Dosage and administration of therapeutic agent comprising substance that recognizes transferrin receptor

Abstract

To provide a therapeutic agent enabling treatment of a malignant tumor.SOLUTION: A therapeutic agent comprising a substance that recognizes a target expressed in an erythroblast, where the therapeutic agent is administered such that, during a dosing period, a serum concentration of the substance in a subject administered with the therapeutic agent is maintained at 10% or more of a maximum value after administration.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to dosage regimens for therapeutic agents containing substances that recognize the transferrin receptor. [Background technology]

[0002] Transferrin receptor (TfR) was first identified on reticulocytes as a cell membrane structure responsible for the intracellular uptake of iron bound to transferrin (Tf). TfR has since been found to be expressed in erythroblasts, placental trophoblast cells, activated lymphocytes, and various tumor cells. For example, high TfR expression has been reported in breast cancer, prostate cancer, lung cancer, pancreatic cancer, colorectal cancer, gastric cancer, bladder cancer, liver cancer, cervical cancer, brain tumors, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, and adult T-cell leukemia. Furthermore, because TfR is highly expressed on the surface of various cancer cells and low in normal cells, it has been recognized as a molecular target for cancer therapy. For example, Patent Document 1 describes an antibody that can specifically recognize the transferrin receptor and an anticancer agent using the antibody.

[0003] On the other hand, NK cell tumors are classified into three types: aggressive NK-cell leukemia (ANKL), extranodal NK / T-cell lymphoma, nasal type (ENKL), and chronic lymphoproliferative disorders of NK cells (CLPD-NK). Aggressive NK-cell leukemia is a malignant lymphoma (blood cancer) derived from NK (natural killer) cells, a type of immune cell. Aggressive NK-cell leukemia is a severe, intractable hematopoietic malignancy that rapidly worsens after onset. Cases are limited to parts of Asia and Latin America, and in Japan, the number of new cases is only a few dozen per year. Therefore, the cause of this ultra-rare disease remains unknown, and the early establishment of an effective standard treatment is needed. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. WO2014 / 073641 Summary of the Invention [Problem to be solved by the invention]

[0005] When anti-transferrin receptor (TfR) antibodies were administered to PDX mice transplanted with cells derived from ANKL patients, extremely high efficacy was observed (PCT / JP2023 / 15337). However, anti-TfR antibodies have the drawback of being cleared extremely quickly when administered to humans or monkeys, resulting in no significant exposure. The objective of the present invention is to determine the dosage regimen of a therapeutic agent that will achieve sufficient exposure to anti-TfR antibodies in subjects. [Means for solving the problem]

[0006] As a result of investigations to solve the above problems, the inventors constructed a kinetic simulation formula based on PK data from a human safety test (Phase 1 clinical trial), and in this simulation, determined a dosage and administration method that would maintain a certain level of exposure.They then actually administered the anti-TfR antibody to monkeys at the determined dosage and administration method, and discovered the optimal dosage and administration method that would maintain a certain level of exposure, thereby completing the present invention.

