Therapeutic agent for polycythemia

Abstract

The present invention addresses the problem of defining an appropriate dose regiment and an appropriate dosage for an anti-TfR antibody, which can suppress promotion of forming red blood cells without inducing a symptom of systemic anemia in a patient with polycythemia. According to the present invention, provided is a therapeutic agent for polycythemia, which includes an antibody recognizing amino acid residues lying between a 629th and a 633th in a human transferrin receptor. After a first administration of the antibody is performed at a dosage of 0.25-20 mg / kg, a second administration is not performed in at least four weeks or more.

Description

Drugs for treating polycythemia

[0001] The present invention relates to dosage regimens for treating polycythemia, including anti-transferrin receptor antibodies.

[0002] The transferrin receptor (TfR) is a Type II membrane protein expressed on the cell surface. By binding to its ligand, transferrin, TfR transports iron into cells, maintaining cell survival and division. While TfR expression is low in many normal cells, it has been reported to be expressed in some cells, such as skin epidermal basal cells and small intestinal epithelial cells (Non-Patent Documents 1-3). Erythroid cells and placental trophoblast cells have a high demand for iron uptake, and therefore have high TfR expression (Non-Patent Documents 4 and 5).

[0003] Polycythemia vera (PV) is a chronic myeloproliferative tumor characterized by an excessive increase in red blood cells. Clinical manifestations of PV include increased blood viscosity and decreased blood flow due to increased red blood cells in the blood. Symptoms of PV include poor organ donation, headache due to hypoxia, dizziness, fatigue, and bleeding due to abnormal platelet function. Furthermore, PV can cause skin pruritus, particularly severe after bathing, due to histamine release caused by increased basophils. Complications of PV include erythromelalgia, which is characterized by asymmetric swelling, pain, and a burning sensation in the hands and feet, thrombosis, and embolism. Some patients progress to acute myeloid leukemia (AML) or myelofibrosis (Non-Patent Document 6).

[0004] The prognosis for PV is better than that of other malignant tumors, with a survival period of approximately 14 years with treatment (Non-Patent Document 7). Currently, there is no cure for PV, and the goal of PV treatment is primarily to reduce the number of red blood cells, control the hematocrit level to 45% or less, and prevent thrombosis. Current treatments for PV include phlebotomy and cytoreductive therapy. However, these treatments have side effects such as iron deficiency and bone marrow suppression, which reduce patients' quality of life. Given the low level of satisfaction with current treatments, there is a need for treatments with fewer side effects and improved patient quality of life.

[0005] Patent Document 1 describes that an anti-human TfR antibody inhibits erythroid colony formation by hematopoietic stem cells derived from PV patients and has a proliferation-suppressing effect on normal erythroid cells, and describes a therapeutic drug for polycythemia using said TfR antibody. Non-Patent Document 8 describes that an anti-human TfR antibody was administered to healthy subjects to evaluate its safety, pharmacokinetics, and pharmacological effects, demonstrating a decrease in blood cell levels and an acceptable safety profile.

[0006] International Publication No. WO2021 / 045184

[0007] P. Ponka, C.N. Lok, The transferrin receptor: role in health and disease, Int. J. Biochem. Cell Biol. 31 (1999) 1111-1137.D.R. Richardson, P. Ponka, The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells, Biochim. Biophys. Acta 1331 (1997) 1- 40.Daniels TR. Delgado T , Rodriguez JA, Helguera G , Penichet ML. The transferrin receptor part I: Biology and targeting with cytotoxic antibodies for the treatment of cancer. Clinical Immunology (2006) 121, 144-158J.E. Levy, O. Jin, Y. Fujiwara, F. Kuo, N.C. Andrews, Transferrin receptor is necessary for development of erythrocytes and the nervous system, Nat. Genet. 21 (1999) 396- 399.Khatun R, Wu Y, Kanenishi K, Ueno M, Tanaka S, Hata T, Sakamoto H., Immunohistochemical study of transferrin receptor expression in the placenta of pre-eclamptic pregnancy., Placenta. 2003;24(8-9):870-6.Butcher C, D’Andrea RJ. Molecular aspects of polycythemia vera (review).. Int J Mol Med. 2000 Sep;6(3):243-52. Tefferi A, Rumi E, Finazzi G, Gisslinger H, Vannucci AM, Rodeghiero F, Randi ML, Vaidya R, Cazzola M, Rambaldi A, Gisslinger B, Pieri L, Ruggeri M, Bertozzi I, Sulai NH, Casetti I, Carobbio A, Jeryczynski G, Larson DR, Müllauer L, Pardani A, Thiele J, Passamonti F, Barbui T. Survival and prognosis among 1545 patients with contemporary polycythemia vera: an international study. .. Leukemia. 2013 Sep;27(9):1874-81Ogama Y, Kumagai Y, Komatsu N, Araki M, Masubuchi N, Akiyoshi H, Matsuura T, Kirisako H, Kyoya A, Nomura F, Ohira Y, Yokokawa T, Yamamoto Y, Phase 1 Clinical Trial of PPMX-T003, a Novel Human Monoclonal Antibody Specific for Transferrin Receptor 1, to Evaluate Its Safety, Pharmacokinetics, andPharmacodynamics, Clinical pharmacology in drug development, 2023 Jun; 12(6), 579-587. ;

[0008] The present invention aims to solve the problem of defining an appropriate dosage regimen for an anti-TfR antibody that can suppress erythropoiesis promotion without causing symptoms of systemic anemia in patients with polycythemia.

[0009] As a result of investigations to solve the above problems, the present inventors determined the dosage regimen for polycythemia patients based on the dosage regimen for healthy subjects in the initial phase 1 clinical trial (P1a) of human safety study in Non-Patent Document 8, and actually administered an anti-TfR antibody to polycythemia patients to confirm safety, pharmacokinetics, and pharmacological effects. As a result, they discovered an optimal dosage regimen for an anti-TfR antibody that can specifically inhibit erythroid differentiation while having almost no effect on other normal organs, and have completed the present invention.

