (SEQ ID NO: 66), wherein X8 is N or I, X9 iS S or T, X10 is I, F, or L, X11 is A or N, and X12 is Q or K.
[Claim 13]
The antibody or immunoreactive fragment thereof of claim 12, further comprising a light chain having light chain complementarity-determining region (CDRL) 1 consisting of the amino acid sequence of X13ASQDIRNYLN
(SEQ ID NO: 69), CDRL2 consisting of the amino acid sequence of YTSRLX14 X15 (SEQ ID NO: 70), and CDRL3 consisting of the amino acid sequence of QQDSKX16 PWT (SEQ
ID NO: 25), wherein Xn is Q or R, X14 is E or H, X15 is S or T, and X16 is H or N.
[Claim 14]
The antibody or immunoreactive fragment thereof of claim 12, having:
CDRH1 consisting of the amino acid sequence of GYFMN
(SEQ ID NO: 5);
CDRH2 consisting of any one amino acid sequence selected from the group consisting 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), and RINPYNGDTFYAQKFQG
(SEQ ID NO: 65); and CDRH3 consisting of the amino acid sequence of LMIFDAYYAMDY (SEQ ID NO: 7) or LGIYDVYHYVMEN (SEQ ID NO:
16).
[Claim 15]
The antibody or immunoreactive fragment thereof of claim 14, further having:
CDRL1 consisting of the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8) or QASQDIRNYLN (SEQ ID NO:
67);
CDRL2 consisting of the amino acid sequence of YTSRLHS (SEQ ID NO: 9) or YTSRLET (SEQ ID NO: 68); and CDRL3 consisting of the amino acid sequence of QQDSKHPWT (SEQ ID NO: 10) or QQDSKNPWT (SEQ ID NO: 17).
[Claim 16]
The antibody or immunoreactive fragment thereof of claim 14, having any one combination of heavy chain CDRs of the following (i) to (vii):
(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 RNNPYNGDSIYNEKFQG (SEQ ID NO: 62), and CDRH3 consisting of the amino acid sequence of LMIFDAYYAMDY (SEQ ID NO: 7);
(iii) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5), CDRH2 consisting of the amino acid sequence of RNNPYNGDSIYAEKFQG (SEQ ID NO: 63), and CDRH3 consisting of the amino acid sequence of LMIFDAYYAMDY (SEQ ID NO: 7);
(iv) 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);
(v) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5), CDRH2 consisting of the amino acid sequence of RINPYNGDTFYNQKFQG (SEQ ID NO: 64), and CDRH3 consisting of the amino acid sequence of LGIYDVYHYVMEN (SEQ ID NO: 16);
(vi) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5), CDRH2 consisting of the amino acid sequence of RINPYNGDTFYAQKFQG (SEQ ID NO: 65), and CDRH3 consisting of the amino acid sequence of LGIYDVYHYVMEN (SEQ ID NO: 16); and (vii) 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).
[Claim 17]
The antibody or immunoreactive fragment thereof of claim 16, further having any one combination of light chain CDRs of the following (a) to (f):
(a) 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);
(b) CDRL1 consisting of the amino acid sequence of QASQDIRNYLN (SEQ ID NO: 67), 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);
(c) CDRL1 consisting of the amino acid sequence of QASQDIRNYLN (SEQ ID NO: 67), CDRL2 consisting of the amino acid sequence of YTSRLET (SEQ ID NO: 68), and CDRL3 consisting of the amino acid sequence of QQDSKHPWT (SEQ ID NO: 10);
(d) 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);
(e) CDRL1 consisting of the amino acid sequence of QASQDIRNYLN (SEQ ID NO: 67), 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); and (f) CDRL1 consisting of the amino acid sequence of QASQDIRNYLN (SEQ ID NO: 67), CDRL2 consisting of the amino acid sequence of YTSRLET (SEQ ID NO: 68), and CDRL3 consisting of the amino acid sequence of QQDSKNPWT (SEQ ID NO: 17).
[Claim 18]
The antibody of any one of claims 1 to 17, which is any of the following:
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 an amino acid sequence of any one selected from 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; and an antibody or an immunoreactive fragment thereof, wherein 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 EQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61.
[Claim 19]
The antibody or immunoreactive fragment thereof of any one of claims 1 to 17, having VH having the amino acid sequence of any one selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53.
[Claim 20]
The antibody or immunoreactive fragment thereof of claim 19, further comprising VL having the amino acid sequence of any one selected from SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61.
[Claim 21]