[0007] That is, according to the present invention, the following inventions are provided. <1> A therapeutic agent comprising a substance that recognizes a target expressed in erythroblasts, wherein the therapeutic agent is administered so that the serum concentration of the substance in a subject to which the therapeutic agent is administered is maintained at 10% or more of the highest value after administration during the administration period. <2> The blood half-life of the substance that recognizes a target expressed in erythroblasts is 24 hours or less when administered in a single dose of 0.25 mg / kg to humans. <1> The therapeutic agent according to any one of the preceding claims. <3> The therapeutic agent is administered so that the serum concentration of the substance in the subject to which the therapeutic agent is administered is maintained at 100 ng / mL or more during the administration period. <1> The therapeutic agent according to any one of the preceding claims. <4> Administered at least once on at least two days out of seven consecutive days <1> The therapeutic agent according to any one of the preceding claims. <5> The single dose of the substance that recognizes a target expressed in erythroblasts is 0.5 mg / kg or more. <1> The therapeutic agent according to any one of the preceding claims. <6> The total weekly dose is 2.5 mg / kg or more. <1> The therapeutic agent according to any one of the preceding claims. <7> The dose of the substance that recognizes a target expressed in erythroblasts is 0.5 mg / kg per administration, and the substance is administered at least once on at least five days out of seven consecutive days. <1> The therapeutic agent according to any one of the preceding claims. <8> The total dose is 40 mg / kg or less, <1> The therapeutic agent according to any one of the preceding claims. <9> It is administered by any of the following administration methods (1) to (3): <1> The therapeutic agent according to any one of the preceding claims. (1) A method of administration comprising a first cycle consisting of 5 consecutive days of administration at 0.5 mg / kg followed by a 2-day rest period, a second cycle consisting of 5 consecutive days of administration at 1.0 mg / kg followed by a 2-day rest period, and a third or subsequent cycle consisting of 5 consecutive days of administration at 2.0 mg / kg followed by a 2-day rest period; (2) A dosing regimen comprising a first cycle of 1.0 mg / kg for 5 consecutive days followed by a 2-day rest period, and a second or subsequent cycle of 2.0 mg / kg for 5 consecutive days followed by a 2-day rest period; or (3) A dosing regimen including the first and subsequent cycles of 2.0 mg / kg for 5 consecutive days followed by a 2-day rest period; <10> The substance that recognizes a target expressed in erythroblasts is a substance that recognizes a transferrin receptor. <1> The therapeutic agent according to any one of the preceding claims. <11> It is a treatment for NK cell tumors. <1> The therapeutic agent according to any one of the preceding claims. <12> The NK cell tumor is aggressive NK cell leukemia or extranodal NK / T-cell lymphoma, nasal type (ENKL). <11> The therapeutic agent according to any one of the preceding claims. <13> The substance that recognizes the transferrin receptor is an antibody that recognizes the transferrin receptor. <10> The therapeutic agent according to any one of the preceding claims. <14> the antibody that recognizes the transferrin receptor is an antibody that recognizes the human transferrin receptor; <13> The therapeutic agent according to any one of the preceding claims. <15> the antibody that recognizes the human transferrin receptor is an antibody that recognizes the amino acids 629 to 633 of the human transferrin receptor, or an antibody that inhibits the binding of other antibodies to the amino acids 629 to 633 of the human transferrin receptor; <13> The therapeutic agent according to any one of the preceding claims. <16> 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; <13> The therapeutic agent according to any one of the preceding claims. <17> The antibody has a heavy chain having SEQ ID NO: 7 and a light chain having SEQ ID NO: 8. <13> The therapeutic agent according to any one of the preceding claims. <18> the antibody is a human antibody or a humanized antibody; <13> The therapeutic agent according to any one of the preceding claims. <19> The antibody is an antibody fragment selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody (scFv), multispecific antibody, disulfide-stabilized V region (dsFv) and CDR-containing peptide; <13> The therapeutic agent according to any one of the preceding claims.

[0008] (A) A therapeutic method comprising administering to a subject in need of treatment a substance that recognizes a target expressed in erythroblasts, wherein the therapeutic agent is administered so that the serum concentration of the substance in the subject to which the therapeutic agent is administered is maintained at 10% or more of the highest value after administration during the administration period. (B) A substance that recognizes a target expressed in erythroblasts for use in the treatment of NK cell tumors and the like, wherein the therapeutic agent is administered so that the serum concentration of the substance in the subject receiving the therapeutic agent is maintained at 10% or more of the peak value after administration during the administration period. (C) Use of a substance that recognizes a target expressed in erythroblasts for the manufacture of a therapeutic agent for NK cell tumors, etc., wherein the therapeutic agent is administered so that the serum concentration of the substance in the subject to which the therapeutic agent is administered is maintained at 10% or more of the peak value after administration during the administration period. [Effects of the Invention]

[0009] The therapeutic agent of the present invention can achieve sufficient exposure to a substance that recognizes a target expressed in erythroblasts, and is useful in the treatment of NK cell tumors and the like. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the results of a toxicity study in which monkeys were given multiple doses of the antibody. [Figure 2] Figure 2 shows the results of a safety test following single administration to humans. [Figure 3] FIG. 3 shows the results of simulating the changes in blood concentration of TfR436 antibody after five consecutive days of administration. [Figure 4] Figure 4 shows the results of simulating the changes in blood concentration of TfR436 antibody when it is administered once every two days. [Figure 5] FIG. 5 shows monkey PK study data. [Figure 6] Figure 6 shows a comparison of the PK measurement data and the kinetic simulation values. DETAILED DESCRIPTION OF THE INVENTION

[0011] Next, the present invention will 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 nomenclature used in connection with 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.

