Therapeutic agent for cardiovascular diseases

Abstract

The present invention addresses the problem of providing a novel therapeutic agent for cardiovascular diseases including vascular remodeling, the therapeutic agent targeting TfR. The present invention provides a therapeutic agent for cardiovascular diseases, the therapeutic agent comprising a molecule that suppresses excessive iron accumulation. The present invention also provides a therapeutic agent for cardiovascular diseases, the therapeutic agent comprising an anti-transferrin receptor (TfR) antibody having inhibitory activity against the binding of transferrin (Tf) and TfR.

Description

Treatment drugs for cardiovascular diseases

[0001] This invention relates to a therapeutic agent for cardiovascular diseases.

[0002] Cardiovascular diseases are a general term for diseases caused by abnormalities in blood circulation, including cardiovascular and cerebrovascular diseases, which account for the top two causes of death worldwide in recent years. Abnormalities seen in the early stages of cardiovascular diseases include hypertension, arteriosclerosis, and vascular diseases such as aortic aneurysms. These vascular diseases worsen and progress, leading to the development of organic diseases. Vascular remodeling occurs in conjunction with vascular diseases and is an important risk factor in patients with vascular diseases. Therefore, attention is being focused on developing treatments for vascular remodeling. While the latest treatments can slow the progression of vascular remodeling, significant clinical challenges remain, such as sudden death caused by aortic lesions like aortic aneurysms. Consequently, new treatment strategies for vascular remodeling are being explored to improve the morbidity and mortality rates of cardiovascular diseases.

[0003] Iron is an essential element for maintaining cellular function, but excess iron increases oxidative stress through the Fenton reaction. Non-patent documents 1 and 2 describe the association of iron excess with risk factors for cardiovascular diseases, including vascular diseases. Non-patent document 3 describes the exacerbation of atherosclerosis. Furthermore, non-patent document 4 reports on the disruption of iron homeostasis in several vascular diseases. Thus, a link between iron and the pathophysiology of cardiovascular diseases, including vascular remodeling, is suggested.

[0004] Transferrin receptors (TfRs) are cell membrane structures that take up transferrin (Tf)-bound iron into cells. Most cells regulate iron uptake by adjusting the expression level of TfRs. While this regulatory function of TfR expression is important for erythrocyte precursors, the role of TfRs in non-hematopoietic tissues, particularly diseased tissues, has been unclear. The present inventors previously reported in Non-Patent Documents 2, 5, and 6 that increased TfR expression was observed in the aorta of hypertensive model rats and chronic kidney disease model rats, indicating that TfRs are involved in vascular remodeling. Furthermore, Non-Patent Document 2 describes increased TfR expression in the pulmonary artery of pulmonary hypertension model rats, and that hypoxia-induced pulmonary vascular modeling was suppressed in TfR heterozygous knockout mice.

[0005] TfR is overexpressed in various cancer cells, and Patent Document 1 describes an anti-TfR antibody that inhibits the survival and proliferation of cancer cells for targeted cancer therapy. Patent Document 2 describes an anti-TfR antibody that inhibits the binding of Tf to TfR and inhibits iron uptake into cells, used for the treatment of iron overload and other conditions. Patent Document 3 describes that a TfR-binding polypeptide that does not inhibit the binding of Tf to TfR can be used as a carrier for delivering drugs to treat cardiovascular diseases, including aortic disease. However, there have been no reports to date on the effects of anti-TfR antibodies on cardiovascular diseases, including vascular remodeling.

[0006] International Publication WO2012 / 153707, International Publication WO2020 / 105621, Japanese Patent Publication No. 2023-100833

[0007] Sawicki KT, et al. Iron Metabolism in Cardiovascular Disease: Physiology, Mechanisms, and Therapeutic Targets. Circ Res. 2023; 132: 379-396. Naito Y, et al. Crosstalk between iron and arteriosclerosis. J Atheroscler Thromb. 2022; 29: 308-314. Vinchi F, et al. Atherosclerosis is aggravated by iron overload and amelliorated by dietary and pharmaceutical iron restriction. Eur Heart J. 2020; 41: 2681-2695. Naito Y, et al. Iron and cardiovascular diseases. J Cardiol. 2021; 77: 160-165. Naito Y, et al. Dietary iron restriction prevents hypertensive cardiovascular remodeling in Dahl salt-sensitive rats. Hypertension. 2011; 57: 497-504. Naito Y, et al. Dietary iron restriction prevents further deterioration of renal damage in a chronic kidney disease rat model. J Hypertens. 2013; 31: 1203-1213.

[0008] The problem to be addressed by this invention is to provide a novel therapeutic agent targeting TfR for cardiovascular diseases, including vascular remodeling.

[0009] As a result of investigations to solve the above problems, the inventors of the present invention found that in vascular remodeling, a process of disease progression in cardiovascular diseases, TfR expression is enhanced in smooth muscle cells, which are constituent cells of blood vessels, and intracellular iron levels increase. They found that vascular remodeling can be avoided by suppressing TfR expression and / or the increase in intracellular iron levels (iron accumulation) in smooth muscle cells. They found that an anti-TfR antibody that inhibits the binding of Tf and TfR reduces the expression level of TfR that is enhanced by AngII in mice, and selectively suppresses AngII-induced vascular remodeling without affecting normal tissues and normal cells. Furthermore, they found that in human vascular smooth muscle cells, the antibody selectively suppresses the increase in intracellular iron levels (iron accumulation) induced by AngII in AngII-stimulated cells, without affecting cells without AngII stimulation, and suppresses AngII-induced protein synthesis, cell proliferation, and cell migration. The present invention was completed based on these findings.

