Antibodies and their use

Abstract

This invention relates to anti-RAMP2 and anti-RAMP3 antibodies that inhibit the tumorigenic effect of adrenomedullin. These antibodies may be used in the treatment of cancer.

Description

[Technical Field]

[0001] This application relates to antibody molecules and their use in therapeutic methods, for example, in methods for treating cancer. In particular, it relates to antibody molecules that suppress the tumorigenic effect of adrenomedullin. [Background technology]

[0002] Adrenomedullin (AM) is a regulatory peptide whose involvement in tumor progression and metastatic responses has been increasingly emphasized in recent years. The literature as a whole supports the view that AM, as a survival factor for tumor cells, can be produced by either the tumor itself or multiple surrounding stromal cells. Generally, AM expression is upregulated by hypoxia, which commonly occurs in (solid) tumors, and overproduction of this peptide is associated with poor patient prognosis. AM acts as an autocrine / paracrine growth factor that prevents apoptosis, increases tumor cell motility and metastatic responses, induces angiogenesis, and suppresses immune surveillance by suppressing the immune system. As described above, AM expression is rapidly activated by hypoxia through a HIF-1α-mediated mechanism and acts as a potent angiogenic factor that promotes neovascularization.

[0003] AM mediates its effects through association with calcitonin-like receptors (CLRs), which are seven-transmembrane receptors belonging to the G protein-coupled receptor (GPCR) family that bind to receptor activity-modifying proteins (RAMPs), which are single-transmembrane proteins. The RAMP family consists of three proteins (RAMP1, RAMP2, and RAMP3) that can interact with GPCRs, leading to significant changes in ligand binding and receptor pharmacology. RAMPs are essential for CLR binding to the cell surface. Depending on the type of RAMP that associates with the CLR, the resulting complex acts as a receptor for either CGRP or AM. By interacting with RAMP1, CLR acquires a high affinity for CGRP, while by interacting with either RAMP2 or RAMP3, CLR acquires a high affinity for AM, forming AM receptor 1 and AM receptor 2, respectively.

[0004] The three RAMPs, each composed of approximately 160 amino acids, share a common structure including a large extracellular N-terminal domain (ECD), a single transmembrane domain (TM), and a very short cytoplasmic C-terminal tail (C-tail); however, they share less than 30% sequence homology. RAMPs are widely distributed throughout the body, but their tissue distribution differs, and the abundance of each isoform depends on the tissue type. In addition, RAMP gene expression is regulated differently under various disease conditions in animal models.

[0005] Recent accumulation of findings by the inventors and other researchers has shown that AM is a major regulator of oncogenesis and tumor progression in numerous cancers, including glioblastoma, prostate cell carcinoma, colon cell carcinoma, lung cell carcinoma, mesothelioma, pheochromocytoma, and renal cell carcinoma. Based on our results, novel treatment protocols and clinical trials based on AM suppression can be conceived in cancer patients, where this remains a major clinical challenge.

[0006] However, to date, no antibodies that can be used to suppress the effects of adremedullin have been found in this field. [Overview of the project]

[0007] This specification describes the production of monoclonal antibodies that specifically bind to RAMP2 or RAMP3 and suppress the protumorogenic effect of adremedullin. These antibodies, therefore, could constitute therapeutic regimens in the fields of cancer and angiogenesis.

[0008] As described herein, the inventors have developed several monoclonal antibodies that are specific to RAMP2 and RAMP3, not only binding strongly to the protein but also suppressing the tumorigenic effects of adremedullin, such as its angiogenesis-promoting effect and its effect of promoting invasion into cancer cells. This is the first demonstration that RAMP2 and RAMP3 antibodies directly suppress the tumorigenic effects of adremedullin, thereby enabling the use of such antibodies as highly specific and low-toxicity effective therapeutic agents in a wide range of applications, including cancer treatment. Therefore, in first view, the present invention provides an antibody molecule that binds to RAMP2 or RAMP3 and suppresses adrenomedullin activity.

[0009] In one embodiment, an antibody molecule is considered to inhibit adrenomedullin activity if it inhibits one or more of the following: (i) angiogenesis, (ii) tumor invasion, such as endothelial tumor invasion, and (iii) chemotaxis.

[0010] As described in the examples, the antibody molecule of the present invention has been shown to induce a significant amount of necrosis in tumors. Therefore, in one embodiment of the present invention, the antibody molecule induces or promotes necrosis in tumor vascular structures or other locations within the tumor.

[0011] In one embodiment, the antibody molecule selectively binds to RAMP2 or RAMP3 without significantly binding to other polypeptides in the body.

[0012] In one embodiment, the antibody molecule binds to the extracellular domain of RAMP2 or RAMP3.

[0013] Thus, in one embodiment of the present invention, the antibody molecule binds to an epitope on the extracellular domain of RAMP2 or RAMP3.

[0014] In one embodiment, the antibody molecule has at least 10 -7 M, for example at least 10 -8 M, or more than that, for example 10 -9 M or more affinity for the target.

[0015] In one embodiment, the antibody molecule comprises an antigen-binding domain comprising at least one, for example two or three, of the CDRs of the VH chain having the amino acid sequence shown in Seq ID No:1 and / or (ii) an antigen-binding domain comprising at least one, for example two or three, of the CDRs of the VL chain having the amino acid sequence shown in Seq ID No:2.

[0016] In another embodiment, the antibody molecule comprises an antigen-binding domain comprising at least one, for example two or three, of the CDRs of the VH chain having the amino acid sequence shown in Seq ID No:33 and / or (ii) an antigen-binding domain comprising at least one, for example two or three, of the CDRs of the VL chain having the amino acid sequence shown in Seq ID No:34.

[0017] In another embodiment, the antibody molecule comprises an antigen-binding domain comprising at least one, for example two or three, of the CDRs of the VH chain having the amino acid sequence shown in Seq ID No:37 and / or (ii) an antigen-binding domain comprising at least one, for example two or three, of the CDRs of the VL chain having the amino acid sequence shown in Seq ID No:38.

[0018] In another embodiment, the antibody molecule comprises an antigen-binding domain comprising at least one, for example two or three, of the CDRs of the VH chain having the amino acid sequence shown in Seq ID No:49 and / or (ii) an antigen-binding domain comprising at least one, for example two or three, of the CDRs of the VL chain having the amino acid sequence shown in Seq ID No:50.

[0019] CDRs can be identified using any suitable system. For example, the positions of such CDRs can be determined by the methods described in Kabat et al., "Sequences of Proteins of Immunological Interest," U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, NIH Publication No. 91-3242, 1991 and www.kabatdatabase.com (http: / / immuno.bme.nwu.edu). Alternative systems that can be used to determine CDRs include the IMGT-specific numbering system (Lefranc M.-P. et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27, 55-77 (2003); Brochet, X., Lefranc, M.-P. and Giudicelli, V., IMGT / V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis, Nucl. Acids Res, 36, W503-508 (2008)). A further variable region numbering system that can be used is the Chothia system (Chothia C and Lesk AM, J. Mol. Biol. 1987, 196, p901; Al-Lazikani et al., (1997) JMB 273, 927-948).

[0020] The VH and VL chains having the amino acid sequences indicated by Seq ID No:1 and Seq ID No:2 are the VL and VH chains of the 1H6 antibody, respectively. In one embodiment, the CDRs of the VH and VL chains of this antibody are as shown in Figure 3.

[0021] The VH and VL chains having the amino acid sequences indicated by Seq ID No:33 and Seq ID No:34 are the VL and VH chains of the 3A12 antibody, respectively. In one embodiment, the CDRs of the VH and VL chains of this antibody are as shown in Figure 4.

[0022] The VH and VL chains having the amino acid sequences indicated by Seq ID No:37 and Seq ID No:38 are the VL and VH chains of the 4C10 antibody, respectively. In one embodiment, the CDRs of the VH and VL chains of this antibody are as shown in Figure 4.

[0023] The VH and VL chains having the amino acid sequences indicated by Seq ID No: 49 and Seq ID No: 50 are the VL and VH chains of the 6C2 antibody, respectively. In one embodiment, the CDRs of the VH and VL chains of this antibody are as shown in Figure 4.

[0024] In one embodiment, the antibody molecule comprises (i) an antigen-binding domain comprising all three CDRs of a VL chain having the amino acid sequence indicated by Seq ID No:1, or a variant of said CDR, and / or (ii) an antigen-binding domain comprising all three CDRs of a VH chain having the amino acid sequence indicated by Seq ID No:2, or a variant of said CDR.

[0025] In another embodiment, the antibody molecule comprises (i) an antigen-binding domain comprising all three CDRs of a VL chain having the amino acid sequence indicated by Seq ID No:33, or a variant of said CDR, and / or (ii) an antigen-binding domain comprising all three CDRs of a VH chain having the amino acid sequence indicated by Seq ID No:34, or a variant of said CDR.

[0026] In another embodiment, the antibody molecule comprises (i) an antigen-binding domain comprising all three CDRs of a VL chain having the amino acid sequence indicated by Seq ID No:37, or a variant of said CDR, and / or (ii) an antigen-binding domain comprising all three CDRs of a VH chain having the amino acid sequence indicated by Seq ID No:38, or a variant of said CDR.

[0027] In another embodiment, the antibody molecule comprises (i) an antigen-binding domain comprising all three CDRs of a VL chain having the amino acid sequence indicated by Seq ID No: 49, or a variant of said CDR, and / or (ii) an antigen-binding domain comprising all three CDRs of a VH chain having the amino acid sequence indicated by Seq ID No: 50, or a variant of said CDR.

[0028] In one embodiment, the antibody molecule comprises an antigen-binding domain having all three CDRs of a VL chain having the amino acid sequence indicated by (i) Seq ID No: 1, and an antigen-binding domain having all three CDRs of a VH chain having the amino acid sequence indicated by (i) Seq ID No: 2.

[0029] In another embodiment, the antibody molecule comprises (i) an antigen-binding domain having all three CDRs of a VL chain having the amino acid sequence indicated by Seq ID No:33, and (ii) an antigen-binding domain having all three CDRs of a VH chain having the amino acid sequence indicated by Seq ID No:34.

[0030] In another embodiment, the antibody molecule comprises (i) an antigen-binding domain comprising all three CDRs of a VL chain having the amino acid sequence indicated by Seq ID No:37, and (ii) an antigen-binding domain comprising all three CDRs of a VH chain having the amino acid sequence indicated by Seq ID No:38.

[0031] In another embodiment, the antibody molecule comprises (i) an antigen-binding domain comprising all three CDRs of the VL chain having the amino acid sequence shown in Seq ID No: 49, and (ii) an antigen-binding domain comprising all three CDRs of the VH chain having the amino acid sequence shown in Seq ID No: 50.

[0032] In one embodiment, the antibody molecule comprises an antibody V L domain or an antibody V H domain, or both.

[0033] In a preferred embodiment, the antibody V L domain comprises the amino acid sequence Seq ID No: 1, and / or the antibody V H domain comprises the amino acid sequence Seq ID No: 2.

[0034] In other specific embodiments, the antibody V L domain comprises the amino acid sequence Seq ID No: 33, and / or the antibody V H domain comprises the amino acid sequence Seq ID No: 31.

[0035] In another embodiment, the antibody V L domain consists of the amino acid sequence Seq ID No: 37, and / or the antibody V H domain consists of the amino acid sequence Seq ID No: 38.

[0036] In another embodiment, the antibody V L domain consists of the amino acid sequence Seq ID No: 49, and / or the antibody V H domain consists of the amino acid sequence Seq ID No: 50.

[0037] The antibody molecule can be an antibody, such as a full-length antibody.

[0038] In an alternative embodiment, the antibody molecule can be an antibody fragment, such as a scFv.

[0039] The antibody molecule of the present invention enables the development of related antibodies that suppress adrenomedullin activity, such as angiogenesis-promoting activity or invasion-promoting activity, and have equivalent or superior binding specificity as needed.

[0040] Accordingly, the scope of the present invention further encompasses antibody molecules comprising at least one, e.g., two or three, CDRs of a VL chain having the amino acid sequence indicated as Seq ID No:1, and / or at least one, e.g., two or three, CDRs of a VH chain having the amino acid sequence indicated as Seq ID No:2, wherein at least one of the CDRs has five or fewer, e.g., four, three, two or one, amino acid substitutions, and the antibody molecule retains the ability to inhibit adrenomedullin activity, e.g., the angiogenic or invasive activity of adrenomedullin.

[0041] Similarly, the present invention further encompasses antibody molecules comprising at least one, for example, two or three VL chain CDRs and / or at least one, for example, two or three VH chain CDRs, wherein at least one of the CDRs has five or fewer, for example, four, three, two or one amino acid substitutions, and the antibody molecule retains the ability to inhibit adrenomedullin activity, for example, the angiogenic or invasive activity of adrenomedullin. (i) The VL chain has the amino acid sequence indicated by Seq ID No: 33, and the VH chain has the amino acid sequence indicated by Seq ID No: 34, or (ii) The VL chain has the amino acid sequence indicated by Seq ID No:37, and the VH chain has the amino acid sequence indicated by Seq ID No:38, or (iii) The VL chain has the amino acid sequence indicated by Seq ID No: 45, and the VH chain has the amino acid sequence indicated by Seq ID No: 46, or (iv) The VL chain has the amino acid sequence indicated by Seq ID No: 49, and the VH chain has the amino acid sequence indicated by Seq ID No: 50, or (v) The VL chain has the amino acid sequence indicated by Seq ID No: 53, and the VH chain has the amino acid sequence indicated by Seq ID No: 54.

[0042] In one embodiment of the first aspect of the present invention, the antibody molecule has the ability to suppress the invasion of tumor cells.

[0043] In other embodiments, the antibody molecule according to the first aspect of the present invention has the ability to suppress angiogenesis.

[0044] In a second embodiment of the present invention, a nucleic acid encoding an antibody molecule according to the first embodiment of the present invention is provided.

[0045] In one embodiment of a second aspect of the present invention, nucleic acids are (i) The nucleotide sequence indicated by VL 1H6 (Sequence ID No: 3) and / or the nucleotide sequence indicated by VH 1H6 (Sequence ID No: 4), (ii) The nucleotide sequence indicated by VL 3A12 (Sequence ID No: 35) and / or the nucleotide sequence indicated by VH 3A12 (Sequence ID No: 36), or (iii) The nucleotide sequence indicated by VL 4C10 (Sequence ID No: 39) and / or the nucleotide sequence indicated by VH 4C10 (Sequence ID No: 40), (iv) Nucleotide sequence indicated by VL 6B8 (Sequence ID No: 47) and / or nucleotide sequence indicated by VH 6B8 (Sequence ID No: 48), (v) The nucleotide sequence indicated by VL 6C2 (Sequence ID No: 51) and / or the nucleotide sequence indicated by VH 6C2 (Sequence ID No: 52), or (vi) It comprises the nucleotide sequence indicated by VL 6D8 (Sequence ID No: 55) and / or the nucleotide sequence indicated by VH 6D8 (Sequence ID No: 56).

[0046] Nucleic acids can be used to provide an antibody molecule according to a first aspect of the present invention. Accordingly, the present invention provides a method for producing an antibody molecule capable of suppressing the tumorigenic activity of adrenomedullin, the method comprising expressing a nucleic acid according to a second aspect of the present invention in a host cell and isolating the antibody molecule from the cell.

[0047] A further aspect of the present invention is a pharmaceutical composition comprising an antibody molecule according to the first aspect of the present invention or a nucleic acid according to the second aspect of the present invention.

[0048] The antibody molecules, nucleic acids, or compositions of the present invention can be used to suppress adrenomedullin activity, such as angiogenesis-promoting activity or invasion-promoting activity, particularly when the adrenomedullin receptor is abnormally expressed. Accordingly, in a further embodiment, the present invention provides a method for suppressing adrenomedullin activity in a biological sample, the method comprising administering an antibody molecule according to the first embodiment of the present invention or a nucleic acid according to the second embodiment to the biological sample.

