JPWO2019054460A1 - Anti-RAMP2 antibody - Google Patents

Abstract

The present invention relates to an anti-RAMP2 antibody having a cAMP inhibitory effect and an HUVEC growth inhibitory effect, and provides an angiogenesis inhibitor containing the antibody as an active ingredient. Specifically, an antibody against receptor activity-regulating protein 2 (RAMP2) or an immunoreactive fragment thereof, which has an inhibitory effect on the growth of vascular endothelial cells in an in vitro proliferation test, or cAMP by animal cells by adding adrenomedulin. The present invention relates to an antibody having an action of suppressing production or an immunoreactive fragment thereof.

Description

The present invention comprises an antibody against a receptor activity-regulating protein 2 (RAMP2) having an angiogenesis-suppressing effect or an immunoreactive fragment thereof, and an antibody against such RAMP2 or an immunoreactive fragment thereof as an active ingredient. Regarding agents.

Adrenomedulin (hereinafter referred to as "AM") is a peptide consisting of 52 amino acids identified from human pheochromocytoma and is known as a vasoactive substance that is mainly secreted from blood vessels and has a function of dilating blood vessels. In addition, various actions such as organ protection action, tissue regeneration action, and anti-inflammatory action have been reported. Receptor activity regulatory protein 2 (RAMP2) is a 1-transmembrane protein that forms a complex with the 7-transmembrane protein calcitonin receptor-like receptor (CRLR) as a co-receptor for AM. It is functioning. Its expression is found in vascular endothelial cells, and by receiving AM in autocline and paracline, intracellular cAMP increases and signals are transmitted to maintain vascular homeostasis.

By inhibiting AM-RAMP2 / CRLR signals, cardiovascular diseases such as blood pressure regulation, and inflammation and edema due to regulation of vascular permeability, cancer / retinal diseases related to angiogenesis, rejection immunity such as organ transplantation, etc. There is a possibility of medicine for various diseases. As such an inhibitor, AM37-52, which is an AM partial sequence peptide (antagonist), has been reported (Patent Document 1). AM37-52 has a weak antagonist activity and a small molecular weight of 5 kDa or less, so that it has poor blood kinetics and is difficult to develop as a pharmaceutical product.

In addition, polyclonal antibodies against RAMP2 and mixtures thereof have been reported so far (Patent Documents 2 and 3), but RAMP2 antibodies (particularly monoclonal antibodies) alone have not effectively suppressed angiogenesis.

International release WO2016 / 183479 International release WO2010 / 012911 International release WO2012 / 029023

In order to obtain an anti-RAMP2 antibody that inhibits the activation of AM-dependent RAMP2 and CRLR complex, particularly an anti-RAMP2 antibody having the ability to suppress the growth of HUVEC (human umbilical vein endothelial cells), the present inventors are humans. A polypeptide fragment encoding the RAMP2 protein was immunized to prepare a polyclonal antibody. However, the antiserum-derived polyclonal antibody did not show cAMP inhibitory ability. Therefore, we attempted to obtain an anti-RAMP2 antibody by immunizing a protein obtained by cell-free synthesis of the extracellular domain of RAMP2. As a result of using a complex protein with CRLR, which is a co-receptor, in addition to RAMP2 protein alone as an antigen, an anti-RAMP2 antibody having excellent cAMP inhibitory ability was obtained. However, these antibodies did not show the effect of suppressing the growth of HUVEC.

Therefore, the present inventors immunized the gene-introduced overexpressing cells as antigens. Since the human RAMP2 gene alone does not function as a receptor for AM peptides, a fibroblast cell line NIH-3T3 cell in which the RAMP2 gene was co-expressed together with the CRLR gene was prepared.

However, the RAMP2 protein has a total length of 175 amino acids (20 kDa), and the extracellular domain is a small molecule of 102 amino acids (12 kDa), and the homology between human RAMP2 and mouse RAMP2 is high, and 62% of the extracellular domains encode the same amino acid. Therefore, it is difficult to establish immunity to the functional domain of human RAMP2, and a good quality antibody could not be obtained by immunization with normal rats, hamsters, or mice. Therefore, we succeeded in obtaining an anti-RAMP2 antibody containing an anti-RAMP2 antibody having excellent functionality by immunizing an MRL / lpr mouse that produces an immune reaction (autoantibody) against itself by hyperimmunization.

In order to screen the obtained hybridoma for an anti-RAMP2 antibody having excellent functionality, Cell ELISA of gene-introduced overexpressing cells was performed. Since the mouse NIH-3T3 cells used as the antigen are falsely positive due to the binding of the antibody to the cells themselves, stable expression cells obtained by gene transfer of human RAMP2-CRLR into hamster ovary cells (CHO-K1). (HCR2-CHOK1) was used for Cell ELISA.

For the candidate antibody selected by Cell ELISA, a signal inhibitory antibody was screened using hCR2-CHOK1 cells using an index as to whether or not the increase in intracellular cAMP due to AM addition was suppressed. Furthermore, the selected signal-inhibiting antibody was screened for an antibody capable of inhibiting the growth of HUVEC by adding AM as a biological activity inhibition test, and succeeded in obtaining an anti-RAMP2 antibody having a cAMP inhibitory effect and an HUVEC growth inhibitory effect.

Therefore, the present invention relates to an anti-RAMP2 antibody having a cAMP inhibitory effect and an HUVEC growth inhibitory effect, and an angiogenesis inhibitor containing the antibody as an active ingredient.
(1) In an in vitro adrenomedulin-added vascular endothelial cell growth test, an antibody that specifically binds to receptor activity-regulating protein 2 (RAMP2) or an immunoreactive fragment thereof, which has an inhibitory effect on vascular endothelial cell growth. ..
(2) The antibody or immunoreactive fragment thereof according to (1), wherein the growth inhibitory effect of the vascular endothelial cells is an IC50 value of 0.5 to 5 nM.
(3) The antibody or immunoreactive fragment thereof according to (1) or (2), which has an action of suppressing cAMP production by animal cells by addition of adrenomedulin.
(4) The antibody according to (3) or its immunoreactivity, wherein the cAMP production inhibitory effect is an IC50 value of 0.03 to 0.5 nM in a cAMP production test using animal cells by adding in vitro adrenomedulin. fragment.
(5) An antibody containing a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 2 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence set forth in SEQ ID NO: 12. It is selected from an antibody containing VH having the amino acid sequence set forth in SEQ ID NO: 14 and VL having the amino acid sequence set forth in SEQ ID NO: 14, and an antibody containing VH having the amino acid sequence set forth in SEQ ID NO: 19 and VL having the amino acid sequence set forth in SEQ ID NO: 21. An antibody or an immunoreactive fragment thereof that competes with any one of the antibodies for binding to RAMP2, or
An antibody containing VH having the amino acid sequence set forth in SEQ ID NO: 2 and VL having the amino acid sequence set forth in SEQ ID NO: 4, VH having the amino acid sequence set forth in SEQ ID NO: 12, and VL having the amino acid sequence set forth in SEQ ID NO: 14. An antibody that binds to the same epitope as any one of the antibodies selected from the antibody containing, and the VH having the amino acid sequence set forth in SEQ ID NO: 19 and the antibody containing VL having the amino acid sequence set forth in SEQ ID NO: 21. Immunoreactive fragment.
(6) The amino acid sequence of LX ₁ IX ₂ DX ₃ YX ₄ YX ₅ MX ₆ X ₇ (SEQ ID NO: 24), where X ₁ is G or M and X ₂ is F or Y. From the amino acid sequence where X ₃ is A or V, X ₄ is absent or H, X ₅ is A or V, X ₆ is D or E, and X ₇ is Y or N. An antibody or an immunoreactive fragment thereof containing a heavy chain having a heavy chain complementarity determining region (CDRH) 3.
(7) The antibody or immunoreactive fragment thereof according to (6), which comprises a heavy chain having CDRH3 consisting of the amino acid sequence of LMIFDAYYAMDY (SEQ ID NO: 7) or LGIYDVYHYVMEN (SEQ ID NO: 16).
(8) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5), CDRH2 consisting of the amino acid sequence of RX ₈ NPYNGDX ₉ X ₁₀ YNQKFKG (SEQ ID NO: 23), and LX ₁ IX ₂ DX ₃ YX ₄ YX ₅ MX ₆ 7. The antibody or immunoreactive fragment thereof according to (6), which comprises a heavy chain having CDRH3 consisting of the amino acid sequence of ₇ (SEQ ID NO: 24).
Here, X ₁ is G or M, X ₂ is F or Y, X ₃ is A or V, X ₄ is absent or H, X ₅ is A or V, and X. ₆ is D or E, X ₇ is Y or N, X ₈ is N or I, X ₉ is S or T, and X ₁₀ is I, F or L.
(9) Further, the light chain complementarity determining regions (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 QQDSKX ₁₁ PWT (SEQ ID NO: 25). The antibody according to (8) or an immunoreactive fragment thereof, which comprises a light chain having CDRL3 consisting of an amino acid sequence.
Here, X ₁₁ is H or N.
(10) (i) CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5),
Do you have CDRH2 consisting of the amino acid sequence of RNNPYNGDSYNQKFKG (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 RINGYNGDTFYNQKFKG (SEQ ID NO: 15) and CDRH3 consisting of the amino acid sequence of LGIYDVYHYVMEN (SEQ ID NO: 16) or CDRH1 consisting of the amino acid sequence of (iii) GYFMN (SEQ ID NO: 5),
The antibody or immunoreactive fragment thereof according to (6), which has CDRH2 consisting of the amino acid sequence of RINGYNGDTLYNQKFKG (SEQ ID NO: 22) and CDRH3 consisting of the amino acid sequence of LGIYDVYHYVMEN (SEQ ID NO: 16).
(11) Further, in the case of (i), CDRL1 consisting of the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8),
It has 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).
In the case of (ii), CDRL1 consisting of the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8),
It has 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).
In the case of (iii), CDRL1 consisting of the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8),
The antibody or immunoreactive fragment thereof according to (10), which comprises 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).
(12) The antibody according to any one of (1) to (11), which is any one antibody selected from the following group or an immunoreactive fragment thereof:
The VH has an amino acid sequence encoded by a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 and a nucleic acid sequence that hybridizes under stringent conditions, and the VL is set forth in SEQ ID NO: 4. An antibody or immunoreactive fragment thereof having an amino acid sequence encoded by a nucleic acid sequence that hybridizes with a nucleic acid sequence encoding an amino acid sequence under stringent conditions;
The VH has an amino acid sequence encoded by a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 12 and a nucleic acid sequence that hybridizes under stringent conditions, and the VL is set forth in SEQ ID NO: 14. An antibody or immunoreactive fragment thereof having an amino acid sequence encoded by a nucleic acid sequence that hybridizes with a nucleic acid sequence encoding an amino acid sequence under stringent conditions;
The VH has an amino acid sequence encoded by a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 19 and a nucleic acid sequence that hybridizes under stringent conditions, and the VL is set forth in SEQ ID NO: 21. An antibody or immunoreactive fragment thereof having an amino acid sequence encoded by a nucleic acid sequence that hybridizes with a nucleic acid sequence encoding an amino acid sequence under stringent conditions;
An antibody or antibody in which VH has an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 2 and VL has an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 4. Its immunoreactive fragment;
An antibody or antibody in which VH has an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 12 and VL has an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 14. The immunoreactive fragment; and VH have an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 19, and VL has 80% or more identity with the amino acid sequence of SEQ ID NO: 21. An antibody having an amino acid sequence or an immunoreactive fragment thereof.
(13) The antibody or immunoreactive fragment thereof according to (11), which is any one antibody or immunoreactive fragment thereof selected from the following group:
An antibody or immunoreactive fragment thereof containing VH having the amino acid sequence set forth in SEQ ID NO: 2 and VL having the amino acid sequence set forth in SEQ ID NO: 4;
An antibody or immunoreactive fragment thereof comprising a VH having the amino acid sequence set forth in SEQ ID NO: 12 and a VL having the amino acid sequence set forth in SEQ ID NO: 14; and a VH having the amino acid sequence set forth in SEQ ID NO: 19 and SEQ ID NO: 21. An antibody containing VL having the described amino acid sequence or an immunoreactive fragment thereof.
(14) A nucleic acid molecule encoding the amino acid sequence of the antibody or immunoreactive fragment thereof according to any one of (1) to (13).
(15) A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 1 and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 3;
A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 11 and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 13; or encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 18. The nucleic acid molecule according to (14), which contains a polynucleotide and a VL-encoding polynucleotide having the nucleotide sequence of SEQ ID NO: 20.
(16) A vector having the nucleic acid molecule according to (14) or (15).
(17) A host cell having the vector according to (16).
(18) The method for producing an antibody or an immunoreactive fragment thereof according to any one of (1) to (13), which comprises culturing the host cell of (17).
(19) A pharmaceutical composition containing the antibody according to any one of (1) to (13) or an immunoreactive fragment thereof as an active ingredient.
(20) The pharmaceutical composition according to (19), which is an angiogenesis inhibitor.
(21) Cancer, cancer metastasis, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, neovascular glaucoma, inflammatory skin disease, rheumatoid arthritis, or degenerative arthritis The pharmaceutical composition according to (19), which is a therapeutic agent or a preventive agent for the above.