[0010] In other words, the present invention provides the following inventions: <1> A therapeutic agent for cardiovascular disease comprising a molecule that suppresses the excessive accumulation of iron. <2> The therapeutic agent for cardiovascular disease according to <1>, wherein the molecule is a molecule having binding inhibitory activity between transferrin (Tf) and transferrin receptor (TfR). <3> The therapeutic agent according to <1> to <2>, wherein the molecule is an antibody, aptamer, or peptide. <4> The therapeutic agent for cardiovascular disease according to <1> to <3>, wherein the molecule is an anti-TfR antibody having binding inhibitory activity between transferrin (Tf) and transferrin receptor (TfR). <5> The therapeutic agent for cardiovascular disease according to <4>, wherein the anti-TfR antibody is an antibody that recognizes amino acids 629 to 633 of human transferrin receptor (TfR). <6> The therapeutic agent for cardiovascular diseases according to <4> to <5>, wherein the anti-TfR antibody is an antibody in which the heavy chain first complementarity-determining region (VH CDR1), heavy chain second complementarity-determining region (VH CDR2), and heavy chain third complementarity-determining region (VH CDR3) are sequence numbers 1, 2, and 3, respectively, and the light chain first complementarity-determining region (VL CDR1), light chain second complementarity-determining region (VL CDR2), and light chain third complementarity-determining region (VL CDR3) are sequence numbers 4, 5, and 6, respectively. <7> The therapeutic agent for cardiovascular diseases according to <4> to <6>, wherein the anti-TfR antibody is an antibody in which the heavy chain has sequence number 7 and the light chain has sequence number 8. <8> The anti-TfR antibody is an antibody in which the heavy chain first complementarity-determining region (VH CDR1), heavy chain second complementarity-determining region (VH CDR2), and heavy chain third complementarity-determining region (VH CDR3) are sequence numbers 9, 10, and 11, respectively, and the light chain first complementarity-determining region (VL CDR1), light chain second complementarity-determining region (VL CDR2), and light chain third complementarity-determining region (VL CDR3) are sequence numbers 12, 13, and 14, respectively, as described in <4>. <9> The anti-TfR antibody is an antibody in which the heavy chain has sequence number 15 and the light chain has sequence number 16, as described in <4> or <8>. <10> The anti-TfR antibody is a human antibody or a humanized antibody, as described in <4> to <9>. <11> The anti-TfR antibody is Fab, Fab', F(ab') ２A therapeutic agent for cardiovascular diseases according to <4> to <6>, <8>, or <10>, wherein the antibody fragment is selected from the group consisting of a single-chain antibody (scFv), a dimerized V region (Diabody), a disulfide-stabilized V region (dsFv), and a peptide containing a CDR. <12> A therapeutic agent for cardiovascular diseases according to any one of <1> to <11>, wherein the molecule reduces the expression level of transferrin receptor (TfR). <13> A therapeutic agent according to any one of <1> to <12>, wherein the cardiovascular disease is a cardiovascular disease. <14> A therapeutic agent according to any one of <1> to <13>, wherein the cardiovascular disease is a disease or condition accompanied by arteriosclerosis. <15> A therapeutic agent according to any one of <1> to <14>, wherein the cardiovascular disease is a disease or condition accompanied by vascular remodeling. <16> The therapeutic agent according to any one of <1> to <15>, wherein the cardiovascular disease is a disease or symptom accompanied by an abnormality in the expression regulation of transferrin receptor (TfR). <17> The therapeutic agent according to any one of <1> to <16>, wherein the cardiovascular disease is a disease or symptom accompanied by an increase in intracellular iron. <1A> A method for treating a cardiovascular disease, comprising administering a therapeutic agent for cardiovascular disease containing a molecule that suppresses excessive iron accumulation to a subject. <2A> The therapeutic method according to <1A>, wherein the molecule is a molecule that has binding inhibitory activity between transferrin (Tf) and transferrin receptor (TfR). <3A> The therapeutic method according to <1A> to <2A>, wherein the molecule is an antibody, aptamer, or peptide. <4A> The therapeutic method according to <1A> to <3A>, wherein the molecule is an anti-TfR antibody that has binding inhibitory activity between transferrin (Tf) and transferrin receptor (TfR). <5A> The therapeutic method according to <4A>, wherein the anti-TfR antibody is an antibody that recognizes the 629th to 633rd amino acids of the human transferrin receptor (TfR).<6A> The treatment method according to <4A> to <5A>, wherein the anti-TfR antibody is an antibody in which the heavy chain first complementarity-determining region (VH CDR1), heavy chain second complementarity-determining region (VH CDR2), and heavy chain third complementarity-determining region (VH CDR3) are sequence numbers 1, 2, and 3, respectively, and the light chain first complementarity-determining region (VL CDR1), light chain second complementarity-determining region (VL CDR2), and light chain third complementarity-determining region (VL CDR3) are sequence numbers 4, 5, and 6, respectively. <7A> The treatment method according to <4A> to <6A>, wherein the anti-TfR antibody is an antibody in which the heavy chain has sequence number 7 and the light chain has sequence number 8. <8A> The treatment method according to <4A>, wherein the anti-TfR antibody is an antibody in which the heavy chain first complementarity-determining region (VH CDR1), heavy chain second complementarity-determining region (VH CDR2), and heavy chain third complementarity-determining region (VH CDR3) are sequence numbers 9, 10, and 11, respectively, and the light chain first complementarity-determining region (VL CDR1), light chain second complementarity-determining region (VL CDR2), and light chain third complementarity-determining region (VL CDR3) are sequence numbers 12, 13, and 14, respectively. <9A> The treatment method according to <4A> or <8A>, wherein the anti-TfR antibody is an antibody in which the heavy chain has sequence number 15 and the light chain has sequence number 16. <10A> The treatment method according to <4A> to <9A>, wherein the anti-TfR antibody is a human antibody or a humanized antibody. <11A> The anti-TfR antibodies are Fab, Fab', and F(ab'). ２The therapeutic method according to <4A> to <6A>, <8A>, or <10A>, wherein the antibody fragment is selected from the group consisting of a single-chain antibody (scFv), a dimerized V region (Diabody), a disulfide-stabilized V region (dsFv), and a peptide containing a CDR. <12A> The therapeutic method according to any one of <1A> to <11A>, wherein the molecule reduces the expression level of transferrin receptor (TfR). <13A> The therapeutic method according to any one of <1A> to <12A>, wherein the cardiovascular disease is a cardiovascular disease. <14A> The therapeutic method according to any one of <1A> to <13A>, wherein the cardiovascular disease is a disease or condition accompanied by arteriosclerosis. <15A> The therapeutic method according to any one of <1A> to <14A>, wherein the cardiovascular disease is a disease or condition accompanied by vascular remodeling. <16A> The treatment method according to any one of <1A> to <15A>, wherein the cardiovascular disease is a disease or symptom accompanied by an abnormal expression regulation of transferrin receptor (TfR). <17A> The treatment method according to any one of <1A> to <16A>, wherein the cardiovascular disease is a disease or symptom accompanied by an increase in intracellular iron. <1B> A molecule for use in the treatment of cardiovascular disease that suppresses excessive iron accumulation. <2B> The molecule according to <1B>, wherein the molecule is a molecule having binding inhibitory activity between transferrin (Tf) and transferrin receptor (TfR). <3B> The molecule according to <1B> to <2B>, wherein the molecule is an antibody, aptamer, or peptide. <4B> The molecule according to <1B> to <3B>, wherein the molecule is an anti-TfR antibody having binding inhibitory activity between transferrin (Tf) and transferrin receptor (TfR). <5B> The molecule described in <4B>, wherein the anti-TfR antibody is an antibody that recognizes the 629th to 633rd amino acids of the human transferrin receptor (TfR).<6B> The molecule according to <4B> to <5B>, wherein the anti-TfR antibody is an antibody in which the heavy chain first complementarity-determining region (VH CDR1), heavy chain second complementarity-determining region (VH CDR2), and heavy chain third complementarity-determining region (VH CDR3) are sequence numbers 1, 2, and 3, respectively, and the light chain first complementarity-determining region (VL CDR1), light chain second complementarity-determining region (VL CDR2), and light chain third complementarity-determining region (VL CDR3) are sequence numbers 4, 5, and 6, respectively. <7B> The molecule according to <4B> to <6B>, wherein the anti-TfR antibody is an antibody in which the heavy chain has sequence number 7 and the light chain has sequence number 8. <8B> The molecule according to <4B>, wherein the anti-TfR antibody is an antibody in which the heavy chain first complementarity-determining region (VH CDR1), heavy chain second complementarity-determining region (VH CDR2), and heavy chain third complementarity-determining region (VH CDR3) are sequence numbers 9, 10, and 11, respectively, and the light chain first complementarity-determining region (VL CDR1), light chain second complementarity-determining region (VL CDR2), and light chain third complementarity-determining region (VL CDR3) are sequence numbers 12, 13, and 14, respectively. <9B> The molecule according to <4B> or <8B>, wherein the anti-TfR antibody is an antibody in which the heavy chain has sequence number 15 and the light chain has sequence number 16. <10B> The molecule according to <4B> to <9B>, wherein the anti-TfR antibody is a human antibody or a humanized antibody. <11B> The anti-TfR antibodies are Fab, Fab', and F(ab'). ２A molecule according to <4B> to <6B>, <8B>, or <10B>, which is an antibody fragment selected from the group consisting of a single-chain antibody (scFv), a dimerized V region (Diabody), a disulfide-stabilized V region (dsFv), and a peptide containing a CDR. <12B> A molecule according to any one of <1B> to <11B>, wherein the molecule reduces the expression level of transferrin receptor (TfR). <13B> A molecule according to any one of <1B> to <12B>, wherein the cardiovascular disease is a cardiovascular disease. <14B> A molecule according to any one of <1B> to <13B>, wherein the cardiovascular disease is a disease or condition accompanied by arteriosclerosis. <15B> A molecule according to any one of <1B> to <14B>, wherein the cardiovascular disease is a disease or condition accompanied by vascular remodeling. <16B> The molecule according to any one of <1B> to <15B>, wherein the cardiovascular disease is a disease or symptom accompanied by dysregulation of transferrin receptor (TfR) expression. <17B> The molecule according to any one of <1B> to <16B>, wherein the cardiovascular disease is a disease or symptom accompanied by an increase in intracellular iron. <1C> Use of a molecule to suppress excessive iron accumulation for the manufacture of a therapeutic agent for cardiovascular disease. <2C> Use according to <1C>, wherein the molecule is a molecule having binding inhibitory activity between transferrin (Tf) and transferrin receptor (TfR). <3C> Use according to <1C> to <2C>, wherein the molecule is an antibody, aptamer, or peptide. <4C> Use according to <1C> to <3C>, wherein the molecule is an anti-TfR antibody having binding inhibitory activity between transferrin (Tf) and transferrin receptor (TfR). <5C> The use described in <4C>, wherein the anti-TfR antibody is an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor (TfR).<6C> The use described in <4C> to <5C>, wherein the anti-TfR antibody is an antibody in which the heavy chain first complementarity-determining region (VH CDR1), heavy chain second complementarity-determining region (VH CDR2), and heavy chain third complementarity-determining region (VH CDR3) are sequence numbers 1, 2, and 3, respectively, and the light chain first complementarity-determining region (VL CDR1), light chain second complementarity-determining region (VL CDR2), and light chain third complementarity-determining region (VL CDR3) are sequence numbers 4, 5, and 6, respectively. <7C> The use described in <4C> to <6C>, wherein the anti-TfR antibody is an antibody in which the heavy chain has sequence number 7 and the light chain has sequence number 8. <8C> The use according to <4C>, wherein the anti-TfR antibody is an antibody in which the heavy chain first complementarity-determining region (VH CDR1), heavy chain second complementarity-determining region (VH CDR2), and heavy chain third complementarity-determining region (VH CDR3) are sequence numbers 9, 10, and 11, respectively, and the light chain first complementarity-determining region (VL CDR1), light chain second complementarity-determining region (VL CDR2), and light chain third complementarity-determining region (VL CDR3) are sequence numbers 12, 13, and 14, respectively. <9C> The use according to <4C> or <8C>, wherein the anti-TfR antibody is an antibody in which the heavy chain has sequence number 15 and the light chain has sequence number 16. <10C> The use according to <4C> to <9C>, wherein the anti-TfR antibody is a human antibody or a humanized antibody. <11C> The anti-TfR antibodies are Fab, Fab', and F(ab'). ２The use according to <4C> to <6C>, <8C>, or <10C>, wherein the antibody fragment is selected from the group consisting of a single-chain antibody (scFv), a dimerized V region (Diabody), a disulfide-stabilized V region (dsFv), and a peptide containing a CDR. <12C> The use according to any one of <1C> to <11C>, wherein the molecule reduces the expression level of transferrin receptor (TfR). <13C> The use according to any one of <1C> to <12C>, wherein the cardiovascular disease is a cardiovascular disease. <14C> The use according to any one of <1C> to <13C>, wherein the cardiovascular disease is a disease or condition accompanied by arteriosclerosis. <15C> The use according to any one of <1C> to <14C>, wherein the cardiovascular disease is a disease or condition accompanied by vascular remodeling. <16C> The use according to any one of <1C> to <15C>, wherein the cardiovascular disease is a disease or condition accompanied by a dysregulation of transferrin receptor (TfR) expression. <17C> The use according to any one of <1C> to <16C>, wherein the cardiovascular disease is a disease or condition accompanied by an increase in intracellular iron levels.

[0011] The present invention provides a novel therapeutic agent for cardiovascular diseases that targets TfR. The therapeutic agent of the present invention is useful in treating cardiovascular diseases and vascular remodeling associated with cardiovascular diseases. The therapeutic agent of the present invention is useful in treating diseases or symptoms involving abnormal regulation of TfR expression in cells and / or excessive accumulation of intracellular iron. The therapeutic agent of the present invention does not affect normal cells or tissues and selectively exerts its effect on the affected area of ​​cardiovascular disease, making it useful in the safe treatment of cardiovascular diseases and vascular remodeling associated with cardiovascular diseases.

[0012] Figure 1 shows the point mutation sites for each TfR mutant fragment. Figure 2 shows the reactivity of TfR436 antibody with soluble wild-type TfR (sTfR) and TfR mutant fragment. Figure 3 shows the Tf-TfR binding inhibitory activity of TfR436 antibody. Figure 4 shows the effect of TfR018 antibody on angiotensin II-induced vascular remodeling in mice. (A) Representative macroscopic findings of the entire aorta from the control group, TfR018 antibody group, AngII group, and AngII + TfR018 antibody group, as well as aortic sections stained by MTC staining and EVG staining (scale bar: macroscopic findings are 1 mm, MTC and EVG staining are 50 μm). (B-D) Quantitative analysis of aortic diameter (B), aortic median thickness (C), and adventitia fibrosis (D) in the control group, AngII group, and AngII + TfR018 antibody group, and in the control group, TfR018 antibody group, AngII group, and AngII + TfR018 antibody group (n=6 in each group). Figure 5 shows the effect of TfR018 antibody on aortic TfR levels in mice administered angiotensin II. (A) Shows the protein levels of TfR and ferritin in the aorta. (B) Shows representative aortic sections immunostained with 3-amino-9-ethylcarbazole (red) for TfR, ferritin, and CD68 in the control group, AngII group, and AngII + TfR018 antibody group (n=5 in each group) (scale bar: low magnification: 100, high magnification: 50 μm). Furthermore, quantitative analysis of TfR, ferritin, and CD68 positive area is shown (n=5 in each group). Figure 6 shows the effect of TfR018 antibody on aortic TfR levels in mice administered angiotensin II. (A) Shows the protein levels of TfR and ferritin in the aorta. (B) Shows representative aortic sections immunostained with 3-amino-9-ethylcarbazole (red) for TfR, ferritin, and CD68 in the control group, TfR018 antibody group, AngII group, and AngII + TfR018 antibody group (n=5 in each group) (scale bar: low magnification: 100, high magnification: 50 μm). Furthermore, quantitative analysis of TfR, ferritin, and CD68 positive area is shown (n=5 in each group).Figure 7 shows the effects of the TfR436 antibody on angiotensin II-induced protein synthesis, proliferation, and migration of human arterial smooth muscle cells. (A) Shows the protein levels of TfR and puromycin in HASMCs. (B) Shows cell proliferation and migration assays in the saline + vehicle group, saline + TfR436 antibody group, AngII + vehicle group, and AngII + TfR436 antibody group (n=11-12 in each group). The upper panel shows representative wound media of each group stained with Diff-Quick. Figure 8 shows intracellular iron ion (Fe) levels in human arterial smooth muscle cells of the saline + vehicle group, saline + TfR436 antibody group, AngII + vehicle group, and AngII + TfR436 antibody group, as stained with Ferro Orange. ２＋ The results of investigating the amount are shown. Figure 9 shows a schematic diagram of the suppression of AngII-induced vascular remodeling by anti-TfR antibody.