[0012] 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.

[0013] 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 in the extracellular domain, truncated forms, mutation forms due to gene mutations or deletions, and forms that have undergone post-translational modifications such as phosphorylation.

[0014] react and reactivity As used herein, "react" and "reactivity" mean the same thing unless otherwise specified. That is, the recognition of an antigen by an antibody. This antigen may be intact TfR expressed on the cell membrane, a truncated form, or a soluble form. It may also be TfR that retains its three-dimensional structure or a 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, and immunoprecipitation.

[0015] Antibodies used in flow cytometry may be labeled with a fluorescent substance such as FITC, biotin, or the like, or may be unlabeled. Depending on the type and whether the antibody is labeled, fluorescently labeled avidin, fluorescently labeled anti-human immunoglobulin antibody, or the like may be used. Reactivity can be assessed by adding a sufficient amount of anti-TfR antibody (usually at a final concentration of 0.01-10 μg / mL) to the sample and comparing the reactivity with that of a negative control antibody and a positive control antibody.

[0016] Substances that recognize targets expressed in erythroblasts Examples of targets expressed in erythroblasts include transferrin receptor, CD235a, hemoglobin A, CD36, EPOR, glycophorin A, carbonicanhydrase 1, CD45, FLI1, and Trim58. Of these, transferrin receptor is preferred. As a substance that recognizes a target expressed in erythroblasts, at least one selected from the group consisting of nucleic acids (nucleic acid aptamers, decoy nucleic acids, etc.), ribozymes, antibodies and fragments thereof, peptides, cyclic peptides, and peptidomimetics can be used. The blood half-life of a substance that recognizes a target expressed in erythroblasts is preferably 24 hours or less, more preferably 18 hours or less, even more preferably 12 hours or less, even more preferably 6 hours or less, and particularly preferably 5 hours or less, when administered as a single dose of 0.25 mg / kg to humans.

[0017] Substances that recognize the transferrin receptor In the present invention, it is preferable to use a substance that recognizes the transferrin receptor. The substance that recognizes the transferrin receptor is not particularly limited, and for example, at least one selected from the group consisting of nucleic acids (nucleic acid aptamers, decoy nucleic acids, etc.), ribozymes, antibodies and fragments thereof, peptides, cyclic peptides, and peptidomimetics can be used. For example, Wilner et al., Molecular Therapy-Nucleic Acids (2012) 1, e21; doi:10.1038 / mtna.2012.14, describes an aptamer that mimics transferrin. Furthermore, Jae H. Lee et al., Eur. J. Biochem. 268, 2004-2012 (2001) describes a peptide that targets the transferrin receptor. Such nucleic acids, peptides, and the like can be used in the present invention.

[0018] antibody In this specification, the following abbreviations (in parentheses) are used according to convention where necessary: 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] Antibodies include, but are not limited to, recombinant antibodies, monoclonal antibodies, polyclonal antibodies, etc. 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 with 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 technology 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, the FR region of an animal species other than human (e.g., mouse or rat) can also be used.

[0023] In one embodiment, the antibody comprises a constant region 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 a human antibody may be any that belongs to human immunoglobulin (hereinafter referred to as hIgG), but those of the hIgG class are preferred, and any subclass within the hIgG class, such as hIgG1, hIgG2, hIgG3, or hIgG4, can also be used. The light chain constant region (CL) may be any that belongs to hIg, and those of the κ class or λ class can be used. Constant regions from 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 sequences shown in SEQ ID NO: 7 for VH and SEQ ID NO: 8 for VL. In the amino acid sequence, one or several (e.g., 1 to 8, preferably 1 to 5, more preferably 1 to 3, and particularly preferably 1 or 2) amino acids have been deleted, added, substituted, and / or inserted. Methods for preparing the amino acid sequence of an antibody that has binding activity to TfR are well known to those skilled in the art, including methods for introducing mutations into proteins.For example, those skilled in the art will be familiar with site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Takagawa, 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 functionally equivalent to antibodies with TfR-binding activity can be prepared by appropriately introducing mutations into the amino acid sequence of an antibody with TfR-binding activity using techniques such as those described in (HJ, HJ (1987) 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 U S A. 82, 488-492). In this way, antibodies with TfR-binding activity that have one or several amino acid mutations in the variable or constant region of the antibody can also be used.