Since the antibody of the present invention or an immunoreactive fragment thereof can bind to RAMP2 expressed on the cell surface and suppress the proliferation of vascular endothelial cells alone, an angiogenesis inhibitor based on a novel mechanism and angiogenesis Provides a therapeutic or prophylactic agent for a disease or disorder that contributes to the onset or exacerbation of the disease.

3 is a graph showing the inhibitory effect of various anti-RAMP2 antibodies (10 nM) on cAMP production at the time of AM addition using hCR2-CHOK1 cells. The vertical axis shows the ratio (%) of the cAMP amount of each group to the cAMP amount (100%) of AM addition / antibody non-administration (control). The horizontal axis indicates the presence or absence of AM and antibody addition, and the added antibody. It is a graph which shows the result of having measured the suppression ability of cAMP production by AM addition of 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody of various concentrations using hCR2-CHOK1 cells. The vertical axis shows the ratio (%) of the amount of cAMP when each antibody was administered to the amount of cAMP (100%) when no antibody was added. The horizontal axis shows the antibody concentration (nM). It is a graph which shows the inhibitory effect of various anti-RAMP2 antibody (10nM) on HUVEC growth by addition of AM. The vertical axis shows the ratio (%) of the number of HUVEC cells in each group to the number of HUVEC cells (100%) to which AM was added / antibody was not administered (control). The horizontal axis indicates the presence or absence of AM and antibody addition, and the added antibody. It is a graph which shows the result of having measured the growth inhibition ability by AM addition of 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody of various concentrations using HUVEC. The vertical axis shows the ratio (%) of the number of HUVEC cells when each antibody was administered to the number of HUVEC cells (100%) when no antibody was added. The horizontal axis shows the antibody concentration (nM). 25H4-4F9 antibody VH (SEQ ID NO: 2) and light chain variable region (SEQ ID NO: 4), 85H7-2B11 antibody VH (SEQ ID NO: 12) and light chain variable region (SEQ ID NO: 14), and 33H4-1G3 antibody The amino acid sequences of VH (SEQ ID NO: 19) and the light chain variable region (SEQ ID NO: 21), and the CDR moiety contained therein are shown. It is a graph which shows the result of having confirmed by Cell ELISA that the acquired antibody is anti-RAMP2-specific. The horizontal axis shows the antibody used. The white bars of the dots show the results using cells expressing human CRLR alone, and the black bars show the cells co-expressing human CRLR and human RAMP2 (hCR2-CHOK1). The vertical axis shows the amount of each antibody bound to these expressing cells (OD450). 25H4-4F9 (black square), 85H7-2B11 (black circle), and 33H4-1G3 (black triangle) obtained in Example 1, and commercially available anti-RAMP2 antibody 4E5 (white circle), 2F5 (white square). ) And B5 (white triangle) are shown in the graph showing the results of analyzing the binding affinity for cell membrane surface proteins by Cell ELISA. The vertical axis represents the binding ratio (%), and the horizontal axis represents the log value of the antibody concentration (nM). For each antibody, the antibody concentration at which the binding ratio was 50% was obtained from the graph and used as the EC50 value. Binding to antigens on the cell membrane surface using 25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1 and commercially available anti-RAMP2 antibodies 4E5, 2F5, and B5 as test antibodies. It is a graph which shows the result of analysis by FACS. The name of each test antibody used is shown at the top of each graph. The vertical axis of each graph shows the number of cells (× 10 ³ cells), the horizontal axis represents the fluorescence intensity of the R-Phycoerythrin-labeled secondary antibody bound to the cell. In the graph, the peak located on the left side indicates the control (in the absence of each test antibody), and the peak located on the right side indicates the presence of each test antibody. The binding of 25H4-4F9 (black triangle), 85H7-2B11 (black circle), and 33H4-1G3 (black square) obtained in Example 1 and the commercially available antibody 4E5 (white circle) to the antigen on the cell surface is biotinylated. It is a graph which shows the result of having analyzed by FACS whether or not it competes with 85H7-2B11 antibody. The vertical axis represents the average value (MFI) of the fluorescence intensity of the fluorescent label bound to the biotinylated 85H7-2B11 antibody bound to the cell, and the horizontal axis represents the concentration (μg / mL) of each antibody used. The results of 85H7-2B11 (black circles) competed with the same antibody as the labeled antibody indicate that the binding is competing. The sequences of the variable regions of the heavy chain and the light chain of the humanized antibody designed based on the sequence of the 25H4-4F9 antibody are shown. Based on the VH sequence h25H4-4F9vh (SEQ ID NO: 2) of the 25H4-4F9 antibody, 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. Further, based on the VL sequence h25H4-4F9vk (SEQ ID NO: 4) of the 25H4-4F9 antibody, 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) was designed. The underlined part shows each CDR sequence. The sequences of the variable regions of the heavy chain and the light chain of the humanized antibody designed based on the sequence of the 85H7-2B11 antibody are shown. Based on the VH sequence h85H7-2B11vh (SEQ ID NO: 12) of the 85H7-2B11 antibody, 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. Further, based on the VL sequence h85H7-2B11vk (SEQ ID NO: 14) of the 85H7-2B11 antibody, h85H7-2B11vk1 (SEQ ID NO: 55), h85H7-2B11vk2 (SEQ ID NO: 57), h85H7-2B11vk3 (SEQ ID NO: 59), and h85H7-2B11vk4 (SEQ ID NO: 61) was designed. The underlined part shows each CDR sequence. AM of 16.7 nM humanized 25H4-4F9 antibody (25H4-4F9v1, 25H4-4F9v2, 25H4-4F9v3, 25H4-4F9v4, 25H4-4F9v5, 25H4-4F9v6, and 25H4-4F9v8) using hCR2-CHOK1 cells. It is a graph which shows the result of having measured the inhibitory ability of cAMP production by addition. The vertical axis shows the ratio (%) of the amount of cAMP when each antibody was administered to the amount of cAMP (100%) when no antibody was added. The horizontal axis shows the presence or absence of AM addition and the antibody used. Using hCR2-CHOK1 cells, 16.7 nM humanized 85H7-2B11 antibody (85H7-2B11v1, 85H7-2B11v2, 85H7-2B11v3, 85H7-2B11v4, 85H7-2B11v5, 85H7-2B11v6, 85H7-2B11v7, and 85H-7. It is a graph which shows the result of having measured the inhibitory ability of cAMP production by the addition of AM of 2B11v8). The vertical axis shows the ratio (%) of the amount of cAMP when each antibody was administered to the amount of cAMP (100%) when no antibody was added. The horizontal axis shows the presence or absence of AM addition and the antibody used. Various concentrations of humanized 25H4-4F9 antibody (25H4-4F9v1, 25H4-4F9v2, 25H4-4F9v3, 25H4-4F9v4, 25H4-4F9v5, 25H4-4F9v6, and 25H4-4F9v8) using hCR2-CHOK1 cells (above). ), And humanized 85H7-2B11 antibody (85H7-2B11v1, 85H7-2B11v2, 85H7-2B11v3, 85H7-2B11v4, 85H7-2B11v5, 85H7-2B11v6, 85H7-2B11v7, and 85H7-2B11v8) (see the figure below). It is a graph which shows the result of having measured the inhibitory ability of cAMP production. In each graph, the vertical axis shows the ratio (%) of the amount of cAMP when each antibody was administered to the amount of cAMP (100%) when no antibody was added. The horizontal axis shows the log value of the antibody concentration (nM). Along with the legend, the IC50 value of each antibody (concentration that inhibits cAMP by 50% in the graph) is shown. Humanized 25H4-4F9 antibody (25H4-4F9v1, 25H4-4F9v2, 25H4-4F9v3, 25H4-4F9v4, 25H4-4F9v5, 25H4-4F9v6, and 25H4-4F9v8) (above), and humanized 85H7-2B11 antibody (85H7). -Cell ELISA was performed using 6.7 nM of -2B11v1, 85H7-2B11v2, 85H7-2B11v3, 85H7-2B11v4, 85H7-2B11v5, 85H7-2B11v6, 85H7-2B11v7, and 85H7-2B11v8) (see the figure below). It is a graph which shows the result of having analyzed the binding affinity with respect to. The vertical axis represents the binding ratio (OD450%), and the horizontal axis represents the antibody used. Humanized 25H4-4F9 antibody (25H4-4F9v1, 25H4-4F9v2, 25H4-4F9v3, 25H4-4F9v4, 25H4-4F9v5, 25H4-4F9v6, and 25H4-4F9v8) (above), and humanized 85H7-2B11 antibody (85H7). -Binding affinity for cell membrane surface proteins by Cell ELISA using -2B11v1, 85H7-2B11v2, 85H7-2B11v3, 85H7-2B11v4, 85H7-2B11v5, 85H7-2B11v6, 85H7-2B11v7, and 85H7-2B11v8) (below). It is a graph which shows the analysis result. The vertical axis represents the binding ratio (%), and the horizontal axis represents the log value of the antibody concentration (nM). For each antibody, the antibody concentration at which the binding ratio was 50% was obtained from the graph and used as the EC50 value. The EC50 value (nM) of each antibody is shown with a legend. It is a graph which shows the result of having analyzed the epitope of an antibody. The vertical axis represents the amount of bound antibody by OD450 (color development intensity of secondary antibody). The horizontal axis shows each peptide derived from the extracellular region of RAMP2 used for binding.

(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 the cell membrane or cell surface. As used herein, an antibody or immunoreactive fragment thereof is "specifically" recognized (bound) to RAMP2 rather than its affinity for other proteins or peptides. It means that they bind with substantially high affinity. Here, "binding with substantially high affinity" means that a specific protein or peptide of interest can be detected separately from other proteins or peptides by a desired measuring device or method. It means high affinity. For example, substantially high affinity means that the intensity detected by ELISA or EIA (for example, fluorescence intensity) is 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more. It may mean that it is 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 100 times or more.

Examples of the binding rate constant (Ka1) in the binding between the antibody of the present invention and RAMP2 include 1 × 10 ⁴ Ms ^-1 or more, 1 × 10 ⁵ Ms ^-1 or more, and 5 × 10 ⁵ Ms ^-1 or more. Can be done. Further, examples of the dissociation rate constant (Kd1) in the binding between the antibody of the present invention and RAMP2 include 1 × 10 ^-3 or less and 1 × 10 ^-4 or less. The binding constant (KD) in the binding between the antibody of the present invention and RAMP2 is, for example, 1 × ^10-8 (M) or less, 5 × ^10-8 (M) or less, 1 × ^10-9 (M) or less, It can be 5 × ^10-9 (M) or less. The binding rate constant (Ka1), dissociation rate constant (Kd1), and binding constant (KD) of the antibody in the present specification are manuals provided by the manufacturer using BIACORE (GE Healthcare Bioscience Co., Ltd., BIACORE-X100). According to this, after fixing the biotinylated RAMP2 on the SA chip, the test antibody is flowed, the binding rate constant Ka1 and the dissociation rate constant Kd1 are measured, and the binding constant KD value can be determined using the dissociation constant Kd1.

The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. In the present invention, a "monoclonal antibody" is an antibody having a substantially uniform structure that reacts with a single antigenic determinant. Further, the antibody of the present invention includes a non-human animal antibody, an antibody having an amino acid sequence of a non-human animal antibody and an amino acid sequence of a human-derived antibody, and a human antibody. Examples of the antibody of a non-human animal include antibodies of mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, sheep, goats, camels, chickens, ducks and the like, preferably for producing hybridomas. It is an animal antibody capable of producing, more preferably a mouse, rat or rabbit antibody. Examples of the antibody having an amino acid sequence of a non-human animal antibody and an amino acid sequence of a human-derived antibody include a human chimeric antibody and a humanized antibody. In the above, the "chimeric antibody" is an antibody derived from a non-human animal and genetically modified so that the constant region of an antibody that specifically binds to RAMP2 has the same constant region as a human antibody. Yes, preferably a human mouse chimeric antibody (see EP0125023). The "humanized antibody" is a primary structure other than the complementary recognition region (CDR) of the H chain and the L chain of an antibody derived from a non-human animal that specifically binds to RAMP2, and has a primary structure corresponding to the human antibody. It is a genetically modified antibody. Here, the CDR is defined as Kabat et al. (“Definence of Proteins of Immunological Interest”, Kabat, E. et al., US Department of Health and Human Services, 1983) or Cha (1983) or Cho. It may be based on any definition of Biol., 196: 901-917). A "human antibody" is a human antibody that is an expression product of a completely human-derived antibody gene. For example, a monoclonal antibody produced using a transgenic animal into which a gene involved in human antibody production has been introduced ( (See European Patent Publication Publication EP0546073) and the like. For example, when the antibody of the present invention is used for diagnosis by treating, preventing, or administering it into the body, the antibody of the present invention is preferably a human / non-human animal chimeric antibody, a humanized antibody, or a human. It is an antibody. Also, preferably, the antibody of the present invention is an antibody that does not exist in nature.