[0013] Next, the present invention will be described in more detail. Definitions and General Art Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have the meanings that are commonly understood by those skilled in the art. In general, the nomenclature and techniques used herein in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization are well known and commonly used in the art.

[0014] The methods and techniques of the present invention are generally carried out in accordance with conventional methods well known in the art, unless otherwise indicated, as described in the various general and more specific references cited and discussed throughout this specification.

[0015] In humans, the transferrin receptor (TfR) is a single-pass transmembrane protein composed of 760 amino acids encoded on human chromosome 3 (SEQ ID NO: 17). This protein is also known as the CD71 antigen and is thought to be involved in iron uptake by cells and cell proliferation. In this invention, TfR is not particularly limited in structure and includes monomers, multimers, the intact form expressed on the cell membrane, the soluble form composed in the extracellular domain, the truncated form, as well as mutation forms due to gene mutations or deletions, and forms that have undergone post-translational modifications such as phosphorylation. The amino acid sequence of human TfR is shown below.Human TfR (SEQ ID NO: 17).

[0016] Reacting and Reactivity In this specification, "reacting" and "reactive" mean the same thing unless otherwise specified. That is, the recognition of an antigen by an antibody. This antigen may be an intact TfR expressed on the cell membrane, a truncated form, or a solubilized form. It may also be a TfR that maintains its three-dimensional structure or a denatured TfR. Methods for examining reactivity include flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), western-blot, fluorescence micrometrometry (FMAT), surface plasmon resonance (BIAcore), immunostaining, and immunoprecipitation.

[0017] The antibodies used in flow cytometry may be labeled with fluorescent substances such as FITC or biotin, or they may be unlabeled. Depending on whether the antibody used is labeled and the type of labeling, fluorescently labeled avidin, fluorescently labeled anti-human immunoglobulin antibodies, etc., may be used. Reactivity can be evaluated by adding a sufficient amount of anti-TfR antibody (usually with a final concentration of 0.01 to 10 μg / mL) to the sample and comparing it with the reactivity of negative and positive control antibodies.

[0018] In this specification, the following abbreviations (in parentheses) are used as necessary, in accordance with convention: heavy chain (H chain), light chain (L chain), heavy chain variable region (VH), light chain variable region (VL), complementarity-determining region (CDR), first complementarity-determining region (CDR1), second complementarity-determining region (CDR2), third complementarity-determining region (CDR3), first complementarity-determining region of the heavy chain (VH CDR1), second complementarity-determining region of the heavy chain (VH CDR2), third complementarity-determining region of the heavy chain (VH CDR3), first complementarity-determining region of the light chain (VL CDR1), second complementarity-determining region of the light chain (VL CDR2), third complementarity-determining region of the light chain (VL CDR3).

[0019] In this specification, the term “antibody” is synonymous with “immunoglobulin” and should be understood as commonly known in the art. Specifically, the term “antibody” is not limited to any particular method of antibody production. For example, the term “antibody” includes, but is not limited to, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Furthermore, antibodies also include antibody fragments, as described below.

[0020] In this specification, the term “human antibody” means any antibody whose variable and constant regions are human sequences. The term also includes antibodies that have sequences derived from human genes but have been modified to, for example, reduce immunogenicity, increase affinity, or remove cysteine ​​which may cause undesirable folding. The term also includes such antibodies produced by recombination in non-human cells that can undergo glycosylation not specific to human cells. These antibodies can be prepared in a variety of ways.

[0021] In this specification, the term "humanized antibody" refers to an antibody of non-human origin in which amino acid residues characteristic of the antibody sequence of a non-human species are substituted with residues found at corresponding positions in a human antibody. This "humanization" process is thought to reduce the immunogenicity of the resulting antibody in humans. It will be understood that non-human antibodies can be humanized using techniques well known in the art. For example, see Winter et al., Immunol. Today 14:43-46 (1993). The antibody in question can be engineered by recombinant DNA technology in which CH1, CH2, CH3, the hinge domain, and / or the framework domain are substituted with the corresponding human sequences. For example, one can refer to WO92 / 02190, and U.S. Patents 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085. In this specification, the term “humanized antibody” includes, to the extent of its meaning, chimeric human antibodies and CDR-transplanted antibodies.

[0022] The sequence of the framework region (FR) in the variable region of an antibody is not particularly limited, as long as it does not substantially affect the specific binding affinity to the corresponding antigen. While it is preferable to use the FR region of a human antibody, it is also possible to use the FR region of an animal species other than human (e.g., mouse or rat).

[0023] In this specification, the term "phage antibody" means an scFv antibody produced by a phage; that is, an antibody fragment containing VH and VL amino acid sequences. This fragment may also contain amino acid sequences as tags, in addition to amino acids as links.

[0024] In one embodiment of the antibody, a constant region is included in addition to the variable region (e.g., an IgG-type antibody). The sequence of the constant region is not particularly limited. For example, the constant region of a known human antibody can be used. The heavy chain constant region (CH) of the human antibody can be any that belongs to human immunoglobulin (hereinafter referred to as hIgG), but the hIgG class is preferred, and any of the subclasses belonging to the hIgG class, such as hIgG1, hIgG2, hIgG3, and hIgG4, can be used. The light chain constant region (CL) can be any that belongs to hIg, and the κ class or λ class can be used. Furthermore, the constant region of an animal species other than human (e.g., mouse or rat) can also be used.

[0025] In this specification, "modified antibody" or "modified antibody" refers to a parent antibody in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence of the variable region (CDR sequence and / or FR sequence). In the present invention, "parent antibody" refers to a TfR436 antibody having the amino acid sequence shown in SEQ ID NO: 7 for VH and SEQ ID NO: 8 for VL, or a TfR018 antibody having the amino acid sequence shown in SEQ ID NO: 15 for VH and SEQ ID NO: 16 for VL. A "modified antibody" or "modified antibody" has one or more amino acids (for example, 1 to 8, preferably 1 to 5, more preferably 1 to 3, and particularly preferably 1 or 2) deleted, added, substituted, and / or inserted into its amino acid sequence. A method well known to those skilled in the art for preparing an amino acid sequence of an antibody whose activity is equivalent to that of a parent antibody is a method of introducing mutations into a protein.For example, a person skilled in the art would know about site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, anDNAkagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100, 468-500, Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ (1987)). By appropriately introducing mutations into the amino acid sequence of the parent antibody using methods such as Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367, Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci US A. 82, 488-492), modified antibodies with equivalent activity to the parent antibody can be prepared. In this way, it is also possible to use antibodies in which one or several amino acids are mutated in the variable or constant region of the antibody, and which have equivalent activity to the parent antibody, i.e., functionally equivalent to the parent antibody.

[0026] In this specification, "activity equivalent to that of the parent antibody" means that the binding activity to TfR, the inhibitory activity of Tf and TfR binding, the suppressive activity of TfR expression, the intracellular iron level reduction activity and / or anti-vascular remodeling activity are equivalent. "Equivalent" does not necessarily mean that the activity is of the same degree; the activity may be enhanced, or it may be reduced as long as it has activity. Examples of antibodies with reduced activity include antibodies having 30% or more activity, preferably 50% or more activity, more preferably 80% or more activity, even more preferably 90% or more activity, and particularly preferably 95% or more activity compared to the original antibody.

[0027] Binding activity refers to the recognition of an antigen. This antigen may be an intact TfR expressed on the cell membrane, a truncated form, or a solubilized form. It may also be a TfR that maintains its three-dimensional structure or a denatured TfR. For example, methods for examining binding activity include flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), western-blot, fluorescence micrometrometry (FMAT), and surface plasmon resonance (BIAcore).

[0028] The Tf-TfR binding inhibitory activity of an antibody can be measured according to the method described in "Example 2 (2) Comparison of TfR436 antibody and other company's antibody in Tf-TfR binding inhibition" below. The TfR solution is dispensed onto a substrate (96-well plate, etc.) and allowed to stand to solidify and block. Next, the HRP-labeled Tf solution is dispensed, and the antibody is added and reacted at room temperature. After that, the substrate is washed, a chromogenic reagent (TMB, etc.) is added and the reaction is carried out, and the absorbance is measured with a plate reader. The Tf-TfR binding inhibitory activity of the antibody can be evaluated by the above procedure.

[0029] TfR expression inhibitory activity means reducing the amount of TfR protein present in tissues or cells, and can be evaluated by measuring the amount of TfR protein in tissues or cells. This TfR may be intact TfR expressed on the cell membrane, truncated form, or solubilized form. It may also be TfR that maintains its three-dimensional structure or denatured TfR. The method for measuring the amount of TfR protein is not particularly limited, but it can be measured by observing and counting TfR-positive cells detected by immunohistochemical methods using an anti-TfR antibody, as described in "Example 3: Inhibition of AngII-induced vascular remodeling and TfR expression by TfR018 antibody (mouse in vivo study)" and "Example 4: Inhibition of AngII-induced protein synthesis, cell proliferation, and cell migration in human arterial smooth muscle cells by TfR436 antibody (in vitro study)," or by Western blot analysis.

[0030] As an example of intracellular iron level reduction activity, intracellular iron ions (Fe ２＋ The activity of reducing the amount of iron ions (Fe) in cells ２＋ This means reducing the amount of iron ions (Fe) inside the cell. ２＋ This can be evaluated by measuring the amount of ferritin. The method for measuring intracellular iron ion levels is not particularly limited, but it can be measured using intracellular iron ion measuring reagents such as FerroOrange, as described in "Example 4: Inhibition of AngII-induced protein synthesis, cell proliferation, and cell migration in human arterial smooth muscle cells by TfR436 antibody (in vitro study)" below. Furthermore, since ferritin reflects the amount of stored iron in cells, it is also possible to indirectly evaluate intracellular iron reduction activity by measuring ferritin reduction activity. The ferritin reduction activity is not particularly limited, but it can be evaluated by measuring the amount of ferritin protein using immunological methods, as described in "Example 3: Inhibition of AngII-induced vascular remodeling and suppression of TfR expression by TfR018 antibody (mouse in vivo study)" below.

[0031] Antivascular remodeling activity refers to the activity of suppressing structural changes in blood vessels, and can be evaluated, for example, by measuring the activity of suppressing the proliferation or migration of vascular smooth muscle cells, and / or the activity of suppressing the dilation of blood vessel diameter, thickening of the media, or fibrosis of the adventitia. The proliferation or migration of vascular smooth muscle cells is not particularly limited, but can be evaluated by the method described in "Example 4: Inhibition of AngII-induced protein synthesis, cell proliferation, and cell migration in human arterial smooth muscle cells by TfR436 antibody (in vitro test)" below. Cell proliferation can be evaluated, for example, by measuring the activity of inhibiting the increase in the number of cultured smooth muscle cells in vitro. Cell migration can be evaluated, for example, by performing a wound healing assay on cultured smooth muscle cells in vitro and measuring the number of migrating cells. The dilation of blood vessel diameter, thickening of the media, or suppression of adventitia fibrosis are not particularly limited, but can be evaluated, for example, by the method described in "Example 3: Suppression of AngII-induced vascular remodeling and suppression of TfR expression by TfR018 antibody (mouse in vivo study)" below. For example, they can be evaluated by macroscopic observation of tissue, measurement of blood vessel diameter, or measurement of media thickness or adventitia fibrosis area after tissue staining.