[0049] In a further embodiment, a method is provided for treating a condition related to adrenomedullin activity in a patient requiring such treatment, the method comprising administering to the patient an antibody molecule according to a first embodiment of the present invention or a nucleic acid according to a second embodiment of the present invention.

[0050] In one embodiment, the state is one related to the abnormal activity of adrenomedullin.

[0051] In the context of this application, an adrenomedullin receptor, such as RAMP2 or RAMP3, is considered abnormally expressed if its expression differs from that of normal healthy cells, for example, if it is expressed at a higher level than normal in cells or tissues, and if this abnormal expression contributes to a disease state.

[0052] Furthermore, an antibody molecule according to the first aspect of the present invention or a nucleic acid according to the second aspect of the present invention is provided for use in pharmaceuticals.

[0053] The present invention further provides an antibody molecule according to a first aspect of the invention or a nucleic acid according to a second aspect of the invention for use in the treatment of conditions associated with adrenomedullin abnormality.

[0054] Furthermore, antibody molecules according to the first aspect of the present invention or nucleic acids according to the second aspect are also provided for use in the preparation of therapeutic agents for conditions related to abnormal activity or abnormal expression of adrenomedullin.

[0055] The present invention may be used to treat any condition associated with abnormal activity of adrenomedullin. For example, conditions to which the present invention may be used include, but are not limited to, diseases and / or cancers associated with excessive, uncontrolled, or inappropriate angiogenesis.

[0056] In certain embodiments of the present invention, the cancer is glioblastoma, renal cancer, prostate cancer, colon cancer, lung cancer, mesothelioma, or pheochromocytoma.

[0057] In one particular embodiment, the cancer is glioblastoma.

[0058] In another specific embodiment, the cancer is kidney cancer. [Modes for carrying out the invention]

[0059] (antibody molecule) In the context of this invention, "antibody molecule" should be understood to mean an immunoglobulin or a part thereof, or any polypeptide having an antibody-binding domain or a binding domain homologous thereto. Antibody molecules include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies and their fragments, as well as chimeric antibodies having an immunoglobulin-binding domain fused with another polypeptide.

[0060] Unchanged (full-length) antibodies consist of an immunoglobulin molecule comprising heavy and light chains, each possessing a variable region called VH and VL, respectively. The variable region comprises three complementarity-determining regions (CDRs, also known as hypervariable regions) and four framework regions (FRs) or scaffolds. The CDRs form a complementary three-dimensional structure with the antigen molecule, determining the antibody's specificity.

[0061] Antibody fragments may retain the binding ability of unchanged antibodies and may be used in place of unchanged antibodies. Therefore, for the purposes of this invention, unless otherwise specifically required in the context, the term “antibody molecule” should be understood to include antibody fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, dAb, and Fv fragments, scFv, bispecific scFv, diabodies, linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng 8(10):1057-1062

[1995] ), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0062] The Fab fragment consists of a full-length L chain (VL and CL) and VH and CH1. The Fab' fragment differs from the Fab fragment in that it has an additional residue at the carboxyl terminus of the CH1 domain containing one or more cysteines derived from the antibody hinge region. The F(ab')2 fragment comprises two disulfide-linked Fab fragments.

[0063] The Fd fragment consists of VH and CH1 domains.

[0064] The Fv fragment consists of the VL and VH domains of a single antibody.

[0065] A single-chain Fv fragment is an antibody fragment linked by a linker and possessing VH and VL domains on which scFv can form an antigen-binding site (see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994)).

[0066] Diabodies are small antibody fragments prepared by constructing scFv fragments with short linkers (approximately 5-10 residues) between the VH and VL domains. As a result, interchain pairing occurs between the V domains, but intrachain pairing does not, resulting in a multivalent fragment, i.e., a fragment with two antigen-binding sites (see, for example, European Patent No. 404097, International Publication No. 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)).

[0067] In one embodiment of the present invention, the antibody molecule is a bispecific antibody molecule having, for example, a first binding site specific to RAMP2 or RAMP3 and a second binding site specific to a second different antigen. In one embodiment, the second binding site is specific to a T cell antigen, such as CD3. Such T cell engager-type bispecific antibodies are described by Baeuerle et al., Drugs of the Future 33:137-147, Kufer et al., Trends Biotechnol. 22:238-244 (2004), and Wolf et al., Drug Discovery Today 10:1237-44 (2005). An example of such an antibody molecule is the BiTE® antibody (Amgen, US), which binds to both cytotoxic T cells and target cells, bringing them into close proximity so that T cells can induce lysis of target cells by granzymes. In one embodiment of the present invention, the antibody molecule is a bispecific antibody molecule having a first binding site specific to RAMP2 or RAMP3 and a second binding site specific to VEGF (vascular endothelial growth factor) or EGFR (epidermal growth factor receptor).

[0068] Furthermore, the fragments also include individual CDRs.

[0069] In this invention, the amino acid sequences of the VH and VL regions of the unchanged anti-RAMP2 antibodies rIgG-k1-1H6, rIgG-k1-1A7, rIgG-k1-1H7, rIgG-k1-1A10, rIgG-k1-2A6, rIgG-k1-2A8, and rIgG-k1-2H3 were identified. The inventors also identified the amino acid sequences of the VH and VL regions of the unchanged anti-RAMP3 antibodies rIgG-k1-3A5, rIgG-k1-3A12, rIgG-k1-4C10, rIgG-k1-5H9, rIgG-k1-6B8, rIgG-k1-6C2, and rIgG-k1-6D8. This invention encompasses antibody molecules having the VH and / or VL sequences of these antibodies.

[0070] The amino acid sequences of these VH and VL regions are shown below, along with the nucleic acid sequences encoding those VH and VL regions.

[0071] (Anti-RAMP2 antibody) RAMP2-rIgG-k1-1H6#4 VL: (SEQ ID NO:1) APVLTQTPASVEAAVGGTVTIKCQAS QSINSW LSWYQQKPGQPPKLLIY QAS KLASGVPSRFKGSGSGTEYTLTISDLKCADAATYYC QSYDYASNSGYVSNP FGGGTEVVVK (SEQ ID NO:3) GCACCTGTGCTGACCCAGACACCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGTATTAATAGTTGGTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACCAGGCATCCAAACTGGCATCTG GGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACACTCTCACCATCAGCGACCTGAAGTGTGCCGATGCTGCCACTTATTATTGTCAAAGCTATGATTATGCTAGTAATAGTGGATATGTGTCTAATCCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA VH: (SEQ ID NO:2) QEHLVESEGGLFKPTDTLTLTCTVS GFSLSSYG VSWVRQAPGKGLEWIGV IGATGSAY YANWAKSRSTITRNTNLNTVTLKVTSLTAADTATYFC ARDDVLTGMDL WGPGTLVTVSS (SEQ ID NO:4) CAGGAGCATCTGGTGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGATACCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATGGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTGGTGCTACTGGCAGCGCATACTACGCGAACTGGGCGAAAAGCCGATCCACCATCACCAGAAACACCAACCTGAACACAGTGACTCTGAAGGTGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGGGATGATGTGCTTACTGGTATGGACCTCTGGGGCCCAGGGACCCTCGTCACCGTCTCTTCA

[0072] RAMP2-rIgG-k1-1A7#1 VL: (SEQ ID NO:5) DGVMTQTPSSVSAAVGSTVTISCQAS QSVFSNNY LSWYQQKPGQPPKLLIY TAS SLASGVPSRFKGSGSGTQFTLTISDLECADAATYYCQC TYGKSGTDTYLNA FGGGTEVVVK (SEQ ID NO:7) GATGGCGTGATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAAGCACAGTCACCATCAGTTGCCAGGCCAGTCAGAGTGTTTTTAGTAACAACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATACTGCATCCAGTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATGCACTTATGGAAAGAGTGGTACTGATACTTATCTAAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA VH: (SEQ ID NO:6) QSVEESGGDLVKPEGSLTLTCTAS GFSFSISYW ICWVRQTPGKGLEW IGCIYGGSSGRP YYASWAKGRFTISKTSSTTVTLQMTSLTAADTAAYFC ATTAYGGYGYGT WGPGTLVTVSS (SEQ ID NO:8) CAGTCGGTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGAGGGATCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCTTCAGTATCAGCTATTGGATATGCTGGGTCCGCCAGACTCCAGGGAAGGGGCTGGAGTGGATCGGATGCATTTATGGTGGTAGTAGTGGTCGTCCTTACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACTAGTCTGACAGCCGCGGACACGGCCGCCTATTTCTGTGCGACCACTGCTTATGGTGGTTATGGTTATGGTACGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0073] RAMP2-rIgG-k1-2A6 VL: (SEQ ID NO:9) DVVMTQTPASVEAAVGGTVTIKCQAS QSISNL LAWYQQKPGQPPKRLIY GAS NLESGVPSRFKGSGSGTQFTLTISDLECADAATYYCQC SYDGSSPNA FGGGTEVVVE (SEQ ID NO:11) GATGTTGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGCAACCTCTTAGCCTGGTATCAACAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTATGGTGCATCCAATCTGGAATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATGTAGTTATGATGGTAGTAGTCCCAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCGAA VH: (SEQ ID NO:10) QSVEESGGDLVKPGASLTLTCTAS GFSFSSRYY MCWVRQAPGKGLEWIGC IFGGSSGNTH YASWAKGRFTIAKTSSTTVTLRMTSLTAADTATYSC VRSDAWGVNL WGPGTLVTVSS (SEQ ID NO:12) CAGTCGGTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCTTCAGTAGCCGCTACTATATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGCTGTATTTTTGGTGGTAGTAGTGGTAATACTCACTACGCGAGCTGGGCGAAAGGCCGCTTCACCATCGCCAAAACCTCGTCGACCACGGTGACTCTGCGAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTCTTGTGTGAGAAGTGATGCCTGGGGTGTTAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0074] RAMP2-rIgG-k1-2A8 VL: (SEQ ID NO:13) APVLTHTAASVSEPVGGIVTINCQAS QSIGSN LAWYQQKSGQPPKLLIY DASN LVSGVPSRFKGSRSGTEYTLTISDLECADAGTYYC QGYYYGSSTSYNLVS FGGGTEVVVK (SEQ ID NO:15) GCACCTGTGCTGACCCATACTGCAGCCTCCGTGTCTGAACCTGTGGGAGGCATAGTCACCATCAACTGCCAGGCCAGTCAGAGCATTGGTAGTAATTTAGCCTGGTATCAGCAGAAATCAGGGCAGCCTCCCAAGCTCCTGATCTATGATGCATCCAATCTGGTATCTGGGGTCCCATCGCGGTTCAAAGGCAGCAGATCTGGGACAGAGTACACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGGCACTTACTACTGTCAAGGCTATTATTATGGTAGTAGTACTAGCTATAATTTAGTGTCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA VH: (SEQ ID NO:14) QSLEESGGGLVQPEGSLTLTCTAS GFDLSRYYY IFWVRQAPGKGLEWIGC IYIGGNIITY YASWAKGRFTISKTSSPAVTLQMTSLTVADTATYFC ARGTGYGGYGDVSL WGPGTLVTVSS (SEQ ID NO:16) CAGTCGTTGGAGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGATCCCTGACACTCACCTGCACAGCTTCTGGATTCGACCTCAGTCGCTACTACTACATATTTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATGCATTTATATTGGTGGTAATATTATCACTTACTACGCGAGCTGGGCGAAAGGCCGCTTCACCATCTCCAAAACCTCGTCGCCCGCGGTGACTCTGCAAATGACCAGCCTGACAGTCGCGGACACGGCCACCTATTTCTGTGCGAGAGGGACTGGTTATGGTGGTTATGGTGATGTTAGCTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0075] RAMP2-rIgG-k1-2H3 VL: (SEQ ID NO:17) AAVMTQTPSPVSGVVGGTVTIKCQAS QSIYGY FSWYQQKPGQPPKLLIY SAS DLASGVPSRFKGSGSGTQFTLTISDLECADAATYYCQC SYDGSSPNA FGGGTEVVVK (SEQ ID NO:19) GCAGCCGTGATGACCCAGACTCCATCCCCCGTGTCTGGAGTTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTTACGGCTACTTTTCTTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTCTGCATCCGATCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATGTAGTTATGATGGTAGTAGTCCCAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA VH: (SEQ ID NO:18) QSVEESGGDLVKPGASLTLTCTAS GFSFSSRYY MCWVRQAPGKGLEWIAC IFGGSSDNSY YARWAKGRFTISKTSSTTVTLRMTSLTAADTATYFC VRSDAWGVNL WGPGTLVTVSS (SEQ ID NO:20) CAGTCGGTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCACGGCCTCTGGATTCTCCTTCAGTAGCCGCTATTATATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGTATTTTTGGTGGTAGTAGTGATAATAGTTATTACGCGAGGTGGGCGAAAGGCCGCTTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCGAATGACCAGTCTGACGGCCGCGGACACGGCCACTTATTTCTGTGTGAGAAGTGATGCCTGGGGTGTTAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0076] RAMP2-rIgG-k1-1H7 VL: (SEQ ID NO:21) AAVLTQTPSSTSGPVGGTVTIKCQAS QNIGSF LSWYQQKPGQPPKLLIY SAS SLATGVPSRFSGSGSGTQFTLTISGVQCDDAATYYC LGFYAYRSDDGTA FGGGTEVVVK (SEQ ID NO:23) GCAGCCGTGCTGACCCAGACTCCATCCTCCACGTCTGGACCAGTGGGAGGCACAGTCACCATCAAATGCCAGGCTAGTCAGAATATTGGTAGTTTCTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTCTGCATCCAGTCTGGCAACTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATTAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGTTTTTATGCTTATAGGAGTGATGACGGTACGGCTTTCGGCGGAGGGACCGAAGTGGTGGTCAAA