The immunoglobulin class of the antibody of the present invention is not particularly limited, and may be any immunoglobulin class (isotype) of IgG, IgM, IgA, IgE, IgD, or IgY, and IgG is preferable. When the antibody of the present invention is IgG, it may be in any subclass (IgG1, IgG2, IgG3, or IgG4). Further, the antibody of the present invention is monospecific, bispecific (bispecific antibody), trispecific (trispecific antibody) (for example, WO1991 / 003493), or multispecific (multispecific antibody). May be good.

It is widely known to those skilled in the art that an antibody is endowed with binding properties in its variable regions (particularly CDRs), and that the binding properties can be utilized even in an antibody fragment that is not a complete antibody. Has been done. In the present specification, the "immune-reactive fragment" is a protein or peptide containing a part (partial fragment) of an antibody, and is a protein or peptide that retains the action (immune-reactivity / binding) of the antibody on an antigen. Means. Examples of such immunoreactive fragments include F (ab') ₂ , Fab', Fab, Fab ₃ , single-chain Fv (hereinafter referred to as “scFv”), and (tandem) bispecific single-chain Fv ( sc (Fv) ₂ ), Single Chain Triple Body, Nanobody, Diverent VHH, Pentavalent VHH, Minibody, (Double Chain) Diabody, Tandem Diabody, Bispecific Tribody, Bispecific Bibody, Dual Affinity Targeting molecule (DART), Tribody (or Tribody), Tetrabody (or [sc (Fv) ₂ ] ₂ ), or (scFv-SA) ₄ ) Disulfide-bound Fv (hereinafter referred to as "dsFv"), compact IgG, Heavy chain antibodies, or polymers thereof, can be mentioned (Nature Biotechnology, 29 (1): 5-6 (2011); Manesh Jean et al., TRENDS in Biotechnology, 25 (7) (2007): 307-. 316; and Christoph stain et al., Antibodies (1): 88-123 (2012)). As used herein, the immunoreactive fragment may be monospecific, bispecific, trispecific, or multispecific. As used herein, the term "antibody" is intended to also include immunoreactive fragments of the antibody, except where such interpretation is inconsistent.

In one aspect, the invention presents the invention 25H4-4F9 antibody, 25H4-4F9v1 antibody, 25H4-4F9v2 antibody, 25H4-4F9v3 antibody, 25H4-4F9v4 antibody, 25H4-4F9v5 antibody, 25H4-4F9v6 antibody, 25H4-4F9v8 antibody, 85H7-. 2B11 antibody, 85H7-2B11v1 antibody, 85H7-2B11v2 antibody, 85H7-2B11v3 antibody, 85H7-2B11v4 antibody, 85H7-2B11v5 antibody, 85H7-2B11v6 antibody, 85H7-2B11v7 antibody, 85H7-2B11v8 antibody, or 33H4-1G3 antibody. Each of these antibodies has a heavy chain (HC) and a light chain (LC) consisting of the amino acid sequences set forth in the SEQ ID NOs shown in Table 2.

In one aspect, the antibody of the invention specifically recognizes the three-dimensional structure of the extracellular region within RAMP2 on the cell surface. For example, the antibody of the present invention is 25H4-4F9 antibody, 25H4-4F9v1 antibody, 25H4-4F9v2 antibody, 25H4-4F9v3 antibody, 25H4-4F9v4 antibody, 25H4-4F9v5 antibody, 25H4-4F9v6 antibody, 25H4-4F9v8 antibody, 85H7-. 2B11 antibody, 85H7-2B11v1 antibody, 85H7-2B11v2 antibody, 85H7-2B11v3 antibody, 85H7-2B11v4 antibody, 85H7-2B11v5 antibody, 85H7-2B11v6 antibody, 85H7-2B11v7 antibody, 85H7-2B11v8 antibody, or 33H4-1G3 antibody Specific recognition of an epitope that may be present in RAMP2 on the cell surface.

In addition, the antibody of the present invention does not recognize the primary structure of the amino acid sequence constituting the extracellular region of RAMP2 as an epitope. Here, not recognizing the primary structure of the amino acid sequence as an epitope means that it does not bind to a peptide or protein consisting of the linear amino acid sequence that is not held (does not form a three-dimensional structure). More specifically, the antibody of the present invention does not bind to any peptide having each amino acid sequence set forth in SEQ ID NOs: 71 to 101, which is an amino acid sequence constituting the extracellular region of RAMP2.

In the present specification, whether or not the test antibody "specifically binds (recognizes)" to an epitope of interest (for example, an epitope to which the above-mentioned specific antibody binds), an antigen, or a peptide is determined. It can be determined by examining the binding of the antibody to the peptide or protein, the antigen, or the peptide, and the binding of the antibody to the other peptide, protein, or other antigen. In particular, since the antibody of the present invention specifically recognizes the three-dimensional structure of the extracellular region in RAMP2 on the cell surface, RAMP2 expressed on the cell surface is used as the protein having the epitope in the above method. Therefore, the binding specificity can be determined. If it binds to a peptide or protein of interest, an antigen of interest, or a peptide of interest but not to another peptide, protein, or other antigen, the test antibody is associated with the epitope, antigen, or peptide of interest. It is determined to specifically bind (recognize).

Further, in the present specification, it can be determined that the test antibody does not bind to a specific peptide or protein by examining the binding of the antibody to the peptide or protein.

In another embodiment, the antibodies of the invention are 25H4-4F9 antibody, 25H4-4F9v1 antibody, 25H4-4F9v2 antibody, 25H4-4F9v3 antibody, 25H4-4F9v4 antibody, 25H4-4F9v5 antibody, 25H4-4F9v6 antibody, 25H4-4F9v8 antibody. , 85H7-2B11 antibody, 85H7-2B11v1 antibody, 85H7-2B11v2 antibody, 85H7-2B11v3 antibody, 85H7-2B11v4 antibody, 85H7-2B11v5 antibody, 85H7-2B11v6 antibody, 85H7-2B11v7 antibody, 85H7-2B11v8 antibody, or 33H4-1G It competes with an antibody (referred to as "25H4-4F9 antibody, etc." in this paragraph) for binding to RAMP2 (particularly RAMP2 on the cell surface). Whether or not the test antibody competes with the 25H4-4F9 antibody or the like in binding to RAMP2 is determined by contacting the labeled 25H4-4F9 antibody or the like with the antigen (RAMP2) together with the test antibody. Can be done. Preferably, as a control, one in which a labeled 25H4-4F9 antibody or the like is independently contacted with the antigen (RAMP2) in the absence of the test antibody can be used. Comparing the labeling amounts of the labeled 25H4-4F9 antibody and the like bound to the antigen, the amount of the labeled 25H4-4F9 antibody and the like bound to the antigen when contacted with the antigen at the same time as the test antibody is absent from the test antibody. If it is less than the amount of the labeled 25H4-4F9 antibody or the like bound to the antigen when contacted with the antigen below, it can be determined that the test antibody competes with the 25H4-4F9 antibody or the like used.

Methods for measuring the binding of an antibody to a protein or peptide include, for example, EIA method, ELISA method, FACS method, co-immunoprecipitation method, pull-down assay method, far western blotting method, homobifunctional crosslinker or heterobifunctional cross. AlphaPPI assay such as cross-linking method using linker, label transfer reaction method, interaction mapping method, surface plasmon resonance method, FRET (Fluororescience resonance assay) method, BIACORE method, AlphaScreen (registered trademark) and AlphaLISA (registered trademark). Various methods such as (PerkinElmer) are well known in the art, and a preferred method can be appropriately adopted depending on the detection of the desired bond.

In one aspect, the invention describes any one in which the VH is selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. It has an amino acid sequence encoded by a nucleic acid sequence that hybridizes with a nucleic acid sequence encoding an amino acid sequence, and has a VL of SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, An antibody having an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence encoding the amino acid sequence described in any one selected from 55, 57, 59, and 61, or an antibody thereof. Regarding immunoreactive fragments. As used herein, hybridizing under stringent conditions means hybridizing under hybridization conditions commonly used by those skilled in the art. For example, it can be determined whether or not to hybridize by the method described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989). For example, the conditions for hybridization are 6 × SSC (0.9M NaCl, 0.09M trisodium citrate) or 6 × SSPE (3M NaCl, 0.2M NaH ₂ PO ₄ , 20 mM EDTA · 2Na, pH 7.4). The conditions may be such that the mixture is hybridized at medium 42 ° C. and then washed with 0.5 × SSC at 42 ° C. Preferably, the present invention comprises a nucleic acid sequence in which VH encodes the amino acid sequence set forth in SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, or 53. It has an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions, and the VL is, in order, SEQ ID NO: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, respectively. Alternatively, it is an antibody or an immunoreactive fragment thereof having an amino acid sequence encoded by a nucleic acid sequence that hybridizes with a nucleic acid sequence encoding the amino acid sequence described in 61 under stringent conditions.

Also in the present invention, VH is 80% with any one amino acid sequence selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. Any one amino acid having an amino acid sequence having the above identity and having a VL selected from SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61. With respect to an antibody having an amino acid sequence having 80% or more identity with the sequence or an immunoreactive fragment thereof. Amino acid sequence identity means the ratio (%) of the number of amino acids of the same type in the amino acid sequence range to be compared between two types of proteins, for example, using a known program such as BLAST or FASTA. Can be decided. As the antibody of the present invention, for example, VH is any one amino acid sequence selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. Has an amino acid sequence having 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity, and the VL is SEQ ID NO: 4, 14, 21, 37, 39, 41. , 43, 55, 57, 59, and 61, and an amino acid sequence having 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity with any one of the amino acid sequences. It may be an antibody having or an immunoreactive fragment thereof. Preferably, the antibody of the present invention has any one amino acid sequence in which VH is selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. And 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more of the amino acid sequences having the same identity, and VL, in that order, are SEQ ID NOs: 4, 14, Any one amino acid sequence selected from 21, 37, 39, 41, 43, 55, 57, 59, and 61 and 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or An antibody having an amino acid sequence having 99% or more identity or an immunoreactive fragment thereof.

Preferably, the present invention relates to the heavy chain (VC) and light chain (LC) amino acid sequences set forth in SEQ ID NOs: Table 2 of the present specification, or the VH and VL amino acid sequences, or CDRH1 to 3. It is an antibody having a combination of amino acid sequences of CDRL1 to 3.

The above-mentioned VH and VL have an amino acid sequence encoded by a nucleic acid sequence that hybridizes with a nucleic acid sequence encoding a specific amino acid sequence under stringent conditions, or an immunoreactive fragment thereof, and VH and VL. , An antibody having an amino acid sequence having 80% or more identity with a specific amino acid sequence or an immunoreactive fragment thereof preferably has the CDRHs and CDRLs described below.

In one aspect, the invention is an antibody comprising a heavy chain having heavy chain complementarity determining regions (CDRH) 3 consisting of the amino acid sequences of LX ₁ IX ₂ DX ₃ YX ₄ YX ₅ MX ₆ X ₇ (SEQ ID NO: 24). Regarding its immunoreactive fragments. Here, X ₁ is G or M, X ₂ is F or Y, X ₃ is A or V, X ₄ is absent or H, X ₅ is A or V, and X. ₆ is D or E and X ₇ is Y or N. Preferably, the present invention is an antibody or immunoreactive fragment thereof comprising a heavy chain having 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 in determining 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).

Preferably, the antibody of the invention or an immunoreactive fragment thereof has CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5).
Also, preferably, the antibody of the present invention or an immunoreactive fragment thereof is derived from the amino acid sequence of RX ₈ NPYNGDX ₉ X ₁₀ YX ₁₁ QKFX ₁₂ G (SEQ ID NO: 66) or RX ₈ NPYNGDX ₉ X ₁₀ YNQKFKG (SEQ ID NO: 23). It has CDRH2. Here, X ₈ is N or I, X ₉ is S or T, X ₁₀ is I, F or L, X ₁₁ is A or N, and X ₁₂ is Q or K. CDRH2 preferably, 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 It has the amino acid sequence of (SEQ ID NO: 65).