[0010] That is, the present invention provides the following: (1) A therapeutic agent for polycythemia comprising an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor, wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not administered for at least four weeks or more. (2) The therapeutic agent for polycythemia according to (1), wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not administered for at least six weeks or more, or at least nine weeks or more. (3) The therapeutic agent for polycythemia according to (1), wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not administered for at least 12 weeks or more. (4) The therapeutic agent for polycythemia according to (1), wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not administered for at least 24 weeks or more. (5) The therapeutic drug for polycythemia according to (1), wherein a first administration of the antibody at a dose of 0.25 mg / kg to 20 mg / kg is followed by a second administration of the antibody at a dose of 0.25 mg / kg to 20 mg / kg at least four weeks or more after the first administration. (6) The therapeutic drug for polycythemia according to any one of (1) to (5), wherein a hematocrit level of 50% or less is maintained for at least four weeks or more after the first administration of the antibody. (7) The therapeutic drug for polycythemia according to any one of (1) to (6), wherein phlebotomy therapy is used in combination with or without phlebotomy therapy. (8) The therapeutic drug for polycythemia according to any one of (1) to (7), wherein the polycythemia is polycythemia vera. (9) The therapeutic agent for polycythemia according to any one of (1) to (8), wherein the antibody has a heavy chain first complementarity determining region (VH CDR1), a heavy chain second complementarity determining region (VH CDR2), and a heavy chain third complementarity determining region (VH CDR3) represented by SEQ ID NOs: 1, 2, and 3, respectively, and a light chain first complementarity determining region (VL CDR1), a light chain second complementarity determining region (VL CDR2), and a light chain third complementarity determining region (VL CDR3) represented by SEQ ID NOs: 4, 5, and 6, respectively. (10) The therapeutic agent for polycythemia according to any one of (1) to (9), wherein the antibody has a heavy chain represented by SEQ ID NO: 7 and a light chain represented by SEQ ID NO: 8.(11) The therapeutic agent for polycythemia according to any one of (1) to (10), wherein the antibody is a human antibody or a humanized antibody. (12) A method for treating polycythemia, comprising administering to a subject an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor, wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not administered for at least four weeks or more. (13) The method for treatment according to (12), wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not administered for at least six weeks or more, or at least nine weeks or more. (14) The method for treatment according to (12), wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not administered for at least 12 weeks or more. (15) The method according to (12), wherein a first administration of the antibody is performed at a dose of 0.25 mg / kg to 20 mg / kg, and a second administration is not performed for at least 24 weeks or more after that. (16) The method according to (12), wherein a first administration of the antibody is performed at a dose of 0.25 mg / kg to 20 mg / kg, and a second administration of the antibody is performed at a dose of 0.25 mg / kg to 20 mg / kg at least 4 weeks or more after that. (17) The method according to any one of (12) to (16), wherein a hematocrit level of 50% or less is maintained for at least 4 weeks or more after the first administration of the antibody. (18) The method according to any one of (12) to (17), wherein phlebotomy treatment is used in combination with or without phlebotomy treatment. (19) The method of any one of (12) to (18), wherein the polycythemia is polycythemia vera. (20) The method of any one of (12) to (19), wherein the antibody is an antibody whose 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 represented by SEQ ID NOs: 1, 2, and 3, respectively, and whose 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 represented by SEQ ID NOs: 4, 5, and 6, respectively.(21) The method of treatment according to any one of (12) to (20), wherein the antibody is an antibody whose heavy chain has SEQ ID NO: 7 and whose light chain has SEQ ID NO: 8. (22) The method of treatment according to any one of (12) to (21), wherein the antibody is a human antibody or a humanized antibody. (23) An antibody for use in treating polycythemia, which recognizes amino acids 629 to 633 of the human transferrin receptor, wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not given for at least 4 weeks or more. (24) The antibody of (23), wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not given for at least 6 weeks or more, or at least 9 weeks or more. (25) The antibody according to (23), wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not for at least 12 weeks or more. (26) The antibody according to (23), wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not for at least 24 weeks or more. (27) The antibody according to (23), wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration of the antibody at a dose of 0.25 mg / kg to 20 mg / kg at least 4 weeks or more later. (28) The antibody according to any one of (23) to (27), wherein a hematocrit level of 50% or less is maintained for at least 4 weeks or more after the first administration of the antibody. (29) The antibody according to any one of (23) to (28), which is administered in combination with phlebotomy therapy or without phlebotomy therapy. (30) The antibody according to any one of (23) to (29), wherein the polycythemia is polycythemia vera. (31) The antibody according to any one of (23) to (30), wherein 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 represented by SEQ ID NOs: 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 represented by SEQ ID NOs: 4, 5, and 6, respectively.(32) The antibody according to any one of (23) to (31), wherein the heavy chain has SEQ ID NO: 7 and the light chain has SEQ ID NO: 8. (33) The antibody according to any one of (23) to (32), wherein the antibody is a human antibody or a humanized antibody. (34) Use of an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor for the manufacture of a therapeutic agent for polycythemia, wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not to be administered for at least 4 weeks or more. (35) The use according to (34), wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not to be administered for at least 6 weeks or more, or at least 9 weeks or more. (36) The use according to (34), wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not for at least 12 weeks or more. (37) The use according to (34), wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not for at least 24 weeks or more. (38) The use according to (34), wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration of the antibody at a dose of 0.25 mg / kg to 20 mg / kg at least 4 weeks or more later. (39) The use according to any one of (34) to (38), wherein a hematocrit level of 50% or less is maintained for at least 4 weeks or more after the first administration of the antibody. (40) The use according to any one of (34) to (39), with or without concomitant phlebotomy therapy. (41) The use according to any one of (34) to (40), wherein the polycythemia is polycythemia vera. (42) The use according to any one of (34) to (41), wherein the antibody is an antibody in which the heavy chain first complementarity determining region (VH CDR1), the heavy chain second complementarity determining region (VH CDR2), and the heavy chain third complementarity determining region (VH CDR3) are represented by SEQ ID NOs: 1, 2, and 3, respectively, and the light chain first complementarity determining region (VL CDR1), the light chain second complementarity determining region (VL CDR2), and the light chain third complementarity determining region (VL CDR3) are represented by SEQ ID NOs: 4, 5, and 6, respectively.(43) The use according to any one of (34) to (42), wherein the antibody is an antibody whose heavy chain has SEQ ID NO: 7 and whose light chain has SEQ ID NO: 8. (44) The use according to any one of (34) to (43), wherein the antibody is a human antibody or a humanized antibody.

[0011] The therapeutic agent for polycythemia of the present invention is useful in treating polycythemia.

[0012] Figure 1 shows the site of point mutation 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 a schematic diagram of the mechanism by which anti-TfR antibody inhibits erythrocyte proliferation / differentiation and reduces hematocrit and hemoglobin levels. Figure 4 shows a schematic diagram of the previously conducted early phase 1 clinical trial (P1a) and the mid-phase 1 clinical trial (P1b) of the present application for the TfR436 antibody. Figure 5 shows the dose escalation process for each polycythemia patient in the mid-phase 1 clinical trial (P1b) (n=6). EoS in the figure means end-of-study. Figure 6 shows the results of observations of the safety of the TfR436 antibody. Figure 7 shows the results of observations of the pharmacokinetic (PK) characteristics of the TfR436 antibody. (a) Comparison of PK between polycythemia patients (Cases 1-6) and healthy subjects, (b) PK trends of the average antibody concentrations administered in Cases 1-6, and (c) PK trends following administration of each antibody in Cases 1-6. Figure 8 shows the results of observations of the pharmacological action of the TfR436 antibody in Case 1. For comparison, the thin dashed line indicates the average value administered to HV at a dose of 0.25 mg / kg (n=6). Inj indicates antibody administration, and PLB indicates phlebotomy. Figure 9 shows the results of observations of the pharmacological action of the TfR436 antibody in Case 2. For comparison, the thin dashed line indicates the average value administered to HV at a dose of 0.25 mg / kg (n=6). Inj indicates antibody administration, and PLB indicates phlebotomy. The thick dashed line indicates the maximum or minimum value in HV. Figure 10 shows the observation results of the pharmacological action of the TfR436 antibody in Case 3. For comparison, the thin dashed line indicates the average value at 0.25 mg / kg (n=6) administration, which was used as a subject for HV. Inj indicates antibody administration, and PLB indicates phlebotomy. Figure 11 shows the changes in Hct after administration of the TfR436 antibody in Cases 1 to 6. (a) shows the absolute value over time, (b) shows the relative change when the value on the day before the first administration is set to 100%, and (c) shows the relative change only when 0.25 mg / kg is administered. Figure 12 shows the changes in Hb after administration of the TfR436 antibody in Cases 1 to 6. (a) shows the absolute value over time, and (b) shows the relative change when the value on the day before the first administration is set to 100%.Figure 13 shows changes in RBC counts after administration of the TfR436 antibody in cases 1 to 6. (a) shows the absolute value over time, and (b) shows the relative change when the value on the day before the first administration is set to 100%. Figure 14 shows changes in MCV after administration of the TfR436 antibody in cases 1 to 6. (a) shows the absolute value over time, and (b) shows the relative change when the value on the day before the first administration is set to 100%. Figure 15 shows changes in reticulocyte counts after administration of the TfR436 antibody in cases 1 to 6. (a) shows the absolute value over time, and (b) shows the relative change when the value on the day before the first administration is set to 100%. Note that the data outside the frame at the time of the third administration in case 2 was entered incorrectly.

[0013] The present invention will now be described in more detail. Definitions and General Techniques Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclatures used and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art.

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

[0015] TfR In humans, transferrin receptor (TfR) is a single-pass transmembrane protein consisting of 760 amino acids encoded by human chromosome 3 (SEQ ID NO: 9). This protein, also known as the CD71 antigen, is thought to be involved in cellular iron uptake and cell proliferation. The TfR of the present invention is not particularly limited in structure, and refers to all TfRs, including monomers, multimers, intact forms expressed in the cell membrane, soluble forms composed of the extracellular domain, truncated forms, mutation forms due to gene mutations or deletions, and forms post-translationally modified by phosphorylation, etc. The amino acid sequence of human TfR is shown below.Human TfR (SEQ ID NO: 9).