[0032] The antibody is not limited by its origin and may be derived from any animal, such as human antibodies, mouse antibodies, or rat antibodies. It may also be a chimeric antibody or a humanized antibody. One preferred form of antibody in this invention is a human antibody.

[0033] Antibodies may differ in amino acid sequence, molecular weight, isoelectric point, and the presence and morphology of glycans depending on the antibody-producing cell, host, or purification method described later. For example, the present invention also includes cases where the amino acid sequence described in this invention undergoes post-translational modification. Furthermore, post-translational modifications at sites other than known post-translational modifications are also included in this invention. In addition, when an antibody is expressed in prokaryotic cells, such as E. coli, a methionine residue is added to the N-terminus of the original antibody's amino acid sequence. Such antibodies may be used in this invention. Post-translational modifications at sites other than known post-translational modifications are also included in this invention.

[0034] Production of Antibodies As the antibodies used in the present invention, either monoclonal antibodies or polyclonal antibodies may be used. Monoclonal antibodies and polyclonal antibodies can be produced by methods known to those skilled in the art. Examples of antibodies include those produced in the blood of animals, those produced by hybridomas, those produced by hosts transformed with an expression vector containing an antibody gene by genetic engineering techniques, those in which an optimal antibody is screened from a clone library by phage display and its gene is produced in CHO cells, or those in which a transgenic mouse producing human antibodies is used to directly obtain human antibodies.

[0035] To produce polyclonal antibodies, an antigen is immunized in an animal such as a rabbit to obtain serum. The obtained serum can be purified by, for example, ammonium sulfate precipitation, protein A column, protein G column, DEAE ion exchange chromatography, affinity column, etc., to prepare polyclonal antibodies.

[0036] To produce monoclonal antibodies, an antigen is immunized in an animal together with an adjuvant if desired. After confirming that the desired antibody level has increased in the serum of the immunized animal, immune cells (such as spleen cells) are collected from the animal and cell-fused with mammalian myeloma cells. The hybridomas obtained by cell fusion can be selected by culturing in a normal selection culture medium, for example, HAT culture medium (culture medium containing hypoxanthine, aminopterin, and thymidine). Thereafter, the limiting dilution method can be carried out to screen hybridomas that produce the target antibody.

[0037] The production of antibodies by the phage display method will be described below. (1) scFv that reacts with an antigen using a phage display library Using the phage display technique, a library containing a repertoire of antibodies with various affinities for TfR can be provided. Then, these libraries can be screened to identify and isolate antibodies against TfR. Preferably, the phage library is a scFv phage display library generated using human VL and VH cDNA prepared from mRNA isolated from human B cells. Methods for preparing and screening such libraries are known in the art. Recover genetic material from phage clones that show reactivity when screened with human TfR as an antigen. By analyzing the genes of the selected phages, the DNA sequences of VH and VL encoding the variable regions of human antibodies that bind to the antigen can be determined. Using the sequence of this scFv, a human antibody can be obtained by IgGizing the scFv.

[0038] (2) IgGization of scFv (production of human antibodies) Prepare expression vectors for the H chain or L chain, express them in host cells, and obtain human antibodies by recovering and purifying the secreted supernatant. Human antibodies can also be obtained by expressing VH and VL in the same vector (tandem type). These methods are well-known, and reference can be made to WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, W093 / 19172, WO95 / 01438, WO95 / 15388, WO97 / 10354, etc.

[0039] Specifically, a full-length heavy chain gene can be obtained by ligating the DNA encoding VH with other DNA molecules encoding the heavy chain constant regions (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (e.g., Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD, but most preferably it is the constant region of IgG1 or IgG2. The IgG1 constant region sequence may be any various alleles or allotypes known to occur between different individuals, such as Gm(1), Gm(2), Gm(3), or Gm(17). These allotypes correspond to naturally occurring amino acid substitutions in the IgG1 constant region.

[0040] Full-length L-chain genes (and Fab light chain genes) can be obtained by ligating the DNA encoding VL with another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (e.g., Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region may be a κ or λ constant region. The κ constant region may be any variety of alleles known to occur between different individuals, such as Inv(1), Inv(2), and Inv(3). The λ constant region can originate from any of the three λ genes.

[0041] An expression vector is prepared by inserting the DNA encoding the H chain or L chain obtained as described above into an expression vector, expressing it in host cells, and obtaining human antibodies by recovering and purifying the secreted supernatant. Examples of expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, and EBV-derived episomes. The expression vector and expression regulatory sequence are selected to be compatible with the host cells used for expression. The antibody light chain gene and antibody heavy chain gene can be inserted into separate vectors, or both genes can be inserted into the same expression vector. The antibody gene is inserted into the expression vector by a standard method (e.g., ligation of complementary restriction sites on the antibody gene fragment with the vector, or blunt-end ligation if no restriction sites exist).

[0042] A suitable vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction sites, engineered to allow for the easy insertion and expression of any VH or VL sequence as described above. In such vectors, splicing typically occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C domain, as well as in splice regions located within the human CH exon. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding region. Recombinant expression vectors can also encode signal peptides that promote the secretion of host cell-derived antibody chains. The antibody chain gene can be cloned into the vector such that the signal peptide is in-frame linked to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0043] Antibody expression vectors may have, in addition to the antibody gene and regulatory sequences, further sequences such as sequences that control vector replication in host cells (e.g., origin of replication) and selection marker genes. Selection marker genes facilitate the selection of host cells into which the vector has been introduced. For example, selection marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate onto host cells into which the vector has been introduced. Preferred selection marker genes include the dehydrofolate reductase (DHFR) gene (used in conjunction with methotrexate selection / amplification in dhfr-host cells), the neomycin phosphotransferase gene (for G418 selection), and the glutamate synthase gene.

[0044] The host cells are transformed using the antibody gene expression vector prepared by the above method. Any cell capable of producing antibodies can be used as the host cell, such as bacteria, yeast, animal cells, insect cells, or plant cells, but animal cells are preferred. Examples of animal cells include Chinese hamster ovary cells CHO / dhfr (-) cells and CHO / DG44 cells, monkey-derived COS cells (A. Wright & S.L. Morrison, J. Immunol. 160, 3393-3402 (1998)), and SP2 / O cells (mouse myeloma) (K. Motmans et al., Eur. J. Cancer Prev. 5, 512-5199 (1996), R.P. Junghans et al., Cancer Res. 50, 1495-1502 (1990)). Furthermore, lipofectin methods (R.W. Malone et al., Proc. Natl. Acad. Sci. USA 86, 6007 (1989), P.L. Felgner et al., Proc. Natl. Acad. Sci. USA 84, 7413 (1987)), electroporation, calcium phosphate methods (F.L. Graham & A.J. van der Eb, Virology 52, 456-467 (1973)), DEAE-Dextran methods, etc., are suitably used for transformation.

[0045] After culturing the transformant, the human antibody is separated from inside the cells of the transformant or from the culture broth. For the separation and purification of the antibody, methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion exchange chromatography, gel filtration chromatography, etc. can be appropriately combined and used.

[0046] Antibody fragments can be prepared based on the antibody fragment antibody or based on the sequence information of the gene encoding the antibody. Examples of antibody fragments include Fab, Fab', F(ab') ２ , scFv, dsFv antibodies.

[0047] Fab is a fragment with a molecular weight of approximately 50,000 composed of an L-chain and a variable region of the H-chain, as well as an H-chain fragment consisting of a CH1 domain and a part of the hinge region, which is obtained by digesting IgG with papain in the presence of cysteine. In the present invention, it can be obtained by digesting the above antibody with papain. Also, a DNA encoding a part of the H-chain and the L-chain of the above antibody can be incorporated into an appropriate vector, and Fab can also be prepared from the transformant transformed using the vector.

[0048] Fab' is a fragment with a molecular weight of approximately 50,000 obtained by cleaving the disulfide bond between the H-chains of the F(ab') ２ described below. In the present invention, it is obtained by digesting the above antibody with pepsin and cleaving the disulfide bond using a reducing agent. Also, similar to Fab, Fab' can also be prepared genetically using the DNA encoding Fab'.

[0049] F(ab') ２ is a fragment with a molecular weight of approximately 100,000 composed of an L-chain and a variable region of the H-chain, as well as an H-chain fragment consisting of a CH1 domain and a part of the hinge region, which is obtained by digesting IgG with pepsin, and the fragment (Fab') is bound by a disulfide bond. In the present invention, it is obtained by digesting the above antibody with pepsin. Also, similar to Fab, F(ab') ２ can also be prepared genetically using the DNA encoding F(ab').

[0050] scFv is an antibody fragment formed by linking the C-terminus of one chain to the N-terminus of the other with a suitable peptide linker, creating a single-chain Fv consisting of a variable H-chain region and a variable L-chain region. Examples of peptide linkers include highly flexible (GGGGS). ３ For example, DNA encoding an scFv antibody can be constructed using DNA encoding the H chain variable region and L chain variable region of the above antibody and DNA encoding the peptide linker. This DNA can then be incorporated into a suitable vector, and scFv can be prepared from a transformant obtained by transforming with this vector.

[0051] dsFv is an Fv fragment obtained by introducing Cys residues at appropriate positions in the H chain variable region and L chain variable region, and stabilizing the H chain variable region and L chain variable region by disulfide bonds. The position of the Cys residue introduction in each chain can be determined based on the three-dimensional structure predicted by molecular modeling. In this invention, for example, the three-dimensional structure can be predicted from the amino acid sequences of the H chain variable region and L chain variable region of the above antibody, DNA encoding the H chain variable region and L chain variable region with mutations introduced based on this prediction can be constructed, incorporated into a suitable vector, and dsFv can be prepared from a transformant transformed using this vector.

[0052] Furthermore, antibody fragments can be polymerized by linking scFv antibodies, dcFv antibodies, etc., using an appropriate linker, or by fusing them with streptavidin.

[0053] A multispecific antibody is one that recognizes TfR, but can also recognize other targets simultaneously. Bispecific antibodies are particularly commonly used. A bispecific antibody is an antibody composed of two molecules: one that recognizes TfR and the other that targets other molecules. For example, a bispecific antibody obtained by chemically crosslinking two monoclonal antibodies (mab) ２Examples include bispecific F(ab')2, quadroma, bsDb (bispecific diversity), scBsDb (single-chain bispecific diversity), scBsTaFv (single-chain bispecific tandem variable domain), Bite (bispecific T cell Engineer antibody), and DNL-F(ab)3 (docl-and-lock trivalent Fab), obtained by chemically crosslinking two Fab fragment molecules (Shim, H. Bispecific Antibodies and Antibody-drug conjugates for cancer therapy: Technical Considerations. Biomolecules 2020, 10, 360) However, the form is not limited to this.