More preferably, the antibody of the invention or an immunoreactive fragment thereof has a light chain complementarity determining region (CDRL) 1 consisting of the amino acid sequence of X ₁₃ ASQDIRNYLN (SEQ ID NO: 69). Here, X ₁₃ is Q or R. CDRL1 preferably has the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8) or QASQDIRNYLN (SEQ ID NO: 67).

Further, the antibody of the present invention or an immunoreactive fragment thereof has CDRL2 consisting of the amino acid sequence of YTSRLX ₁₄ X ₁₅ (SEQ ID NO: 70). Here, X ₁₄ is E or H, and X ₁₅ is S or T. CDRL2 preferably has the amino acid sequence of YTSRLHS (SEQ ID NO: 9) or YTSRLET (SEQ ID NO: 68).

In addition, the antibody of the present invention or an immunoreactive fragment thereof has CDRL3 consisting of the amino acid sequence of QQDSKKX ₁₆ PWT (SEQ ID NO: 25). Here, X ₁₆ is H or N. CDRL3 preferably has the amino acid sequence of QQDSKHPWT (SEQ ID NO: 10) or QQDSKNPWT (SEQ ID NO: 17).

In one example, the antibody of the invention has a combination of heavy chain CDRs of any one of (i)-(vii) below:
(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 of SEQ ID NO: 65, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 16;
(Vii) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence of RINGYNGDTLYNQKFKG (SEQ ID NO: 22), and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 16.

Furthermore, the antibody of the present invention may have a combination of any one of the following light chain CDRs (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;
(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;
(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.

For example, the present invention includes an antibody having the following combination of CDRs or an immunoreactive fragment thereof:

In the present specification, amino acids are represented by one letter notation. Specifically, A is alanine, L is leucine, R is arginine, K is lycine, N is aspartic acid, M is methionine, D is aspartic acid, F is phenylalanine, C is cysteine, P is proline, and Q is glutamine. S stands for serine, E stands for glutamine, T stands for methionine, G stands for glycine, W stands for tryptophan, H stands for histidine, Y stands for tyrosine, I stands for isoleucine, and V stands for valine.

The antibody of the present invention and an immunoreactive fragment thereof have an inhibitory effect on the growth of vascular endothelial cells in an in vitro adrenomedulin-added vascular endothelial cell growth test. Whether or not the test antibody or its immunoreactive fragment has an inhibitory effect on the proliferation of vascular endothelial cells in an in vitro adrenomedulin-added vascular endothelial cell proliferation test was pre-cultured in an appropriate medium for several hours. By adding the test antibody or an immunoreactive fragment thereof to the vascular endothelial cells, adding AM to a final concentration of 50 to 200 nM, and observing the proliferation of the vascular endothelial cells after culturing for 2 to 7 days. It can be carried out. For the proliferation of vascular endothelial cells, the number of cells may be counted, or OD450 or the like, which is an index of the number of cells, may be measured. When the number of vascular endothelial cells cultured in the presence of the test antibody or its immunoreactive fragment is smaller than the number of vascular endothelial cells cultured without the addition of the test antibody or its immunoreactive fragment, the subject is concerned. The test antibody or an immunoreactive fragment thereof is determined to have an inhibitory effect on the proliferation of vascular endothelial cells in an in vitro adrenomedulin addition-induced vascular endothelial cell proliferation test. Further, the number of vascular endothelial cells cultured without adding the test antibody or its immunoreactive fragment is 100, and the number of vascular endothelial cells cultured in the presence of the test antibody or its immunoreactive fragment is 50. The concentration of the test antibody or an immunoreactive fragment thereof may be determined as an IC50 value (M). For example, the antibody of the present invention has an IC50 value of 35 nM or less, 30 nM or less, 25 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1.6 nM or less. Good. The lower limit of the IC50 value does not need to be set in particular, but 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 0.8 nM or more. The IC50 value may be in the range of any combination of these upper limit values and lower limit values, and is, for example, 0.1 to 35 nM, 0.8 to 32.7 nM, 0.2 to 30 nM, 0. It may be 3 to 25 nM, 0.4 to 20 nM, 0.5 to 15 nM, 0.5 to 10 nM, 0.5 to 5 nM, 0.8 to 1.6 nM. Further, in the case of these IC50 values, even if it is determined that the test antibody or its immunoreactive fragment has a vascular endothelial cell growth inhibitory effect in a vascular endothelial cell growth test by addition of in vitro adrenomedulin. Good.

Preferably, the antibody of the present invention or an immunoreactive fragment thereof is used in an in vitro adrenomedulin-added vascular endothelial cell growth test in which cAMP by animal cells by adrenomedulin addition is added to the growth inhibitory effect of vascular endothelial cells. It has the effect of suppressing production. Whether or not the test antibody or an immunoreactive fragment thereof has an effect of suppressing cAMP production by animal cells due to adrenomedulin addition is determined by animal cells that produce cAMP by adrenomedulin addition (for example, hCR2-CHOK1 cells). Can be determined by culturing overnight in a suitable medium, adding the test antibody or immunoreactive fragment thereof, adding AM at a final concentration of 50-300 pM, and measuring cAMP. The cAMP can be measured using a commercially available kit (for example, LANCE Ultra cAMP kit (PerkinElmer), etc.). If the cAMP production by the test antibody or its immunoreactive fragment-added cells is lower than that of the antibody-free cells, the antibody or its immunoreactive fragment is produced by the animal cells by adrenomedulin addition. It is determined that it has an effect of suppressing cAMP production. Further, the cAMP produced by the cells cultured without adding the test antibody or the immunoreactive fragment thereof is 100, and the cAMP produced by the cells cultured in the presence of the test antibody or the immunoreactive fragment thereof is 50. The concentration of the test antibody or an immunoreactive fragment thereof may be determined as an IC50 value (M). For example, the antibody of the present invention has an IC50 value of 2 nM or less, 1.5 nM or less, 1 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.15 nM or less. You may. The lower limit of the IC50 value does not have to be set in particular, but may be, for example, 0.01 nM or more, 0.02 nM or more, or 0.03 nM or more. Further, the IC50 value may be in the range of any combination of these upper limit values and lower limit values, and for example, 0.01 to 2 nM, 0.01 to 1.5 nM, 0.01 to 1 nM, 0. It may be 01 to 0.5 nM, 0.03 to 1.35 nM, or 0.03 to 0.15 nM. Further, in the case of these IC50 values, it may be determined that the test antibody or an immunoreactive fragment thereof has an effect of suppressing cAMP production by animal cells due to the addition of adrenomedulin.

Preferably, the antibody of the invention or an immunoreactive fragment thereof binds to cells that co-express human CRLR and human RAMP2 in vitro. In the present specification, the "cell co-expressing human CRLR and human RAMP2" is a stable expression cell obtained by introducing a cell gene into human CRLR and human RAMP2, and is a cell co-expressing human CRLR and human RAMP2. If there is, it is not particularly limited, but preferably, a stable expression cell line (hCR2-CHOK1 or hCR2-) obtained by gene-introducing human CRLR and human RAMP2 into hamster ovary cells (CHO-K1) or NIH3T3 cells. NIH3T3). A cell co-expressing human CRLR and human RAMP2 can be obtained by incorporating human CRLR (NM_005795) and human RAMP2 (NM_005854) into a vector for gene transfer in tandem and introducing the gene into a target cell. Stable expression strains can be obtained by selective culturing in the presence of a drug corresponding to the drug resistance gene previously inserted into the vector.

For example, the antibody of the present invention may have a binding affinity (EC50) of 3.5 nM or less according to a Cell ELISA test using cells co-expressing human CRLR and human RAMP2. For example, when the immunoglobulin class of the test antibody is IgG, the Cell ELISA test can be performed by the following method. Cells co-expressing human CRLR and human RAMP2 in a suitable medium were added to a 96-well culture plate at 1 × 10 ³ to 1 × 10 ⁵ cells / well and cultured overnight, followed by the addition of 4% paraformaldehyde solution. The cells are fixed by allowing them to stand at 4 ° C. for several tens of minutes, washed, and then blocked with 1% BSA-PBS for 1 hour or longer. Each purified test antibody is appropriately diluted and added, a labeled anti-IgG antibody is reacted as a secondary antibody, a reagent matching the label is reacted, and the luminescence intensity and color development intensity of the label are measured. The amount of binding can be determined by. The EC50 value can be determined as the antibody concentration that binds to 50% of the maximum binding amount. For example, the antibody of the present invention has an EC50 value of 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. There may be. The lower limit of the EC50 value does not need to be set in particular, 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 in the range of any combination of these upper limit values and lower limit values, for example, 0.01 to 4 nM, 0.03 to 3 nM, 0.05 to 2 nM, or 0.14. It may be ~ 3.19 nM. Further, in the case of these EC50 values, it may be determined that the test antibody or an immunoreactive fragment thereof binds to cells co-expressing human CRLR and human RAMP2 in vitro.

(Nucleic acid molecule)
In another aspect, the invention relates to a nucleic acid molecule having a polynucleotide encoding an antibody of the invention or an immunoreactive fragment thereof described above. Specifically, the nucleic acid molecule of the present invention is any one selected as VH from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. Any one having a polynucleotide encoding the amino acid according to one and selected as VL from SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61. It may be a nucleic acid molecule having a polynucleotide encoding the amino acid sequence described in 1. For example, the nucleic acid molecule of the present invention is a nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2 as VH and SEQ ID NO: 4 as VL; the amino acid set forth in SEQ ID NO: 27 as VH and SEQ ID NO: 37 as VL. Nucleic acid molecule having a polynucleotide encoding a sequence; Nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 29 as VH and SEQ ID NO: 37 as VL; SEQ ID NO: 29 as VH and SEQ ID NO: 39 as VL. Nucleic acid molecule having a polynucleotide encoding the amino acid sequence of SEQ ID NO: 31 as VH and Nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 39 as VH; SEQ ID NO: 31 as VH and SEQ ID NO: 41 as VL. Nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in the above; Nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 33 as VH and SEQ ID NO: 41 as VL; Sequence as SEQ ID NO: 35 and VL as VH Nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in No. 43; Nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 12 as VH and SEQ ID NO: 14 as VL; SEQ ID NO: 45 and VL as VH. Nucleic acid molecule having the polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 55; Nucleic acid molecule having the polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 12 as VH and SEQ ID NO: 14 as VL; And a nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 55 as VL; a nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 47 as VH and SEQ ID NO: 57 as VL; sequence as VH. Nucleic acid molecule having the polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 57 as numbers 49 and VL; nucleic acid molecule having the polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 49 and SEQ ID NO: 59 as VH; VH Nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 59 as SEQ ID NO: 51 and VL; nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 51 as VH and SEQ ID NO: 61 as VL. Allocation as VH with SEQ ID NO: 53 and VL A nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in column number 61; and a nucleic acid molecule having a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 19 as VH and SEQ ID NO: 21 as VL. ..

For example, the nucleic acid molecule of the present invention may contain a polynucleotide having the following sequence: the nucleotide sequence set forth in SEQ ID NO: 1 and the nucleotide sequence set forth in SEQ ID NO: 3; the nucleotide sequence set forth in SEQ ID NO: 26. , And the nucleotide sequence set forth in SEQ ID NO: 36; the nucleotide sequence set forth in SEQ ID NO: 28, and the nucleotide sequence set forth in SEQ ID NO: 36; the nucleotide sequence set forth in SEQ ID NO: 28, and the nucleotide sequence set forth in SEQ ID NO: 38; The 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. Nucleotide sequence and nucleotide sequence set forth in SEQ ID NO: 54; nucleotide sequence set forth in SEQ ID NO: 46 and nucleotide sequence set forth in SEQ ID NO: 54; nucleotide sequence set forth in SEQ ID NO: 46, and nucleotide sequence set forth in SEQ ID NO: 56. The nucleotide sequence set forth in SEQ ID NO: 48 and the nucleotide sequence set forth in SEQ ID NO: 56; the nucleotide sequence set forth in SEQ ID NO: 48 and the nucleotide sequence set forth in SEQ ID NO: 58; the nucleotide sequence set forth in SEQ ID NO: 50 and the sequence. The nucleotide sequence set forth in No. 58; the nucleotide sequence set forth in SEQ ID NO: 50 and the nucleotide sequence set forth in SEQ ID NO: 60; the nucleotide sequence set forth in SEQ ID NO: 52 and the nucleotide sequence set forth in SEQ ID NO: 60; or the nucleotide sequence set forth in SEQ ID NO: 18; The nucleotide sequence of SEQ ID NO: 20 and the nucleotide sequence of SEQ ID NO: 20.