[0025] As used herein, "having activity equivalent to that of a parent antibody" means that the binding activity to TfR 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 decreased as long as the antibody still has activity. Examples of antibodies with decreased 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 binding activity refers to the recognition of an antigen. This antigen may be intact TfR expressed on the cell membrane, a truncated form, or a soluble form. It may also be TfR that retains 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 blotting, fluorescence microanalysis (FMAT), and surface plasmon resonance (BIAcore).

[0027] 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 antibody in Tf-TfR binding inhibition in International Publication WO2023 / 204181." A TfR solution is dispensed onto a substrate (e.g., a 96-well plate) and allowed to stand, solidified, and blocked. An HRP-labeled Tf solution is then dispensed, and an antibody is added and allowed to react at room temperature. The substrate is then washed, and a color reagent (e.g., TMB) is added and allowed to react, and the absorbance is measured using a plate reader. The above procedures allow the antibody's Tf-TfR binding inhibitory activity to be evaluated.

[0028] 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.

[0029] 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.

[0030] Antibody production The antibody used in the present invention may be either a monoclonal antibody or a polyclonal antibody, which can be prepared by methods known to those skilled in the art. Antibodies include those produced in the blood of animals, those produced by hybridomas, those produced by hosts transformed using genetic engineering techniques with expression vectors containing antibody genes, those produced by screening optimal antibodies from clone libraries using phage display and then producing the genes in CHO cells, and those produced directly from transgenic mice that produce human antibodies.

[0031] 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.

[0032] To produce monoclonal antibodies, animals are 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.

[0033] The production of antibodies by phage display is described below. (1) Antigen-reactive scFv from a phage display library Using phage display technology, libraries containing repertoires 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 when screened using TfR as an antigen. By analyzing the genes of the selected phages, the DNA sequences of the VH and VL regions encoding the variable regions of human antibodies that bind to the antigen can be determined. Using the scFv sequences, the scFvs can be converted into IgG to obtain human antibodies.

[0034] (2) Conversion of scFv to IgG (production of human antibodies) Human antibodies are obtained by constructing expression vectors for the H or L chain, expressing them in host cells, and recovering and purifying the secreted supernatant. Human antibodies can also be obtained by expressing VH and VL in the same vector (tandem). 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.

[0035] Specifically, a full-length heavy chain gene can be obtained by ligating the VH-encoding DNA 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, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDapartment 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 that of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD, with IgG1 or IgG2 constant regions being most preferred. The IgG1 constant region sequence may be any of the various alleles or allotypes known to occur between 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.

[0036] A full-length L chain gene (as well as a Fab light chain gene) can be obtained by ligating 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, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDapartment 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.

[0037] The DNA encoding the H or L 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 human antibodies. 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. Expression vectors 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 genes are 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).

[0038] Convenient vectors encode functionally complete human CH or CL immunoglobulin sequences with appropriate restriction sites engineered to facilitate the 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).

[0039] 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 selectable marker genes. 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.

[0040] Host cells are transformed with the antibody gene expression vector constructed by the above method. Host cells can be any cell capable of producing antibodies, including bacteria, yeast, animal cells, insect cells, and 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. Marone 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.

[0041] After culturing the transformant, human antibodies are isolated from the cells of the transformant or from the culture medium. Antibody isolation and purification can be achieved by using an appropriate combination of methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion exchange chromatography, and gel filtration chromatography.

[0042] antibody fragment Antibody fragments can be produced based on antibodies or on the sequence information of the genes encoding the antibodies, including Fab, Fab', F(ab')2, scFv, and dsFv antibodies.

[0043] 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, it can be obtained by papain digestion of the above antibody. 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.

[0044] 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 F(ab')2, as described below. In the present invention, it can be obtained by digesting the above-mentioned antibody with pepsin and cleaving the disulfide bond using a reducing agent. Alternatively, like Fab, Fab' can also be prepared by genetic engineering using DNA encoding it.

[0045] 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 part of the hinge region are linked by disulfide bonds. In the present invention, it is obtained by digesting the above-mentioned antibody with pepsin. Furthermore, like Fab, it can also be prepared by genetic engineering using DNA encoding F(ab')2.