[0054] Aptamers are single-stranded nucleic acids composed of approximately 20 to 60 bases and can be configured to have a sequence-specific higher-order structure. Aptamers have the ability to bind to specific molecules such as proteins and can inhibit the function of such proteins or cells. Aptamers can have diversity and specificity similar to antibodies. The aptamers in this invention may be DNA aptamers, RNA aptamers, or DNA or RNA aptamers modified by sequence re-randomization, substitution with artificial or modified bases, etc. The aptamers in this invention target TfR, preferably human TfR, and more preferably human TfR1, and can bind specifically to TfR. The aptamers in this invention can bind to TfR, for example, through a ligand-binding domain between TfR and Tf, and can have TfR-Tf binding inhibitory activity. The aptamers in this invention can suppress dysregulation of TfR expression in cells, for example, overexpression of Tfr. The aptamers in the present invention can suppress the excessive accumulation of iron in cells. Aptamers having the desired sequence and higher-order structure can be chemically synthesized by known methods. For example, by repeatedly selecting and amplifying an aptamer sequence that binds to TfR, for example, at the ligand-binding domain between TfR and Tf, using a method such as SELEX (Systematic Evolution of Ligands by Exponential Enrichment), aptamers having specific inhibitory activity against the binding of transferrin (Tf) to the transferrin receptor (TfR) can be screened and obtained. The aptamers in the present invention can recognize amino acids of TfR in the same way as the anti-TfR antibodies described later.

[0055] Peptides In the present invention, peptides (or polypeptides) typically refer to peptides and proteins having a length of about 4 amino acids or more. Peptides in the present invention may consist of artificially designed sequences or may be peptides of biological origin. Peptides in the present invention may be natural peptides, synthetic peptides, recombinant peptides, etc. The peptide fragments described above are also included in the peptides of the present invention. Peptides in the present invention may target TfR, preferably human TfR, more preferably human TfR1, and may bind specifically to TfR. Peptides in the present invention may bind to TfR, for example, via a ligand-binding domain between TfR and Tf, and may have inhibitory activity against the binding of TfR and Tf. Peptides in the present invention may suppress dysregulation of TfR expression in cells, for example, overexpression of Tfr. Peptides in the present invention may suppress excessive accumulation of iron in cells. The peptides in this invention may be modified as long as their binding activity to TfR, inhibitory activity between Tf and TfR, suppressive activity against TfR expression, intracellular iron reduction activity and / or anti-vascular remodeling activity are maintained at an equivalent level, and mutations may be introduced by introducing mutations into the proteins described above as "antibodies". For example, the peptide in this invention may be a partial peptide of an anti-TfR antibody having inhibitory activity between transferrin (Tf) and transferrin receptor (TfR), as described later. Such peptides may be obtained by chemical synthesis, or by incorporating a part of the gene encoding the anti-TfR antibody described later into an expression vector and expressing it, or by degrading the anti-TfR antibody described later using a protease or the like. For example, the peptide in this invention may recognize the same TfR amino acids as the anti-TfR antibody described later.

[0056] The present invention provides a therapeutic agent for cardiovascular diseases comprising a molecule that suppresses the excessive accumulation of iron. In one embodiment, the molecule that suppresses the excessive accumulation of iron is a molecule having binding inhibitory activity between transferrin (Tf) and the transferrin receptor (TfR), and may be, for example, an antibody, aptamer, or peptide. In one example, a therapeutic agent for cardiovascular diseases comprising a molecule that suppresses the excessive accumulation of iron may reduce the expression level of the transferrin receptor (TfR). In one embodiment, the present invention provides a method for treating cardiovascular diseases comprising administering a therapeutic agent for cardiovascular diseases comprising a molecule that suppresses the excessive accumulation of iron to a subject. In another embodiment, the present invention provides a molecule that suppresses the excessive accumulation of iron for use in the treatment of cardiovascular diseases. In yet another embodiment, the present invention relates to the use of a molecule that suppresses the excessive accumulation of iron for the manufacture of a therapeutic agent for cardiovascular diseases. In one embodiment, the present invention provides a therapeutic agent for cardiovascular diseases (hereinafter sometimes referred to as "the therapeutic agent of the present invention") comprising an anti-TfR antibody having binding inhibitory activity between transferrin (Tf) and the transferrin receptor (TfR). In one embodiment, the therapeutic agent of the present invention contains an anti-TfR antibody as an active ingredient that binds to TfR. In one embodiment, the therapeutic agent of the present invention is a therapeutic agent for cardiovascular diseases in which the anti-TfR antibody has binding inhibitory activity between Tf and TfR and reduces the expression level of TfR. In one embodiment, the anti-TfR antibody of the therapeutic agent of the present invention reduces intracellular iron levels. In one embodiment, the anti-TfR antibody of the therapeutic agent of the present invention suppresses the proliferation and / or migration of vascular smooth cells. In one embodiment, the anti-TfR antibody of the therapeutic agent of the present invention suppresses vascular remodeling. In one embodiment, the present invention provides a method for treating cardiovascular diseases, comprising administering a therapeutic agent for cardiovascular diseases, comprising an anti-TfR antibody having binding inhibitory activity between Tf and TfR, to a subject. In another embodiment, the present invention provides an anti-TfR antibody having binding inhibitory activity between Tf and TfR for use in the treatment of cardiovascular diseases. In yet another embodiment, the present invention relates to the use of an anti-TfR antibody having binding inhibitory activity between Tf and TfR for the manufacture of a therapeutic agent for cardiovascular diseases.

[0057] In one embodiment, the anti-TfR antibody of the therapeutic agent of the present invention is an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor (TfR). In one example, the anti-TfR antibody recognizes amino acids 629 to 633 of human TfR and binds to TfR, thereby inhibiting the binding of Tf to TfR and reducing the expression level of TfR.

[0058] In one embodiment, the anti-TfR antibody of the therapeutic agent of the present invention is an antibody in which the heavy chain first complementarity-determining region (VH CDR1), heavy chain second complementarity-determining region (VH CDR2), and heavy chain third complementarity-determining region (VH CDR3) are sequence numbers 1, 2, and 3, respectively, and the light chain first complementarity-determining region (VL CDR1), light chain second complementarity-determining region (VL CDR2), and light chain third complementarity-determining region (VL CDR3) are sequence numbers 4, 5, and 6, respectively. In another embodiment, the anti-TfR antibody of the therapeutic agent of the present invention is an antibody in which the heavy chain has sequence number 7 and the light chain has sequence number 8. For example, an anti-TfR antibody in which VH CDR1, 2, and 3 are sequence numbers 1, 2, and 3 respectively, and VL CDR1, 2, and 3 are sequence numbers 4, 5, and 6 respectively, or an anti-TfR compound in which the heavy chain has sequence number 7 and the light chain has sequence number 8, can inhibit the binding of Tf to TfR and reduce the expression level of TfR by binding to TfR.

[0059] In one embodiment, the anti-TfR antibody of the therapeutic agent of the present invention is an antibody in which the heavy chain first complementarity-determining region (VH CDR1), heavy chain second complementarity-determining region (VH CDR2), and heavy chain third complementarity-determining region (VH CDR3) are sequence numbers 9, 10, and 11, respectively, and the light chain first complementarity-determining region (VL CDR1), light chain second complementarity-determining region (VL CDR2), and light chain third complementarity-determining region (VL CDR3) are sequence numbers 12, 13, and 14, respectively. In another embodiment, the anti-TfR antibody of the therapeutic agent of the present invention is an antibody in which the heavy chain has sequence number 15 and the light chain has sequence number 16. In one example, an anti-TfR antibody in which VH CDR1, 2, and 3 are sequence numbers 9, 10, and 11, respectively, and VL CDR1, 2, and 3 are sequence numbers 12, 13, and 14, respectively, or an anti-TfR compound in which the heavy chain has sequence number 15 and the light chain has sequence number 16, can inhibit the binding of Tf to TfR and reduce the expression level of TfR by binding to TfR.

[0060] In one embodiment, the anti-TfR antibody of the therapeutic agent of the present invention is a human antibody or a humanized antibody. For example, a human anti-TfR antibody or a humanized anti-TfR antibody having activity to inhibit the binding of Tf to TfR can inhibit the binding of Tf to TfR and reduce the expression level of TfR by binding to TfR.

[0061] In one embodiment, the anti-TfR antibody of the therapeutic agent of the present invention is an antibody fragment selected from the group consisting of Fab, Fab', F(ab')2, a single-chain antibody (scFv), a dimerized V region (Diabody), a disulfide-stabilized V region (dsFv), and a peptide containing CDR. In one example, these antibody fragments can inhibit the binding of Tf to TfR and reduce the expression level of TfR by binding to TfR.

[0062] In this specification, "cardiovascular disease" broadly refers to diseases caused by abnormalities in blood circulation, and includes cardiovascular diseases and cerebrovascular diseases. "Cardiovascular disease" refers to heart and vascular diseases caused by abnormalities in the heart, myocardium, and blood vessels and / or blood flow connected to the heart. Specifically, examples include heart diseases such as ischemic heart disease, hypertensive heart disease, cardiac hypertrophy, cardiomyopathy, myocarditis, congenital heart disease, valvular heart disease, and heart failure, and vascular diseases such as aortic aneurysm, aortic dissection, aortic disease, coronary artery disease, arteriosclerosis, vascular disease, pulmonary hypertension, obstructive arteriosclerosis, Takayasu's arteritis, arrhythmia, and hypertension. "Cerebrovascular disease" refers to diseases caused by abnormalities in the blood vessels and / or blood flow of the brain. Specifically, examples include stroke, cerebral arteriovenous malformation, trigeminal neuralgia, facial spasm, and moyamoya disease. Other conditions such as chronic kidney disease and renal failure are also included in cardiovascular diseases. The present invention provides therapeutic drugs for these cardiovascular diseases. In one embodiment, the present invention provides a therapeutic agent for cardiovascular diseases comprising a molecule that suppresses excessive iron accumulation, and the present invention provides a therapeutic agent for cardiovascular diseases in which the cardiovascular disease is a cardiovascular disease.

[0063] In this specification, "arteriosclerosis" means a condition in which blood vessels harden and their elasticity and / or flexibility is reduced, and includes atherosclerosis, Mönkeberg-type arteriosclerosis (medial sclerosis), arteriolar sclerosis, etc. It also includes hypertensive arteriosclerosis, arteriosclerosis associated with dyslipidemia, and arteriosclerosis associated with diabetes. In one embodiment, a therapeutic agent for cardiovascular disease containing a molecule that suppresses excessive iron accumulation, and the therapeutic agent of the present invention provide a therapeutic agent for cardiovascular disease in which the cardiovascular disease is a disease or symptom accompanied by arteriosclerosis.