Furthermore, the present invention includes a vector having the nucleic acid molecule. Such a vector is not particularly limited as long as it is a vector that can be used for antibody expression, and can be selected depending on the host using an appropriate viral vector, plasmid vector, or the like. In another aspect, the invention relates to a host cell containing the vector. The host cell is not particularly limited as long as it is a host cell that can be used for antibody expression, and includes mammalian cells (mouse cells, rat cells, rabbit cells, human cells, etc.), yeasts, and microorganisms (Escherichia coli, etc.). Can be mentioned.

In another aspect, the present invention relates to a pharmaceutical composition containing the above-mentioned antibody of the present invention or an immunoreactive fragment thereof as an active ingredient. The target disease of the pharmaceutical composition of the present invention is a disease or disorder in which angiogenesis contributes to the onset or exacerbation, for example, cancer, cancer metastasis, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, and the like. Examples include diabetic retinopathy, retinal vein occlusion, angiogenic glaucoma, inflammatory skin disease, rheumatoid arthritis, or degenerative arthritis. Therefore, the pharmaceutical composition of the present invention can be a preventive agent, a therapeutic agent, a progression inhibitor or an ameliorating agent for these diseases or disorders. Alternatively, the present invention relates to the use of the antibody of the present invention or an immunoreactive fragment thereof for producing the pharmaceutical composition.

The pharmaceutical composition of the present invention may be any oral or parenteral preparation as long as it can be administered to a patient. Examples of the composition for parenteral administration include injections, nasal drops, suppositories, patches, ointments and the like. An injection is preferred. Dosage forms of the pharmaceutical composition of the present invention include, for example, liquid preparations and lyophilized preparations. When the pharmaceutical composition of the present invention is used as an injection, if necessary, a solubilizing agent such as propylene glycol and ethylenediamine, a buffer material such as phosphate, an isotonic agent such as sodium chloride and glycerin, and a sulfite. Additives such as stabilizers such as stabilizers, preservatives such as phenol, and painkillers such as lidocaine (see "Pharmaceutical Additives Dictionary" Yakuji Nippo Co., Ltd., "Handbook of Pharmaceutical Expiments Fifth Edition" APhA Publications Co., Ltd.) .. When the pharmaceutical composition of the present invention is used as an injection, examples of the storage container include ampoules, vials, prefilled syringes, pen-type syringe cartridges, and drip bags.

(Acquisition of antibody)
The antibody of the present invention uses cells expressing human CRLR and human RAMP2 as an immunogen, and if necessary, an immunostimulant (for example, mineral oil or aluminum precipitate and heat-killed bacteria or lipopolysaccharide, complete adjuvant of Freund). , Or, with Freund's incomplete adjuvant, etc.), it can be produced by immunizing a non-human animal that produces an immune response. The immune animal is not particularly limited as long as it is an animal capable of producing hybridomas such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, sheep, goats, chickens and ducks, but mice or animals are preferable. It is a rat, more preferably a mouse, and most preferably an MRL / lpr mouse. The cell that serves as the immunogen is preferably a cell of the same species as the immune animal because it suppresses the production of antibodies to antigens other than the target antigen. The immunogen is administered to animals once, for example, 1 × 10 ⁶ cells or several times at appropriate intervals (usually once every 1 to 6 weeks for a total of 2 to 10 injections) subcutaneously. It can be done by injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection or footpad injection. One to two weeks after the last immunization, blood is collected from the orbit or tail vein of the immunized animal, and the antibody titer is measured using the serum. The antibody of the present invention can be obtained by purifying from the serum of an animal showing a sufficient antibody titer.

Monoclonal antibodies can be obtained by culturing hybridomas obtained by fusing antibody-producing cells obtained from immunosensitizers immunized by the above method with myeloma cells (myeloma cells). Examples of the fusion method include the method of Milstain et al. (Galfre, G. & Milstein, C. (1981) Methods Enzymol., 73: 3-46). The antibody-producing cells used can be collected from the spleen, pancreas, lymph nodes, and peripheral blood of a mouse or rat that has been immunized by the above method and whose serum has a sufficient antibody titer. The myeloma cells used are, for example, cells derived from mammals such as mice, rats, guinea pigs, hamsters, rabbits and humans, and are not particularly limited as long as they are cells capable of in vitro proliferation. Examples of such cells include P3-X63Ag8 (X63) (Nature, 256,495,1975) and P3 / NS1 / 1-1-Ag4-1 (NS1) (Eur. J. Immunol., 6, 292, 1976). ), P3X63Ag8U1 (P3U1) (Curr. Top. Microbiol. Immunol., 81, 1, 1978), P3X63Ag 8.653 (653) (J. Immunol., 123, 1548, 1979), Sp2 / 0-Ag14 (Sp2 / O) (Nature, 276,269,1978), Sp2 / O / FO-2 (FO-2) (J. Immunol. Methods, 35,1,1980), SP2ab and the like can be mentioned, preferably an antibody. It is a cell derived from an allogeneic animal as the producing cell, and more preferably a cell derived from an animal of the same lineage as the antibody producing cell.

After culturing, the culture supernatant is collected, and a clone that binds to RAMP2 is selected by Cell ELISA using cells co-expressing human CRLR and human RAMP2 (for example, hCR2-CHOK1 cells). The selected clone is monocellularized by repeating the limiting dilution method 1 to 5 times, and among the antibodies produced by the monocellularized clone, the candidate antibody selected by Cell ELISA is further selected. , A signal-inhibiting antibody is screened based on whether or not the increase in intracellular cAMP of cells co-expressing human CRLR and human RAMP2 (for example, hCR2-CHOK1 cells) due to the addition of AM is suppressed. As a biological activity inhibition test, an antibody capable of inhibiting the growth of HUVEC by adding AM can be screened for the selected signal-inhibiting antibody, and an anti-RAMP2 antibody having a cAMP inhibitory effect and an HUVEC growth inhibitory effect can be obtained.

The obtained antibody can be purified to a uniform level. For the separation and purification of the antibody, the separation and purification methods used for ordinary proteins can be used. For example, an antibody can be separated and purified by appropriately selecting and combining column chromatography such as affinity chromatography, filter, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and the like. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Examples of the column used for affinity chromatography include a protein A column and a protein G column. Further, regardless of the antibody class, RAMP2 (or human CRLR and human RAMP2) solid-phase column, ion exchange chromatography, hydrophobic interaction chromatography and the like can also be used.

Alternatively, for example, an antibody that binds to RAMP2 can be obtained by using an antibody phage library (Tomitsuka et al., Nature Genet., 15, 146-156 (1997)). When an antibody phage library is used, for example, cells expressing human CRLR and human RAMP2 are immobilized on a solid phase, the phage antibody library is reacted, unbound phages are washed and removed, and then bound. The desired clone can be obtained by collecting the phage (panning).

Alternatively, with reference to the amino acid sequence described herein, the amino acid sequence of the antibody of interest or an immunoreactive fragment thereof is designed, DNA encoding the designed amino acid sequence is prepared, and incorporated into an expression vector. It can be obtained by screening an antibody having high specificity for RAMP2 according to the above-mentioned method from the antibody obtained by introducing the vector into an appropriate host cell and expressing it.

(Production of human chimeric antibody)
When the antibody of the present invention is a human chimeric antibody, DNA encoding VH and VL of a non-human animal monoclonal antibody that specifically recognizes RAMP2 is prepared, and this is expressed by binding to the constant region cDNA of human immunoglobulin. It can be obtained by incorporating the vector into a vector and introducing the vector into an appropriate host cell for expression (Morrison, S.L. et al., Proc. Natl. Acad. Sci. USA, 81, 6851-6855, 1984). ..

(Production of humanized antibody)
When the antibody of the present invention is a humanized antibody, the amino acid sequence encoding the CDR of VH and VL of a non-human animal monoclonal antibody that specifically recognizes RAMP2 is transplanted into the Flamework Region (FR) of VH and VL of human antibody. It can be obtained by constructing a DNA encoding the V region, binding the constructed DNA to the constant region cDNA of a human-derived immunoglobulin, incorporating the vector into an expression vector, and introducing the vector into an appropriate host cell for expression. Can (L. Riohmann et al., Nature, 332, 323, 1988: Kettreborough, CA et al., Protein Eng., 4,773-783, 1991; Clark M., Immunol. Today., 21, 397-402, See 2000). The CDRs of the non-human animal monoclonal antibody are the amino acid sequences predicted from the DNA sequences encoding the VH and VL of the non-human animal monoclonal antibody obtained by the above method, and the total amino acid sequences of the VH and VL of the known antibody. Can be obtained by comparison. The amino acid sequence of a known antibody can be obtained from, for example, the amino acid sequence of an antibody registered in a database such as a protein data bank. The FR of the humanized antibody is not particularly limited as long as the antibody after transplantation exerts the effect of the present invention, but preferably, the variable region of the humanized antibody (hereinafter referred to as "V region") is used. The FR of a human antibody having a three-dimensional structure similar to the V region of the non-human animal monoclonal antibody from which the CDR is derived, or the human antibody FR having high identity with the amino acid sequence of the FR of the non-human animal monoclonal antibody used. In humanized antibodies, some of the amino acids that make up FR derived from human antibodies (particularly amino acids that are sterically located close to the CDR) are non-human animal monoclonal antibodies from which the CDR is derived, if necessary. It may be substituted with the FR sequence of (see Queen et al., US Pat. No. 5,585,089). The DNA sequence encoding the V region of the humanized antibody to be used is designed as a DNA sequence corresponding to the amino acid sequence in which the CDR amino acid sequence of the non-human animal monoclonal antibody and the FR amino acid sequence of the human antibody are linked. The DNA encoding the V region of the humanized antibody can be prepared by a method well known to those skilled in the art based on the designed DNA sequence.

(Human antibody)
Human antibodies can be obtained, for example, by using human antibody phage libraries or human antibody-producing transgenic mice (Tomitsuka et al., Nature Genet., 15, 146-156 (1997)). When using a human antibody phage library, for example, cells expressing human CRLR and human RAMP2 are immobilized on a solid phase, the phage antibody library is reacted, unbound phages are washed and removed, and then binding is performed. The desired clone can be obtained by collecting the obtained phage (panning). The human antibody-producing transgenic mouse is a mouse in which the Ig gene of a human antibody has been introduced into a mouse in which the endogenous immunoglobulin (Ig) gene has been knocked out. Using a human antibody-producing transgenic mouse as an immune animal, RAMP2 is specifically immunized with an antigen (preferably a cell expressing human CRLR and human RAMP2) according to the antibody production method of the present invention described above. A human antibody to be recognized can be obtained.

(Nucleic acid, vector, host cell)
The nucleic acid of the present invention can be cloned from a hybridoma producing the antibody obtained above, or by appropriately designing a nucleic acid sequence based on the amino acid sequence of the antibody obtained above or an immunoreactive fragment thereof. Obtainable. The vector of the present invention can be obtained by appropriately incorporating the obtained nucleic acid into a vector suitable for expression. In addition to the nucleic acid of the present invention, the vector of the present invention may contain regions necessary for expression (promoter, enhancer, terminator, etc.). In addition, the host cell of the present invention can be obtained by introducing the vector of the present invention into an appropriate cell line (for example, animal cells, insect cells, plant cells, microorganisms such as yeast and Escherichia coli).

(Pharmaceutical composition (therapeutic agent, preventive agent) and therapeutic method)
The antibody of the present invention or an immunoreactive fragment thereof can be used as a pharmaceutical composition by purifying it if necessary and then formulating it according to a conventional method. The present invention also includes the use of the antibody of the present invention or an immunoreactive fragment thereof for producing a pharmaceutical composition. Alternatively, the present invention includes the use of the antibody of the present invention or an immunoreactive fragment thereof for the treatment or prevention of a disease or disorder in which angiogenesis contributes to the onset or exacerbation of angiogenesis. Furthermore, the present invention is a method for treating a disease or disorder in which angiogenesis contributes to the onset or exacerbation of angiogenesis, which comprises administering an effective amount of the antibody of the present invention or an immunoreactive fragment thereof to a patient in need thereof. Regarding preventive methods.

For example, the pharmaceutical composition (therapeutic agent or prophylactic agent) of the present invention can be used as an injection, and can be used as an intravenous injection, a subcutaneous injection, an intradermal injection, an intramuscular injection, an intravitreal injection, or an infusion. Includes dosage forms such as agents. Such an injection can be prepared according to a known method, for example, by dissolving, suspending or emulsifying the above antibody or the like in a sterile aqueous or oily solution usually used for an injection. As the aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose, sucrose, mannitol, and other auxiliary agents can be used, and suitable solubilizing agents such as alcohol (eg, ethanol) can be used. ), Polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80, polysorbate 20, HCO-50 (polyoxyethylene (50 mol) added of hydride detergent oil)] and the like can be added. .. As the oily liquid, for example, sesame oil, soybean oil and the like can be used, and benzyl benzoate, benzyl alcohol and the like can be added as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule, vial or syringe. In addition, a lyophilized preparation is prepared by adding an appropriate excipient to the antibody of the present invention or an immunoreactive fragment thereof, and dissolved in water for injection, physiological saline, etc. at the time of use to prepare an injection solution. You can also. In general, oral administration of proteins such as antibodies is difficult because they are decomposed by the digestive organs, but oral administration may be possible depending on the ingenuity of antibody fragments, modified antibody fragments and dosage forms. Examples of the preparation for oral administration include capsules, tablets, syrups, granules and the like.