[0077] VH: (SEQ ID NO:22) QSLEESGGDLVKPEGSLTLTCTAS GFSFSISYW ICWVRQTPGKGLEW IGCIYGGSSGRP YYASWAKGRFTISKTSSTTVTLQMTSLTAADTAAYFC ATTAYGGYGYGT WGPGTLVTVSS (SEQ ID NO:24) CAGTCGTTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGAGGGATCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCTTCAGTATCAGCTATTGGATATGCTGGGTCCGCCAGACTCCAGGGAAGGGGCTGGAGTGGATCGGATGCATTTATGGTGGTAGTAGTGGTCGTCCTTACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACTAGTCTGACAGCCGCGGACACGGCCGCCTATTTCTGTGCGACCACTGCTTATGGTGGTTATGGTTATGGTACGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0078] RAMP2-rIgG-k1-1A10 VL: (SEQ ID NO:25) AAVMTQTPSSLSAAVGGTVTIKCQAS QSIRNQ VSWYQQKPGQRPKLLIY RAS TLESGVPSRFKGSGSGTDFTLTISDLECADAATYYCQC TYGSSSSNRYGNT FGGGTEVAVK (SEQ ID NO:27) GCAGCCGTGATGACCCAGACTCCATCCTCCCTGTCTGCCGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGTATTAGGAATCAAGTATCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGAATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATGCACTTATGGTAGTAGTAGTAGTAATCGTTATGGAAATACTTTCGGCGGAGGGACCGAGGTGGCGGTCAAA VH: (SEQ ID NO:26) QSLEESGGGLVQPEGSLTLTCTAS GFSFSSSYY MCWVRQAPGKGLEWIGS IYTLSGSTA YASWAKGRFTISKTSSTTVTLQMTSLTAADTAMYFC ARDSDGWGVSFNL WGPGTLVTVSS (SEQ ID NO:28) CAGTCGTTGGAGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGATCCCTGACACTCACCTGCACAGCTTCTGGATTCTCCTTCAGTAGCAGCTACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATCCATTTATACTCTTAGTGGTAGCACTGCCTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACAGCCGCGGACACGGCCATGTATTTCTGTGCGAGAGATAGTGATGGCTGGGGTGTCAGTTTTAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0079] (Anti-RAMP3 antibody) RAMP3-rIgG-k1-3A5 VL: (SEQ ID NO:29) DVVMTQTPASVSAAVGATVTIKCQAS ETIGNN LAWYQQKPGQPPKVLMF LAS TLASGVPSRFKGSRSGTEYTLTISGVQCDDAATYYC QSYLGTSYA FGGGTEVVVK (SEQ ID NO:31) GATGTTGTGATGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGCCACAGTCACCATCAAGTGCCAGGCCAGTGAGACCATTGGTAATAATTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGGTCCTGATGTTTCTGGCATCCACACTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTAGATCTGGGACAGAGTACACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAAGCTATCTGGGTACTAGTTATGCTTTCGGCGGAGGGACCGAGGTGGTCGTCAAA VH: (SEQ ID NO:30) QSLEESGGGLVQPEGTLTLTCTVS GFSFSSNYW ICWVRQAPGKGLEW IACIVTNSGTTY YTSWAKGRLTLSKTSSTTVTLQMTSLTAADTATYFC ARDNNSVTGVEFNL WGPGTLVTVSS (SEQ ID NO:32) CAGTCGTTGGAGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGAACCCTGACACTCACCTGCACAGTCTCTGGATTCTCCTTCAGTAGCAACTACTGGATATGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTGTTACTAATAGTGGTACTACTTACTACACGAGCTGGGCGAAAGGCCGACTCACCCTCTCCAAAACCTCATCGACCACGGTGACTCTACAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGAGATAATAATAGTGTGACTGGTGTTGAGTTTAATTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0080] RAMP3-rIgG-k1-3A12 VL: (SEQ ID NO:33) DPMLTQTPSSKSVPVGGTVTINCQSS ESVYDNNW LAWFQQKPGQPPKRLIY SAS TLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYC TGYKSRTTDEIA FGGGTEVVVK (SEQ ID NO:35) GACCCTATGCTGACCCAGACTCCATCTTCCAAGTCTGTCCCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTGAGAGTGTTTATGATAACAACTGGTTAGCCTGGTTTCAGCAGAAGCCAGGGCAGCCTCCCAAGCGCCTGATCTATTCTGCATCCACTCTGGCATCTGGGGTCTCATCGCGATTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTACAGGATATAAAAGCCGTACTACTGATGAGATTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA VH: (SEQ ID NO:34) QEQLEESEGGLFKPTDTLTLTCTVS GFSLSSYG VSWVRQAPGNGLEYIGF IGKTGSAY YASWAKSRSTITRNTTLNTVTLKMTSLTVADTATYFC ARLGPGSIYYFDI WGPGTLVTVSS (SEQ ID NO:36) CAGGAGCAGCTGGAGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGATACCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATGGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAATACATCGGATTCATTGGTAAAACTGGTAGCGCATACTACGCGAGCTGGGCGAAAAGCCGATCCACCATCACCAGAAACACCACCCTGAACACGGTGACTCTGAAAATGACCAGTCTGACAGTCGCGGACACGGCCACCTATTTCTGTGCGAGATTGGGACCTGGTAGTATTTACTATTTTGACATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0081] RAMP3-rIgG-k1-4C10 VL: (SEQ ID NO:37) DGVMTQTPSSTSAAVGSTVTISCQST QSIINNNW LAWYQQKPGHSPKLLIY DAS TLASGVPSRFSGSGSGTQFTLTISDLECDDAATYYC AARYSGDIYT FGGGTEVVVR (SEQ ID NO:39) GATGGCGTGATGACCCAGACTCCATCTTCCACGTCTGCAGCTGTGGGAAGCACAGTCACCATCAGTTGCCAGTCCACTCAGAGTATTATTAATAATAATTGGTTAGCCTGGTATCAGCAGAAACCAGGGCACTCTCCTAAGCTCCTGATCTACGATGCATCCACTCTGGCATCTGGGGTCCCATCACGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTGCAGCCCGTTATAGTGGTGATATTTATACTTTCGGCGGAGGGACCGAGGTGGTGGTCAGA VH: (SEQ ID NO:38) QEQLEESGGGLVKPTDTLTLTCTVS GFSLSTNS IFWVRQAPGNGLEWIGA ISYAGGTA YASWAKSRSTITRNTNLNTVTLKMTSLTAADTATYFC GRGYVGYSRSKGDI WGPGTLVTVSS (SEQ ID NO:40) CAGGAGCAGCTGGAGGAATCCGGGGGAGGCCTGGTCAAGCCAACGGATACCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTACCAATTCAATTTTCTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAATGGATCGGAGCCATTAGTTATGCTGGTGGCACAGCCTACGCGAGCTGGGCGAAAAGCCGATCCACCATCACCAGAAACACCAACCTGAACACGGTGACTCTGAAGATGACTAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGGGAGAGGTTATGTTGGTTATAGTCGTAGTAAGGGGGATATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0082] RAMP3-rIgG-k1-5H9 VL: (SEQ ID NO:41) DVVMTQTPASVEAAVGGTVTIKCQAT ESIASW LAWYQQKAGQPPKLLIY EAS KLESGVPSRFKGSGSGTEFTLTISDLECADAATYYC QSYLYSSSNTYGNS FGGGTEVVVK (SEQ ID NO:43) GATGTTGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCACTGAGAGCATTGCCAGTTGGTTAGCCTGGTATCAGCAGAAAGCAGGGCAGCCTCCCAAGCTCCTGATCTACGAAGCATCCAAACTGGAATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAAAGCTATCTTTATAGTAGCAGTAATACTTATGGTAATTCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA VH: (SEQ ID NO:42) QSLEESEGGLFKPTDTLTVTCIVS GLSLSRNA MSWVRQAPGSGLEWIGV IVTYGDTY YASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFC IGGLDI WGPGTLVTVSS (SEQ ID NO:44) CAGTCGTTGGAGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGATACCCTGACAGTCACCTGTATAGTCTCTGGACTCTCCCTCAGTAGGAATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAGCGGGCTGGAATGGATCGGAGTCATTGTGACTTATGGTGACACATACTACGCGAGCTGGGCGAAAAGCCGATCCACCATCACCAGAAACACCAACGAGAACACGGTGACTCTGAAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTTTGTATTGGTGGTCTTGACATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0083] RAMP3-rIgG-k1-6B8 VL: (SEQ ID NO:45) DVMMTQTPASVSEPVGGTVTIKCQAS QNIGNN LAWYQQKPGQPPKLLIY LTS TLASGVPSRFKGSRSGTEFTLTISDLECADAATYFC QSYLYTTYA FGGGTEVVVK (SEQ ID NO:47) GATGTCATGATGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAACATTGGTAATAATTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATCTGACATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTAGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTTCTGTCAAAGCTATTTATATACTACTTATGCTTTCGGCGGAGGGACCGAGGTGGTCGTCAAA VH: (SEQ ID NO:46) QSLEESGGGLVKPEGSLTLTCTAS GFFFDRSYW ICWVRQTPAKGLEW IACIVVSSGATY YASWAKGRLTLSKTSSTTVTLQMTSLTAADTAIYFC ARDSSSTSGVEFNL WGPGTLVTVSS (SEQ ID NO:48) CAGTCGTTGGAGGAGTCCGGGGGAGGCCTGGTCAAGCCTGAGGGATCCCTGACACTCACCTGCACAGCCTCTGGTTTCTTCTTCGATAGAAGCTACTGGATATGCTGGGTCCGCCAGACTCCAGCAAAGGGGCTGGAGTGGATCGCATGCATTGTTGTTAGCAGTGGTGCTACTTACTACGCGAGCTGGGCGAAAGGCCGACTCACCCTCTCCAAAACCTCGTCGACCACGGTGACTCTACAAATGACCAGTCTGACAGCCGCGGACACGGCCATCTATTTCTGTGCGAGAGATAGTAGTAGTACTAGTGGTGTTGAGTTTAATTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0084] RAMP3-rIgG-k1-6C2 VL: (SEQ ID NO:49) DGVMTQTPSSVSAAVGGTVTISCQSS QSVYKNNY LSWFQQKPGQPPKLLIY EIS KLESGVPDRFSGSGSGTQFTLTISGVQCDDAATYYC LGGYDDDSDTS FGGGTEVVVK (SEQ ID NO:51) GATGGCGTGATGACCCAGACTCCATCGTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTCAGAGTGTTTATAAGAACAACTACTTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACGAAATATCCAAACTGGAATCTGGGGTCCCAGATAGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGGTTATGATGATGATTCTGATACTTCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA VH: (SEQ ID NO:50) QEQLEESEGGLFKPTDTLTLTCTVS GFSLSVYA LTWVRQAPGNGLEWIGA IGNSGNTY YASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFC ARGYVRINV WGPGTLVTVSS (SEQ ID NO:52) CAGGAGCAGCTGGAGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGATACCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTGTCTATGCTTTAACCTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAGTGGATCGGAGCCATTGGTAATAGTGGTAACACGTACTACGCGAGCTGGGCGAAAAGCCGATCCACCATCACCAGAAACACCAACGAGAACACGGTGACTCTGAAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGAGGATATGTCCGAATTAACGTTTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCG

[0085] RAMP3-rIgG-k1-6D8 VL: (SEQ ID NO:53) DVVMTQTPASVSEPVGGTVTIKCQAS QNIGNN LAWYQQKPGHPPKLLIYLAST LAS GVPSRFKGSRSGTEFTLTISDLECADAATYYC QSYLYTTYA FGGGTEVVVK (SEQ ID NO:55) GATGTTGTGATGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAACATTGGTAATAATTTAGCCTGGTATCAGCAGAAACCAGGGCACCCTCCCAAGCTCCTGATCTATCTGGCATCTACTCTGGCATCTGGGGTCCCATCGCGATTCAAAGGCAGTAGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAAAGCTATTTGTATACTACTTATGCTTTCGGCGGAGGGACCGAGGTGGTCGTCAAA VH: (SEQ ID NO:54) QSVEESGGDLVRPEGSLTLTCTAS GFSLSSSYW ICWVRQAPGKGLEW IACIVSNTGTTY YASWAKGRLTLSKTSSTTVTLQMTSLTAADTATYFC ARDNNSKSGVEFNL WGPGTLVTVSS (SEQ ID NO:56) CAGTCGGTGGAGGAGTCCGGGGGAGACCTGGTCAGGCCTGAGGGATCCCTGACACTCACCTGCACAGCTTCTGGATTCTCCCTCAGTAGCAGCTACTGGATATGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTAGAGTGGATCGCATGCATTGTTAGTAATACTGGTACCACTTACTAC GCGAGCTGGGCGAAAGGCCGACTCACCCTCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGAGATAATAGTAAAAGTGGTGTTGAGTTTAATTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0086] As described above, the antibody molecules of the present invention include rIgG-k1-1H6, rIgG-k1-1A7, rIgG-k1-1H7, rIgG-k1-1A10, rIgG-k1-2A6, rIgG-k1-2A8, rIgG-k1-2H3, rIgG-k1-3A5, rIgG-k1-3A12, rIgG-k1-4C10, rIgG-k1-5H9, rIgG-k1-6B8, rIgG-k1-6C2, rIgG-k1-6D8 antibodies, as well as variants that maintain the function or activity of adremedullin, such as the ability to suppress the pro-angiogenic or pro-invasive activity of adremedullin, although these are not limited to the specific VH, VL, and CDR sequences disclosed herein.

[0087] In certain accessions, the antibody molecule is a humanized version of the rIgG-k1-1H6, rIgG-k1-1A7, rIgG-k1-1H7, rIgG-k1-1A10, rIgG-k1-2A6, rIgG-k1-2A8, rIgG-k1-2H3, rIgG-k1-3A5, rIgG-k1-3A12, rIgG-k1-4C10, rIgG-k1-5H9, rIgG-k1-6B8, rIgG-k1-6C2, or rIgG-k1-6D8 antibody.

[0088] In one embodiment, the present invention comprises an antibody molecule having the ability to suppress adrenomedullin activity, for example, the angiogenic or invasive activity of adrenomedullin, comprising at least one, e.g., two or three VL chain CDRs and / or at least one, e.g., two or three VH chain CDRs, (i) The VL chain has the amino acid sequence indicated by Seq ID No:1, and the VH chain has the amino acid sequence indicated by Seq ID No:2, or (ii) The VL chain has the amino acid sequence indicated by Seq ID No: 5, and the VH chain has the amino acid sequence indicated by Seq ID No: 6, or (iii) The VL chain has the amino acid sequence indicated by Seq ID No: 9, and the VH chain has the amino acid sequence indicated by Seq ID No: 10, or (iv) The VL chain has the amino acid sequence indicated by Seq ID No: 13, and the VH chain has the amino acid sequence indicated by Seq ID No: 14, or (v) The VL chain has the amino acid sequence indicated by Seq ID No: 17, and the VH chain has the amino acid sequence indicated by Seq ID No: 18, or (vi) The VL chain has the amino acid sequence indicated by Seq ID No: 21, and the VH chain has the amino acid sequence indicated by Seq ID No: 22, or (vii) The VL chain has the amino acid sequence indicated by Seq ID No: 25, and the VH chain has the amino acid sequence indicated by Seq ID No: 26, or (viii) The VL chain has the amino acid sequence indicated by Seq ID No: 29, and the VH chain has the amino acid sequence indicated by Seq ID No: 30, or (ix) The VL chain has the amino acid sequence indicated by Seq ID No: 33, and the VH chain has the amino acid sequence indicated by Seq ID No: 34, or (x) The VL chain has the amino acid sequence indicated by Seq ID No: 37, and the VH chain has the amino acid sequence indicated by Seq ID No: 38, or (xi) The VL chain has the amino acid sequence indicated by Seq ID No: 41, and the VH chain has the amino acid sequence indicated by Seq ID No: 42, or (xii) The VL chain has the amino acid sequence indicated by Seq ID No: 45, and the VH chain has the amino acid sequence indicated by Seq ID No: 46, or (xiii) The VL chain has the amino acid sequence indicated by Seq ID No: 49, and the VH chain has the amino acid sequence indicated by Seq ID No: 50, or (xiv) The VL chain has the amino acid sequence indicated by Seq ID No: 53, and the VH chain has the amino acid sequence indicated by Seq ID No: 54.

[0089] In another embodiment, the present invention includes an antibody molecule comprising at least one, for example, two or three VL chain CDRs and at least one, for example, two or three VH chain CDRs, wherein the antibody molecule possesses the ability to suppress adrenomedullin activity, for example, the angiogenic or invasive activity of adrenomedullin. (i) The VL chain has the amino acid sequence indicated by Seq ID No:1, and the VH chain has the amino acid sequence indicated by Seq ID No:2, or (ii) The VL chain has the amino acid sequence indicated by Seq ID No: 5, and the VH chain has the amino acid sequence indicated by Seq ID No: 6, or (iii) The VL chain has the amino acid sequence indicated by Seq ID No: 9, and the VH chain has the amino acid sequence indicated by Seq ID No: 10, or (iv) The VL chain has the amino acid sequence indicated by Seq ID No: 13, and the VH chain has the amino acid sequence indicated by Seq ID No: 14, or (v) The VL chain has the amino acid sequence indicated by Seq ID No: 17, and the VH chain has the amino acid sequence indicated by Seq ID No: 18, or (vi) The VL chain has the amino acid sequence indicated by Seq ID No: 21, and the VH chain has the amino acid sequence indicated by Seq ID No: 22, or (vii) The VL chain has the amino acid sequence indicated by Seq ID No: 25, and the VH chain has the amino acid sequence indicated by Seq ID No: 26, or (viii) The VL chain has the amino acid sequence indicated by Seq ID No: 29, and the VH chain has the amino acid sequence indicated by Seq ID No: 30, or (ix) The VL chain has the amino acid sequence indicated by Seq ID No: 33, and the VH chain has the amino acid sequence indicated by Seq ID No: 34, or (x) The VL chain has the amino acid sequence indicated by Seq ID No: 37, and the VH chain has the amino acid sequence indicated by Seq ID No: 38, or (xi) The VL chain has the amino acid sequence indicated by Seq ID No: 41, and the VH chain has the amino acid sequence indicated by Seq ID No: 42, or (xii) The VL chain has the amino acid sequence indicated by Seq ID No: 45, and the VH chain has the amino acid sequence indicated by Seq ID No: 46, or (xiii) The VL chain has the amino acid sequence indicated by Seq ID No: 49, and the VH chain has the amino acid sequence indicated by Seq ID No: 50, or (xiv) The VL chain has the amino acid sequence indicated by Seq ID No: 53, and the VH chain has the amino acid sequence indicated by Seq ID No: 54.