[0064] In this specification, "vascular remodeling" broadly refers to the change in the structure of blood vessels in response to hemodynamic changes such as blood flow and blood pressure, and vascular damage caused by various stimuli such as oxidized LDL. Specifically, this includes, but is not limited to, the proliferation or migration of vascular endothelial cells and vascular smooth muscle cells, and / or the widening of blood vessel diameter, thickening of the tunica media, or fibrosis of the tunica adventitia. Furthermore, it also includes cardiac remodeling such as myocardial cell hypertrophy and interstitial fibrosis due to hemodynamic load caused by hypertension, and pulmonary vascular remodeling such as excessive cell proliferation and thickening of the intima and media of the pulmonary artery in pulmonary hypertension. In one embodiment, a therapeutic agent for cardiovascular disease containing a molecule that suppresses excessive iron accumulation, and the therapeutic agent of the present invention, provide a therapeutic agent for cardiovascular disease in which the cardiovascular disease is a disease or symptom accompanied by vascular remodeling.

[0065] In one embodiment, a therapeutic agent for cardiovascular diseases containing a molecule that suppresses excessive iron accumulation, and the therapeutic agent of the present invention, provide a therapeutic agent for cardiovascular diseases in which the cardiovascular disease is a disease or condition accompanied by dysregulation of transferrin receptor (TfR) expression. Examples of cardiovascular diseases accompanied by dysregulation of transferrin receptor (TfR) expression include, but are not limited to, hypertensive organ disorders of the aorta, kidneys, lungs, etc. In one embodiment, a therapeutic agent for cardiovascular diseases containing a molecule that suppresses excessive iron accumulation, and the therapeutic agent of the present invention, provide a therapeutic agent for cardiovascular diseases in which the cardiovascular disease is a disease or condition accompanied by overexpression of TfR and / or an increase in intracellular iron levels.

[0066] A therapeutic agent for cardiovascular disease containing a molecule that suppresses excessive iron accumulation, and the therapeutic agent of the present invention, preferably contains a molecule that suppresses excessive iron accumulation or an anti-TfR antibody, in addition to a physiologically acceptable diluent or carrier, which may be a mixture with other drugs. Suitable carriers include, but are not limited to, physiological saline, phosphate-buffered saline, phosphate-buffered saline glucose solution, and buffered saline. Alternatively, the antibody may be freeze-dried and reconstituted by adding the above-mentioned buffered aqueous solution as needed. Possible forms of administration include oral administration in the form of tablets, capsules, granules, powders, syrups, etc., or parenteral administration by injection (subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), percutaneous, transmucosal, nasal, transpulmonary, suppositories, etc. The therapeutic agent of the present invention may be administered alone or in combination with other drugs.

[0067] The dosage of the cardiovascular disease treatment drug containing a molecule that suppresses excessive iron accumulation, and the therapeutic drug of the present invention, varies depending on symptoms, age, weight, etc. However, for oral administration, the amount of antibody for adults is usually about 0.01 mg to 1000 mg per day, which can be administered in one dose or in several divided doses. For parenteral administration, about 0.01 mg to 1000 mg can be administered by subcutaneous injection, intramuscular injection, or intravenous injection.

[0068] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

[0069] In Examples 1, 2, and 4 below, the TfR436 antibody described in paragraphs 0090 and 0091 of International Publication WO2014 / 073641 was used as the anti-TfR antibody.

[0070] The CDR sequences of the TfR436 antibody are shown below. VH CDR1: SYGMH (SEQ ID NO: 1) VH CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 2) VH CDR3: DSNFWSGYYYSPVDV (SEQ ID NO: 3) VL CDR1: TRSSGSIASNSVQ (SEQ ID NO: 4) VL CDR2: YEDTQRPS (SEQ ID NO: 5) VL CDR3: QSYDSAYHWV (SEQ ID NO: 6)

[0071] The VH and VL sequences of the TfR436 antibody are shown below. TfR436 antibody VH (SEQ ID NO: 7) DVQLVQSGGGVVQPGRSLRLSCAASGFPFKSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRGEDTAVYYCARDSNFWSGYYSPVDVWGQGTTVTVSS

[0072] TfR436 antibody VL (SEQ ID NO: 8) NFMLTQPHSVSESPGKTVTISCTRSSGSIASNSVQWYQQRPGSAPITVYYEDTQRPSGVPDRFGSIDSSSSNSASLTISGLQTEDEADYYCQSYDSAYHWVFGGGTKLAVL

[0073] Example 1: Identification of the binding site of the TfR436 antibody. The TfR436 antibody did not cross-react with mouse TfR, but showed cross-reactivity with hamster TfR. The amino acid sequences of the transferrin (Tf) binding site (amino acids 569-760) in TfR were aligned. In the human TfR sequence, amino acids that were the same as those in hamster and different from those in mouse were picked out. Point mutation was performed on the picked-out amino acids as shown in Figure 1 to prepare a soluble TfR mutant fragment.

[0074] (1) Preparation of soluble wild-type TfR (sTfR) and TfR mutant fragment (MF1-MF7) Human TfR extracellular domain (amino acids 89-760), or the base sequences encoding TfR mutant fragment (MF1-MF7) and AAARGGPEQKLISEEDLNSAVDHHHHHH (Sequence ID 18), as shown in Figure 1, were totally synthesized. Synthesized genes were inserted into the multi-cloning sites of a vector incorporating the neomycin resistance gene and the DHFR gene into the expression vector pCAGGS (see Niwa et al. Gene. 1991 Dec 15;108(2):193-9), thereby creating the pCAGGS-Neo-DHFR-sTFR-myc-his expression plasma. Expi293 cells (Invitrogen) were transfected with the plasma using Expifectamine (Invitrogen) at 37°C and 8% CO2. ２ The cells were cultured at 135 rpm for 5 days. The culture supernatant was then collected by centrifugation, and sTfR or MF1-MF7 was purified using 20 mM Imidazole / DPBS as the binding buffer and 500 mM Imidazole / DPBS as the elution buffer, with AKTA prime (GE Healthcare) connected to a HisTrapHP (GE Healthcare) column. The eluted proteins were re-buffered using Zeba spin column (Thermo Scientific) with 30 mM HEPES, 5% trehalose, and pH 7.2.

[0075] (2) Identification of the binding site of the TfR436 antibody The purified sTfR or MF1 to MF7 described above were diluted with PBST (Phosphate Buffered Saline with Tween 20, TaKaRa) to prepare seven different dilutions starting from 600 ng / mL using 3-fold dilutions. Then, 100 μL / well of the dilution was dispensed onto Ni-NTA HisSorb Strips 96-well plate (QIAGEN), placed on a shaker, and allowed to react at room temperature. After 1 hour, the plates were washed five times with PBST Buffer, and 100 μL / well of TfR436 antibody (1 ug / mL) was dispensed onto a shaker, placed on a shaker, and allowed to react at room temperature for 1 hour. Subsequently, the samples were washed five times with PBS-T Buffer, dispensed into 100 μL / well of 50,000-fold diluted secondary antibody F(ab')2 Fragment Anti-Human IgG Fcγ (Jackson Immuno Research), and reacted at room temperature for 1 hour. After washing five times with PBST Buffer, 100 μL / well of TMB Soluble Reagent (High Sensitivity) (ScyTek) was dispensed and reacted at room temperature in the dark for 3 minutes. Then, 100 μL / well of TMB Stop Buffer (ScyTek) was added, and the mixture was shaken in a shaker for 1 minute. The absorbance at 450 nm (ref. 620 nm) was measured using a plate reader.

[0076] As shown in Figure 2, the TfR436 antibody showed decreased reactivity with TfR mutant fragment MF5, but no decrease in reactivity with other mutant fragments. In other words, substituting the 629th, 630th, and 633rd amino acids of TfR with other amino acids prevents the TfR436 antibody from recognizing TfR. This suggests that amino acids 629th to 633rd are the recognition epitopes of the TfR436 antibody.

[0077] Example 2: Comparison of TfR436 antibody and comparative antibody in Tf-TfR binding inhibition (1) Production of comparative antibody A24 Patent document US2008 / 0193453 describes the A24 antibody against human TfR. To compare the TfR436 antibody with this antibody, deposited Hybridoma was obtained and the antibody was produced. Specifically, Hybridoma was placed in a medium of RPMI1640 (GIBCO) and 10% FBS at a cell concentration of 1-2 x 10⁻⁶ ５ Wrap the mixture so that it reaches / mL, and heat at 37°C in 5% CO2. ２ Cells were cultured in an incubator. After expansion culture, cells were harvested by centrifugation, washed twice with PBS, and then further cultured in serum-free medium cosmedium005 (CosmoBio) and 0.5% Nutridoma-CS (Roche) until the volume reached 550 mL. Five days after the cells reached confluence, the culture supernatant was collected by centrifugation.

[0078] The recovered supernatant was applied to a protein A carrier (Ab-Capcher Extra: Protenova), and the antibody bound to protein A was eluted with 0.1 M glycine hydrochloride buffer (pH 2.7), followed by rapid neutralization with 1 M Tris hydrochloride buffer (pH 8.5). Then, buffer exchange to PBS was performed using an UltraCel ultrafiltration disk (Merck Millipore).

[0079] (2) Comparison of TfR436 antibody and other company's antibody in Tf-TfR binding inhibition The sTfR described in Example 1 was prepared to 5.0 μg / mL by PBST, and the diluted solution was dispensed at 100 μL / well into MaxiSorp 96-well plate (Nunc) and allowed to stand overnight at 4°C to solidify. The next day the solid phase was discarded and the sample was blocked by standing at room temperature in 200 μL / well of 100% Block Ace (DS Pharma Biomedical). After 1 hour, the mixture was washed five times with PBST Buffer, then 50 μL / well of HRP-labeled Tf (2 ug / mL) was dispensed, followed by 50 μL / well of TfR436 antibody, A24 antibody (10 μg / mL to 2-fold dilution series), or holo-Tf (Sigma) (300 μg / mL to 2-fold dilution series). After reacting at room temperature for 1 hour, the mixture was washed five times with PBST Buffer, and 100 μL / well of TMB Soluble Reagent (High Sensitivity) was dispensed and reacted at room temperature in the dark. After 25 minutes, 100 μL / well of TMB Stop Buffer was added and the mixture was shaken in a shaker for 1 minute. The absorbance at 450 nm (ref. 620 nm) was measured using a plate reader.

[0080] As shown in Figure 3, the TfR436 antibody completely inhibited Tf-TfR binding at a much lower dose (100 ng / mL). On the other hand, the A24 antibody could not completely inhibit Tf-TfR binding even at a dose of 10 μg / mL, inhibiting only 50% of Tf-TfR binding. This suggests that the TfR436 antibody is superior in inhibiting Tf-TfR binding.

[0081] Example 3: Suppression of AngII-induced vascular remodeling and TfR expression by TfR018 antibody (mouse in vivo study) Since TfR436 antibody is specific to human TfR and does not cross-react with mouse TfR, in the mouse in vivo study of Example 3, TfR018 antibody, a phage antibody derived from a different phage clone, was used as the anti-TfR antibody. TfR018 antibody is described in paragraphs 0083 and 0084 of International Publication WO2012 / 153707. TfR018 antibody is human TfR reactive and also cross-reacts with mouse TfR.