The pharmaceutical composition of the present invention is preferably prepared in a dosage form of a dosage unit suitable for the dose of the active ingredient. Examples of the dosage form of such a dosage unit include injections (ampoules, vials, prefilled syringes), and usually 5 to 500 mg, 5 to 100 mg, 10 to 250 mg of the antibody of the present invention or immunity thereof per dosage unit dosage form. It may contain a reactive fragment.

Administration of the antibody or pharmaceutical composition (therapeutic or prophylactic agent) of the present invention may be local or systemic. The administration method is not particularly limited, and is administered parenterally or orally as described above. Parenteral routes of administration include intraocular, subcutaneous, intraperitoneal, blood (intravenous or intraarterial) or cerebrospinal fluid injections or infusions, preferably intraocular or blood. Is. The pharmaceutical composition (therapeutic agent or prophylactic agent) of the present invention may be administered temporarily, or may be administered continuously or intermittently. For example, administration can be continued for 1 minute to 2 weeks. In addition, the antibody of the present invention may be administered alone or in combination with other drugs. Alternatively, the antibody of the present invention may be an antibody-drug conjugate (ADC) to which the other drug is bound. As the other drug, a drug known to have a therapeutic effect on a disease or disorder in which angiogenesis contributes to the onset or exacerbation of angiogenesis can be used.

The dose of the pharmaceutical composition of the present invention is not particularly limited as long as it can obtain a desired therapeutic effect or preventive effect, and can be appropriately determined depending on symptoms, sex, age and the like. The dose of the pharmaceutical composition of the present invention can be determined, for example, by using the therapeutic effect or preventive effect of a disease or disorder in which angiogenesis contributes to the onset or exacerbation as an index. For example, when used for the prevention and / or treatment of patients with diseases or disorders in which angiogenesis contributes to the onset or exacerbation, the single dose of the active ingredient of the pharmaceutical composition of the present invention is usually 0. About 01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 10 times a month, preferably about 1 to 5 times a month. To some extent, it is convenient to administer by intravenous injection. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased or the number of administrations may be increased depending on the symptoms.

Hereinafter, the present invention will be described in more detail based on Examples. However, the present invention is not limited to these examples. All documents cited throughout the present application are incorporated herein by reference. The present application also claims priority from Japanese Patent Application No. 2017-175311. All the contents of Japanese Patent Application No. 2017-175311, for which the present application claims priority, are incorporated herein by reference.

(Example 1) Production of anti-RAMP2 antibody In producing a hybridoma, cells used as an antigen were prepared by the following method. Human CRLR (NM_005795) and human RAMP2 (NM_005854) were tandemly integrated into Thermo's pCHO1.0 vector and gene-transduced into NIH-3T3 cells (ATCC: CRL-1658) using Lipofectamine 2000 (Thermo) (hCR2). -NIH3T3). In order to prepare a stable expression strain, selective culture was carried out in D-MEM medium (Wako Pure Chemical Industries, Japan) containing 10 μg / mL Puromycin and 100 nM methotrexate.

The obtained hCR2-NIH3T3 cells were prepared so as to be 1 × 10 ⁶ cells per MRL / lpr mouse, and were intraperitoneally administered once a week for 5 weeks to immunize with an antigen. Serum before and after immunization was collected, and an increase in antigen-specific antibody titer was confirmed by Cell ELISA. After confirming the increase in antibody titer, 1 × 10 ⁶ cells of hCR2-NIH3T3 cells were re-immunized as an antigen as final immunization.

For cell fusion, SP2ab cell line was used as mouse myeloma cells, and cells derived from spleen and lymph nodes were fused by the PEG method and seeded on a 96-well culture plate. From the day after the fusion, 10% FBS-TIL medium containing HAT was added, and the medium was exchanged every 3 days to prepare a hybridoma.

To screen for anti-human RAMP2 antibody-producing strains, hCRLR / hRAMP2-expressing CHO-K1 cells (hCR2-CHOK1) prepared in the same manner as NIH-3T3 cells were seeded on 96-well culture plates. After immobilization with 4% PFA and blocking with 1% BSA-PBS, the culture supernatant was added to react Goat Anti-mouse-IgG-HRP (Southern Biotech) as a secondary antibody. Positive wells were picked up by color development by TMB solution (eBioscience) and used as candidate hybridomas.

Of the candidate hybridomas, 3 strains were acclimated to ASF104 medium (Ajinomoto Co., Ltd., Japan), the culture supernatant was collected, and IgG antibody was purified with Protein A derivative Ab-Capcher Extra (Protenova Co., Ltd., Japan) to anti-RAMP2 Antibodies, 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody were obtained.

(Example 2) cAMP suppression test Using hCR2-CHOK1 cells, a cAMP suppression test of the obtained anti-RAMP2 antibody was carried out. As a control antibody, known commercially available antibodies (Novus bio, 4E5, CatNo. NBP2-01853; Abnova, 2F5, CatNo. H000010266-M05; and Santacruz, B5, CatNo. sc-365240) were used. A Half area 96well plate was used for the measurement. 2500 cells of hCR2-CHOK1 cells were cultured overnight in D-MEM / Ham's F-12 (Wako Pure Chemical Industries, Japan) containing 10% FBS, and the final concentrations were 0.001 nM, 0.01 nM, 0.1 nM, 1 nM. Each antibody (25H4-4F9, 85H7-2B11, 33H4-1G3, 4E5, 2F5, and B5) was added so as to be 10, 10 nM, 100 nM, and 1000 nM, and human AM with a final concentration of 150 pM (Peptide Institute, Inc. , Japan) was added. The cAMP was measured using the LANCE Ultra cAMP kit (PerkinElmer) according to the manufacturer's manual. As controls, cAMP production by antibody-free / AM-added and antibody-free / AM-free hCR2-CHOK1 cells was similarly measured.

The results showing the suppression of cAMP when 10 nM of the antibody was added are shown in FIG. FIG. 1 shows the ratio of the cAMP concentration in each antibody-added group when the cAMP concentration in the case of no antibody addition is 100. The control antibody hardly suppressed cAMP production, whereas the 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody suppressed cAMP production to the same extent as in the AM-free group. In addition, the relationship between the concentration of each antibody and the cAMP suppression rate is shown in FIG. The IC50 values for cAMP suppression of the 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody were 0.15 nM, 0.03 nM, and 0.045 nM, respectively.

(Example 3) Growth suppression test of HUVEC HUVEC (KURABO) in 2% FBS-containing Humania-EG2 medium excluding growth factors was seeded on a 96-well culture plate at 2500 cells per well and adhered by pre-culture for 3 hours. It was. Each antibody (25H4-4F9, 85H7-2B11, 33H4-1G3, 4E5, 2F5) has a final concentration of 0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, and 1000 nM. , And B5) were added, AM with a final concentration of 100 nM was added, and the growth of HUVEC was observed after 96 hours to perform a growth suppression test of HUVEC. Using Cell Counting Kit-8 (DOJINDO, Inc.) for the measurement of cell number, 2% FBS containing Humedia-EG2 solution diluted 10-fold in medium minus growth factors was 100μL added, for 4 hours developed with CO ₂ incubator , Measured as OD450.

The result showing the growth inhibition when 10 nM of the antibody was added is shown in FIG. FIG. 3 shows the ratio of OD450 (number of HUVEC cells) in each antibody-added group when OD450 (number of HUVEC cells) in the case of no antibody was set to 100. The control antibody hardly suppressed the growth of HUVEC, whereas the 25H4-4F9 antibody, the 85H7-2B11 antibody, and the 33H4-1G3 antibody suppressed the growth to about 30%. In addition, the relationship between the concentration of each antibody and the HUVEC growth inhibition rate is shown in FIG. The IC50 values for HUVEC growth inhibition of 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody were 1.2 nM, 0.8 nM, and 1.6 nM, respectively.

(Example 4) Sequence analysis RNA was extracted from cell pellets of each antibody-producing hybridoma using an SV-Total RNA Isolation kit (Promega), and cDNA was synthesized by a Transscriptor First Strand cDNA Synthesis Kit (Roche). The antibody sequence of the hybridoma was analyzed. A double primer was designed from the CDR sequences of existing mice, and the sequences of the heavy chain and the light chain were amplified using Ex Taq polymerase manufactured by Takara Bio Inc. The amino acid sequence was determined from the obtained base sequence, and the CDR regions were identified by the KABAT method.

VH (SEQ ID NO: 2) and VL (SEQ ID NO: 4) of the determined 25H4-4F9 antibody, VH (SEQ ID NO: 12) and VL (SEQ ID NO: 14) of the 85H7-2B11 antibody, and VH (SEQ ID NO: 14) of the 33H4-1G3 antibody. The sequences of No. 19) and VL (SEQ ID NO: 21) are shown in FIG. In addition, each determined CDR sequence is shown below.
25H4-4F9 antibody heavy chain CDR sequences:
CDRH1: GYFMN (SEQ ID NO: 5)
CDRH2: RNNPYNGDSIYNQKFKG (SEQ ID NO: 6)
CDRH3: LMIFDAYYAMDY (SEQ ID NO: 7)
25H4-4F9 antibody light chain CDR sequences:
CDRL1: RASQDIRNYLN (SEQ ID NO: 8)
CDRL2: YTSRLHS (SEQ ID NO: 9)
CDRL3: QQDSKHPWT (SEQ ID NO: 10)

85H7-2B11 antibody heavy chain CDR sequences:
CDRH1: GYFMN (SEQ ID NO: 5)
CDRH2: RINGYNGDTFYNQKFKG (SEQ ID NO: 15)
CDRH3: LGIYDVYHYVMEN (SEQ ID NO: 16)
85H7-2B11 antibody light chain CDR sequences:
CDRL1: RASQDIRNYLN (SEQ ID NO: 8)
CDRL2: YTSRLHS (SEQ ID NO: 9)
CDRL3: QQDSKNPWT (SEQ ID NO: 17)

33H4-1G3 antibody heavy chain CDR sequences:
CDRH1: GYFMN (SEQ ID NO: 5)
CDRH2: RINGYNGDTLYNQKFKG (SEQ ID NO: 22)
CDRH3: LGIYDVYHYVMEN (SEQ ID NO: 16)
33H4-1G3 antibody light chain CDR sequences:
CDRL1: RASQDIRNYLN (SEQ ID NO: 8)
CDRL2: YTSRLHS (SEQ ID NO: 9)
CDRL3: QQDSKNPWT (SEQ ID NO: 17)

(Example 5) Confirmation of anti-RAMP2-specific antibody It was confirmed by Cell ELISA that the obtained antibody was anti-RAMP2-specific. Specifically, human CRLR and human RAMP2 were gene-introduced into hamster ovary cells (CHO-K1) according to Example 1. The binding property of the antibody was compared between the cells into which human CRLR and human RAMP2 were transgeniced and the cells into which only human CRLR was transgenic.

The results are shown in FIG. The 25H4-4F9 antibody, 85H7-2B11 antibody, and 33H4-1G3 antibody hardly bound to CHO-K1 cells expressing only human CRLR, and to CHO-K1 cells co-expressing human CRLR and human RAMP2. Indicates that they specifically bind.

(Example 6) Analysis of binding affinity for cell membrane surface protein by Cell ELISA 96-well culture plate (BD-FALCON) in 1 × 10 ⁴ cells / well of hCR2-CHOK1 cells in DMEM / F-12 medium containing 10% FCS. Was added to and cultured overnight. Equal amounts of 4% paraformaldehyde solution (MUTO chemistry) were added and allowed to stand at 4 ° C. for 20 minutes to fix cells, washed with PBST, and then blocked with 1% BSA-PBS for 1 hour or longer. .. As the antibody, 25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1 and commercially available anti-RAMP2 antibodies 4E5, 2F5, and B5 were used. Each purified antibody was diluted 1/2 step from 67.5 nM and added, and Goat Anti-mouse-IgG-HRP (Southern Biotech) was reacted as a secondary antibody. After color development by adding TMB solution of (eBioscience, Inc.), the reaction was stopped by addition of _H 2 SO ₄ solution 2N. OD450 was measured using iMark (BioRAD). From the obtained binding data, the binding affinity (EC50) was calculated using GraphPad Prism7.