[0090] In yet another embodiment, the present invention includes an antibody molecule comprising all three CDRs of the VL chain and all three CDRs of the VH chain, wherein the antibody molecule possesses the ability to suppress adrenomedullin activity, for example, the angiogenic or invasive activity of adrenomedullin. (i) The VL chain has the amino acid sequence indicated by Seq ID No:1, and the VH chain has the amino acid sequence indicated by Seq ID No:2, or (ii) The VL chain has the amino acid sequence indicated by Seq ID No: 5, and the VH chain has the amino acid sequence indicated by Seq ID No: 6, or (iii) The VL chain has the amino acid sequence indicated by Seq ID No: 9, and the VH chain has the amino acid sequence indicated by Seq ID No: 10, or (iv) The VL chain has the amino acid sequence indicated by Seq ID No: 13, and the VH chain has the amino acid sequence indicated by Seq ID No: 14, or (v) The VL chain has the amino acid sequence indicated by Seq ID No: 17, and the VH chain has the amino acid sequence indicated by Seq ID No: 18, or (vi) The VL chain has the amino acid sequence indicated by Seq ID No: 21, and the VH chain has the amino acid sequence indicated by Seq ID No: 22, or (vii) The VL chain has the amino acid sequence indicated by Seq ID No: 25, and the VH chain has the amino acid sequence indicated by Seq ID No: 26, or (viii) The VL chain has the amino acid sequence indicated by Seq ID No: 29, and the VH chain has the amino acid sequence indicated by Seq ID No: 30, or (ix) The VL chain has the amino acid sequence indicated by Seq ID No: 33, and the VH chain has the amino acid sequence indicated by Seq ID No: 34, or (x) The VL chain has the amino acid sequence indicated by Seq ID No: 37, and the VH chain has the amino acid sequence indicated by Seq ID No: 38, or (xi) The VL chain has the amino acid sequence indicated by Seq ID No: 41, and the VH chain has the amino acid sequence indicated by Seq ID No: 42, or (xii) The VL chain has the amino acid sequence indicated by Seq ID No: 45, and the VH chain has the amino acid sequence indicated by Seq ID No: 46, or (xiii) The VL chain has the amino acid sequence indicated by Seq ID No: 49, and the VH chain has the amino acid sequence indicated by Seq ID No: 50, or (xiv) The VL chain has the amino acid sequence indicated by Seq ID No: 53, and the VH chain has the amino acid sequence indicated by Seq ID No: 54.

[0091] The CDR sequences of the rIgG-k1-1H6, rIgG-k1-1A7, rIgG-k1-1H7, rIgG-k1-1A10, rIgG-k1-2A6, rIgG-k1-2A8, rIgG-k1-2H3, rIgG-k1-3A5, rIgG-k1-3A12, rIgG-k1-4C10, rIgG-k1-5H9, rIgG-k1-6B8, rIgG-k1-6C2, and rIgG-k1-6D8 antibody molecules are shown in Figures 3 and 4.

[0092] The present invention also extends to variants in which one or more CDR sequences of the antibody molecules of the above embodiments are modified. The modified amino acid residues in the amino acid sequence of the CDR variant are 30% or less, more preferably 20% or less, and most preferably 10% or less of the total number of amino acids in the entire CDR. Such variants can be provided using the teachings of this application and the art known to those skilled in the art. The CDR can be supported in a framework structure comprising the antibody heavy chain or light chain sequence or a portion thereof. Preferably, such CDR is located at a location corresponding to the naturally occurring CDR positions of the VH and VL domains. The location of such CDR can be determined by the method described in Kabat et al., "Sequences of Proteins of Immunological Interest," U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, NIH Publication No. 91-3242, 1991, and www.kabatdatabase.com http: / / immuno.bme.nwu.edu.

[0093] In one such embodiment, the antibody molecule comprises an antibody molecule having at least one, for example, two or three VL chain CDRs and / or at least one, for example, two or three VH chain CDRs, (a) (i) The VL chain has the amino acid sequence indicated by Seq ID No:1, and the VH chain has the amino acid sequence indicated by Seq ID No:2. (ii) The VL chain has the amino acid sequence indicated by Seq ID No: 5, and the VH chain has the amino acid sequence indicated by Seq ID No: 6. (iii) The VL chain has the amino acid sequence indicated by Seq ID No:9, and the VH chain has the amino acid sequence indicated by Seq ID No:10. (iv) The VL chain has the amino acid sequence indicated by Seq ID No: 13, and the VH chain has the amino acid sequence indicated by Seq ID No: 14. (v) The VL chain has the amino acid sequence indicated by Seq ID No: 17, and the VH chain has the amino acid sequence indicated by Seq ID No: 18. (vi) The VL chain has the amino acid sequence indicated by Seq ID No: 21, and the VH chain has the amino acid sequence indicated by Seq ID No: 22. (vii) The VL chain has the amino acid sequence indicated by Seq ID No: 25, and the VH chain has the amino acid sequence indicated by Seq ID No: 26. (viii) The VL chain has the amino acid sequence indicated by Seq ID No: 29, and the VH chain has the amino acid sequence indicated by Seq ID No: 30. (ix) The VL chain has the amino acid sequence indicated by Seq ID No:33, and the VH chain has the amino acid sequence indicated by Seq ID No:34. (x) The VL chain has the amino acid sequence indicated by Seq ID No:37, and the VH chain has the amino acid sequence indicated by Seq ID No:38. (xi) The VL chain has the amino acid sequence indicated by Seq ID No: 41, and the VH chain has the amino acid sequence indicated by Seq ID No: 42, or (xii) The VL chain has the amino acid sequence indicated by Seq ID No: 45, and the VH chain has the amino acid sequence indicated by Seq ID No: 46. (xiii) The VL chain has the amino acid sequence indicated by Seq ID No: 49, and the VH chain has the amino acid sequence indicated by Seq ID No: 50, or (xiv) The VL chain has the amino acid sequence indicated by Seq ID No: 53, and the VH chain has the amino acid sequence indicated by Seq ID No: 54. (b) at least one of the CDRs has five or fewer amino acid substitutions, for example, four, three, two, or one, and the antibody molecule retains the ability to inhibit adrenomedullin activity, for example, the angiogenic or invasive activity of adrenomedullin.

[0094] Furthermore, the framework region of the variable region can also be modified alternatively or additionally. Such changes to the framework region may improve antibody stability and reduce antibody immunogenicity.

[0095] The antibody molecules of the present invention include “chimeric” antibodies in which, to the extent that they exhibit desired biological activity, a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies (see U.S. Patent No. 4,816,567, Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). The chimeric antibodies of interest as used herein include “primatized” antibodies comprising a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and a human constant region sequence.

[0096] In certain embodiments of the present invention, the antibody molecule according to the present invention or used in the present invention is a humanized antibody. The humanized antibody molecule used in the present invention can be produced by any suitable method.

[0097] For example, in certain embodiments of the present invention, the antibody molecule is a humanized version of the rIgG-k1-6D8 antibody.

[0098] In one such embodiment, the humanized antibody has a heavy chain variable sequence selected from the VH humanized variants indicated by Seq ID No: 65, 66, 67, 68, and 69 and / or a light chain variable sequence selected from the VL humanized variants indicated by Seq ID No: 70, 71, 72, 73, and 74. These VH and VL sequences are shown below.

[0099] >VH1 (Sequence ID No: 65) EVQLVESGGGLVQPGGSLRLSCAASGFSLSS SYWIC WVRQAPGKGLEWIA CIVSNTGTTY YASWAKGRFTLSKDTSKTTLTLQMNSLRAEDTAVYFCAR DNNSKSGVEFNL WGQGTLVTVSS >VH2 (Sequence ID No: 66) ELQLVESGGAVVQPGESLRLSCAASGFSLSS SYWIC WVRQAPGRGLEWVA CIVSNTGTTY YASWAKGRLTISKDTSSNTLYLQMNSLRAEDTAMYFCAR DNNSKSGVEFNL WGQGTLVTVSS >VH3 (Sequence ID No: 67) ELQLVESGGAVVQPGESLRLSCAASGFSLSS SYWIC WVRQAPGRGLEWVG CIVSNTGTTY YASWAKGRLTISKTSSNTLYLQMNSLRAEDTAMYFCAR DNNSKSGVEFNL WGQGTLVTVSS >VH4 (Sequence ID No: 68) EVQLVESGGDLAQPGGSLRLSCTVSGFSLSS SYWIC WVRQAPGKGLEWVA CIVSNTGTTY YASWAKGRLTISRDNSKNTVYLQMTSLRAEDTALYFCAR DNNSKSGVEFNL WGQGTLVTVSS >VH5 (Sequence ID No: 69) QVQLVQSGGEVKQPGASVKVSCTASGFSLSS SYWIC WVRQAPGKGLEWMG CIVSNTGTT YYASWAKGRFVLSKDTSASTATLQISSLKAADTATYFCAR DNNSKSGVEFNL WGPGTLVTVSS

[0100] Humanized variant of the VL domain: >VL1 (Sequence ID No: 70) DIQMTQSPSTLSASVGDRVTITC QASQNIGNNLA WYQQKPGHAPKLLIY LASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QSYLYTTYA FGQGTKLEIK >VL2 (Sequence ID No: 71) DIQMTQSPSSLSASVGDRVTIKC QASQNIGNNLA WYQQKPGKVPKLLIY LASTLAS GVPSRFKGSRSGTDFTLTISSLQPEDVATYYC QSYLYTTYA FGGGTKVEIK >VL3 (Sequence ID No: 72) DVVMTQSPSTVSASVGDRVTLKC QASQNIGNNLA WYQQKPGHPPKLLIY LASTLAS GVPDRFSGSGSGTDFTLTISSLEAADAATYYC QSYLYTTYA FGQGTKVEIK >VL4 (Sequence ID No: 73) EIVMTQSPATLSVSPGERATLKC QASQNIGNNLA WYQQKPGHPPRLLIY LASTLAS GIPARFKGSRSGTEFTLTISDLESADAATYYC QSYLYTTYA FGGGTEVVVK >VL5 (Sequence ID No: 74) DIQMTQSPSSLSASVGDRVTIKC QASQNIGNNLA WYQQKPGHPPKLLIY LASTLAS GVPSRFKGSRSGTDFTLTISDLEPADAATYYC QSYLYTTYA FGGGTEVVVK

[0101] In one such embodiment, the humanized antibody has a heavy chain variable region sequence selected from a VH humanized variant having the amino acid sequences shown in SEQ ID NOs. 65, 66, 67, 68, and 69, and a light chain variable region sequence selected from a VL humanized variant having the amino acid sequences shown in SEQ ID NOs. 70, 71, 72, 73, and 74.

[0102] To address the problem of unwanted immune responses to non-human therapeutic antibodies administered to individuals, many methods have been developed for generating humanized antibodies. These methods typically involve substituting specific components of a non-human antibody with equivalent portions derived from a human antibody. For example, such methods generate chimeric antibodies that possess a non-human variable region linked to a human-derived constant region.

[0103] Alternatively, antibodies can be humanized using techniques such as "CDR grafting" or Composite Human Antibody technology (trademark) (Abzena, Cambridge, UK). Composite Human Antibody is entirely human-derived and comprises multiple human variable region sequence segments from different human antibodies. Such techniques can be used to produce fully humanized monoclonal antibodies that exhibit single binding specificity, with both the variable region framework and the CDR region derived from human germline immunoglobulin sequences. This is in contrast to humanized antibodies produced by "CDR grafting," in which a complementation-determining region (CDR) derived from a mouse antibody is grafted onto a framework receptor sequence provided by regions of human antibody light and heavy chain variable domains. This results in an antibody in which the non-human component is only the grafted mouse CDR region, while retaining the binding specificity of the mouse antibody.

[0104] In CDR grafting, the therapeutic effect of the resulting humanized antibody may be impaired. For example, simple transplantation of the CDR region has often been observed to reduce the therapeutic effect of the antibody due to decreased antibody binding affinity. To address this problem, reverse mutations can be used, which involve substituting amino acid residues at specific positions in the framework sequence (outside the CDR sequence) to improve the binding specificity of the humanized antibody.

[0105] Typically, in CDR grafting technology, the CDR of a donor antibody is selected using one of the following methods: Kabat, IMGT, or Chothia. Usually, a reverse mutation of a specific residue outside the CDR sequence is required to restore satisfactory affinity. As described in International Publication No. 2014 / 072741, a method combining the IMGT and Kabat methods to define the CDR sequence allows for the easy acquisition of high-affinity humanized antibodies.

[0106] Therefore, in embodiments of the present invention, a humanized antibody molecule may be used, which has a heavy chain, and its complementarity-determining region (CDR) is derived from a non-human donor antibody. (i) The first and last amino acid residues of the humanized antibody molecule CDRH1 are the first amino acid residue and the last amino acid residue of the donor antibody CDRH1 as defined by the IMGT method and Kabat method, respectively, and CDRH1 comprises intermediate amino acid residues of the donor CDRH1 between the first and last amino acid residues. (ii) The first and last amino acid residues of the CDRH3 of the humanized antibody molecule are the first amino acid residue and the last amino acid residue of the CDRH3 of the donor antibody as defined by the IMGT method and the Kabat method, respectively, and the CDRH3 comprises intermediate amino acid residues of the donor CDRH3 between the first and last amino acid residues, and (iii) The CDRH2 of the humanized antibody molecule corresponds to the CDRH2 of the donor antibody as defined by the Kabat method.

[0107] The term "derived from" is intended to encompass not only substances derived from a physical source, but also substances that are structurally identical to the reference source but do not originate from it. Therefore, CDRs derived from non-human donor antibodies do not necessarily need to be purified or isolated from the donor antibody or antibody framework.

[0108] (Production of antibody molecules) The antibody molecules of the present invention and antibody molecules for use in this invention can be produced either naturally or synthetically by any suitable method. Such methods may include, for example, conventional hybridoma technology (Kohler and Milstein (1975) Nature, 256:495-499), recombinant DNA technology (see, e.g., U.S. Patent No. 4,816,567), or phage display technology using antibody libraries (see, e.g., Clackson et al., (1991) Nature, 352:624-628 and Marks et al., (1992) Bio / Technology, 10:779-783). Other antibody production techniques are described in Antibodies: A Laboratory Manual, edited by Greenfield et al., Cold Spring Harbor Laboratory, 2012.

[0109] The antibody molecules of the present invention can be prepared by standard synthesis techniques known to those skilled in the art, such as liquid-phase peptide synthesis or solid-phase peptide synthesis. Alternatively, the antibody molecules can be prepared in solution using liquid-phase peptide synthesis techniques, and further by a combination of solid-phase, liquid-phase, and solution chemistry.

[0110] The present invention further extends to generating the antibody molecule of the present invention by expressing a nucleic acid encoding at least one amino acid sequence, either alone or in combination with one or more other amino acid sequences, in a suitable expression system. As a result, a desired peptide or polypeptide can be encoded. For example, the antibody molecule of the present invention can be provided by expressing a nucleic acid encoding an amino acid light chain and a second nucleic acid encoding an amino acid heavy chain.