[0016] React and Reactivity In this specification, unless otherwise specified, "react" and "reactivity" have the same meaning. That is, an antibody recognizes an antigen. This antigen may be intact TfR expressed on the cell membrane, or a truncated or solubilized form. It may also be TfR that retains its three-dimensional structure, or denatured TfR. Methods for examining reactivity include flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), Western blotting, fluorescence microanalysis (FMAT), surface plasmon resonance (BIAcore), immunostaining, immunoprecipitation, etc.

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

[0018] Antibodies Where necessary, the following conventional abbreviations (in parentheses) are used herein: 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] As used herein, the term "antibody" is synonymous with immunoglobulin and should be understood as commonly known in the art. Specifically, the term antibody is not limited by any particular method of producing the antibody. 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] As used herein, the term "human antibody" refers to any antibody in which the variable and constant region sequences are human sequences. The term encompasses antibodies that have sequences derived from human genes but that have been altered to, for example, reduce potential immunogenicity, increase affinity, or remove cysteines that may cause undesired folding. The term also encompasses such antibodies that have been recombinantly produced in non-human cells, which can provide glycosylation not typical of human cells. These antibodies can be prepared in a variety of ways.

[0021] As used herein, the term "humanized antibody" refers to an antibody of non-human origin in which amino acid residues characteristic of the antibody sequence of the non-human species are replaced by residues found at the corresponding positions in human antibodies. This "humanization" process is believed to reduce the immunogenicity of the resulting antibody in humans. It will be understood that antibodies of non-human origin can be humanized using techniques well known in the art. See, for example, Winter et al., Immunol. Today 14:43-46 (1993). The antibody of interest can be engineered by recombinant DNA techniques to replace the CH1, CH2, CH3, hinge domain, and / or framework domain with the corresponding human sequence. See, for example, WO 92 / 02190, and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085. As used herein, the term "humanized antibody" includes within its meaning chimeric human antibodies and CDR-grafted 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 to the corresponding antigen. Although 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 one embodiment of the antibody, the antibody comprises a constant region in addition to the variable region (e.g., an IgG 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 may be any one that belongs to human immunoglobulin (hereinafter referred to as hIgG), but those of the hIgG class are preferred. Furthermore, any subclass of the hIgG class, such as hIgG1, hIgG2, hIgG3, or hIgG4, can be used. The light chain constant region (CL) may be any one that belongs to hIg, and those of the κ class or λ class can be used. Constant regions of animal species other than humans (e.g., mice and rats) can also be used.

[0024] As used herein, a "modified antibody" or "modified antibody" refers to an antibody in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of the variable region (CDR sequence and / or FR sequence) of a parent antibody. In the present invention, a "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. A "modified antibody" or "modified antibody" has one or several (e.g., 1 to 8, preferably 1 to 5, more preferably 1 to 3, particularly preferably 1 or 2) amino acids deleted, added, substituted, and / or inserted in the amino acid sequence. Methods for introducing mutations into proteins are 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.For example, those skilled in the art will be familiar with site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Kagawa, 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 (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456). Modified antibodies with activity equivalent to that of the parent antibody can be prepared by appropriately introducing mutations into the amino acid sequence of the parent antibody using techniques such as those described in, for example, "HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367" and "Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 82, 488-492." In this way, antibodies with one or several amino acid mutations in the variable or constant region of the antibody and with activity equivalent to that of the parent antibody, i.e., antibodies functionally equivalent to the parent antibody, can also be used.

[0025] As used herein, "having activity equivalent to that of a parent antibody" means that the activity of binding to TfR, the activity of reducing hematocrit, the activity of reducing hemoglobin levels, and / or the activity of inhibiting erythrocyte differentiation is equivalent. "Equivalent" does not necessarily mean that the activity is at the same level; the activity may be enhanced, or the activity may be reduced as long as the activity is still present. Examples of antibodies with reduced activity include antibodies that have 30% or more of the activity of the original antibody, preferably 50% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0026] The term "binding activity" refers to the recognition of an antigen. This antigen may be intact TfR expressed on the cell membrane, or a truncated or solubilized form. Furthermore, it may be 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 microanalysis (FMAT), and surface plasmon resonance (BIAcore).

[0027] The hematocrit value-reducing activity, hemoglobin content-reducing activity, and / or erythrocyte differentiation inhibitory activity of an antibody can be measured according to the method described below in "Example 2(4) Confirmation of Pharmacological Action." The hematocrit value (Hct) and hemoglobin content (Hb) of blood collected from a patient administered the antibody can be measured using known methods (e.g., conventional blood tests) to evaluate the hematocrit value-reducing activity and hemoglobin content-reducing activity. Furthermore, the red blood cell count (RBC) can be measured, and the mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), and mean corpuscular hemoglobin concentration (MCHC) can be calculated. The erythrocyte differentiation inhibitory effect can be evaluated by examining the decrease in hematocrit value and hemoglobin content, the increase in red blood cell count, and the decrease in MCV and MCHC observed in microcytic anemia.

[0028] The method for measuring Hct is not particularly limited as long as it can measure the proportion of red blood cell volume relative to a certain blood volume, but examples include the microhematocrit method, the Wintrobe method, the electrical resistance method, a method measuring specific gravity, and a method measuring the total red blood cell volume in the whole circulating blood using RI. The method for measuring Hb is not particularly limited as long as it can measure the amount of hemoglobin contained in a certain amount of blood, but examples include the cyanmethemoglobin method, the azidomethemoglobin method, the SLS-Hb method, and the alkaline hematin method. The method for measuring RBC is not particularly limited as long as it can measure the number of red blood cells contained in a certain amount of blood, but for example, it can be measured using an automated blood cell analyzer. Furthermore, by dividing Hct and Hb by the number of RBCs, the mean volume (MCV) and mean hemoglobin (MCH) per red blood cell can be calculated. Furthermore, by dividing Hb by Hct, the Hb concentration per red blood cell can be calculated. The calculation formula is shown below. MCV=Hct(%) / RBC(10 ６ / μL)×10 MCH=Hb(g / dL) / RBC(10 ６ / μL)×10 MCHC=Hb(g / dL) / Hct(%)×100

[0029] The antibody is not limited by its origin and may be derived from any animal, such as a human antibody, a mouse antibody, or a rat antibody. It may also be a chimeric antibody or a humanized antibody. One preferred embodiment of the antibody of the present invention is a human antibody.

[0030] Antibodies may differ in amino acid sequence, molecular weight, isoelectric point, or the presence or absence or morphology of sugar chains depending on the antibody-producing cells, host, or purification method described below. For example, the present invention also includes cases in which the amino acid sequences described in the present invention are modified after translation. Furthermore, post-translational modifications at sites other than known post-translational modifications are also included in the present invention. Furthermore, when antibodies are expressed in prokaryotic cells, such as Escherichia coli, a methionine residue is added to the N-terminus of the amino acid sequence of the original antibody. Such antibodies may be used in the present invention. Post-translational modifications at sites other than known post-translational modifications are also included in the present invention.

[0031] Antibody Production The antibodies used in the present invention may be either monoclonal or polyclonal. Monoclonal and polyclonal antibodies can be produced by methods known to those skilled in the art. Examples of antibodies include those produced in animal blood, those produced by hybridomas, those produced by hosts transformed by genetic engineering techniques with expression vectors containing antibody genes, those produced by screening optimal antibodies from clone libraries by phage display and then producing the genes in CHO cells, and those produced directly from transgenic mice that produce human antibodies.

[0032] To produce polyclonal antibodies, animals such as rabbits are immunized with an antigen to obtain serum, which can then be purified using, for example, ammonium sulfate precipitation, a protein A column, a protein G column, DEAE ion exchange chromatography, or an affinity column to prepare polyclonal antibodies.