[0082] The CDR sequences of the TfR018 antibody are shown below. VH CDR1: DYAMH (SEQ ID NO: 9) VH CDR2: GINWNGGSTDYADSVEG (SEQ ID NO: 10) VH CDR3: DYADLGSGSDY (SEQ ID NO: 11) VL CDR1: SGSRSNIGSNYVH (SEQ ID NO: 12) VL CDR2: RNDQRPS (SEQ ID NO: 13) VL CDR3: ASWDDKMSGRL (SEQ ID NO: 14)

[0083] The VH and VL sequences of the TfR018 antibody are shown below. TfR018 antibody VH (SEQ ID NO: 15) EVQLVESGGGLLVQPGRSLRLSCAASGFTFGDYAMHWVRQSPGKGLEWVSGINNWNGGSTDYADSVEGRFTISRDNAANSLFLQMNSLRAEDTAIYYCARDYADLGSGSDYWGQGTLVTVSS

[0084] TfR018 antibody VL (SEQ ID NO: 16) QSVLTQPPSASGTPGQRVTISCSGSRSNIGSNYVHWYQQLPGAAPKLLLIYRNDQRPSGVPDRFSGSRSDTSAFLAISGLRSSEDEADYYCASWDDKMSGRLFGGGTKVTVL

[0085] First, to investigate the effect of anti-TfR antibodies on angiotensin II (AngII)-induced vascular remodeling, mice administered either AngII or physiological saline were given anti-TfR antibodies (TfR018 antibodies) intraperitoneally five days a week for two weeks. AngII is known to promote arteriosclerosis. Subsequently, systolic blood pressure and hematological parameters were evaluated, and histological analysis was performed. Western blot analysis was also performed to evaluate the effect of anti-TfR antibodies on aortic TfR expression levels. Details are shown below.

[0086] Male C57 / BL6J mice aged 8-10 weeks were subcutaneously administered either angiotensin II (AngII; 1,000 ng / kg / min, Peptide Laboratories, Inc., Osaka, Japan) or physiological saline for two weeks using an osmotic minipump. To evaluate the effect of anti-TfR antibodies on vascular remodeling, mice administered AngII or physiological saline were intraperitoneally administered either a vehicle or anti-TfR antibody (TfR018 antibody; 6 mg / kg, Perseus Proteomics Co., Ltd., Tokyo, Japan) five days a week for two weeks. The mice were housed in a temperature-controlled facility with a 12-hour light-dark cycle and given free access to food and water. After two weeks, the mice were euthanized under isoflurane anesthesia. Tissue was extracted, rapidly frozen in liquid nitrogen, and stored at -80°C. In Example 3, the control group consisted of mice administered with physiological saline via vehicle injection, the TfR018 antibody group consisted of mice administered with physiological saline via TfR018 antibody, the AngII group consisted of mice administered with AngII via vehicle injection, and the AngII + TfR018 antibody group consisted of mice administered with AngII via TfR018 antibody. All animal experiments were conducted in accordance with the guidelines of the National Institutes of Health (NIH). All experimental procedures were approved by the Animal Experiment Committee of Hyogo College of Medicine (Protocol #23-079A).

[0087] (Evaluation of systolic blood pressure and hematological parameters) Systolic blood pressure (SBP) was measured using a non-invasive tail cuff system (MK-2000, Muromachi Machinery Co., Ltd., Tokyo, Japan). Peripheral blood cell counts were measured according to a previous report (Naito Y, et al. Iron Deficiency Induces Heart Failure with Ectopic Cardiac Calcification in Mice with Metabolic Syndrome. Circ Heart Failure. 2022; 15: e009034.).

[0088] (Histological Analysis) Aortic tissue was embedded in Tissue-Tech® O.C.T. Compound (Sakura FineTech Japan Co., Ltd.) and cut into 10 μm thick sections. Serial sections of the aorta were stained with Masson's trichrome (MTc) and Elastica-van Gieson (EVG) stains. Arterial medial thickness and adventitia fibrosis were quantified by measuring the medial thickness and adventitia fibrosis area of ​​the thoracic aorta using ImageJ software. Aortic sections were also immunohistochemically stained with rabbit anti-TfR1 primary antibody (abcam, Cambridge, UK; dilution 1:100), rabbit anti-ferritin primary antibody (abcam; dilution 1:100), and rat CD68 primary antibody (abcam; dilution 1:200). Immunostaining was visualized using avidin-biotin-peroxidase conjugate and a 3-amino-9-ethylcarbazole substrate. Quantification of TfR, ferritin, and CD68-positive areas was performed by counting the number of TfR, ferritin, and CD68-positive cells in 10 randomly selected regions.

[0089] (Western blot analysis) Protein homogenates were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Protein expression levels were detected using an enhanced chemiluminescence kit. The antibodies used were anti-TfR1 antibody (Invitrogen, Waltham, MA, USA; dilution 1:1000), anti-ferritin antibody (abcam; dilution 1:1000), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Cell Signaling Technology, Inc, Danvers, MA, USA; dilution 1:1000). In the control group, TfR018 antibody group, AngII group, and AngII + TfR018 antibody group (n=5 in each group), the expression levels of each protein were standardized based on the expression level of the GAPDH protein, and the relative values ​​of the protein amounts are shown in the graph.

[0090] Table 1. Physiological and hematological parameters of all groups and 2 weeks after administration of physiological saline or AngII.

[0091]

[0092] In Table 1, * indicates p < 0.05 compared to the control group, ¶ indicates p < 0.05 compared to the TfR018 antibody group, and # indicates p < 0.05 compared to the AngII group. Each group was studied with n = 6. SBP represents systolic blood pressure, HW represents cardiac weight, and TL represents tibia length.

[0093] Regarding the effects of the TfR018 antibody on physiological and hematological parameters, there was no difference in body weight between the experimental groups. The heart weight / tibial length ratio increased in the AngII group compared to the control group, and this increase was suppressed by the administration of the TfR018 antibody, but it was not statistically significant. Regarding the effects on hematological parameters, serum hemoglobin and hematocrit levels decreased in the AngII + TfR018 antibody group (Table 1).

[0094] As shown in Table 1, SBP increased after AngII administration. TfR018 antibody administration did not affect SBP after AngII or saline administration. However, a significant difference in AngII-induced vascular remodeling was observed in the AngII + TfR018 antibody group. Representative macroscopic findings of the entire aorta shown in Figure 4A, and aortic sections stained with MTC and EVG, showed vascular remodeling in the AngII group compared to the control group. However, this vascular remodeling was suppressed by the administration of TfR018 antibody. In particular, the dilation of the aortic diameter caused by AngII was suppressed by the administration of TfR018 antibody (Figure 4B). Furthermore, as shown in Figures 4C and 4D, histological evaluation revealed that the AngII group showed thickening of the median and fibrosis of the adventitia, but these were suppressed by the administration of TfR018 antibody. On the other hand, these effects of the TfR018 antibody on vascular remodeling were not observed in the TfR018 antibody group (Figures 4A-D).

[0095] Next, to evaluate the effect of anti-TfR antibodies on the amount of TfR in the aorta, the amount of TfR protein in the aorta was measured in the control group, the AngII group, and the AngII + TfR018 antibody group, and the results are shown in Figures 5 and 6. Compared to the control group, the amount of TfR in the aorta increased in the AngII group, but this increase was suppressed in the AngII + TfR018 antibody group (Figures 5A and 6A). Notably, the amount of ferritin protein in the aorta also increased in the AngII group, and this increase was suppressed by the administration of TfR018 antibody (Figures 5A and 6A). Furthermore, immunohistochemical analysis revealed that the TfR018 antibody reduced the increase in TfR and ferritin-positive cells in the aorta of the AngII group, as well as the CD68 inflammatory cell infiltration, mainly in the adventitious lesion area (Figures 5B and 6B). On the other hand, these effects of the TfR018 antibody were not observed in the TfR018 antibody group (Figures 6 and B). These results suggest that the TfR018 antibody suppresses AngII-induced vascular remodeling in mice. In addition, administration of the TfR018 antibody does not affect normal cells or tissues, suggesting that it may selectively exert its effects on cardiovascular disease-affected areas and potentially be safe to administer. In other words, anti-TfR antibodies do not exert vascular remodeling inhibitory effects (e.g., inhibitory effects on aortic diameter expansion, median thickness thickening, adventitia fibrosis, TfR expression increase, and ferritin increase) on normal cells with low TfR expression levels. On the other hand, they selectively act on degenerated tissues or cells (e.g., tissues or cells causing vascular remodeling) where TfR expression is high due to elevated TfR expression outside the normal range, and thus exert therapeutic effects.

[0096] Example 4: Inhibition of AngII-induced protein synthesis, cell proliferation, and cell migration in human arterial smooth muscle cells by TfR436 antibody (in vitro study) Since the anti-TfR antibody inhibited AngII-induced vascular remodeling in mice, we then evaluated TfR expression in human arterial smooth muscle cells (HASMCs) by AngII stimulation with or without the addition of a human-specific anti-TfR antibody (TfR436 antibody). Furthermore, we investigated the effect of the anti-TfR antibody on AngII-induced protein synthesis, proliferation, and migration in vitro. In addition, to investigate the involvement of iron in the mechanisms of AngII-induced proliferation and migration in these HASMCs, we measured intracellular iron ions (Fe ２＋ We investigated the amount of iron in cells using FerroOrange staining, which specifically detects iron. Specifically, the results are as follows.

[0097] Human arterial smooth muscle cells (HASMCs) were purchased from Lonza, Inc. (Walkersville, MD, USA). The HASMCs were grown in SmBM cell culture medium supplemented with SmGM-2® Bulletkit® (Lonza, Inc.) containing 5% FBS before the experiment. The cells were incubated in a humidified cell culture incubator at 37°C and 5% CO2. ２ The cells were maintained and used for 3-4 passages. The cell culture medium was replaced with serum-free medium for 24 hours. Subsequently, HASMCs were pretreated with or without the addition of anti-TfR antibody (PPMX-T003 (identical to the TfR436 antibody of the present invention); 1000 ng / mL, Perseus Proteomics Co., Ltd., Tokyo, Japan). After 24 hours, HASMCs were treated with 0.1 μM AngII for 24 hours, either in the presence or absence of TfR436 antibody. Cell proliferation was evaluated by counting cells using Cell Counting Kit-8 (Dojin Chemical Research Institute Co., Ltd., Kumamoto, Japan) according to the manufacturer's instructions. Furthermore, intracellular iron ions (Fe ２＋ The amount was evaluated by performing FerroOrange staining using FerroOrange® (registered trademark) (Dojin Chemical Research Institute Co., Ltd.) in accordance with the manufacturer's instructions.

[0098] Cell migration was evaluated using a wound healing assay. HASMCs were cultured in serum-free medium for 24 hours, then incubated for 24 hours in or without TfR436 antibody (1000 ng / mL). Subsequently, cells were treated with 0.1 μM AngII, wounds were created using a yellow pipette tip in or without TfR436 antibody, and the cells were cultured for 24 hours. The wound medium was then stained using Diff-Quick (Sysmex Corporation, Kobe, Japan) and photographed. Cells that migrated from the edge of the dish were counted in 10 microscopic fields per dish.