[0111] Conventional hybridoma techniques typically involve immunizing mice or other animals with an antigen to induce the production of lymphocytes capable of binding to the antigen. The lymphocytes are isolated and fused with myeloma cell lines to form hybridoma cells, which are then cultured under conditions that suppress the proliferation of the parent myeloma cells but allow the proliferation of antibody-producing cells. Hybridomas can be subjected to genetic mutations, which may or may not alter the binding specificity of the antibodies produced. Synthetic antibodies can be produced using techniques known to those skilled in the art (see, for example, Knappik et al., J.Mol.Biol.(2000)296, 57-86 and Krebs et al., J.Immunol.Meth.(2001)2154 67-84).

[0112] The binding members, VH, VL, or CDR, and even FR, can be modified using any suitable technique known to those skilled in the art. For example, variable VH and / or VL domains can be generated by introducing a CDR, such as CDR3, into a VH or VL domain lacking such a CDR. Marks et al., (1992) Bio / Technology, 10: 779-783, describe a shuffling technique for creating a repertoire of VH variable domains lacking CDR3 and combining them with CDR3 of a specific antibody to generate novel VH regions. Novel VH and VL domains comprising the CDR-derived sequence of the present invention can be generated using a similar technique.

[0113] Accordingly, in one embodiment of the present invention, the present invention provides a method for producing an antibody molecule having specificity for RAMP2. The method includes (a) providing a starting repertoire of nucleic acids encoding a variable domain, wherein the variable domain comprises a substituted CDR1, CDR2, or CDR3, or the nucleic acid lacks a region encoding such a CDR; (b) combining the repertoire with a donor nucleic acid encoding the CDR amino acid sequence of an anti-RAMP2 variable domain, wherein the anti-RAMP2 variable domain has an amino acid sequence indicated as Seq ID No: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, or 26, and as a result the donor nucleic acid is inserted into the CDR region in the repertoire to provide a productive repertoire of nucleic acids encoding the variable domain; (c) expressing the nucleic acids of the productive repertoire; (d) selecting an antigen-binding fragment specific to RAMP2; and (e) recovering the specific antigen-binding fragment or the nucleic acid encoding it. This method may include an optional step of testing whether an antibody molecule has the ability to inhibit adrenomedullin activity.

[0114] In another embodiment, the present invention provides a method for producing an antibody molecule having specificity to RAMP3, the method comprising: (a) providing a starting repertoire of nucleic acids encoding a variable domain, wherein the variable domain comprises a substituted CDR1, CDR2, or CDR3, or the nucleic acid lacks a region encoding such a CDR; (b) combining the repertoire with a donor nucleic acid encoding the CDR amino acid sequence of an anti-RAMP3 variable domain, wherein the anti-RAMP3 variable domain has an amino acid sequence indicated as Seq ID No: 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, or 54, thereby providing a productive repertoire of nucleic acids encoding the variable domain by inserting the donor nucleic acid into the CDR region in the repertoire; (c) expressing the nucleic acids of the productive repertoire; (d) selecting an antigen-binding fragment specific to RAMP3; and (e) recovering the specific antigen-binding fragment or the nucleic acid encoding it. This method may include an optional step of testing whether an antibody molecule has the ability to inhibit adrenomedullin activity.

[0115] Other techniques for generating the mutant antibody molecules of the present invention include, for example, random mutagenesis of genes encoding VH or VL domains using error-induced PCR (Gram et al., 1992, PNAS 89 3576-3580). In addition or alternatively, CDRs can be targeted for mutagenesis using molecular evolutionary methods (described in Barbas et al., 1991 PNAS 3809-3813 and Scier 1996 J Mol Biol 263 551-567).

[0116] After generating such mutants, antibodies, and fragments, they can be tested to determine whether they bind to RAMP2 or RAMP3, and whether they have the ability to suppress the activity of adrenomedullin.

[0117] As described herein, the inventors have demonstrated that the antibody molecule of the present invention has anti-angiogenic and / or anti-invasive effects. Therefore, it is possible to use the antibody molecule of the present invention as a therapeutic agent containing it as an active ingredient.

[0118] Therefore, in one embodiment of the present invention, the antibody molecule is a "naked" antibody molecule. A "naked" antibody molecule is an antibody molecule that is not conjugated with an "effective therapeutic agent".

[0119] In the context of this application, "effective therapeutic agent" refers to a molecule or atom that is conjugated to an antibody portion (including antibody fragments, CDRs, etc.) to generate a conjugate. Examples of such "effective therapeutic agents" include drugs, toxins, radiosensitizers, radioisotopes, molecules that convert prodrugs into active drugs, cytotoxic chemotherapeutic agents, cytotoxic peptides, immunomodulators, chelating agents, boron compounds, dyes, nanoparticles, etc.

[0120] In one embodiment of the present invention, the antibody molecule is in the form of an immunoconjugate, comprising an antibody fragment conjugated with an "active therapeutic agent".

[0121] Methods for generating immunoconjugates are known to those skilled in the art. For example, see U.S. Patent No. 5,057,313, Shih et al., Int. J. Cancer 41: 832-839 (1988); Shih et al., Int. J. Cancer 46: 1101-1106 (1990), Wong, Chemistry Of Protein Conjugation And Cross-Linking (CRC Press 1991); Upeslacis et al., “Modification of Antibodies by Chemical Methods” in Monoclonal Antibodies: Principles And Applications, edited by Birch et al., pp. 187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterization of Synthetic Peptide-Derived Antibodies,” in Monoclonal Antibodies: Production, Engineering And Clinical Application, edited by Ritter et al., pp. 60-84 (Cambridge University Press 1995).

[0122] The antibody molecule of the present invention may be further modified. For example, the antibody molecule may be glycosylated, polyethylene glycolated, or conjugated to albumin or a non-proteinoid polymer. The antibody molecule may be in the form of an immunoconjugate.

[0123] In one embodiment of the present invention, the antibody molecule is defucosized. The ADCC activity of the antibody has been found to depend on the amount of fucose bound to the antibody. Defucosized therapeutic antibodies have been shown to be more potent than their corresponding fucoseized antibodies (Mori et al., Cytotechnology (2007) 55:109-114). Therefore, in certain situations, such as when ESCSR is expressed in the cancer endothelium of blood vessels nourishing tumors, defucosized antibodies may be useful in enhancing ADCC targeting of such blood vessels. Many techniques exist for generating defucosized antibodies by those skilled in genetic engineering and molecular biology. These include, but are not limited to, post-production fucose removal via incubation of antibodies with α-1,6-fucosidase; genetic engineering to reduce or eliminate fucoseization in mammalian cells by mutations in the fucose transferase genes FUT8 or GMD; inhibition of fucose addition by transfection of the glycosyl-N-transferase gene GnTIII; or disruption of the FUT8 and / or GMD genes using siRNA (see, for example, Jefferis R. Trends Pharmacol Sci. 2009 Jul;30(7):356-62; Yamane-Ohnuki N, Satoh M. MAbs. 2009 May;1(3):230-6; Mori K, et al., Cytotechnology. 2007 Dec;55(2-3):109-14).

[0124] The antibody molecules of the present invention can be labeled. Available labeling methods may include radiolabeling, enzymatic labeling such as horseradish peroxidase, alkaline phosphatase, and biotin. As described in the examples, the antibodies of the present invention have been shown to bind to cancer cells. Therefore, labeled antibody molecules may be useful in tumor imaging, tumor detection, diagnosis, or prognostic imaging.

[0125] In fact, another aspect of the present invention provides a method for imaging neovascular structures in tumors of an individual, the method being: The steps include administering the antibody molecule of the present invention to an individual, wherein the antibody molecule has a labeling site, The method includes the step of detecting the location of a marked site within an individual.

[0126] Further aspects of the present invention include a method for detecting tumor cells, such as solid tumors, in the cells, tissues, or organs of an individual, the method being: The present invention comprises the steps of administering the antibody molecule to cells, tissues, organs, or an organism in which the antibody molecule has a labeling site, The process includes the step of detecting the presence and / or location of a marked site in a cell, tissue, organ, or organism.

[0127] The method may be carried out in vivo, ex vivo, or in vitro, as appropriate.

[0128] The method may be used to diagnose the presence of a tumor or to monitor tumor progression and / or response to a particular treatment regimen. For example, the method may be repeated at time intervals during the patient's treatment period to determine differences in the size, vascularization, and other characteristics of any detected tumors.

[0129] The ability of an antibody molecule to inhibit adrenomedullin activity can be tested using any suitable method. For example, the ability of an antibody molecule to inhibit angiogenesis can be tested using any suitable assay known in the field. For example, assays described in the examples, such as those using Matrigel, can be used, for example, by seeding HUVEC cells in Matrigel with an antibody molecule and a suitable control. Tubular vessel formation can be monitored and quantified by measuring, for example, the decrease in branching point number, number of vessels, mean vessel length, and maximum vessel length compared to a control.

[0130] The ability of an antibody molecule to inhibit tumor cell invasion can be tested using any suitable invasion assay known in the field. For example, such ability can be tested using a modified Boyden chamber. An antibody molecule is considered to inhibit tumor cell invasion if it has the ability to inhibit invasion by a statistically significant amount. For example, in one embodiment, the antibody molecule has an invasion inhibitory ability of at least 10%, e.g., at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a suitable control antibody.

[0131] The ability of an antibody molecule to inhibit the chemotactic activity of adrenomedullin can be tested using a HUVEC migration assay. In such an assay, an antibody molecule is considered to inhibit the chemotactic activity of adrenomedullin if it has the ability to inhibit the migration of HUVEC cells to the endothelial cell supplement by a statistically significant amount. For example, in one embodiment, the antibody molecule has at least 10%, e.g., at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% chemotactic activity inhibitory ability compared to a suitable control antibody. A suitable control antibody may be, for example, a control antibody of the same isotype that does not inhibit the chemotactic effect of adrenomedullin, e.g., an IgG1 isotype control antibody.

[0132] Other assays include wound assays. The degree of "wound" closure is assessed blindly under a microscope by an independent evaluator and can be quantified, for example, using a calibrated eyepiece micrometer. The degree of closure in antibody-treated slides can be compared to time-matched control slides, and the percentage of wound closure inhibition compared to the time-matched control can be calculated.

[0133] In certain embodiments of the present invention, the antibody molecule of the present invention has the ability to induce or promote necrosis in vascular structures or other locations within a tumor. The ability of the antibody molecule to induce or promote necrosis can be evaluated by any suitable means. For example, such ability can be evaluated by histological examination of tumor tissue obtained from a xenograft model of the cancer under investigation. For example, as described in the examples, a xenograft model using a glioblastoma U87MG tumor can be used, and necrotic cells can be identified and evaluated by caspase-3 staining.

[0134] In one embodiment, an antibody molecule is considered to induce or promote apoptosis if the area percentage of necrotic cells in tumor tissue sections obtained from animals treated with the antibody molecule is more than twice, for example, more than four times, more than five times, more than seven times, more than ten times, more than twelve times, or more than fifteen times, compared to the area percentage of necrotic cells in tumor tissue sections obtained from animals treated with a control, for example, PBS or a control antibody.

[0135] (nucleic acid) The nucleic acids used in this invention are DNA or RNA, which can be produced by recombinant, synthetic, or any means available to those skilled in the art, such as cloning by standard techniques.

[0136] As described above, the present invention encompasses nucleic acids encoding the VL or VH chain of the antibody molecule of the present invention. Therefore, in one embodiment, the nucleic acid of the present invention is (i) Nucleotide sequence indicated by VL 1H6 (Sequence ID No: 3) and / or nucleotide sequence indicated by VH 1H6 (Sequence ID No: 4); (ii) The nucleotide sequence indicated by VL 1A7 (Sequence ID No: 7) and / or the nucleotide sequence indicated by VH 1A7 (Sequence ID No: 8); (iii) The nucleotide sequence indicated by VL 2A6 (Sequence ID No: 11) and / or the nucleotide sequence indicated by VH 2A6 (Sequence ID No: 12); (iv) The nucleotide sequence indicated by VL 2A8 (Sequence ID No: 15) and / or the nucleotide sequence indicated by VH 2A8 (Sequence ID No: 16); (v) Nucleotide sequence indicated by VL 2H3 (Sequence ID No: 19) and / or nucleotide sequence indicated by VH 2H3 (Sequence ID No: 20); (vi) Nucleotide sequence indicated by VL 1H7 (Sequence ID No: 23) and / or nucleotide sequence indicated by VH 1H7 (Sequence ID No: 24); (vii) Nucleotide sequence indicated by VL 1A10 (Sequence ID No: 27) and / or nucleotide sequence indicated by VH 1A10 (Sequence ID No: 28); (viii) Nucleotide sequence indicated by VL 3A5 (Sequence ID No: 31) and / or nucleotide sequence indicated by VH 3A5 (Sequence ID No: 32); (ix) Nucleotide sequence indicated by VL 3A12 (Sequence ID No: 35) and / or nucleotide sequence indicated by VH 3A12 (Sequence ID No: 36); (x) Nucleotide sequence indicated by VL 4C10 (Sequence ID No: 39) and / or nucleotide sequence indicated by VH 4C10 (Sequence ID No: 40); (xi) Nucleotide sequence indicated by VL 5H9 (Sequence ID No: 43) and / or nucleotide sequence indicated by VH 5H9 (Sequence ID No: 44); (xii) Nucleotide sequence indicated by VL 6B8 (Sequence ID No: 47) and / or nucleotide sequence indicated by VH 6B8 (Sequence ID No: 48); (xiii) The nucleotide sequence indicated by VL 6C2 (Sequence ID No: 51) and / or the nucleotide sequence indicated by VH 6C2 (Sequence ID No: 52), or (xiv) May comprise the nucleotide sequence indicated by VL 6D8 (Sequence ID No: 55) and / or the nucleotide sequence indicated by VH 6D8 (Sequence ID No: 56).

[0137] Nucleic acids can be inserted into any suitable vector. Vectors comprising the nucleic acids of the present invention form further embodiments of the present invention. In one embodiment, the vector is an expression vector, and the nucleic acid is operably linked to a regulatory sequence that enables the expression of the nucleic acid in a host cell. Various vectors can be used. For example, suitable vectors include viruses (e.g., vaccinia virus, adenovirus, etc.), baculoviruses, yeast vectors, phages, chromosomes, artificial chromosomes, plasmids, or cosmid DNA.

[0138] Vectors can be used to introduce the nucleic acids of the present invention into host cells. Various host cells can be used for the expression of the nucleic acids of the present invention. Suitable host cells for use in the present invention may be prokaryotes or eukaryotes. These include bacteria, such as Escherichia coli, yeast, insect cells, and mammalian cells. Usable mammalian cell lines include Chinese hamster ovary cells, baby hamster kidney cells, NSO mouse melanoma cells, monkey and human cell lines and their derivatives, and many others.

[0139] Host cell lines that regulate, modify, and / or specifically process gene product expression may be used. Such processing may include glycosylation, ubiquitination, disulfide bond formation, and general post-translational modifications.

[0140] Therefore, the present invention also provides a host cell comprising one or more nucleic acids or vectors of the present invention.

[0141] Furthermore, the present invention also encompasses a method for producing the antibody molecule of the present invention. The method includes culturing a host cell comprising the nucleic acid of the present invention under conditions in which an antibody molecule is expressed from the nucleic acid, and isolating and / or purifying the antibody molecule as necessary.

[0142] For details on known techniques and protocols for nucleic acid manipulation, such as preparation of nucleic acid constructs, mutagenesis, sequencing, DNA delivery and gene expression into cells, and protein analysis, see, for example, Current Protocols in Molecular Biology, 5th ed., edited by Ausubel et al., John Wiley & Sons, 2005 and Molecular Cloning: a Laboratory Manual: 3 rd See edition Sambrook et al., Cold Spring Harbor Laboratory Press, 2001.

[0143] (treatment) "Treatment" includes any system that may be beneficial to humans or non-human animals. Treatment may be for an existing condition or may be preventive (preventive treatment). Treatment may include curative, mitigating, or preventive effects.

[0144] The antibody molecules and nucleic acids of the present invention can be used to treat a variety of conditions and disorders. These include neoplastic diseases / cancer and other diseases associated with angiogenesis.