The antibody or immunoreactive fragment thereof of claim 20, comprising any one combination of VH and VL
selected from the group of:
VH having the amino acid sequence of SEQ ID NO: 2 and VL having the amino acid sequence of SEQ ID NO: 4;
VH having the amino acid sequence of SEQ ID NO: 27 and VL having the amino acid sequence of SEQ ID NO: 37;
VH having the amino acid sequence of SEQ ID NO: 29 and VL having the amino acid sequence of SEQ ID NO: 37;
VH having the amino acid sequence of SEQ ID NO: 29 and VL having the amino acid sequence of SEQ ID NO: 39;
VH having the amino acid sequence of SEQ ID NO: 31 and VL having the amino acid sequence of SEQ ID NO: 39;
VH having the amino acid sequence of SEQ ID NO: 31 and VL having the amino acid sequence of SEQ ID NO: 41;
VH having the amino acid sequence of SEQ ID NO: 33 and VL having the amino acid sequence of SEQ ID NO: 41;
VH having the amino acid sequence of SEQ ID NO: 35 and VL having the amino acid sequence of SEQ ID NO: 43;
VH having the amino acid sequence of SEQ ID NO: 12 and VL having the amino acid sequence of SEQ ID NO: 14;
VH having the amino acid sequence of SEQ ID NO: 45 and VL having the amino acid sequence of SEQ ID NO: 55;
VH having the amino acid sequence of SEQ ID NO: 47 and VL having the amino acid sequence of SEQ ID NO: 55;
VH having the amino acid sequence of SEQ ID NO: 47 and VL having the amino acid sequence of SEQ ID NO: 57;

VH having the amino acid sequence of SEQ ID NO: 49 and VL having the amino acid sequence of SEQ ID NO: 57;
VH having the amino acid sequence of SEQ ID NO: 49 and VL having the amino acid sequence of SEQ ID NO: 59;
VH having the amino acid sequence of SEQ ID NO: 51 and VL having the amino acid sequence of SEQ ID NO: 59;
VH having the amino acid sequence of SEQ ID NO: 51 and VL having the amino acid sequence of SEQ ID NO: 61;
VH having the amino acid sequence of SEQ ID NO: 53 and VL having the amino acid sequence of SEQ ID NO: 61;
and VH having the amino acid sequence of SEQ ID NO: 19 and VL having the amino acid sequence of SEQ ID NO: 21.
[Claim 22]
A nucleic acid molecule encoding an amino acid sequence of the antibody or immunoreactive fragment thereof of any one of claims 1 to 21.
[Claim 23]
The nucleic acid molecule of claim 22, comprising any one polynucleotide selected from the group of:
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: 26, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 36;

a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO: 28, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 36;
a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO: 28, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 38;
a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO: 30, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 38;
a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO: 30, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 40;
a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO: 32, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 40;
a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO: 34, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 42;
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;
a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO: 44, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 54;
a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO: 46, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 54;

a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO: 46, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 56;
a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO: 48, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 56;
a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO: 48, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 58;
a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO: 50, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 58;
a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO: 50, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 60;
a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO: 52, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO: 60; and 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.
[Claim 24]
A vector comprising the nucleic acid molecule of claim 22 or 23.
[Claim 25]
A host cell comprising the vector of claim 24.
[Claim 26]

A method for producing the antibody or immunoreactive fragment thereof of any one of claims 1 to 21, comprising culturing the host cell of claim 25.
[Claim 27]
A pharmaceutical composition comprising the antibody or immunoreactive fragment thereof of any one of claims 1 to 21 as an active ingredient.
[Claim 28]
The pharmaceutical composition of claim 27, which is an angiogenesis inhibitor.
[Claim 29]
The pharmaceutical composition of claim 27, 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.