[0033] To produce monoclonal antibodies, an animal is immunized with an antigen, optionally with an adjuvant. After confirming that the desired antibody level is elevated in the serum of the immunized animal, immune cells (such as spleen cells) are collected from the animal and fused with mammalian myeloma cells. Hybridomas obtained by cell fusion can be selected by culturing them in a conventional selective medium, such as HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Hybridomas producing the desired antibody can then be screened by limiting dilution.

[0034] The production of antibodies using phage display is described below. (1) Antigen-reactive scFv from a phage display library. Using phage display technology, libraries containing a repertoire of antibodies with varying affinities for TfR can be provided. These libraries can then be screened to identify and isolate antibodies against TfR. Preferably, the phage library is an scFv phage display library generated using human VL and VH cDNAs prepared from mRNA isolated from human B cells. Methods for preparing and screening such libraries are known in the art. Genetic material is recovered from phage clones that show reactivity to human TfR as an antigen. Genetic analysis of the selected phage allows the determination of the VH and VL DNA sequences encoding the variable regions of the human antibody that binds to the antigen. Using this scFv sequence, the scFv can be converted into IgG, thereby obtaining a human antibody.

[0035] (2) Conversion of scFv to IgG (Preparation of Human Antibodies) Expression vectors for the H chain or L chain are prepared, expressed in host cells, and the secreted supernatant is collected and purified to obtain human antibodies. 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, WO93 / 19172, WO95 / 01438, WO95 / 15388, WO97 / 10354, etc.

[0036] Specifically, a full-length heavy chain gene can be obtained by linking a VH-encoding DNA to other DNA molecules encoding 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 ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG1 or IgG2 constant region. The IgG1 constant region sequence may be any of the various alleles or allotypes known to occur among different individuals, such as Gm(1), Gm(2), Gm(3), and Gm(17). These allotypes correspond to naturally occurring amino acid substitutions in the IgG1 constant region.

[0037] Full-length L chain genes (as well as Fab light chain genes) can be obtained by linking the VL-encoding DNA to 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 encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. The kappa constant region can be any of the various alleles known to occur between different individuals, such as Inv(1), Inv(2), or Inv(3). The lambda constant region can be derived from any of the three lambda genes.

[0038] The DNA encoding the heavy or light chain obtained as described above is inserted into an expression vector to produce an expression vector, which is then expressed in host cells. The secreted supernatant is then collected and purified to obtain a human antibody. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, and EBV-derived episomes. The expression vector and expression regulatory sequences 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 using standard methods (e.g., ligation of the vector with complementary restriction sites on the antibody gene fragment, or blunt-end ligation if no restriction sites are present).

[0039] A convenient vector encodes a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction sites engineered to allow for 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 at splice regions present within the human CH exon. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding region. The recombinant expression vector can also encode a signal peptide that facilitates secretion of the antibody chain from host cells. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0040] In addition to the antibody genes and control sequences, antibody expression vectors may contain additional sequences, such as sequences that control replication of the vector in host cells (e.g., origins of replication) and a selectable marker gene. The selectable marker gene facilitates selection of host cells into which the vector has been introduced. For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on host cells into which the vector has been introduced. Preferred selectable marker genes include the dehydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification), the neomycin phosphotransferase gene (for G418 selection), and the glutamate synthetase gene.

[0041] Host cells are transformed with the antibody gene expression vector prepared by the above method. Host cells may be any cell capable of producing antibodies, 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, 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)). For transformation, the lipofectin method (R.W.M. Solone et al., Proc. Natl. Acad. Sci. USA 86, 6007 (1989), P.L. Felgner et al., Proc. Natl. Acad. Sci. USA 84, 7413 (1987)), electroporation, the calcium phosphate method (F.L. Graham & A.J. van der Eb, Virology 52, 456-467 (1973)), the DEAE-Dextran method, etc. are preferably used.

[0042] After culturing the transformant, the human antibody is isolated from the cells of the transformant or from the culture medium. Isolation and purification of the antibody can be performed by appropriately combining methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion exchange chromatography, and gel filtration chromatography.

[0043] Antibody fragments Antibody fragments can be produced based on antibodies or on the sequence information of the gene encoding the antibody. Antibody fragments include Fab, Fab', F(ab') ２ , scFv, and dsFv antibodies.

[0044] Fab is a fragment with a molecular weight of approximately 50,000, which is obtained by papain digestion of IgG in the presence of cysteine ​​and is composed of an L chain, an H chain variable region, and an H chain fragment consisting of a CH1 domain and part of the hinge region. In the present invention, the above antibody can be obtained by papain digestion. Alternatively, Fab can be prepared from a transformant transformed with an appropriate vector by incorporating DNA encoding a part of the H chain and L chain of the above antibody into the vector.

[0045] Fab' is F(ab') ２ Fab' is a fragment with a molecular weight of approximately 50,000 that can be obtained by cleaving the disulfide bond between the H chains of the antibody. In the present invention, it can be obtained by digesting the above antibody with pepsin and cleaving the disulfide bond using a reducing agent. Furthermore, like Fab, Fab' can also be prepared by genetic engineering using DNA encoding it.

[0046] F(ab') ２ is a fragment with a molecular weight of approximately 100,000, obtained by digesting IgG with pepsin, in which fragments (Fab') consisting of an L chain, an H chain variable region, and an H chain fragment consisting of a CH1 domain and a part of the hinge region are linked by disulfide bonds. In the present invention, this is obtained by digesting the above antibody with pepsin. Similarly to Fab, F(ab') ２ It can also be prepared by genetic engineering using DNA encoding the above.

[0047] An scFv is an antibody fragment in which an Fv consisting of an H-chain variable region and an L-chain variable region is linked to the C-terminus of one chain and the N-terminus of the other chain via an appropriate peptide linker to form a single chain. Examples of peptide linkers include the 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-mentioned antibody and DNA encoding a peptide linker, and then inserted into an appropriate vector, followed by transformation with the vector to prepare an scFv from the transformant.

[0048] A dsFv is an Fv fragment in which Cys residues have been introduced at appropriate positions in the H-chain variable region and L-chain variable region, stabilizing the H-chain variable region and the L-chain variable region through disulfide bonds. The positions of the Cys residues to be introduced in each chain can be determined based on the three-dimensional structure predicted by molecular modeling. In the present invention, for example, the three-dimensional structure is predicted from the amino acid sequences of the H-chain variable region and L-chain variable region of the above-mentioned antibody, and DNA encoding the H-chain variable region and L-chain variable region into which mutations have been introduced based on such prediction is constructed. This DNA is then inserted into an appropriate vector, and a dsFv can be prepared from a transformant transformed with the vector.

[0049] Furthermore, antibody fragments can also be multimerized by linking scFv antibodies, dcFv antibodies, etc. using an appropriate linker, or by fusing streptavidin.

[0050] Multispecific antibodies As long as one antibody recognizes TfR, it may also be a multispecific antibody that simultaneously recognizes other targets. Bispecific antibodies are particularly commonly used. A bispecific antibody is an antibody that combines a molecule that recognizes TfR on one side and a molecule that targets another molecule on the other side. Examples of such antibodies include bispecific diabodies (mab)2 obtained by chemically crosslinking two molecules of monoclonal antibodies, bispecific F(ab')2 obtained by chemically crosslinking two molecules of Fab fragments, quadroma, bsDb (bispecific diabody), scBsDb (single-chain bispecific diabody), scBsTaFv (single-chain bispecific tandem variable domain), Bite (bispecific T cell engager antibody), and DNL-F(ab)3 (docl-and-lock trivalent Fab) (Shim, H. Bispecific Antibodies and Antibody-Drug Conjugates for Cancer Therapy: Technical Considerations. Biomolecules 2020, 10, 360), but the form is not limited thereto.