[0046] 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 via a suitable peptide linker at the C-terminus of one chain to the N-terminus of the other to form a single chain. Examples of peptide linkers that can be used include the highly flexible (GGGGS)3. For example, DNA encoding an scFv antibody can be constructed using DNA encoding the H-chain and L-chain variable regions of the antibody and DNA encoding the peptide linker, and this can be inserted into an appropriate vector. The vector can then be used to transform the resulting transformant, from which the scFv can be prepared.

[0047] 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. Furthermore, antibody fragments can also be multimerized by linking scFv antibodies, dcFv antibodies, etc. using an appropriate linker, or by fusing streptavidin.

[0048] 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. Bispecific antibodies are antibodies that combine two molecules, one of which recognizes TfR and the other of which targets another molecule. Examples include bispecific (mab)2, obtained by chemically crosslinking two monoclonal antibodies; bispecific F(ab')2, obtained by chemically crosslinking two 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: Technological Considerations. Biomolecules 2020, 10, 360). However, the forms of these antibodies are not limited to these.

[0049] NK cell tumors NK cell tumors include three types: aggressive NK-cell leukemia (ANKL), extranodal NK / T-cell lymphoma, nasal type (ENKL), and chronic lymphoproliferative disorders of NK cells (CLPD-NK).

[0050] Pharmaceutical Compositions and Formulations The therapeutic agent for NK cell tumors of the present invention can be provided as a pharmaceutical composition or formulation. The therapeutic agent for NK cell tumors of the present invention can be used to treat NK cell tumors in a subject in need of such treatment.

[0051] The therapeutic agent for NK cell tumors of the present invention may contain a physiologically acceptable diluent or carrier in addition to the substance that recognizes the transferrin receptor. Suitable carriers include, but are not limited to, physiological saline, phosphate-buffered saline, phosphate-buffered saline glucose solution, and buffered saline. Alternatively, the substance that recognizes the transferrin receptor may be lyophilized (freeze-dried) or frozen and then reconstituted when needed by adding an aqueous buffer solution such as those described above. Examples of dosage forms include oral administration using tablets, capsules, granules, powders, syrups, etc., or parenteral administration using injections (subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), transdermal, transmucosal, nasal, pulmonary, suppositories, etc. Examples of dosage forms include oral administration using tablets, capsules, granules, powders, syrups, etc., or parenteral administration using injections (subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), transdermal, transmucosal, nasal, pulmonary, suppositories, etc.

[0052] Dosage In the present invention, a therapeutic agent is administered so that the serum concentration of a "substance that recognizes a target expressed in erythroblasts" in a subject receiving the therapeutic agent is maintained at 10% or more of its maximum post-administration value during the administration period. Preferably, the serum concentration of the substance in the subject is maintained at 20% or more of its maximum post-administration value, more preferably, the serum concentration of the substance in the subject is maintained at 30% or more of its maximum post-administration value, even more preferably, the serum concentration of the substance in the subject is maintained at 40% or more of its maximum post-administration value, and particularly preferably, the serum concentration of the substance in the subject is maintained at 50% or more of its maximum post-administration value.

[0053] Preferably, the therapeutic agent is administered so that the serum concentration of the substance in the subject receiving the therapeutic agent is maintained at 100 ng / mL or higher, more preferably 500 ng / mL or higher, more preferably 1000 ng / mL or higher, even more preferably 2000 ng / mL or higher, even more preferably 2500 ng / mL or higher, and even more preferably 3000 ng / mL or higher, during the administration period.

[0054] The therapeutic agent of the present invention is preferably administered at least once on two or more days (preferably three or more, four or more, or five or more) out of seven consecutive days. As an example, one course can consist of five consecutive days of administration followed by two days of rest. The seven consecutive days described above constitute one course, and the above course can be administered multiple times. Administration can be carried out for two or more courses (for example, two to ten courses, or more).

[0055] The single dose of a substance that recognizes a target expressed in erythroblasts is not particularly limited, but is preferably 0.5 mg / kg or more, and examples thereof include 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, or 2.0 mg / kg. The single dose can be any dose between 0.5 mg / kg and 1.0 mg / kg, any dose between 1.0 mg / kg and 1.5 mg / kg, any dose between 1.5 mg / kg and 2.0 mg / kg, or any dose between 2.0 mg / kg and 3.0 mg / kg.