[0145] "Therapy of cancer" includes the treatment of conditions caused by cancerous growth and / or vascularization, and includes the treatment of neoplastic growth or malignant tumors. Examples of tumors that can be treated using the present invention include, for example, sarcomas including osteogenic sarcomas and soft tissue sarcomas, carcinomas such as breast cancer, lung cancer, bladder cancer, thyroid cancer, prostate cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, liver cancer, uterine cancer, prostate cancer, cervical cancer and ovarian cancer, non-small cell lung cancer, hepatocellular carcinoma, lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma, neuroblastoma, melanoma, myeloma, Wilms' tumor, astrocytoma, glioma and retinoblastoma.

[0146] In one particular embodiment, the antibody molecule or nucleic acid of the present invention may be used to treat glioblastoma.

[0147] In other embodiments of the present invention, the antibody molecule or nucleic acid of the present invention may be used for the treatment of renal cancer.

[0148] The present invention may be particularly useful for the treatment of existing cancers and for preventing cancer recurrence after initial treatment or surgery.

[0149] The antibody molecules, nucleic acids, and compositions of the present invention may also be used to treat other disorders mediated by or related to angiogenesis. Such conditions include, for example, benign tumors, various autoimmune diseases, genetic diseases, and ocular diseases.

[0150] The methods of the present invention may be used to treat angiogenesis-mediated disorders, including hemangiomas, solid tumors, leukemia, metastatic reactions, telangiectasia, psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis, Crohn's disease, plaque neovascularization, coronary collateral circulation, cerebral collateral circulation, arteriovenous malformations, ischemic limb angiogenesis, corneal diseases, iris neovascularization, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, arthritis, diabetic neovascularization, macular degeneration, peptic ulcers, Helicobacter-related diseases, fractures, keloids, and angiogenesis.

[0151] The antibody molecules, nucleic acids, and methods of the present invention can also be used to treat inflammation associated with angiogenesis, such as various arthritis, including rheumatoid arthritis and osteoarthritis.

[0152] Furthermore, these methods provide combination therapy with the compounds described herein and other agents useful for treating the disorder. Such agents include, for example, cyclooxygenase-2 (COX-2) inhibitors known to those skilled in the art.

[0153] Blood vessels within the synovial membrane of joints can undergo angiogenesis. Endothelial cells form new vascular networks and release factors and reactive oxygen species that cause pannus proliferation and cartilage destruction. These factors are thought to be actively involved in rheumatoid arthritis and osteoarthritis. Activation of chondrocytes by angiogenesis-related factors contributes to joint destruction and also promotes new bone formation. The antibody molecules, nucleic acids, and methods described herein can be used as therapeutic interventions to prevent bone destruction and new bone formation.

[0154] Pathological angiogenesis is also thought to be involved in chronic inflammation. Examples of disorders that can be treated using the methods described herein include ulcerative colitis, Crohn's disease, and arteriosclerosis.

[0155] Because adrenomedullin has vasodilatory activity, the antibody molecule of the present invention can be used to treat conditions or diseases characterized by excessive vasodilation, or conditions in which vasoconstriction may be beneficial.

[0156] In the later stages of sepsis, adrenomedullin is known to constitute a risk factor strongly associated with mortality in patients with septic shock. In one embodiment, the antibody molecule of the present invention may be used for the treatment of the later stages of sepsis and septic shock.

[0157] Other conditions in which antibodies against adrenomedullin have been described (see, for example, International Publication No. 2004 / 097423, International Publication No. 2006 / 027147A1, PCT / EP2005 / 012844) and conditions in which the antibody molecule of the present invention may be used in one embodiment include, for example, cardiovascular diseases, infectious diseases, skin diseases, neurological diseases, urinary tract diseases, endocrine diseases, metabolic diseases, gastrointestinal diseases, hematological diseases, and respiratory diseases.

[0158] (Pharmaceutical composition) Antibody molecules and nucleic acids can be administered as pharmaceutical compositions. The pharmaceutical compositions according to the present invention and the pharmaceutical compositions used in accordance with the present invention, in addition to the active ingredient, may contain pharmaceutically acceptable excipients, carriers, buffer stabilizers, or other materials known to those skilled in the art (e.g., Remington: The Science and Practice of Pharmacy, 23) rd (See edition, Adejare A et al., Academic Press, 2020). Such materials may include buffers such as acetic acid, tris, phosphoric acid, citric acid and other organic acids, antioxidants, preservatives, proteins (e.g., serum albumin, gelatin or immunoglobulin), hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine, carbohydrates, chelating agents, modifiers and surfactants.

[0159] The pharmaceutical composition may also contain one or more additional active compounds as needed, depending on the specific indication being treated, preferably having complementary activity that does not have adverse effects on the activity of the binding molecule, nucleic acid or composition of the present invention. For example, in the treatment of cancer, in addition to the anti-RAMP2 or anti-RAMP3 antibody molecule of the present invention, the formulation may comprise additional antibodies that bind to different epitopes of anti-RAMP2 or anti-RAMP3, or antibodies against other targets such as adrenomedullin or growth factors that affect the growth of certain cancers, and / or chemotherapeutic agents.

[0160] Active ingredients (e.g., antibody molecules and / or chemotherapeutic agents) may be administered via microspheres, microcapsules, liposomes, or other microparticle delivery systems. For example, active ingredients may be encapsulated in microcapsules prepared in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, for example, by coacervation technology or interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules, poly(methyl methacrylate) microcapsules. For further details, see Remington: the Science and Practice of Pharmacy, 23. rd See edition, edited by Adejare A et al., Academic Press, 2020.

[0161] Sustained-release formulations can be used for the delivery of active ingredients. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies, in the form of molded articles, e.g., films, suppositories, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid.

[0162] As described above, the nucleic acids of the present invention can also be used in a treatment method. The nucleic acids of the present invention can be delivered to the target cells using any suitable technique known to those skilled in the art. The nucleic acids (which may be contained within a vector if necessary) can be delivered to the cells of a patient using in vivo or ex vivo techniques. As in vivo techniques, transfection by viral vectors (such as adenovirus, herpes simplex virus type I, adeno-associated virus, etc.) and lipid-based systems (lipids useful for lipid-mediated transfer of genes include, for example, DOTMA, DOPE, and DC-Chol) can be used (see, for example, Anderson et al., Science 256: 808-813 (1992). Also see WO 93 / 25673).

[0163] In ex vivo techniques, the nucleic acids are introduced into cells isolated from the patient, and the modified cells are administered either directly to the patient or encapsulated, for example, within a porous membrane (see, for example, U.S. Patent Nos. 4,892,538 and 5,283,187). Techniques available for introducing nucleic acids into living cells can include retroviral vectors, liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like.

[0164] The binding molecule, agent, product or composition can be administered locally to the tumor site or other desired site, or delivered in a manner that targets the tumor or other cells. Targeted therapy can be used to specifically deliver the active ingredient to a specific cell type by using a targeting system such as an antibody or a cell-specific ligand. Targeting may be desirable for various reasons, for example, when the agent has unacceptable toxicity, when an excessive dosage is required, or when it cannot enter the target cells.

[0165] (Dosage) The antibody molecules, nucleic acids, or compositions of the present invention are preferably administered to an individual in a "therapeutably effective amount," that is, an amount sufficient to provide a benefit to the individual. The optimal dosage and actual administration regimen may be determined by a physician and depend on several factors, including the condition being treated, its severity, the age, sex, and weight of the patient being treated, the agent used, and the route of administration, and are left to the physician's discretion.

[0166] As a general guideline, antibody dosages can be administered in the range of 1 ng / kg to 500 mg / kg of patient body weight.

[0167] The present invention will be further described in the following non-limiting embodiments. Please refer to the accompanying drawings. [Brief explanation of the drawing]

[0168]

Figure 1A

Example

[0169] (Example 1 Preparation of Antibody)

[0170] (Expression and Purification of RAMP2 Variant) DNAs encoding the amino acid sequences of several RAMP2 variants (see Table 1) were synthesized and cloned into the mammalian transient expression plasmid pETE V1 (Fusion antibodies plasmid). The RAMP2 variants were expressed using a HEK-based transient expression system, and the resulting antibody-containing cell culture supernatant was clarified by centrifugation and filtration. The RAMP2 variants were purified from the cell culture supernatant via affinity chromatography (using a state-of-the-art AKTA chromatography device). The successfully purified product was dialyzed / buffer-exchanged into a phosphate-buffered saline aqueous solution. The purity of the RAMP2 variant was determined by reducing and denaturing sodium dodecyl sulfate polyacrylamide gel and was determined to exceed 95%. The bacterial endotoxin level was determined using the Endosafe®-PTS system and Endosafe® PTS cartridges (manufactured by Charles River Laboratories). The protein concentration was determined by measuring the absorbance at 280 nm and calculated using a standard theoretical extinction coefficient (determined by Expasy protoparm).

[0171] Table 1 - Amino Acid Sequences of RAMP2 Constructs: (The signal peptide is underlined) >RAMP2_IMM_V1 (Sequence ID No: 57) MGWTLVFLFLLSVTAGVHS QPLPTTGTPGSEGGTVKNYETAVQFCWNHYKDQMDPIEKDWCDWAMISRPYSTLRDCLEHFAELFDLGFPNPLAERIIFETHQIHFANCSLVQPTFSDPPEDVGGSHRRLDKIEDERNLHEDFVFMKTIQRCNKGEGSLSLLNCKEIRSQFEGFVKDIMLNKEEPKKEINFEMQKGDQDPQIAAHLISEASSKSSSVLQWAKKGYYTMSNTLVTLENGKQLKVKRQGFYYIYAQVTFCSNQEPSSQAPFIASLCLKSSGGSERILLRAANARSSSKTCEQQSIHLGGVFELQADASVFVN VTDASQVNHGTGFTSFGLLKLRGSHHHHHH ** >RAMP2_Scr_V1 (Sequence ID No: 58) MGWTLVFLFLLSVTAGVHS QPLPTTGTPGSEGGTVKNYETAVQFCWNHYKDQMDPIEKDWCDWAMISRPYSTLRDCLEHFAELFDLGFPNPLAERIIFETHQIHFANCSLVQPTFSDPPEDVGSGAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ** >RAMP2_IMM_ECD_V1 (Sequence ID No: 59) MGWTLVFLFLLSVTAGVHSQPLPTTGTPGSEGGTVKNYETAVQFCWNHYKDQMDPIEKDWCDWAMISRPYSTLRDCLEHFAELFDLGFPNPLAERIIFETHQIHFANCSLVQPTFSDPPEDV RGSHHHHHH ** >RAMP2_IMM_V2 (Sequence ID No: 60) MGWTLVFLFLLSVTAGVHS QPLPTTGTPGSEGGTVKNYETAVQFCWNHYKDQMDPIEKDWCDWAMISRPYSTLRDCLEHFAELFDLGFPNPLAERIIFETHQIHFANCSLVQPTFSDPPEDVGGSPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVR TARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK **

[0172] (Expression and purification of RAMP3 variants) DNA encoding the amino acid sequences of several RAMP3 variants (see Table 2) was synthesized and cloned into the mammalian transient expression plasmid pETE V1 (Fusion antibody plasmid). RAMP3 variants were expressed using an HEK-based transient expression system, and the resulting antibody-containing cell culture supernatant was clarified by centrifugation and filtration. RAMP3 variants were purified from the cell culture supernatant via affinity chromatography (using a state-of-the-art AKTA chromatography system). The purified product was dialyzed / buffered with phosphate-buffered saline. The purity of the RAMP3 variants was determined to be over 95% by reduction and denaturation of sodium dodecyl sulfate polyacrylamide gel. Bacterial endotoxin levels were determined using the Endosafe®-PTS system and Endosafe®-PTS cartridge (Charles River Laboratories). Protein concentration was determined by measuring absorbance at 280 nm and calculated using the standard theoretical extinction coefficient (determined by Expasy protoparm).

[0173] Table 2 - Amino acid sequence of RAMP3 construct: (Signal peptides are underlined.) >RAMP3_IMM_V1 (Sequence ID No: 61) MGWTLVFLFLLSVTAGVHS RAGGCNETGMLERLPLCGKAFADMMGKVDVWKWCNLSEFIVYYESFTNCTEMEANVVGCYWPNPLAQGFITGIHRQFFSNCTVDRVHLEDPPDEVGGSHRRLDKIEDERNLHEDFVFMKTIQRCNKGEGSLSLLNCKEIRSQFEGF VKDIMLNKEEPKKEINFEMQKGDQDPQIAAHLISEASSKSSSSVLQWAKKGYYTMSNTLVTLENGKQLKVKRQGFYYIYAQVTFCSNQEPSSQAPFIASLCLKSSGGSERILLRAANARSSSKTCEQQSIHLGGVFELQADASVFVN VTDASQVNHGTGFTSFGLLKLRGSHHHHHH** >RAMP3_Scr_V1 (Sequence ID No: 62) MGWTLVFLFLLSVTAGVHS RAGGCNETGMLERLPLCGKAFADMMGKVDVWKWCNLSEFIVYYESFTNCTEMEANVVGCYWPNPLAQGFITGIHRQFFSNCTVDRVHLEDPPDEVGSGAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ** >RAMP3_IMM_ECD_V1 (Sequence ID No: 63) MGWTLVFLFLLSVTAGVHS RAGGCNETGMLERLPLCGKAFADMMGKVDVWKWCNLSEFIVYYESFTNCTEMEANVVGCYWPNPLAQGFITGIHRQFFSNCTVDRVHLEDPPDEVRGSHHHHHH ** >RAMP3_IMM_V2 (Sequence ID No: 64) MGWTLVFLFLLSVTAGVHS RAGGCNETGMLERLPLCGKAFADMMGKVDVWKWCNLSEFIVYYESFTNCTEMEANVVGCYWPNPLAQGFITGIHRQFFSNCTVDRVHLEDPPDEVGGSPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK**

[0174] (Immune and antibody response) Three rabbits were immunized three times at 3-week intervals with RAMP2 Imm V2 (2.35 mg / ml, lot 030419) and three rabbits with RAMP3 Imm V2 (3.08 mg / ml, lot 030419). In the first immunization, 200 μg of antigen was used in complete Freund's adjuvant, and in subsequent immunizations, the antigen was used in incomplete Freund's adjuvant. Three weeks after the third immunization, the animals received a booster immunization by intravenously administering half of the antigen in PBS and the remaining half subscapularly in incomplete Freund's adjuvant. The animals were euthanized three days after the final booster immunization. Splenocytes were isolated from the spleen within 3 hours and frozen in liquid nitrogen for storage.

[0175] Serum was collected on day 0 and on day 10 after the second or third immunization. Specific antibody titers were measured by ELISA, and target proteins (RAMP2 Scr or RAMP3 Scr, respectively) were coated onto plates (Nunc MaxiSorp, Thermo; 1 μg / ml in PBS). RAMP2 IMM V2 immunized rabbits were also tested with RAMP2 ECD (coated with 1 μg / ml in PBS, lot 130219). Serum from all rabbits was also tested against unrelated proteins (plates coated with 2% BSA in PBS) for non-specific binding evaluation, and no reaction with BSA was detected.

[0176] (Antibody production using HybridFree technology) (Panning of spleen cells and preparation of a complete antibody library) Panning experiments were performed on RAMP2 Scr or RAMP3 Scr (5 μg / ml) immobilized in Nunc® MaxiSorp® 96-well plates. Splenocytes (1 × 10⁶ per well) were obtained from RAMP2 Imm V2 immunized rabbits No. 53 and No. 54, or RAMP3 Imm V2 immunized rabbits No. 7 and No. 8. 4A total of 24 reactions were performed for both target antigens. After incubation for 45 minutes, the wells were washed with PBS to remove unbound cells. RNA was isolated, and cDNA was synthesized using SuperScript® IV First-Strand Synthesis System for RT-PCR (Invitrogen), which was used for amplification of VH and VL.