[0051] Pharmaceutical Compositions and Formulations The therapeutic agent for polycythemia of the present invention comprises an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor. In one embodiment, the antibody that recognizes amino acids 629 to 633 of the human transferrin receptor is preferably 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 represented by SEQ ID NOs: 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 represented by SEQ ID NOs: 4, 5, and 6, respectively. In one embodiment, the antibody that recognizes amino acids 629 to 633 of the human transferrin receptor is preferably an antibody in which the heavy chain has SEQ ID NO: 7 and the light chain has SEQ ID NO: 8. In one embodiment, the antibody that recognizes amino acids 629 to 633 of the human transferrin receptor is a human antibody or a humanized antibody.

[0052] Pharmaceutical compositions and preparations containing the therapeutic agent for polycythemia of the present invention are also included within the scope of the present invention. The therapeutic agent for polycythemia of the present invention can be used for the treatment of polycythemia.

[0053] Polycythemia is a condition in which the blood volume is increased, either absolute or relative. Because the majority of blood cells are red blood cells, polycythemia is essentially the same concept as erythrocytosis. Polycythemia vera includes polycythemia vera, relative polycythemia, and secondary polycythemia. Polycythemia vera is a myeloproliferative neoplasm in which the proliferation of hematopoietic stem cells, caused by an acquired genetic abnormality, results in an absolute increase in the number of red blood cells in the blood and circulating blood volume. In polycythemia vera, white blood cells and platelets are often increased, resulting in an increase in total blood cell count. Relative polycythemia is a condition in which the total number of red blood cells per unit volume of blood is increased relative to the total number of red blood cells due to a decrease in plasma, the liquid component that normally accounts for more than half of blood's total volume. Secondary polycythemia, also known as secondary erythrocytosis, is a condition in which an increase in the amount of red blood cells occurs due to an increase in the amount of erythropoietin, a hematopoietic factor, caused by some cause, resulting in a reaction of erythropoiesis. Polycythemia vera is preferred as the polycythemia of the present invention. In one aspect, the present invention provides a therapeutic agent for polycythemia, wherein the polycythemia is polycythemia vera.

[0054] The therapeutic agent for polycythemia of the present invention may or may not be used in combination with other therapeutic methods for polycythemia. Other therapeutic methods for polycythemia include phlebotomy, chemotherapy (e.g., anticancer drugs), and antiplatelet agents. Phlebotomy is a treatment in which blood is drawn and discarded. Examples of chemotherapeutic agents include hydroxycarbamide (hydroxyurea, trade name: Hydrea), ranimustine (trade name: Cymerin), busulfan (trade name: Mablin Powder), and ruxolitinib (trade name: Jakavi). Examples of antiplatelet agents include low-dose aspirin. Preferably, the therapeutic agent for polycythemia of the present invention can be used in combination with hydroxyurea.

[0055] Pharmaceutical compositions and formulations containing the therapeutic agents for polycythemia of the present invention preferably contain, in addition to the antibody, a physiologically acceptable diluent or carrier, and 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 lyophilized (freeze-dried) and reconstituted as needed by adding an aqueous buffer solution such as those described above. Administration forms include oral administration in the form of tablets, capsules, granules, powders, syrups, etc., and parenteral administration in the form of injections (subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), transdermal, transmucosal, nasal, pulmonary, suppositories, etc. The pharmaceutical compositions of the present invention may be administered alone or in combination with other drugs.

[0056] Dosage and Administration Method In the present invention, the therapeutic agent for polycythemia comprises administering a first dose of an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor to a subject at a dose of 0.25 mg / kg to 50 mg / kg. The first dose is preferably 0.25 mg / kg to 40 mg / kg, 0.25 mg / kg to 30 mg / kg, 0.25 mg / kg to 20 mg / kg, 0.25 mg / kg to 15 mg / kg, 0.25 mg / kg to 10 mg / kg, or 0.25 mg / kg to 5 mg / kg, more preferably 0.25 mg / kg to 2 mg / kg, and most preferably 0.25 mg / kg to 1 mg / kg. For example, the first dose may be 0.25 mg / kg, 0.4 mg / kg, 0.64 mg / kg, or 1.0 mg / kg. In one embodiment, the first dose may be 0.25 mg / kg.

[0057] The antibody can be administered in conjunction with phlebotomy, and in this case, can be administered 1 to 10 days before or after phlebotomy in a subject, preferably 1 to 5 days before or after phlebotomy, more preferably 1 to 3 days before or after phlebotomy, even more preferably 1 to 3 days after phlebotomy, and most preferably 1 day after phlebotomy.

[0058] In the present invention, after a first administration of an antibody to a subject, a second administration is not performed for at least 4 weeks or more, preferably at least 6 weeks or more, more preferably at least 9 weeks or more, even more preferably at least 12 weeks or more, even more preferably at least 24 weeks or more, or most preferably at least 52 weeks or more. In one embodiment, the therapeutic agent for polycythemia of the present invention, after a single administration, does not require a second administration to the subject and / or does not require phlebotomy treatment for at least 4 weeks or more, 6 weeks or more, 9 weeks or more, 12 weeks or more, 24 weeks or more, or 52 weeks or more after the administration.

[0059] In the present invention, after the first administration of the antibody to a subject, the hematocrit level is maintained at 50% or less for at least 4 weeks or more, preferably at least 6 weeks or more, more preferably at least 9 weeks or more, even more preferably at least 12 weeks or more, even more preferably at least 24 weeks or more, and most preferably at least 52 weeks or more. In one embodiment, a single administration of the therapeutic agent for polycythemia of the present invention maintains the hematocrit level at 50% or less for at least 4 weeks or more, 6 weeks or more, 9 weeks or more, 12 weeks or more, 24 weeks or more, or 52 weeks or more after the administration.

[0060] In the present invention, the second administration of the antibody can be performed at least 4 weeks or more, preferably at least 6 weeks or more, more preferably at least 9 weeks or more, even more preferably at least 12 weeks or more, even more preferably at least 24 weeks or more, or most preferably at least 52 weeks or more after the first administration of the antibody.

[0061] The second or subsequent administration can be administered to a subject at a dose of 0.008 mg / kg to 50 mg / kg. The second or subsequent administration dose is preferably 0.05 mg / kg to 40 mg / kg, 0.05 mg / kg to 30 mg / kg, 0.05 mg / kg to 20 mg / kg, 0.05 mg / kg to 10 mg / kg, or 0.05 mg / kg to 5 mg / kg, more preferably 0.1 mg / kg to 2.5 mg / kg, and most preferably 0.25 mg / kg to 1 mg / kg. For example, the second or subsequent administration dose can be 0.25 mg / kg, 0.4 mg / kg, 0.64 mg / kg, or 1.0 mg / kg. The second or subsequent administration dose of the antibody can be the same as the first administration, or can be increased or decreased, taking into consideration the symptoms, disease progression, and severity in the subject after the first administration of the antibody. As an example, the first dose may be 0.25 mg / kg, the second dose may be 0.4 mg / kg, the third dose may be 0.64 mg / kg, and the fourth dose may be 1.0 mg / kg.

[0062] The second or subsequent administration of the antibody can be administered in conjunction with phlebotomy, in which case it can be administered 1 to 10 days before or after the phlebotomy in the subject, preferably 1 to 5 days before or after the phlebotomy, more preferably 1 to 3 days before or after the phlebotomy, even more preferably 1 to 3 days after the phlebotomy, and most preferably 1 day after the phlebotomy.

[0063] In one embodiment, the therapeutic agent for polycythemia is administered to a subject at a dose of 0.25 mg / kg to 20 mg / kg, using a method in which a first administration of an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor is administered, followed by a second administration not given for at least four weeks or more. In one embodiment, the therapeutic agent for polycythemia is administered to a subject at a dose of 0.25 mg / kg to 20 mg / kg, using a method in which a first administration of an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor is administered, followed by a second administration not given for at least nine weeks or more. In one embodiment, the therapeutic agent for polycythemia is administered to a subject at a dose of 0.25 mg / kg to 20 mg / kg, using a method in which a second administration of an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor is administered, followed by a second administration not given for at least 12 weeks or more. In one aspect, the therapeutic agent for polycythemia is administered to a subject by a first administration of an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not for at least 24 weeks.