[0099] Protein synthesis in HASMCs was evaluated using a modified Surface Sensing of Translation (SUNSET) method, in accordance with prior literature (Cicalese S, et al. Endoplasmic reticulum chemical chaperone 3-hydroxy-2-naphthoic acid reduces angiotensin II-induced vascular remodeling and hypertension in vivo and protein synthesis in vitro. J Am Heart Assoc. 2022; 11: e028201). The cell culture medium was replaced with serum-free medium for 24 hours. Next, HASMCs were incubated in or without TfR436 antibody (1000 ng / mL). After 24 hours, the cells were treated with 0.1 μM AngII in or without TfR436 antibody and cultured for 24 hours. After the experiment, the cells were pulsed with 5 μM puromycin (Sigma-Aldrich, St. Louis, Missouri, USA) for 1 hour. Western blot analysis was performed on the cells using mouse anti-puromycin primary antibody (Sigma-Aldrich; dilution 1:1000). In the saline + vehicle, saline + TfR436 antibody, AngII + vehicle, and AngII + TfR436 antibody groups (n=4 in each group), the TfR and puromycin protein levels of HASMCs were standardized relative to the GAPDH protein level, and the relative values ​​are shown in the graph.

[0100] As shown in Figure 7A, the TfR436 antibody clearly suppressed TfR expression in HASMCs. In the in vivo mouse experiment of Example 3, the amount of TfR in the aorta of the AngII group increased, but in the in vitro experiment, AngII stimulation did not increase the amount of TfR in vascular smooth muscle cells (Figure 7A). This suggests that the increase in TfR levels due to AngII administration in the in vivo experiment is not solely dependent on AngII. Furthermore, the SUNSET assay showed that the TfR436 antibody did not alter the fundamental rate of protein synthesis, but reduced the AngII-induced protein synthesis enhancement (Figure 7A). In addition, the TfR436 antibody suppressed AngII-induced cell proliferation and cell migration in HASMCs (Figure 7B). These results demonstrate that the TfR436 antibody suppresses AngII-induced protein synthesis, cell proliferation, and cell migration in vascular smooth muscle cells. Furthermore, FerroOrange staining, performed to investigate the involvement of iron, revealed that AngII increases the intracellular iron content of HASMCs, while the TfR436 antibody suppresses the increase in intracellular iron content mediated by AngII (Figure 8).

[0101] (Statistical Analysis) In Examples 3 and 4, values ​​were expressed as mean ± standard error. The assumption of normality was evaluated by the Shapiro-Wilk test. Statistical analysis was performed using Tukey's test with one-way ANOVA (in vivo experiment in Example 3) and two-way ANOVA (in vitro experiment in Example 4). Statistical analysis was performed using GraphPad Prism 9. A probability value < 0.05 was considered statistically significant.

[0102] (Discussion) In this specification, it has been demonstrated that anti-TfR antibodies suppress AngII-induced vascular remodeling in vivo and reduce intracellular iron levels and inhibit the proliferation and migration of AngII-induced vascular smooth muscle cells in vitro. TfR is essential for hematopoiesis of red blood cells. Therefore, in Example 3 of this application, a slight decrease in serum hemoglobin and hematocrit levels was observed in the AngII + TfR018 antibody group. On the other hand, the role of TfR in non-hematopoietic tissues, especially diseased tissues, has not been fully clarified. However, as explained in the background, there have been reports on the relationship between iron and TfR and the pathophysiology of cardiovascular diseases, including vascular remodeling, such as the proliferation and migration of smooth muscle cells. TfR is universally expressed in most cells and takes up iron into cells through interaction with transferrin. Under conditions of low iron levels, TfR expression in cells increases, and under conditions of high iron levels, TfR expression in cells decreases. However, in Example 3 of this application, both the amount of TfR and ferritin in the aorta increased in the AngII group. This suggests that dysregulation of TfR is involved in the development of vascular remodeling in the AngII group. The anti-TfR antibody suppressed the increase in the amount of TfR and ferritin in the aorta induced by AngII administration. On the other hand, no effect of TfR018 antibody administration was observed in the TfR018 antibody group, which was not administered AngII. Iron is a cofactor of enzymes involved in cell proliferation. Vascular remodeling is a progressive disorder characterized by thickening of the media, which is caused by the proliferation of vascular smooth muscle cells. Taking these factors together, it is thought that the anti-TfR antibody suppresses the expression of TfR in the aorta and inhibits iron uptake in the aorta via TfR, thereby suppressing the increase in the amount of iron in aortic smooth muscle cells, and thereby suppressing smooth muscle cell proliferation and vascular remodeling in AngII-administered mice (Figure 9). Furthermore, anti-TfR antibodies do not show any cell proliferation inhibitory effect on normal cells with low TfR expression levels (e.g., several hundred TfR cells per cell).Therefore, it is thought that anti-TfR antibodies selectively act only on mutant tissues or cells that are in a state where TfR expression is elevated outside the normal range, such as during disease exacerbation (e.g., tens of thousands of TfR cells / cell), and the resulting increase in intracellular iron levels is causing tissue degeneration such as increased cell proliferation and associated vascular remodeling. This allows the antibodies to suppress TfR expression, inhibit the increase in intracellular iron levels, and suppress cell proliferation, thereby exerting a therapeutic effect. Furthermore, iron is known to be associated with inflammation (Winn NC, et al. Regulation of tissue iron homeostasis: the macrophage “ferrostat”. JCI Insight. 2020; 5: e132964.). In Example 3 of this application, the anti-TfR antibody suppressed CD68-positive inflammatory cell infiltration, mainly in the outer membrane lesions, in mice administered AngII. This suggests that these anti-inflammatory effects may also contribute to the suppression of vascular remodeling in the AngII + TfR018 group.

[0103] Previous studies have reported that AngII directly affects vascular remodeling independently of blood pressure elevation. In Example 3 of this application, the anti-TfR antibody suppressed the development of AngII-induced vascular remodeling in mice administered with AngII without any blood pressure-lowering effect. This indicates that the suppression of AngII-induced vascular remodeling by the therapeutic agent of the present invention is independent of blood pressure. Hypertension is a major risk factor for cardiovascular disease. Many antihypertensive treatments have been developed, and there is evidence that antihypertensive treatment prevents cardiovascular disease. On the other hand, it has also been reported that even when blood pressure is well controlled with antihypertensive treatment, patients still show a risk of developing cardiovascular disease. Therefore, a new therapeutic strategy of suppressing vascular remodeling may help break the chain of hypertension that causes vascular remodeling, thereby preventing the development of cardiovascular disease. As described herein, TfR can be a therapeutic target for vascular remodeling. In fact, in cancer treatment, TfR has been studied extensively as an attractive target, and it has been reported that TfR targeting is effective for the delivery of many therapeutic agents. Furthermore, a Phase I clinical trial using PPMX-T003 (identical to the TfR436 antibody in this specification), a humanized anti-TfR1 monoclonal antibody, demonstrated acceptable safety. This specification demonstrates in vivo the preventive effect of anti-TfR antibodies against the development of vascular remodeling in mice administered with AngII. Furthermore, the inhibitory effect of anti-TfR antibodies on the proliferation and migration of vascular smooth muscle cells induced by AngII was shown in vitro. It was also shown that anti-TfR antibodies selectively suppress the dysregulation of TfR expression in smooth muscle cells, which are vascular constituent cells, and the resulting increase in intracellular iron levels (iron overgrowth), observed in vascular remodeling, a process of disease progression in cardiovascular diseases. This invention is the first report of an in vivo and in vitro inhibitory effect of anti-TfR antibodies against cardiovascular diseases, particularly vascular remodeling. Anti-TfR antibodies have the potential to become a new therapeutic agent for cardiovascular diseases, including vascular remodeling.

Claims

1. A drug for treating cardiovascular diseases that contains molecules that suppress excessive iron accumulation.

2. The therapeutic agent for cardiovascular disease according to claim 1, wherein the molecule is a molecule having binding inhibitory activity between transferrin (Tf) and transferrin receptor (TfR).

3. The therapeutic agent for cardiovascular disease according to claim 1, wherein the molecule is an antibody, an aptamer, or a peptide.

4. The therapeutic agent for cardiovascular disease according to claim 1, wherein the molecule is an anti-TfR antibody having binding inhibitory activity between transferrin (Tf) and transferrin receptor (TfR).

5. The therapeutic agent for cardiovascular disease according to claim 4, wherein the anti-TfR antibody is an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor (TfR).

6. The therapeutic agent for cardiovascular disease according to claim 4, wherein the anti-TfR antibody is an antibody in which the heavy chain first complementarity-determining region (VH CDR1), heavy chain second complementarity-determining region (VH CDR2), and heavy chain third complementarity-determining region (VH CDR3) are sequence numbers 1, 2, and 3, respectively, and the light chain first complementarity-determining region (VL CDR1), light chain second complementarity-determining region (VL CDR2), and light chain third complementarity-determining region (VL CDR3) are sequence numbers 4, 5, and 6, respectively.

7. The therapeutic agent for cardiovascular disease according to claim 4, wherein the anti-TfR antibody is an antibody in which the heavy chain has SEQ ID NO: 7 and the light chain has SEQ ID NO:

8.

8. The therapeutic agent for cardiovascular disease according to claim 4, wherein the anti-TfR antibody is an antibody in which the heavy chain first complementarity-determining region (VH CDR1), heavy chain second complementarity-determining region (VH CDR2), and heavy chain third complementarity-determining region (VH CDR3) are sequence numbers 9, 10, and 11, respectively, and the light chain first complementarity-determining region (VL CDR1), light chain second complementarity-determining region (VL CDR2), and light chain third complementarity-determining region (VL CDR3) are sequence numbers 12, 13, and 14, respectively.

9. The therapeutic agent for cardiovascular disease according to claim 4, wherein the anti-TfR antibody is an antibody in which the heavy chain has SEQ ID NO: 15 and the light chain has SEQ ID NO:

16.

10. The therapeutic agent for cardiovascular disease according to claim 4, wherein the anti-TfR antibody is a human antibody or a humanized antibody.

11. The anti-TfR antibody is Fab, Fab', F(ab') ２ The therapeutic agent for cardiovascular disease according to claim 4, wherein the antibody fragment is selected from the group consisting of a single-chain antibody (scFv), a dimerized V region (Diabody), a disulfide-stabilized V region (dsFv), and a peptide containing a CDR.

12. The therapeutic agent for cardiovascular disease according to any one of claims 1 to 11, wherein the molecule reduces the expression level of transferrin receptor (TfR).

13. The therapeutic agent according to any one of claims 1 to 11, wherein the cardiovascular disease is a cardiovascular disease.

14. The therapeutic agent according to any one of claims 1 to 11, wherein the cardiovascular disease is a disease or condition accompanied by arteriosclerosis.

15. The therapeutic agent according to any one of claims 1 to 11, wherein the cardiovascular disease is a disease or condition accompanied by vascular remodeling.

16. The therapeutic agent according to any one of claims 1 to 11, wherein the cardiovascular disease is a disease or condition accompanied by a dysregulation of transferrin receptor (TfR) expression.

17. The therapeutic agent according to any one of claims 1 to 11, wherein the cardiovascular disease is a disease or condition accompanied by an increase in intracellular iron content.

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