[0177] Dedicated VH and VL primers were designed using rabbit sequences stored in IMGT® [http: / / www.imgt.org / IMGTrepertoire / Accessed Aug 2015] and literature data: Kodangatti S. et al., The functional repertoire of rabbit antibodies and antibody discovery via next-generation sequencing. MAbs. 2014 May-June;6(3):628-36. Lavinder JJ. et al., Systematic characterization and comparative analysis of the rabbit immunoglobulin repertoire.PLoS One. June 2014 30;9(6). Kivi G. et al., HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species. January 2016.

[0178] Amplified VH and VL were purified, and a total of 20 cloning reactions were performed for each target. These were then cloned into a two-cassette expression plasmid encoding rabbit IgG-κ1 using ligase-independent cloning (LIC). The resulting antibody library pool was grown in E. coli strain DH5α. Plasmid DNA was purified and transfected into CHOEBNALT85-1E9 cells. Antibodies were transiently expressed in serum-free medium for 48 hours in a 24-well format. Cell culture supernatants were tested for binding to RAMP2 Scr and RAMP2 ECD or RAMP3 Scr by ELISA (wells coated with 1 μg / ml PBS).

[0179] result: From rabbits No. 53 and No. 54, a pool of 17 ELISA-positive samples for RAMP2 (tested for Scr and ECD proteins) was identified. From rabbits No. 7 and No. 8, a pool of 16 ELISA-positive samples for RAMP3 (tested with Scr protein) was identified.

[0180] (Expression of complete antibody library clones, validation of VH / VL sequences) Eight ELISA-positive pools for both targets were selected, and single clone isolation was performed. Single clones were selected in LB-Amp solid medium, and colonies were grown overnight in liquid medium at 37°C on a shaker, using 96-well microtiter plates (2-4 96-well plates per target). Plasmid DNA was isolated and transfected into CHOEBNALT85-1E9 cells for transient antibody expression. 48-72 hours after transfection, the supernatant was analyzed by ELISA for RAMP2 Scr or RAMP3 Scr, respectively.

[0181] ELISA-positive rIgG-κ clones for RAMP2 Scr and RAMP3 Scr were identified from each selection pool, and sequence analysis was performed on 17 RAMP2-specific clones and 32 RAMP3-specific clones.

[0182] result: Seven unique anti-RAMP2 antibody clones (1A7, 1H6, 1H7, 1A10, 2A6, 2A8, 2H3) were identified. Seven unique anti-RAMP3 antibody clones (3A5, 3A12, 4C10, 5H9, 6B8, 6C2, 6D8) were identified.

[0183] The sequence alignments and phylogenetic trees of the isolated and selected antibody clones are shown in Figures 1 and 2.

[0184] Antibodies are clustered, with identical or similar VH and VL sequences grouped together, and CDRs are shown in blue on the consensus sequence. For clarity, only last-selection clones with different CDRs (with at least 5 amino acid differences in the CDR) are shown in the alignment. Comparisons of heavy and light chain CDRs of anti-RAMP2 and anti-RAMP3 antibodies are shown in Figures 3 and 4, respectively.

[0185] (Expression of selected clones in a 24-well format) The isolated antibody clones were subcloned, the plasmid miniprep DNA was isolated, and CHOEBNALT85-1E9 cells were transfected in a 24-well format for transient antibody generation. Specific binding of the supernatant to the screening antigens (RAMP2 Scr and RAMP2 ECD or RAMP3 Scr) was confirmed by ELISA 48 hours post-transfection, and the sequences of the full heavy and light chains were verified by sequencing.

[0186] result: All seven isolated and selected anti-RAMP2 antibody clones (1A7, 1H6, 1H7, 1A10, 2A6, 2A8, 2H3) showed specific reactions to RAMP2 (equivalent ELISA signals in Scr and ECD) and retained their unique VH / VL sequences after subcloning. All seven isolated and selected anti-RAMP3 antibody clones (3A5, 3A12, 4C10, 5H9, 6B8, 6C2, 6D8) showed a specific response to RAMP3 and retained their unique VH / VL sequences even after subcloning.

[0187] (Generation of RAMP2 and RAMP3-specific rabbit IgG-κ1 antibodies in 6 wells) Fourteen developed rabbit IgG-κ1 antibody clones (seven clones against RAMP2 and seven clones against RAMP3) were transfected into CHOEBNALT85-1E9 cells and transiently expressed in a 6-well format (2 ml of culture medium). Ten days after transfection, the cells were removed by centrifugation (300 rcf, 5 minutes), and 1.5 mL of each supernatant was dispensed into screw-cap vials under sterile conditions.

[0188] The generated antibody supernatant was tested by ELISA using plates coated with RAMP2 ECD or RAMP3 Scr.

[0189] result: The titers of all seven isolated anti-RAMP2 antibody clones were less than 8 ng / mL. The titers of the anti-RAMP3 antibody clones ranged from 8 to 500 ng / mL.

[0190] Immunomodule (Thermo Scientific®) was coated with RAMP2 ECD or RAMP3 Scr and incubated in PBS at 1.0 μg / ml, 100 μl / well, at 4°C for 20 hours. Supernatants of anti-RAMP2 and anti-RAMP3 rabbit IgG-κ1 antibody clones were added to the wells at different concentrations (8–500 ng / mL; 100 μL / well, in PBS-0.05% Tween 20–1% BSA) and incubated on a shaker at room temperature (RT) for 60 minutes. As a secondary reagent, goat anti-rabbit IgG HRP conjugate (Invitrogen; 1:20,000 in 1% BSA-PBS-0.05% Tween 20, 100 μL / well) was used directly in ELISA (incubated at RT for 60 minutes), TMB substrate was added for 10 minutes for color development, and the reaction was stopped with 0.5 M H2SO4. Between each incubation step, the wells were washed four times with PBS-Tween 20.

[0191] (Example 2: In vitro characterization of the prepared antibody)

[0192] (material and method) (cell culture) Human umbilical vein endothelial cells (HUVECs) purchased from Lonza (Paris, France) were cultured in a humidified incubator at 37°C under air / 5% CO2 conditions using EBM-2 medium (Lonza) supplemented with hydrocortisone (1 μg / ml), bovine brain extract (12 μg / ml), epidermal growth factor (10 ng / ml), and 2% fetal bovine serum (FBS; Life Technologies). HUVECs were confirmed to be mycoplasma-negative and cultured for up to 5 passages. U87MG, HEP3B, DU145, PC3, LnCAP, O786, JHH6, SKHEP1, and Caki-1 were obtained from the American Type Culture Collection. These cell lines were maintained in T-25 culture flasks at 37°C under a humidified 5% CO2 atmosphere in RPMI 1640 medium supplemented with 10% FBS (#26140079, Gibco FBS qualified USA origin, Life Technologies), 2 mM glutamine (PAA Laboratories), 100 units / ml penicillin, and 100 μg / ml streptomycin (PAA Laboratories). Cell lines with 5 to 40 passages were used and cultured for up to 6 weeks. Cell counts were measured using a hemocytometer.

[0193] (HUVEC cell angiogenesis assay) HUVEC cells (7 × 10 4The seeds were pre-incubated for 30 minutes with RAMP2 Ab clones (1A7_#1;1H6_#4;1H7_#5;1A10_#7;2A6_#9;2A8_#12 and 2H3_#13) 60 μg / mL, or RAMP3 Ab clones (3A5_#15;3A12_#17;4C10_#19;H9_#22;6B8_#24;6C2_#25 and 6D8_#28) 60 μg / mL, or RAMP2(B-5)sc-365240 60 μg / mL or RAMP3(G-1)sc-365313 60 μg / mL obtained from Santa Cruz Biotechnology, Inc. (Texas, USA), and then seeded into wells coated with 300 μl of BD Matrigel (BD Biosciences) 8.5 mg / ml solution. In this tube formation assay, adrenomedullin (AM) (Bachem, Bubendorf, Switzerland) 2.10 is added to a medium containing 0.5% FBS. -7 M was added with and without RAMPs antibody. The plates were incubated at 37°C for 5 hours, fixed with methanol-free 4% paraformaldehyde, and then microscopic images were collected for analysis of junctions and node formation. For quantitative image analysis, the inventors used WimTube analysis developed by WIMASIS Image analysis (www.wimasis.com), defining skeletal regions where three or more tubes converge as total branching points and background regions surrounded (or nearly surrounded) by tubular structures as total loops for evaluation.

[0194] (Cell invasion assay) The invasiveness was measured using a Matrigel invasive chamber (24 wells, BD Biosciences). A membrane (8 μm) was coated with Matrigel (50 μg, BD Biosciences), and adrenomedullin (AM) (Bachem, Bubendorf, Switzerland) 1.10 -7 M was placed in a lower chamber containing 0.6 ml of RPMI 1640 medium supplied as a chemottractant. 1.10 U87MG or Caki-1 cells were added. 5The cells were pre-incubated for 120 minutes with either RAMP2 Ab clone (1H6_#4) 60 μg / mL or RAMP3 Ab clone (3A5_#15;3A12_#17;4C10_#19;6B8_#24;6C2_#25 and 6D8_#28) 60 μg / mL, and then seeded onto an insert suspended in 0.3 ml of serum-free RPMI. After incubation for 24 hours, with or without RAMPs Ab, non-invasive cells were removed from the top of the filter. Invading cells were fixed and stained with Diff Quik Detection Kit (Sigma-Aldrich, Saint-Louis, USA). The mean number of invasive cells per field of view was assessed by counting 9 randomly selected fields of view under a light microscope (400x magnification).

[0195] (Cell proliferation assay) Cell proliferation was determined by the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). The reaction in which yellow water-soluble tetrazolium MTT is converted to purple insoluble formazan is catalyzed by mitochondrial dehydration enzymes and can be used to estimate the number of viable cells. U87MG, HEP3B, DU145, PC3, LnCAP, O786, JHH6, SKHEP1, and Caki-1 cells were placed in 3 × 10⁶ wells of a 96-well tissue culture plate. 3 After seeding at a cell / well density, pre-incubate for 120 minutes after 24 hours with RAMP2 Ab clone (1H6_#4) 60 μg / mL or RAMP3 Ab clone (3A5_#15;3A12_#17;4C10_#19;6B8_#24;6C2_#25 and 6D8_#28) 60 μg / mL, then add adrenomedullin (AM) (Bachem, Bubendorf, Switzerland) 1.10 in each well. -7M or 10% SFB was added. 48 hours after drug exposure, the cells were incubated at 37°C for 4 hours with 0.4 mg / ml MTT. After incubation, the supernatant was removed, the cell pellet was resuspended in 0.1 ml DMSO, and the absorbance at 560 nm was measured using a MultiSKan EX microplate reader (Thermo Scientific, France). Wells containing RPMI-1640 were used as negative controls. Experiments were performed in triple or quadruple replication, and unless otherwise specified, at least three independent runs were conducted. Results are expressed as the mean of at least three independent runs and as SEM values, unless otherwise specified.

[0196] (statistical analysis) Results were expressed as the mean ± SEM of at least three independent experiments. Statistical analysis was performed using Prism 9.2 for MS Windows software (Graph Pad Software) with two-way ANOVA and Bonferroni post-hoc tests. A p-value of 0.05 or less was considered statistically significant.

[0197] (result) (HUVEC cell angiogenesis assay) To investigate the ability of RAMPs Ab clones to suppress AM-induced endothelial cell angiogenesis, the inventors measured the ability of HUVEC cells to inhibit angiogenesis in an in vitro Matrigel assay in the presence of AM. In the presence of 0.5% FBS, HUVECs remained spherical and isolated on the Matrigel bed, with no cell spread observed. These data indicate that 5-hour treatment of 9 out of 14 RAMP Ab clones evaluated blocked the AM-induced morphogenesis effect on HUVEC differentiation into vascular structures (with a cutoff of over 50%), a phenomenon necessary for endothelial cell budding and tube formation in vivo. On the other hand, evaluation of total branching points and total loops in HUVEC structure using two commercially available anti-RAMP2 and anti-RAMP3 antibodies developed by Santa Cruz Biotechnology, Inc. failed to block the AM-induced angiogenesis effect (Figure 5).

[0198] (Cell invasion assay) To investigate whether RAMP2 and RAMP3 Ab clones affect the motility of glioblastoma and renal cell carcinoma cells, U87MG and Caki-1 cells were incubated in a Boyden chamber with 0.1 μM AM in the presence of RAMPs Ab for 2 hours. The number of cells that migrated to the underside of the Transwell membrane increased in the AM group compared to the control group (Figure 2). When U87MG cells were pre-incubated with 1H6_4, 3A12_17, and 4C10_19 antibodies at 60 μg / ml each, and Caki-1 cells were pre-incubated with 3A12_17 and 6C2_25 antibodies at 60 μg / ml each for 120 minutes, invasion was significantly suppressed compared to control cells after 24 hours of incubation at 37°C in the presence of AM, demonstrating the blockade of the AM response by these antibody clones (Figure 6).

[0199] (Cell proliferation assay) In vitro tumor cell proliferation was evaluated using a panel of cancer cell lines belonging to prostate models (DU145 and PCC3), hepatocellular carcinoma (SKHEP1, JHH6, and HEP3B), and renal cell carcinoma (Caki-1). Cells were treated with various RAMPs Ab clones at 60 μg / ml for 48 hours, with and without AM and SFB. Under evaluation conditions, no inhibition of cell proliferation by RAMPs Ab clone treatment was observed (Figure 7).

[0200] (Example 3) (Hypomorphic model) The experimental protocol was approved by the French Ministry of Higher Education and Scientific Research under the number APAFIS #33619-2021102212389984 v3. Male 6-8 week old NMRI nude mice with thymus deficiency were given 0.5 × 10⁶ ions in 200 μl of phosphate-buffered saline (PBS). 6 Individual U87MG cells were subcutaneously injected into the right flank. Two days after subcutaneous cell injection, the animals were randomly divided into four groups (5 animals per group) and treated with one of the following methods. • Vehicle (PBS, intraperitoneal (ip), continuous administration) • 3A12_#17 (anti-RAMP3), 16.5 mg / kg (approx. 330 μg / animal), ip, administered 3 times weekly (tiw) • 6B8_#24 (anti-RAMP3), 16.5 mg / kg (approx. 330 μg / animal), ip, administered 3 times weekly (tiw) • 6D8_#28 (anti-RAMP3), 16.5 mg / kg (approx. 330 μg / animal), ip, administered 3 times weekly (tiw)

[0201] Mice are diagnosed when they exhibit obvious symptoms such as lethargy or a hunched posture, or when the tumor volume reaches 1300-1400 mm². 3 The animals were euthanized when they reached a certain size. Tumor size measurements were recorded three times a week (Monday, Wednesday, and Friday) from the start of treatment until the end of the study.

[0202] Where possible, the control and Ab-treated mice were euthanized 4 hours after the final treatment. The tumors were surgically removed and immediately divided into two. One was immediately rapid-frozen and stored at -80°C. The other was fixed overnight in 10% neutral buffered formalin (NBF) at 4°C, followed by standard paraffin embedding. In addition, adjacent normal tissue was also collected, immediately rapid-frozen, and stored at -80°C.

[0203] (statistical analysis) The mean and standard errors of tumor volume and body weight were determined for all experimental groups. Statistical analysis was performed using GraphPad Prism version 8.0 software (GraphPad software, Inc., La Jolla, California, USA). After ANOVA, Dunnett's multiple comparison test was performed to evaluate whether there were any statistically significant differences between the treatment group and the control group.

[0204] (result) Evaluation of the in vivo effects of three RAMPs mAbs in subcutaneous mouse glioblastoma U87MG tumors. The U87MG cell line, a model of human glioblast cell lines with epithelial morphology, was selected to evaluate three RAMPs mAbs (3A12_#17, 6B8_#24, and 6D8_#28) as monotherapy agents.