[0064] In one aspect, the therapeutic agent for polycythemia is administered to a subject by a first administration of an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration of the antibody at a dose of 0.25 mg / kg to 20 mg / kg at least four weeks or more after the first administration of the antibody that recognizes amino acids 629 to 633 of the human transferrin receptor. In one aspect, the therapeutic agent for polycythemia maintains a hematocrit level of 50% or less for at least four weeks or more after the first administration of the antibody that recognizes amino acids 629 to 633 of the human transferrin receptor at a dose of 0.25 mg / kg to 20 mg / kg.

[0065] In one embodiment, the therapeutic agent for polycythemia of the present invention is administered in the above-mentioned administration method in combination with or without phlebotomy therapy.

[0066] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0067] In the following examples, the TfR436 antibody described in paragraphs 0090 and 0091 of International Publication WO2014 / 073641 was used. The TfR436 antibody is also sometimes referred to as PPMX-T003, which can be used interchangeably to refer to the same anti-TfR antibody.

[0068] The CDR sequences of the TfR436 antibody are as follows: VH CDR1: SYGMH (SEQ ID NO: 1) VH CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 2) VH CDR3: DSNFWSGYYSPVDV (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)

[0069] The VH and VL sequences of the TfR436 antibody are shown below: TfR436 VH (SEQ ID NO: 7) DVQLVQSGGGVVQPGRSLRLSCAASGFPFKSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRGEDTAVYYCARDSNFWSGYYSPVDVWGQGTTVTVSS

[0070] TfR436 VL (SEQ ID NO: 8) NFMLTQPHSVSESPGKTVTISCTRSSGSIASNSVQWYQQRPGSAPITVIYEDTQRPSGVPDRFSGSIDSSSNSASLTISGLQTEDEADYYCQSYDSAYHWVFGGGTKLAVL

[0071] Example 1: Identification of the binding site of the TfR436 antibody The TfR436 antibody did not cross-react with mouse TfR, but did cross-react with hamster TfR. The amino acid sequence of the Transferrin (TF) binding site (amino acids 569-760) in TfR was aligned. Amino acids identical to those in the hamster TfR sequence but different from those in the mouse TfR sequence were selected. The selected amino acids were subjected to point mutation as shown in Figure 1 to generate soluble TfR mutant fragments.

[0072] (1) Preparation of soluble wild-type TfR (sTfR) and TfR mutant fragments (MF1 to MF7) The base sequences encoding the human TfR extracellular domain (amino acids 89 to 760), or the TfR mutant fragments (MF1 to MF7) shown in Figure 1 and AAARGGPEQKLISEEDLNSAVDHHHHHH (SEQ ID NO: 10), respectively, were totally synthesized. The neomycin resistance gene and DHFR gene were inserted into the expression vector pCAGGS (Non-Patent Document 2: Niwa et al. 1991), and the synthesized genes were inserted into the multicloning site of the vector to prepare the pCAGGS-Neo-DHFR-sTFR-myc-his expression plasmid. The plasmid was transfected into Expi293 cells (Invitrogen) using Expifectamine (Invitrogen), and the cells were cultured at 37°C, 8% CO2, and 135 rpm for 5 days. The culture supernatant was then collected by centrifugation, and sTfR or MF1-MF7 was purified using an AKTA prime (GE Healthcare) connected to a HisTrap HPP (GE Healthcare) column, with 20 mM imidazole / DPBS as the binding buffer and 500 mM imidazole / DPBS as the elution buffer. The eluted proteins were buffer-exchanged to 30 mM HEPES, 5% trehalose, pH 7.2, using a Zeba spin column (Thermo Scientific).

[0073] (2) Identification of the binding site of the TfR436 antibody. The purified sTfR or MF1 to MF7 was diluted with PBST (Phosphate Buffered Saline with Tween 20, TaKaRa) to prepare seven 3-fold dilutions starting from 600 ng / mL. The diluted solution was then dispensed at 100 μL / well into a Ni-NTA HisSorb Strips 96-well plate (QIAGEN), placed on a shaker, and incubated at room temperature. After 1 hour, the plate was washed five times with PBST buffer, and TfR436 antibody (1 μg / mL) was dispensed at 100 μL / well. The plate was then placed on a shaker and incubated at room temperature for 1 hour. Thereafter, the plate was washed five times with PBS-T buffer, and 100 μL / well of a 50,000-fold diluted secondary antibody, F(ab′)2 Fragment Anti-Human IgG Fcγ (Jackson ImmunoResearch), was dispensed 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 plate was shaken on a shaker for 1 minute. The absorbance at 450 nm (ref. 620 nm) was measured using a plate reader.

[0074] As shown in Figure 2, the results showed that the TfR436 antibody showed reduced reactivity with the TfR mutant fragment MF5, but no reduced reactivity with other mutant fragments. In other words, when the amino acids at positions 629, 630, and 633 of TfR were substituted with other amino acids, the TfR436 antibody could no longer recognize TfR. This suggests that amino acids 629 to 633 are the epitope recognized by the TfR436 antibody.

[0075] Example 2: Confirmation of safety, pharmacokinetic properties, and pharmacological effects of TfR436 antibody in patients with polycythemia (1) Clinical trial design A clinical trial was designed as follows to determine the safety and pharmacokinetic profile of TfR436 antibody (PPMX-T003) in patients with polycythemia, and also to explore its preliminary pharmacodynamic effects. (1-1) Study subjects and inclusion criteria Study subjects were patients with polycythemia who had been clinically diagnosed with polycythemia and were being treated with regular phlebotomy alone for 4 to 9 weeks. During the clinical trial, it was difficult to recruit patients diagnosed with PV according to the WHO 2008 / 2016 criteria (hemoglobin >16.5 g / dL for men and >16.0 g / dL for women, and hematocrit >49% for men and >48% for women). Therefore, the criteria were modified to include JAK2 mutation-negative patients undergoing clinical phlebotomy or patients with elevated EPO levels. However, patients with secondary polycythemia were excluded. Furthermore, patients receiving cytoreductive drugs such as hydroxyurea or ruxolitinib, except for supplemental aspirin, were excluded. The mid-phase Phase 1 clinical trial (P1b) of this application was an open-label, dose-escalation study without a cohort structure, and its schematic diagram is shown in Figure 4. The clinical trial was conducted on six Japanese patients with polycythemia aged 20 to 75 years. The subjects in cases 1 to 4 and 6 were JAK2 V617F(-), and the subject in case 5 was JAK2 V617F(+). For exon 12, at least in patients in cases 2 and 4 to 6, JAK2 ex12(-) was found.

[0076] (1-2) Dosage Regimen and Dose Because disease progression varies from patient to patient, an intra-individual dose-escalation design was used, as shown in Figure 5. The initial dose was set at 0.25 mg / kg, as lower-than-normal hemoglobin levels were observed in a previous Phase 1 clinical trial (P1a) conducted in healthy volunteers (Healthy Volunteers; HV, n = 40) (Figure 4, left panel). The Phase 1 clinical trial was conducted in five cohorts (each group consisting of eight volunteers, including two placebo subjects) at doses of 0.008 mg / kg, 0.04 mg / kg, 0.08 mg / kg, 0.16 mg / kg, and 0.25 mg / kg, using a double-blind method (each group consisted of eight volunteers, including two placebo subjects). One of the six patients in the cohort receiving the highest dose developed Grade 1 anemia. A single dose of TfR436 antibody was administered intravenously over one hour one day after a scheduled phlebotomy. If a subsequent phlebotomy (PLB) was scheduled, the dose was increased depending on the patient's adverse event (AE) profile during the study. However, in Case 2, after the second antibody administration, the patient was not permitted to undergo treatment for three PLBs due to patient reasons, so the administration after PLB was postponed three times. The third antibody administration was then administered after the sixth PLB. The primary endpoint was the incidence and severity of treatment-related adverse events, and the key secondary endpoint was meeting the criteria for 12 weeks or more. If a patient became phlebotomy-free for 12 weeks or more, the end of observation (EoS) was reached and no further observation was required.