[0056] In one example of the present invention, the single dose of a substance that recognizes a target expressed in erythroblasts is 0.5 mg / kg and can be administered once or more times (e.g., once, twice, or three times each day) on five or more days out of seven consecutive days, or it can be administered once every two days.

[0057] After the first course of administration, the dose may be increased in the second and subsequent courses. After the first course of administration, the dose may be reduced in the second and subsequent courses. After the first course of administration, the second and subsequent courses may be administered at the same dose as in the first course. As an example, the single dose of a substance that recognizes a target expressed in erythroblasts may be 0.5 mg / kg for the first course and 1.0 mg / kg for the second course and thereafter.

[0058] Specific examples of administration methods include any of the following. (1) A method of administration comprising a first cycle consisting of 5 consecutive days of administration at 0.5 mg / kg followed by a 2-day rest period, a second cycle consisting of 5 consecutive days of administration at 1.0 mg / kg followed by a 2-day rest period, and a third or subsequent cycle consisting of 5 consecutive days of administration at 2.0 mg / kg followed by a 2-day rest period; (2) A dosing regimen comprising a first cycle of 1.0 mg / kg for 5 consecutive days followed by a 2-day rest period, and a second or subsequent cycle of 2.0 mg / kg for 5 consecutive days followed by a 2-day rest period; or (3) A dosing regimen including the first and subsequent cycles of 2.0 mg / kg for 5 consecutive days followed by a 2-day rest period; The above are merely illustrative examples, and the single dose is not limited to 0.5 mg / kg, 1.0 mg / kg, or 2.0 mg / kg, and may be changed to other doses (e.g., 0.75 mg / kg, 1.5 mg / kg, 2.5 mg / kg, etc.). A dose of 0.5 mg / kg may also be administered twice a day. Modifications are possible, such as administering every two days or completing the first course of administration.

[0059] In the present invention, the total weekly dose of a substance that recognizes a target expressed in erythroblasts is preferably 2.5 mg / kg or more.

[0060] In the present invention, it is preferable that the total dose of substances that recognize targets expressed in erythroblasts is 40 mg / kg or less. For example, one course consists of five administrations of 2 mg / kg, and if this is administered a total of four times (first to fourth courses), the total dose will be 40 mg / kg. Alternatively, if the first course is administered five times at 0.5 mg / kg, the second course is administered five times at 1.0 mg / kg, and the third to fifth courses are administered five times at 2.0 mg / kg x 3 courses, the total dose will be 37.5 mg / kg.

[0061] 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. [Example]

[0062] In the following examples, the TfR436 antibody described in paragraphs 0090 and 0091 of International Publication WO2014 / 073641 was used.

[0063] 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: 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)

[0064] The VH and VL sequences of the TfR436 antibody are shown below. TfR436 VH (SEQ ID NO: 7) DVQLVQSGGGVVQPGRSLRLSCAASGFPFKSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRGEDTAVYYCARDSNFWSGYYSPVDVWGQGTTVTVSS

[0065] TfR436 VL (SEQ ID NO: 8) NFMLTQPHSVSESPGKTVTISCTRSSGSIASNSVQWYQQRPGSAPITVIYEDTQRPSGVPDRFSGSIDSSSNSASLTISGLQTEDEADYYCQSYDSAYHWVFGGGTKLAVL

[0066] The identification of the binding site of the TfR436 antibody, comparison of the TfR436 antibody and comparative antibodies in inhibiting Tf-TfR binding, and PDX mouse test data are included in Examples 1, 2, and 13 of International Publication No. WO 2023 / 204181, which are incorporated herein by reference.

[0067] Example 1: Measurement of blood concentration of TfR antibody The concentration of TfR antibodies in blood was measured by ELISA. Plasma or serum samples were used, and the measurements were performed by diluting them so that the measurement results fell within the range of a calibration curve using a known concentration of TfR436 antibody as the standard substance. For the measurements, a solid-phase plate was prepared with immobilized anti-TfR436 antibody (supplied by Perseus Proteomics, Inc.), and the standard substance and sample were added to this plate, followed by reaction and washing. After reaction and washing, the plate was reacted with HRP-labeled anti-TfR436 antibody (prepared fresh), followed by colorimetric quantification using TMB.