[0205] RAMPs Abs was administered intraperitoneally at a dose of 16.5 mg / kg three times a week, starting 48 hours after cell injection and continuing until euthanasia of the animals. Tumor size was recorded three times a week (Monday, Wednesday, Friday), and the tumor volume was 1300-1400 mm². 3 The animals were euthanized for ethical reasons once they reached a certain stage. Tumor growth in animals treated with Abs 3A12_#17 or 6B8_#24 was similar to that of the control group. On the other hand, animals treated with 6D8_#28 showed a significant suppression of tumor growth compared to the control group on day 23 (Figure 8).

[0206] In the Abs-treated mice, no signs of toxicity, including changes in body weight, were observed throughout the experimental period compared to the control group (Figure 9).

[0207] (Analysis of tumor tissue) (Necrosis) Tumor tissue samples taken from animals provided further evidence of significant necrosis throughout the tumor tissue. Paraffin-fixed tissue slides obtained from each individual animal were stained with anti-Caspase 3 antibody and imaged (the method was the same as for the apoptosis analysis described below, but necrotic cells were identified visually).

[0208] In control animals treated with PBS alone, a small proportion of necrotic cells were observed that were uniformly distributed throughout the tissue sections.

[0209] Tissue sections from animals treated with antibodies showed large, clearly defined necrotic areas, varying in degree. Furthermore, significant necrosis was observed in vascular-related cells even within tumor tissue where other parts were healthy. Representative images from each treatment group are shown below. Necrotic cells are stained red / brown.

[0210] Figure 10 shows a pair of representative microscopic images of U87MG tumor tissue sections stained for necrosis (brown = necrotic cells), the PBS control group. Figure 11 shows representative microscopic images of U87MG tumor tissue sections stained for necrosis (brown = necrotic cells), the RAMP3 #28 treated group. Left: A large, clearly defined area of ​​progressively increasing necrosis. Right: A close-up of vascular-related cells in healthy tissue, showing necrotic epithelial cells. Figure 12 shows representative microscopic images of U87MG tumor tissue sections stained for necrosis (brown = necrotic cells), the RAMP3 6B8 #24 treated group. Left: A large, clearly defined area of ​​progressively increasing necrosis. Right: A close-up of vascular-related cells in healthy tissue, showing necrotic epithelial cells. Figure 13 shows representative microscopic images of U87MG tumor tissue sections stained for necrosis (brown = necrotic cells), specifically the RAMP3 3A12#17 treated group. Left: A large area of ​​progressively increasing necrosis with less defined boundaries compared to other treated groups. Right: A close-up of vascular-related cells in healthy tissue, showing necrotic epithelial cells.

[0211] Several differences in necrosis patterns were observed among the three antibodies. RAMP3 6D8#28 treated tissue showed the largest area of ​​complete necrosis, and significant necrosis was also observed in vascular cells within healthy tissue, but more pronounced in areas adjacent to the continuum necrotic region. RAMP3 6B8#24 showed a slightly smaller continuum necrotic region, but the extent of vascular necrosis appeared greater than with 6D8#28. RAMP3 3A12#17 showed a smaller necrotic region and similarly less vascular necrosis than the other two treatment groups. The results are summarized in Table 1 below.

[0212] [Table 1]

[0213] (apoptosis) Additional sets of fixed tissue sections were also used to further investigate activated apoptotic activity within cells. Briefly, apoptotic levels were assessed in formalin-fixed, paraffin-embedded U87 xenografts and kidney tissue samples, and immunochemical analysis was performed using the Vectastain Elite ABC Universal kit (Vector Laboratories, California, USA) as previously described (Berenguer et al., Clin, Cancer Res. 2013, 19, 6138-6150). A rabbit monoclonal antibody (purified IgG) (1:400; Cell Signaling, France) prepared against active caspase-3 was used. Paraffin-embedded samples (4 micron sections) were tested for the presence of active caspase-3 after antigen retrieval by heating in citrate buffer (pH 6) at 97°C for 40 minutes. Incubation was maintained overnight at 4°C, and then the samples were treated with a second layer containing biotinylated anti-rabbit antiserum (Histostain plus, Zymed). The following treatments were performed using an avidin-biotin peroxidase complex kit (Histostain plus, Zymed). Note that endogenous biotin and endogenous peroxidase were neutralized by a biotin blocking system (Dako) and 3% H2O2, respectively.

[0214] Overall, the staining patterns were very similar to the necrosis staining described above, with large clumps of apoptosis and dead cells, as well as vascular necrosis, throughout the tissue sections. RAMP3 6D8#28 showed the largest stained cell clumps and also had significant staining in the blood vessels adjacent to the large clumps. RAMP3 6B8#24 showed slightly smaller clumps and a more widespread distribution of apoptosis in vascular cells throughout the tumor section, while RAMP3 3A12#17 showed a generally low level of apoptosis staining. A series of representative images from each of the four treatment groups are shown in Figures 14–21. Figure 14 shows a representative microscopic image of a U87MG tumor tissue section stained for caspase-3 (brown = apoptotic cells) - PBS control group. Figure 15 shows a representative microscopic image of a U87MG tumor tissue section stained for caspase-3 (brown = apoptotic cells) - RAMP3 6D8#28 treatment group. Left - Large, clearly defined areas where apoptosis and cell death increase stepwise. Right - Close-up of vascular-related cells in healthy tissue, showing apoptotic epithelial cells. Figure 16 shows a representative microscopic image of a U87MG tumor tissue section stained for caspase-3 (brown = apoptotic cells) - RAMP3 6B8#24 treatment group. Left - A large, clearly defined area where apoptosis and cell death increase stepwise. Right - Close-up of vascular-related cells in healthy tissue, showing apoptotic epithelial cells. Figure 17 shows a representative microscopic image of a U87MG tumor tissue section stained for caspase-3 (brown = apoptotic cells) - RAMP3 3A12#17 treatment group. Left - A large area with less defined boundaries than other treatment groups, where apoptotic cells increase stepwise. Right - Close-up of vascular-related cells in healthy tissue, showing apoptotic epithelial cells. Figures 18–21 show low-magnification images of tissue sections of U87MG xenografts taken from mice treated with PBS vehicle controls Ab6D8#28, Ab6B8#24, and Ab3A12#17.

[0215] Similar to the necrosis staining described above, RAMP3 6D8#28 appears to have the strongest effect on apoptosis of tumor cells. Table 2 summarizes the caspase 3 staining.

[0216] [Table 2]

[0217] (Off-target effects - Renal vascular structure) In addition to a detailed analysis of tumor tissue sections, samples were also taken from highly vascularized areas of kidney tissue within the treated animals and analyzed for the presence of apoptosis in the same manner as described above. Apoptotic cells were found to be relatively uniformly distributed throughout the tissue sections, but no observable differences in pattern or frequency were observed between the control group and each of the three treatment groups in this study. This data supports the idea that the treatment is highly specific and safe for the normal vascular function of the treated animals. The observations are summarized in Table 3 below.

[0218] [Table 3]

[0219] Our findings demonstrate that an antibody against RAMP3 (clone 6D8_#28) reduced U87 tumor growth compared to the control group without any toxic effects during the treatment period. Furthermore, each treated animal showed substantial necrotic areas and activated apoptosis within the tumor and throughout the tumor vascular structure, although this was not reflected in the overall tumor volume. RAMP3 clone 6D8#28 again showed the greatest effect in this analysis.

[0220] The inventors' findings indicate that each of the antibodies Abs 3A12_#17, 6B8_6B8#24, and 6D8_#28 induced tumor necrosis in animals treated with a control vehicle without exhibiting any toxic effects during the treatment period, and that the 6D8_#28 antibody further reduced the growth of U87 tumors compared to the control group.

[0221] (Example 4: Humanization process of anti-RAMP3 antibody 6D8#28)

[0222] (Stage 1: Humanized Design) Humanized antibody designs were created using the variable region sequence of the rabbit antibody 6D8#28. The variable chain was humanized by grafting the CDR sequence onto an appropriate mature human antibody sequence. The receptor human framework was selected based on key residues crucial for protein structure, stability, and function, identified by our proprietary platform. All of these receptor sequences were derived from mature human IgG of human origin, not from phage display or other technologies. As a result, the humanized sequences are expected to be non-immunogenic and retain the standard structure of the CDR loop. The CDR was identified using the IMGT and Kabat antibody numbering systems. These two numbering systems identified different residues of the rabbit antibody as belonging to the CDR, and Fusion's humanization technology uses an IMGT / Kabat composite CDR sequence for optimal retention of the CDR loop structure. Humanized variants were obtained by grafting the Kabat / IMGT composite CDR from the rabbit 6D8#28 antibody onto the receptor framework sequence. The humanized sequences were screened for risks such as T cell epitopes, glycosylation, and deamidation. For each variable chain, five variants were designed using different human donor sequences. These are shown in Figures 22A and 22B. This resulted in the creation of a matrix of 25 human antibodies for expression. The three-dimensional structures of the designed humanized antibodies were also constructed using protein structure prediction software. This software utilizes an algorithm developed by combining a comparison sequence with a standard loop structure database with ab initio structure prediction. This allows for the construction of accurate, complete three-dimensional atomic models of proteins with unknown structures.

[0223] Stage 2: The DNA encoding the humanized variant to be expressed was optimized for codon use, synthesized, and cloned into a pETE expression vector.

[0224] Stage 3: CHO cells were adapted to serum-free conditions and individually transfected with expression vectors for each of the 25 human variants, using combinations of heavy and light chain pairs, at appropriate cell densities. Antibodies were then purified by affinity chromatography, with a target yield exceeding 100 μg. After purification, the antibodies were dialyzed to PBS before QC analysis. Antibody purity and size were analyzed by reduction and denaturation SDS-PAGE analysis, and quantification was performed by UV spectroscopy.

[0225] All references herein are incorporated herein by reference. Various modifications and variations to the embodiments described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art are intended to be encompassed by the invention.

Claims

1. An antibody molecule that binds to RAMP2 or RAMP3 and inhibits adrenomedullin activity, The antibody molecule comprises at least one, for example, two or three VL chain CDRs and / or at least one, for example, two or three VH chain CDRs, (i) The VL chain has the amino acid sequence indicated by Sep ID No: 53, and the VH chain has the amino acid sequence indicated by Sep ID No: 54, or (ii) The VL chain has the amino acid sequence indicated by Sep ID No: 33, and the VH chain has the amino acid sequence indicated by Sep ID No: 34, or (iii) The VL chain has the amino acid sequence indicated by Sep ID No: 45, and the VH chain has the amino acid sequence indicated by Sep ID No: 46, or (iv) The VL chain has the amino acid sequence indicated by Seq ID No: 1, and the VH chain has the amino acid sequence indicated by Seq ID No: 2, or (v) The VL chain has the amino acid sequence indicated by Seq ID No: 5, and the VH chain has the amino acid sequence indicated by Seq ID No: 6, or (vi) The VL chain has the amino acid sequence indicated by Seq ID No: 9, and the VH chain has the amino acid sequence indicated by Seq ID No: 10, or (vii) The VL chain has the amino acid sequence indicated by Sep ID No: 13, and the VH chain has the amino acid sequence indicated by Sep ID No: 14, or (viiii) The VL chain has the amino acid sequence indicated by Seq ID No: 17, and the VH chain has the amino acid sequence indicated by Seq ID No: 18, or (ix) The VL chain has the amino acid sequence indicated by Seq ID No: 21, and the VH chain has the amino acid sequence indicated by Seq ID No: 22, or (x) The VL chain has the amino acid sequence indicated by Seq ID No: 25, and the VH chain has the amino acid sequence indicated by Seq ID No: 26, or (xi) The VL chain has the amino acid sequence indicated by Seq ID No: 29, and the VH chain has the amino acid sequence indicated by Seq ID No: 30, or (xi) The VL chain has the amino acid sequence indicated by Seq ID No: 37, and the VH chain has the amino acid sequence indicated by Seq ID No: 38, or (xiiii) The VL chain has the amino acid sequence indicated by Sep ID No: 41, and the VH chain has the amino acid sequence indicated by Sep ID No: 42, or (xiv) The VL chain has the amino acid sequence indicated by Sep ID No: 49, and the VH chain has the amino acid sequence indicated by Sep ID No:

50. antibody molecule.

2. The antibody molecule according to claim 1, wherein three or fewer amino acid substitutions, for example, two or one amino acid substitutions, are performed in at least one of the CDRs.

3. The antibody molecule according to claim 1 or 2, wherein the antibody molecule comprises at least one CDR of the VL chain and at least one CDR of the VH chain.

4. The antibody molecule according to claim 3, wherein the antibody molecule comprises all three CDRs of the VL chain and all three CDRs of the VH chain.

5. The antibody molecule according to claim 4, wherein the antibody molecule comprises all three CDRs of the VL chain having the amino acid sequence indicated by Sep ID No: 53 and all three CDRs of the VH chain having the amino acid sequence indicated by Sep ID No:

54.

6. The antibody molecule according to claim 5, wherein the VL chain has the amino acid sequence indicated by Sep ID No: 53, and the VH chain has the amino acid sequence indicated by Sep ID No:

54.

7. The antibody molecule according to claim 4, wherein the antibody molecule comprises all three CDRs of the VL chain having the amino acid sequence indicated by Sep ID No: 33 and all three CDRs of the VH chain having the amino acid sequence indicated by Sep ID No:

34.

8. The antibody molecule according to claim 7, wherein the VL chain has the amino acid sequence indicated by Sep ID No: 33, and the VH chain has the amino acid sequence indicated by Sep ID No:

34.

9. The antibody molecule according to claim 4, comprising all three CDRs of the VL chain having the amino acid sequence indicated by Sep ID No: 45 and all three CDRs of the VH chain having the amino acid sequence indicated by Sep ID No:

46.

10. The antibody molecule according to claim 9, wherein the VL chain has an amino acid sequence indicated by Sep ID No: 45, and the VH chain has an amino acid sequence indicated by Sep ID No:

46.

11. The antibody molecule according to claim 5, having a heavy chain variable region sequence selected from the VH amino acid sequences indicated by Sep ID No: 65, 66, 67, 68, and 69, and a light chain variable region sequence selected from the VL amino acid sequences indicated by Sep ID No: 70, 71, 72, 73, and 74.

12. An antibody molecule that binds to RAMP2 or RAMP3 and inhibits adrenomedullin activity.

13. The antibody molecule according to any one of claims 1 to 12, wherein the antibody molecule induces or promotes necrosis of tumor cells.

14. A nucleic acid encoding an antibody molecule according to any one of the preceding claims.

15. A pharmaceutical composition comprising an antibody molecule according to any one of claims 1 to 13 or a nucleic acid according to claim 14.

16. A method for inducing or promoting necrosis in a patient's tumor, the method comprising administering to the patient an antibody molecule according to any one of claims 1 to 13, a nucleic acid according to claim 14, or a composition according to claim 15.

17. A method for treating a condition related to angiogenesis in a patient requiring treatment, the method comprising administering to the patient an antibody molecule according to any one of claims 1 to 13, a nucleic acid according to claim 14, or a composition according to claim 15.

18. An antibody molecule according to any one of claims 1 to 13 or a nucleic acid according to claim 14 for use in pharmaceuticals.

19. An antibody molecule according to any one of claims 1 to 13 or a nucleic acid according to claim 14 for use in the treatment of conditions related to angiogenesis.

20. The antibody molecule or nucleic acid according to claim 19, wherein the aforementioned state is a state related to abnormal adrenomedullin activity.

21. The condition is cancer, the method according to claim 17, the antibody molecule according to any one of claims 18 to 20, or the nucleic acid according to any one of claims 18 to 20.

22. The method according to claim 21, an antibody molecule or nucleic acid, wherein the cancer is glioblastoma, renal cancer, prostate cancer, colon cancer, lung cancer, mesothelioma or pheochromocytoma.

23. The method according to claim 22, an antibody molecule, or a nucleic acid, wherein the cancer is glioblastoma.

24. The method according to claim 22, an antibody molecule, or a nucleic acid, wherein the cancer is renal cancer.

25. A method for producing an antibody molecule capable of inhibiting angiogenesis, the method comprising expressing the nucleic acid described in claim 14 in a host cell and isolating the antibody molecule from the cell.

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