[0077] (2) Safety observations: In safety observations, lymphocyte counts, CRP, fever, and subjective symptoms were monitored. Lymphocyte counts and CRP were measured by subjecting blood collected from patients to standard blood tests.

[0078] As shown in Figure 5, which shows the dose escalation progress for each patient in Cases 1 to 6, all six patients in Cases 1 to 6 completed the final administration, and five of them (Cases 1 to 4 and 6) reached EoS without requiring PLB for 12 weeks or more. Three patients (Cases 1, 3, and 6) became PLB-free immediately after the initial single administration (0.25 mg / kg). Two patients (Cases 2 and 4) required regular PLB until they reached the doses of 0.64 mg / kg and 1.0 mg / kg, respectively. The remaining patient (Case 5) requested an alternative treatment at Week 7 (Day 49) after administration of 1.0 mg / kg. AEs after the specified observation period are being followed up, and the final results after LPO will be reported.

[0079] Figure 6 summarizes the safety observation results and shows the changes in lymphocytes and CRP after administration of the TfR436 antibody. To date, no serious adverse events (SAEs) have been observed in the six patients who received the drug. Sixty-four AEs were observed, regardless of whether they were causally related to the administered TfR436 antibody, of which 47 were causally related to the TfR436 antibody. Of the AEs that were causally related, seven were observed in two or more patients. These AEs were: (i) elevated CRP, (ii) lymphopenia, (iii) fatigue, (iv) low-grade fever, (v) neutrophilia, (vi) infusion-related reactions (IRR), and (vii) transient serum iron. Six adverse events thought to be related to IRR resolved within one week. In one patient (Case 2), whose dose was increased to 0.64 mg / kg before the endpoint was reached, the patient complained of fever and fatigue within 24 hours of administration, accompanied by mild AEs of marked fatigue and mild fever. The AEs of fever reaching 37.6°C within 24 hours of administration, a CRP increase of approximately 5 mg / dL on the third day after administration, and lymphopenia on the second day after administration (which resolved seven days later) are typical IRRs observed with other antibody therapeutics. Furthermore, dose-dependent transient lymphopenia on the second day after administration and a dose-dependent transient CRP increase within one week after administration following administration of the TfR436 antibody were similarly observed in previous studies of HVs. Regarding fever, transient fevers up to 38.5°C were observed in three of the six HVs. AEs in this clinical trial were mild in severity, similar to those observed in previous HV studies.

[0080] (3) Evaluation of Pharmacokinetic Properties Next, blood antibody levels were measured at intervals after administration of the TfR436 antibody to examine the half-life of TfR. Blood antibody levels were measured using ELISA. These were compared with the changes in blood antibody levels (mean values ​​of n = 6 for each cohort) observed in previous studies targeting HV, in which TfR was administered at doses of 0.04 mg / kg to 0.25 mg / kg.

[0081] As shown in Figure 7, the pharmacokinetics of TfR436 antibody in patients with polycythemia were dose-dependent and similar between patients with polycythemia and HV. The half-life of the TfR436 antibody was 15 hours. Because the half-life of general antibodies is longer than two weeks, the TfR436 antibody exhibited pharmacokinetics different from that of conventional antibody therapeutics. The target molecule of the TfR436 antibody is highly expressed in the bone marrow. It is likely that the administered TfR436 antibody was rapidly consumed by its target, erythrocytes and BFU-C / CFU-C progenitor cells in the bone marrow, which highly express TfR. Furthermore, when comparing the half-life of the TfR436 antibody at 1.0 mg / kg in Case 4 (a JAK2 mutation-negative patient) and Case 5 (a JAK2 mutation-positive patient), the half-life was 25 hours in Case 4 and 5 hours in Case 5, indicating that the TfR436 antibody was consumed more rapidly in the JAK2 mutation-positive patient in Case 5. This is thought to indicate that in JAK2 mutation-positive patients, the expression of the target TfR is increased in the erythroblast lineage, resulting in rapid absorption and degradation of the TfR436 antibody.

[0082] (4) Confirmation of pharmacological action After administration of TfR436, hematocrit value (Hct), hemoglobin content (Hb), red blood cell count (RBC), and reticulocyte count were measured by a routine blood test, and mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated from these measurements.

[0083] As shown in Figure 8, in Case 1, RBC, Hct, and Hb decreased after administration of the TfR436 antibody, then gradually returned to normal. MCV, MCH, and MCHC continued to decrease gradually. The clinical trial was completed 84 days after administration of 0.25 mg / kg of the TfR436 antibody, without the need for PLB for more than 16 weeks. Currently, 52 weeks have passed since administration of the TfR436 antibody, without PBL. For comparison, Figures 8 to 10 also show the results of administration of 0.25 mg / kg of TfR against HV (mean values ​​of n = 6). As shown in Figure 10, in Case 3, RBC, Hct, and Hb temporarily decreased after administration of the TfR436 antibody, then gradually returned to normal. There were no significant differences in MCV, MCH, and MCHC. The clinical trial was terminated 84 days after administration of 0.25 mg / kg of TfR436 antibody, without the need for PLB for at least 12 weeks. As shown in Figure 9, in Case 2, the increase in Hct was suppressed for a longer period with increasing doses of TfR436 antibody. Hb decreased. The decrease in MCV and increase in RBC suggested microcytic hematopoiesis and the resulting increase in RBC. The decrease in MCV, MCH, and MCHC continued, and the low MCV and MCHC suggested iron deficiency anemia. The clinical trial was terminated 84 days after administration of 0.64 mg / kg of TfR436 antibody, without PLB, and the endpoint was reached 84 days after administration.

[0084] Furthermore, Figures 11 to 15 show the changes in Hct, Hb, RBC, MCV, and reticulocyte counts after administration of the TfR436 antibody in Cases 1 to 6. A decrease in Hct, which indicates the percentage of red blood cells in the blood, was confirmed with administration of the TfR436 antibody. A decrease in MCV, an index of the average size of each red blood cell, was also confirmed. Therefore, it was confirmed that red blood cells in the blood were smaller, and that the percentage of red blood cells in the blood was also reduced. Meanwhile, the increase in RBC counts is thought to be due to the decrease in Hb caused by the action of the TfR436 antibody, which is thought to be due to the inhibition of iron uptake during the red blood cell production process. This suggests that the resulting reduction in the size of each red blood cell is a state of microcytic hematopoiesis. Furthermore, the reticulocyte count decreased on the second day after administration of the TfR436 antibody, rebounded on the seventh day, and stabilized three weeks later (on the 21st day). This is thought to be a rebound response to transient iron depletion of the erythroid lineage, representing a three-week fluctuation in the number of days required for erythroid lineage differentiation. Combined with MCV, this is presumed to be a reaction to increased microcytic hematopoiesis and iron deficiency anemia. Initially, a single administration of the TfR436 antibody was predicted to suppress differentiation to MEP-BFU-CFU-Erytyroblast for three weeks. However, in this clinical trial, a longer-than-expected suppression trend of approximately three months was observed. This suggests that the TfR436 antibody exerts a long-term inhibitory effect on cell populations undergoing clonally proliferating growth.

[0085] As shown in Figures 8-15, administration of TfR436 antibody reduced hematocrit and hemoglobin levels, and even increased red blood cell counts. The observed effects, including hematocrit, hemoglobin, and other parameters, were consistent with those of microcytic anemia. These findings suggest that TfR436 antibody is effective in erythroid differentiation in the bone marrow. TfR436 antibody may specifically inhibit erythroid differentiation by controlling iron influx into target erythroid cells with high TfR expression, while leaving other normal organs with low TfR expression levels unaffected. Therefore, TfR436 antibody is expected to be an alternative treatment to PLB for patients with polycythemia. These results also suggest that TfR436 antibody may be useful for treating various cancer patients while managing anemia.

Claims

1. A therapeutic drug for polycythemia comprising an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor, the therapeutic drug being administered a first time at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration for at least four weeks.