As shown in FIG. 7, 25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1 showed clearly superior binding activity as compared with the commercially available antibodies 4E5, 2F5, and B5. .. The vertical axis was calculated as a ratio to the maximum binding amount, with the maximum binding amount (OD value) of the antibody obtained in the same test as 100%. The EC50 representing the binding affinity of each antibody was 0.40 nM for 25H4-4F9, 0.14 nM for 85H7-2B11, and 0.30 nM for 33H4-1G3. Since all of the commercially available antibodies had low binding ability, a normal binding curve could not be drawn, and the EC50 values of 4E5 were 37.7 nM or more and 2F5 and B5 were 66.7 nM or more.

(Example 7) Binding analysis to cell membrane surface protein by FACS Each antibody (25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1 and a commercially available anti-RAMP2 antibody, 4E5, 2F5, And B5), the binding ability of hCR2-CHOK1 cells to the membrane surface RAMP2 protein was measured with a flow cytometer (ACEA). 3 × 10 ⁵ cells of exfoliated hCR2-CHOK1 cells and 50 μL of 3 μg / mL antibody solution were added to a 96-well V-bottom plate (Thermo Fisher Scientific) and allowed to stand on ice for 1 hour to bind the antibody. R-Phycoerythrin-conjugated Affinipure F (ab') ₂ Fragment Goat Anti-Mouse IgG (1% BSA / 0.05% NaN ₃ / PBS) washed 3 times and adjusted to 2.5 μg / mL. H + L) (Jackson Immunoreesearch Laboratories) was added, and the mixture was allowed to stand for 30 minutes under shading on ice to bind a secondary antibody. After washing once, the mixture was suspended in 500 μL of FACS buffer, and the fluorescence was measured by FACS in the same manner as in Example 7.

As shown in FIG. 8, 25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1 all bound to the cell membrane surface protein. Of the commercially available antibodies, 4E5 was observed to bind to the cell membrane surface protein, but the commercially available antibodies 2F5 and B5 were not observed to bind.

(Example 8) Competitive analysis of epitopes by FACS Biotinylated 85H7-2B11 and non-biotinylated antibodies (25H4-4F9, 85H7-2B11, and 33H4-1G3 obtained in Example 1 and commercially available antibody 4E5) Was simultaneously reacted with hCR2-CHOK1 to perform a competitive test against the antigen. The 85H7-2B11 antibody was biotinylated using Biotin Labeling kit-NH ₂ (Dojindo) according to the manufacturer's manual. Add biotinylated 85H7-2B11 antibody 3 μg / mL and non-biotinylated antibody 0, 0.1, 0.3, 1, 3, 10 and 30 μg / mL to hCR2-CHOK1 cells and allow for 1 hour on ice The antibody was bound under competitive conditions. After washing three times, Streptavidin-Fluorescein Isothiocyanate (Biolegend) prepared at 2.5 μg / mL was added, and the mixture was allowed to stand for 30 minutes under shading on ice to bind the secondary antibody. After one wash, it was suspended in 500 μL FACS buffer and fluorescence was measured by FACS in the same manner as in Example 7.

As shown in FIG. 9, 25H4-4F9, 85H7-2B11, and 33H4-1G3 competed with 85H7-2B11 for binding to the antigen, whereas the commercial antibody 4E5 did not compete with 85H7-2B11.

(Example 9) Design of humanized antibody For 25H4-4F9 and 85H7-2B11 antibody, humanized antibody (humanized 25H4-4F9, humanized 85H7-2B11 antibody) was designed. The design of the humanized sequence was carried out according to the method devised by Xoma corporation (US Patent 5,766,886; MODEFIED ANTIBODY VARIABLE DOMAINS, patent expiration date). A homology search was performed on the human immunoglobulin GERMLINE database (NCBI: IGBLAST) using the amino acid sequences of 25H4-4F9 mouse antibody heavy chain and VL or 85H7-2B11 mouse antibody heavy chain and VL, and the amino acid showing the highest homology. Sequence (25H4-4F9 heavy chain: IGHV1-69-2 * 01, IGHV1-18 * 04 and IGHJ6 * 01, 25H4-4F9 light chain: IKG1-33 * 01 and IKGJ2 * 01, 85H7-2B11 heavy chain: IGHV1- 46 * 01, IGHV1-18 * 04 and IGHJ6 * 01, 85H7-2B11 light chain: IKGV1-33 * 01 and IKGJ2 * 01, except for one VL amino acid sequence of 25H4-4F9 and 85H7-2B11. Since they are homologous, the same sequence was selected as the framework) as the framework for humanized antibodies. For each amino acid residue of these frameworks, there are three stages (HIGH, MODERATE, LOW) of risk of loss of antibody three-dimensional structure and antigen binding when the mouse sequence is changed to the human sequence. Evaluate and convert the LOW risk amino acid residue to the human type amino acid sequence first, and then convert the MODERATE and HIGH risk amino acid residues, and 5 types (vh1 to vh5) of the heavy chain of each antibody. For the light chain, four types of sequences (vk1 to vk4) were designed. Furthermore, a total of eight types of humanized antibodies were prepared for each antibody by combining the heavy chain and the light chain.

The designed VH and VL sequences of 25H4-4F9 are shown in FIG. 10, and the designed 85H7-2B11 VH and VL sequences are shown in FIG. The combinations of the heavy chain and light chain and the sequences of VH, VL, and CDR in the prepared humanized antibody are shown in Table 2 below.

(Example 10) Preparation of recombinant humanized antibody A gene sequence encoding a heavy chain (SEQ ID NO: 102, 104, 106, 108, 110, 120, 122, 124, 126, and 128) and 128 for preparing a recombinant antibody. The gene sequences encoding the light chain (SEQ ID NOs: 112, 114, 116, 118, 130, 132, 134, and 136) were cloned into the mammalian expression vector pcDNA3.4 (Thermo). Vectors cloned from heavy and light chains were mixed in a weight ratio of 1: 1 and transfected into mammalian cells CHO-S (Thermo) using 10 μg to 30 μg of vector DNA. Transfection was carried out using ExpiFectamineTMCHO Transfection Kit (Thermo), and 10 mL to 30 mL of cell suspension was subjected to swirling culture in a flask for 14 days. Cells were removed by centrifugation from the culture medium cultured for 14 days, and the culture supernatant passed through a 0.45 μm filter was purified with Protein A resin (Amsphere A3; JSR Life Sciences). The Protein A resin and the culture supernatant were mixed overnight at 4 ° C. to bind the recombinant antibody, and then washed with 20 mM phosphate buffer pH 7.0. Recombinant antibody bound to the resin was eluted with an Elution buffer of 0.1 M Glycine-HCl pH 3.2 and neutralized with 1/50 volume of 1 M Tris-HCl pH 9.0. After neutralization, Buffer was replaced with phosphate buffer using a 30 kDa ultrafiltration membrane (Amicon Ultra), and the absorbance at 280 nm was measured to determine the concentration of recombinant antibody.

(Example 11) cAMP suppression test of humanized antibody A cAMP suppression test of humanized antibody was carried out using hCR2-CHOK1 cells. Similar to the measurement of mouse antibody activity, a Half area 96well plate manufactured by PerkinElmer was used for the measurement, and 2500 cells of hCR2-CHOK1 cells were contained in D-MEM / Ham's F-12 (Wako Pure Chemical Industries, Ltd.) containing 10% FBS. Incubate overnight in (Japan), add each antibody (humanized 25H4-4F9, humanized 85H7-2B11, 4E5, 2F5, and B5) to human AM with a final concentration of 150 pM (Peptide Institute, Japan). Was added. The cAMP was measured using the LANCE Ultra cAMP kit (PerkinElmer) according to the manufacturer's manual. As controls, cAMP production by antibody-free / AM-added and antibody-free / AM-free hCR2-CHOK1 cells was similarly measured.

12 and 13 show the cAMP suppression rate when 16.7 nM of humanized 25H4-4F9 antibody and humanized 85H7-2B11 antibody were added, respectively. The cAMP suppression rate was shown as the ratio of the cAMP concentration in each antibody-added group when the cAMP concentration in the case of no antibody addition was 100. The commercially available antibodies (4E5, 2F5, and B5) used as controls hardly suppressed cAMP production, whereas the humanized 25H4-4F9 antibody and the humanized 85H7-2B11 antibody were equivalent to those in the AM-free group. It suppressed cAMP production.

In addition, the relationship between the concentration of each antibody and the cAMP suppression rate is shown in FIG. The IC50 values for cAMP suppression of humanized 25H4-4F9 antibody and humanized 85H7-2B11 antibody were 0.17 nM for h25H4-4F9v1, 0.14 nM for h25H4-4F9v2, 0.26 nM for h25H4-4F9v3, and h25H4-, respectively. 4F9v4 is 0.17nM, h25H4-4F9v5 is 0.50nM, h25H4-4F9v6 is 1.35nM, h25H4-4F9v8 is 1.07nM, and h85H7-2B11v1 is 0.07nM, h85H7-2B11v2 is 0.11nM, h85H7- 2B11v3 was 0.08 nM, h85H7-2B11v4 was 0.13 nM, h85H7-2B11v5 was 0.14 nM, h85H7-2B11v6 was 0.18 nM, h85H7-2B11v7 was 0.12 nM, and h85H7-2B11v8 was 0.18 nM.

(Example 12) Binding affinity analysis of humanized antibody by Cell ELISA 1 × 10 hCR2-CHOK1 cells in DMEM / F-12 medium (Wako Pure Drug) containing 10% FCS on a 96-well plate (BD-FALCON). ^It was added at ⁴ cells / well and cultured overnight. An equal amount of 4% paraformaldehyde solution (MUTO chemistry) was added, the cells were allowed to stand at 4 ° C. for 20 minutes to fix the cells, washed with PBST, and then blocked with 1% BSA-PBS for 1 hour or longer. It was. The purified antibody was diluted 1/10 from 67 nM and added, and 100 μL of Goat-Anti-Human-IgG-Fc-Fragment-HRP-conjuged (Bethyl) as a secondary antibody was added and reacted. I let you. TMB solution (eBioscience, Inc.) were developed by to measure the OD450 using iMark (BioRAD Co.) to stop the reaction _H 2 SO ₄ solution 2N. For the obtained binding data, the binding affinity (EC50) was calculated using GraphPad Prism7.

As shown in FIG. 15, the binding when the antibody was added at 6.7 nM was clearly superior in humanized 25H4-4F9 and humanized 85H7-2B11 as compared with the commercially available antibodies 4E5, 2F5, and B5. ..

In addition, the binding at each concentration is shown in FIG. The binding activity (EC50) of each antibody is 0.73 nM for h25H4-4F9v1, 1.05 nM for h25H4-4F9v2, 0.83 nM for h25H4-4F9v3, 1.83 nM for h25H4-4F9v4, and 2.32 nM for h25H4-4F9v5. , H25H4-4F9v6 is 3.19 nM, h25H4-4F9v8 is 3.03 nM, and h85H7-2B11v1 is 0.18 nM, h85H7-2B11v2 is 0.24 nM, h85H7-2B11v3 is 0.23 nM, and h85H7-2B11v4 is 0.22 nM. , H85H7-2B11v5 was 0.25 nM, h85H7-2B11v6 was 0.24 nM, h85H7-2B11v7 was 0.23 nM, and h85H7-2B11v8 was 0.20 nM.

(Example 13) HUVEC growth inhibitory test of humanized antibody The HUVEC growth inhibitory ability of the humanized antibody was examined by the same method as in Example 3. HUVEC was seeded on 96-well culture plates at 2,500 cells / well and adhered by pre-culture for 3 hours. Then, humanized 25H4-4F9 and humanized 85H7-2B11 at each concentration were added, AM with a final concentration of 100 nM was added, and the growth of HUVEC was observed after 96 hours to confirm the growth inhibitory ability of HUVEC. .. Cell Counting Kit-8 (DOJINDO) was used to measure the number of cells. 100 μL of a color-developing solution diluted 10-fold with Humania-EG2 medium containing 2% FBS excluding growth factors was added, and the color was developed in a CO ₂ incubator for 4 hours and measured as OD450.

The HUVEC growth inhibition rate (IC50) was calculated from the ratio of the added concentration of each antibody, with OD450 (number of HUVEC cells) in the case of no antibody being added as 100. As a result, the IC50 values of the humanized 25H4-4F9 antibody and the humanized 85H7-2B11 antibody were 16.0 nM for h25H4-4F9v1, 10.9 nM for h25H4-4F9v2, 5.1 nM for h85H7-2B11v1, and h85H7, respectively. -2B11v2 is 9.5nM, h85H7-2B11v3 is 1.5nM, h85H7-2B11v4 is 1.4nM, h85H7-2B11v5 is 21.4nM, h85H7-2B11v6 is 14.8nM, h85H7-2B11v7 is 32.7nM, h85H7-2B11v Was 22.6 nM.