2. The method for treating polycythemia according to claim 1, wherein the antibody is administered at a first dose of 0.25 mg / kg to 20 mg / kg, and then not administered at least six weeks or at least nine weeks after the first dose.

3. The method for treating polycythemia according to claim 1, wherein the antibody is administered at a first dose of 0.25 mg / kg to 20 mg / kg, and then not administered at a second dose for at least 12 weeks.

4. The method of claim 1, wherein the antibody is administered at a first dose of 0.25 mg / kg to 20 mg / kg, and then not administered at a second dose for at least 24 weeks.

5. The method for treating polycythemia according to claim 1, wherein the antibody is administered a first time at a dose of 0.25 mg / kg to 20 mg / kg, followed at least four weeks later by a second time administration of the antibody at a dose of 0.25 mg / kg to 20 mg / kg.

6. The method for treating polycythemia according to any one of claims 1 to 5, wherein the hematocrit value is maintained at 50% or less for at least four weeks or more after the first administration of the antibody.

7. A therapeutic agent for polycythemia according to any one of claims 1 to 6, which is used in combination with phlebotomy therapy or without phlebotomy therapy.

8. A therapeutic agent for polycythemia according to any one of claims 1 to 7, wherein the polycythemia is polycythemia vera.

9. A therapeutic agent for polycythemia according to any one of claims 1 to 8, wherein the antibody has a heavy chain first complementarity determining region (VH CDR1), a heavy chain second complementarity determining region (VH CDR2), and a heavy chain third complementarity determining region (VH CDR3) represented by SEQ ID NOs: 1, 2, and 3, respectively, and a light chain first complementarity determining region (VL CDR1), a light chain second complementarity determining region (VL CDR2), and a light chain third complementarity determining region (VL CDR3) represented by SEQ ID NOs: 4, 5, and 6, respectively.

10. The method of any one of claims 1 to 9, wherein the antibody has a heavy chain having SEQ ID NO: 7 and a light chain having SEQ ID NO:

8.

11. The method of any one of claims 1 to 10, wherein the antibody is a human antibody or a humanized antibody.

12. A method for treating polycythemia comprising administering to a subject an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor, wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, and then a second administration is not administered for at least four weeks.

13. The method of claim 12, wherein a first administration of the antibody at a dose of 0.25 mg / kg to 20 mg / kg is not followed by a second administration for at least 6 weeks or at least 9 weeks.

14. The method of claim 12, wherein a first dose of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second dose not administered for at least 12 weeks.

15. The method of claim 12, wherein a first administration of the antibody at a dose of 0.25 mg / kg to 20 mg / kg is not followed by a second administration for at least 24 weeks or more.

16. The method of treatment according to claim 12, comprising administering a first dose of the antibody at a dose of 0.25 mg / kg to 20 mg / kg, followed at least four weeks later by administering a second dose of the antibody at a dose of 0.25 mg / kg to 20 mg / kg.

17. A method of treatment according to any one of claims 12 to 16, in which the hematocrit level is maintained at 50% or less for at least four weeks or more after the first administration of the antibody.

18. A method of treatment according to any one of claims 12 to 17, with or without phlebotomy therapy.

19. A method of treatment according to any one of claims 12 to 18, wherein the polycythemia is polycythemia vera.

20. A method for treatment according to any one of claims 12 to 19, wherein the antibody has a heavy chain first complementarity determining region (VH CDR1), a heavy chain second complementarity determining region (VH CDR2), and a heavy chain third complementarity determining region (VH CDR3) that are represented by SEQ ID NOs: 1, 2, and 3, respectively, and a light chain first complementarity determining region (VL CDR1), a light chain second complementarity determining region (VL CDR2), and a light chain third complementarity determining region (VL CDR3) that are represented by SEQ ID NOs: 4, 5, and 6, respectively.

21. A method of treatment according to any one of claims 12 to 20, wherein the antibody is an antibody whose heavy chain has SEQ ID NO:7 and whose light chain has SEQ ID NO:

8.

22. The method of treatment of any one of claims 12 to 21, wherein the antibody is a human antibody or a humanized antibody.

23. An antibody that recognizes amino acids 629 to 633 of the human transferrin receptor for use in treating polycythemia, wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not to be administered for at least 4 weeks.

24. The antibody of claim 23, wherein a first administration of the antibody at a dose of 0.25 mg / kg to 20 mg / kg is not followed by a second administration for at least 6 weeks or more, or at least 9 weeks or more.

25. The antibody of claim 23, wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second administration not administered for at least 12 weeks or more.

26. The antibody of claim 23, wherein a first dose of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by a second dose not administered for at least 24 weeks or more.

27. The antibody of claim 23, wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed at least four weeks later by a second administration of the antibody at a dose of 0.25 mg / kg to 20 mg / kg.

28. The antibody of any one of claims 23 to 27, wherein the hematocrit level is maintained at 50% or less for at least four weeks after the first administration of the antibody.

29. The antibody of any one of claims 23 to 28, with or without phlebotomy therapy.

30. The antibody of any one of claims 23 to 29, wherein the polycythemia is polycythemia vera.

31. The antibody according to any one of claims 23 to 30, wherein the heavy chain first complementarity determining region (VH CDR1), the heavy chain second complementarity determining region (VH CDR2), and the heavy chain third complementarity determining region (VH CDR3) are represented by SEQ ID NOs: 1, 2, and 3, respectively, and the light chain first complementarity determining region (VL CDR1), the light chain second complementarity determining region (VL CDR2), and the light chain third complementarity determining region (VL CDR3) are represented by SEQ ID NOs: 4, 5, and 6, respectively.

32. The antibody of any one of claims 23 to 31, wherein the antibody has a heavy chain having SEQ ID NO:7 and a light chain having SEQ ID NO:

8.

33. The antibody of any one of claims 23 to 32, wherein the antibody is a human antibody or a humanized antibody.

34. Use of an antibody that recognizes amino acids 629 to 633 of the human transferrin receptor for the manufacture of a therapeutic agent for polycythemia, comprising administering a first dose of the antibody at a dose of 0.25 mg / kg to 20 mg / kg, followed by not administering a second dose for at least four weeks.

35. The use of claim 34, wherein a first administration of the antibody at a dose of 0.25 mg / kg to 20 mg / kg is not followed by a second administration for at least 6 weeks or more, or at least 9 weeks or more.

36. The use of claim 34, wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed by no second administration for at least 12 weeks or more.

37. The use of claim 34, comprising administering a first dose of the antibody at a dose of 0.25 mg / kg to 20 mg / kg, followed by no second dose for at least 24 weeks or more.

38. The use according to claim 34, wherein a first administration of the antibody is administered at a dose of 0.25 mg / kg to 20 mg / kg, followed at least four weeks later by a second administration of the antibody at a dose of 0.25 mg / kg to 20 mg / kg.

39. The use of any one of claims 34 to 38, wherein the hematocrit level is maintained at 50% or less for at least four weeks or more after the first administration of the antibody.

40. The use according to any one of claims 34 to 39, with or without phlebotomy therapy.

41. The use according to any one of claims 34 to 40, wherein the polycythemia is polycythemia vera.

42. The use according to any one of claims 34 to 41, wherein the antibody has a heavy chain first complementarity determining region (VH CDR1), a heavy chain second complementarity determining region (VH CDR2), and a heavy chain third complementarity determining region (VH CDR3) that are represented by SEQ ID NOs: 1, 2, and 3, respectively, and a light chain first complementarity determining region (VL CDR1), a light chain second complementarity determining region (VL CDR2), and a light chain third complementarity determining region (VL CDR3) that are represented by SEQ ID NOs: 4, 5, and 6, respectively.

43. The use according to any one of claims 34 to 42, wherein the antibody is an antibody whose heavy chain has SEQ ID NO:7 and whose light chain has SEQ ID NO:

8.

44. The use of any one of claims 34 to 43, wherein the antibody is a human antibody or a humanized antibody.

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