(Example 14) Antibody epitope analysis In order to determine whether or not a specific epitope primary sequence recognized by the 85H7-2B11 antibody is present, 12 amino acids are overlapped in the extracellular region of the human RAMP2 protein 3 Peptide fragments (SEQ ID NOs: 71 to 101) consisting of the following 31 amino acids shifted by amino acids were prepared, and the binding ability of antibodies to these peptides was examined by the ELISA method. A sufficient amount of 1.0 μg / mL was used for the solid phase of the peptide and protein, and the 85H7-2B11 antibody was reacted at 20 ng / mL for binding. The Goat Anti-mouse-IgG-HRP as secondary antibody and reacted at 50.0ng / mL, the bound antibody and developed with TMB solution was measured OD450 after stopping the reaction with _H 2 SO ₄ solution 2N.
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)
DWAMISRPYSTRRDS (SEQ ID NO: 85)
MISRPYSTLRDSLEH (SEQ ID NO: 86)
RPYSTLRDSLEFAE (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)
PNPLAERIIIFETHQI (SEQ ID NO: 94)
LAERIIFETHQIHFA (SEQ ID NO: 95)
RIIFENTHQIHFANSS (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)

As shown in FIG. 17, a sufficient amount of 85H7-2B11 antibody was bound to an equal amount of human RAMP2 protein. On the other hand, the 85H7-2B11 antibody did not bind to any human RAMP2 peptide fragment. From this, it was shown that the 85H7-2B11 antibody recognizes an epitope composed of a higher-order structure such as a three-dimensional structure rather than a specific primary structure of the RAMP2 protein. Further, from the results of Example 8, since the antibodies of the present invention compete with each other in binding to the antigen, all the antibodies of the present invention have a higher-order structure such as a three-dimensional structure rather than a specific primary structure of the RAMP2 protein. It was thought to recognize the constituent epitopes. From these results, it was shown that in order to suppress the proliferation of HUVEC, it is important to recognize an epitope composed of an extracellular three-dimensional structure rather than a specific primary structure of RAMP2 protein.

Since this antibody can specifically suppress the function of human RAMP2, which is a human adrenomedulin receptor, it can provide a method for preventing, diagnosing or treating a disease related to RAMP2, or a prophylactic, diagnostic or therapeutic agent for the disease. It is expected to play an important role in drug development.

Claims (29)

An antibody or an immunoreactive fragment thereof that specifically binds to receptor activity regulatory protein 2 (RAMP2), which has an inhibitory effect on vascular endothelial cells in a vascular endothelial cell growth test by adding in vitro adrenomedulin. The antibody or immunoreactive fragment thereof according to claim 1, wherein the growth inhibitory effect of the vascular endothelial cells is an IC50 value of 35 nM or less. The antibody or immunoreactive fragment thereof according to claim 1 or 2, which has an action of suppressing cAMP production by animal cells by addition of adrenomedulin. The antibody or immunoreactive fragment thereof according to claim 3, wherein the cAMP production inhibitory effect is an IC50 value of 2 nM or less in a cAMP production test using animal cells by adding in vitro adrenomedulin. The antibody or immunoreactive fragment thereof according to any one of claims 1 to 4, which binds to a cell co-expressing human CRLR and human RAMP2. The antibody or immunoreactive fragment thereof according to claim 5, wherein the binding affinity (EC50) according to the Cell ELISA test using cells co-expressing human CRLR and human RAMP2 is 3.5 nM or less. Heavy chain variable region (VH) having the amino acid sequence according to any one selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. And an antibody comprising a light chain variable region (VL) having the amino acid sequence according to any one selected from SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61. , And an antibody or immunoreactive fragment thereof that competes for binding to RAMP2, or
VH and SEQ ID NOs: 4, 14 having the amino acid sequence according to any one selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. , 21, 37, 39, 41, 43, 55, 57, 59, and 61, an antibody that binds to the same epitope as an antibody containing VL having the amino acid sequence according to any one of, or an immune reaction thereof. Sex fragment. The antibody or immunoreactive fragment thereof according to any one of claims 1 to 7, which does not recognize the primary structure of the amino acid sequence constituting the extracellular region of RAMP2. The antibody or immunoreactive fragment thereof according to claim 8, which does not bind to all of the peptides consisting of the respective amino acid sequences of SEQ ID NOs: 71 to 101. The amino acid sequence represented by LX ₁ IX ₂ DX ₃ YX ₄ YX ₅ MX ₆ X ₇ (SEQ ID NO: 24), where X ₁ is G or M and X ₂ is F or Y. Amino acid sequences, where X ₃ is A or V, X ₄ is absent or H, X ₅ is A or V, X ₆ is D or E, and X ₇ is Y or N. The antibody or immunoreactive fragment thereof according to any one of claims 1 to 9, which comprises a heavy chain having a heavy chain complementarity determining region (CDRH) 3 comprising. The antibody or immunoreactive fragment thereof according to 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). Further, heavy chains have CDRH2 comprising the amino acid sequence of GYFMN CDRHl comprising the amino acid sequence of (SEQ ID NO: 5), and _{RX 8 NPYNGDX 9 X 10 YX 11} QKFX 12 G ( SEQ ID NO: 66), according to claim 10 or claim 11. The antibody or immunoreactive fragment thereof,
Here, X ₈ is N or I, X ₉ is S or T, X ₁₀ is I, F or L, X ₁₁ is A or N, and X ₁₂ is Q or K. Furthermore, the light chain complementarity determining regions (CDRL) 1 consisting of the amino acid sequence of X ₁₃ ASQDIRNYLN (SEQ ID NO: 69), CDRL2 consisting of the amino acid sequence of YTSRLX ₁₄ X ₁₅ (SEQ ID NO: 70), and QQDSKX ₁₆ PWT (SEQ ID NO: 25). The antibody according to claim 12 or an immunoreactive fragment thereof, which comprises a light chain having CDRL3 consisting of the amino acid sequence of).
Here, X ₁₃ is Q or R, X ₁₄ is E or H, X ₁₅ is S or T, and X ₁₆ is H or N. CDRH1 consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5),
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 from RINPYNGDTFYAQKFQG (SEQ ID NO: 65) The antibody of claim 12 or an immune reaction thereof having CDRH2 consisting of any one amino acid sequence selected from the group consisting of, and CDRH3 consisting of the amino acid sequence of LMIFDAYYAMDY (SEQ ID NO: 7) or LGIYDVYHYVMEN (SEQ ID NO: 16). Sex fragment. Further, CDRL1 consisting of the amino acid sequence of RASQDIRNYLN (SEQ ID NO: 8) or QASQDIRNYLN (SEQ ID NO: 67),
The antibody according to claim 14, which has 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). Or an immunoreactive fragment thereof. The antibody or immunoreactive fragment thereof according to claim 14, which has a combination of one of the following heavy chain CDRs (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 RNNPYNGDSYNQKFKG (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 RNNPYNGDSYNEKFQG (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 RINGYNGDTFYNQKFKG (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 RINGYNGDTFYNQKFQG (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 RINGYNGDTFYAQKFQG (SEQ ID NO: 65) and CDRH3 consisting of the amino acid sequence of LGIYDVYHYVMEN (SEQ ID NO: 16);
(Vii) CDRH1, consisting of the amino acid sequence of GYFMN (SEQ ID NO: 5),
CDRH2 consisting of the amino acid sequence of RINGYNGDTLYNQKFKG (SEQ ID NO: 22) and CDRH3 consisting of the amino acid sequence of LGIYDVYHYVMEN (SEQ ID NO: 16). The antibody or immunoreactive fragment thereof according to claim 16, further comprising a combination of any one of the following light chain CDRs (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);
(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). The antibody according to any one of claims 1 to 17, which is any of the following:
The nucleic acid sequence in which VH encodes the amino acid sequence set forth in any one selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. It has an amino acid sequence encoded by a nucleic acid sequence that hybridizes with and stringent conditions, and VL is SEQ ID NO: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61. An antibody or immunoreactive fragment thereof having an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence encoding the amino acid sequence described in any one selected from.
VH has 80% or more identity with any one amino acid sequence selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. It has an amino acid sequence and the VL is 80% or more with any one amino acid sequence selected from SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61. An antibody or an immunoreactive fragment thereof having an amino acid sequence having the same identity. 1. Claim 1 having a VH having the amino acid sequence according to any one selected from SEQ ID NOs: 2, 12, 19, 27, 29, 31, 33, 35, 45, 47, 49, 51, and 53. The antibody or immunoreactive fragment thereof according to any one of claims 17. 19 of claim 19, further comprising a VL having the amino acid sequence according to any one selected from SEQ ID NOs: 4, 14, 21, 37, 39, 41, 43, 55, 57, 59, and 61. Antibodies or immunoreactive fragments thereof. The antibody or immunoreactive fragment thereof according to claim 20, which comprises any one combination of VH and VL selected from the following groups:
VH having the amino acid sequence set forth in SEQ ID NO: 2 and VL having the amino acid sequence set forth in SEQ ID NO: 4;
VH having the amino acid sequence set forth in SEQ ID NO: 27 and VL having the amino acid sequence set forth in SEQ ID NO: 37;
VH having the amino acid sequence set forth in SEQ ID NO: 29 and VL having the amino acid sequence set forth in SEQ ID NO: 37;
VH having the amino acid sequence set forth in SEQ ID NO: 29 and VL having the amino acid sequence set forth in SEQ ID NO: 39;
VH having the amino acid sequence set forth in SEQ ID NO: 31 and VL having the amino acid sequence set forth in SEQ ID NO: 39;
VH having the amino acid sequence set forth in SEQ ID NO: 31 and VL having the amino acid sequence set forth in SEQ ID NO: 41;
VH having the amino acid sequence set forth in SEQ ID NO: 33 and VL having the amino acid sequence set forth in SEQ ID NO: 41;
VH having the amino acid sequence set forth in SEQ ID NO: 35 and VL having the amino acid sequence set forth in SEQ ID NO: 43;
VH having the amino acid sequence set forth in SEQ ID NO: 12 and VL having the amino acid sequence set forth in SEQ ID NO: 14;
VH having the amino acid sequence set forth in SEQ ID NO: 45 and VL having the amino acid sequence set forth in SEQ ID NO: 55;
VH having the amino acid sequence set forth in SEQ ID NO: 47 and VL having the amino acid sequence set forth in SEQ ID NO: 55;
VH having the amino acid sequence set forth in SEQ ID NO: 47 and VL having the amino acid sequence set forth in SEQ ID NO: 57;
VH having the amino acid sequence set forth in SEQ ID NO: 49 and VL having the amino acid sequence set forth in SEQ ID NO: 57;
VH having the amino acid sequence set forth in SEQ ID NO: 49 and VL having the amino acid sequence set forth in SEQ ID NO: 59;
VH having the amino acid sequence set forth in SEQ ID NO: 51 and VL having the amino acid sequence set forth in SEQ ID NO: 59;
VH having the amino acid sequence set forth in SEQ ID NO: 51 and VL having the amino acid sequence set forth in SEQ ID NO: 61;
VH having the amino acid sequence set forth in SEQ ID NO: 53 and VL having the amino acid sequence set forth in SEQ ID NO: 61;
VH having the amino acid sequence set forth in SEQ ID NO: 19 and VL having the amino acid sequence set forth in SEQ ID NO: 21. A nucleic acid molecule encoding the amino acid sequence of the antibody or immunoreactive fragment thereof according to any one of claims 1 to 21. The nucleic acid molecule of claim 22, which comprises any one polynucleotide selected from the following group:
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 1 and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 3;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 26, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 36;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 28, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 36;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 28, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 38;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 30 and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 38;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 30 and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 40;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 32, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 40;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 34, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 42;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 11 and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 13;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 44, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 54;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 46, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 54;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 46, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 56;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 48, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 56;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 48, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 58;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 50, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 58;
A polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 50, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 60;
A polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 52, and a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 60; and encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 18. A polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 20. A vector having the nucleic acid molecule according to claim 22 or 23. A host cell having the vector according to claim 24. The method for producing an antibody or an immunoreactive fragment thereof according to any one of claims 1 to 21, which comprises culturing the host cell according to claim 25. A pharmaceutical composition containing the antibody according to any one of claims 1 to 21 or an immunoreactive fragment thereof as an active ingredient. The pharmaceutical composition according to claim 27, which is an angiogenesis inhibitor. Therapeutic agents for cancer, cancer metastasis, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, neovascular glaucoma, inflammatory skin disease, rheumatoid arthritis, or osteoarthritis The pharmaceutical composition according to claim 27, which is a preventive agent.