[0068] Example 2: Toxicity study of multiple doses of antibody in monkeys To confirm the pharmacokinetics of TfR436 antibody administered to cynomolgus monkeys at doses of 1, 5, and 30 mg / kg (6 mL / kg, 1-hour administration) once weekly for 4 weeks, serum TfR436 concentrations were measured. Blood samples were taken immediately before and after the first administration, 6 and 24 hours, and 8, 15, 22, and 29 days after each administration. Blood concentrations were measured at each time point and plotted as shown in Figure 1.

[0069] Example 3: Safety test after single administration to humans To confirm the pharmacokinetics of TfR436 antibody after single administration (6 mL / kg, 1-hour administration) to humans at doses of 0.008, 0.04, 0.08, 0.16, and 0.25 mg / kg, serum TfR436 concentrations were measured. Blood samples were taken immediately before administration, immediately after administration, and 2, 3, 4, 5, 9, and 13 hours, and 2, 3, 7, 14, and 21 days after administration. The blood concentration at each point was measured and plotted as shown in Figure 2. The results in Figure 2 show that the blood half-life of the TfR436 antibody was 4.3 hours after a single dose of 0.25 mg / kg was administered to humans. Since the half-life of antibody drugs is generally around three weeks, this indicates that the blood half-life of the TfR436 antibody is shorter than that of general antibody drugs.

[0070] Example 4: Human dynamics simulation Using the data from Example 3, a model equation describing the pharmacokinetics of TfR436 was constructed using Napp (nonlinear least squares software) (Drug Metab Dispos 42: 726-734, 2014; J Pharm Sci 102(1):237-249, 2013; Pharm Res. 2016 Feb;33(2):269-282). @At 0: y1=Dose / Vd, y2=0, @From 0: y1'=(-(CL+Vmax / (Km+y1))*y1-k12*y1*Vd+k21*y2) / Vd, y2'=k12*Vd*y1-k21*y2, y1: TfR436 concentration in the central compartment (ng / mL) y2: Amount of TfR436 in the peripheral compartment (ng / kg) fixed parameters: Dose: dose (ng / kg), CL: non-saturable clearance 0.283 (mL / min / kg) *CL is taken from the Herceptin interview form (CL when Herceptin is administered at 8.0 mg / kg).

[0071] The above formula was input into Napp, and the free parameter values ​​were estimated by fitting using the nonlinear least squares method with the actual measured TfR436 blood concentration data from clinical trials in healthy individuals. The results are shown in the table below.

[0072] [Table 1]

[0073] Free parameters Vd: Volume of distribution (mL / kg) CL: clearance (mL / min / kg) Vmax: maximum reaction velocity (ng / min / kg) k12,k21: Distribution rate constant ( / min) Km: Michaelis constant (ng / mL)

[0074] The parameter values ​​obtained from the estimation were input into Napp, and the blood concentration profile after five consecutive days of TfR436 administration (0.5, 1.0, 1.5, 2.0, 3.0 mg / kg) was simulated. The results are shown in Figure 3. Figure 4 shows the simulated results of the blood concentration transition when administered once every two days (1.0 mg / kg).

[0075] Example 5: Monkey PK Study Data To confirm the pharmacokinetics of TfR436 antibodies, serum TfR436 concentrations were measured in male cynomolgus monkeys (4 groups, 2 monkeys per group) after repeated intravenous administration (6 mL / kg, 1-hour infusion) once daily for 5 days. All groups received 4 courses of TfR436 antibodies once daily for 5 days, followed by a 2-day rest period. Blood samples were collected on the first day of administration (immediately after administration, 3, 6, and 12 hours after administration), days 2, 5, 8, 12, 19, and 22 (before administration and immediately after administration), day 26 (immediately after administration, 3, 6, and 12 hours after administration), and days 29, 31, and 34. The circles in the upper panel of Figure 5 indicate the blood sampling points, and the blood concentration measurements are shown in the lower panel of Figure 5.

[0076] Example 6: Clinical trial data of ANKL administered multiple times in humans PK was measured over a course of 5 consecutive days of administration followed by 2 days of rest. Blood samples were taken immediately after administration and 4, 8, and 12 hours after administration on the first day, immediately before administration and immediately after administration on the second day, immediately before administration on the third day, immediately before administration on the fourth day, and immediately before administration and immediately after administration on the fifth day. From the second course onwards, blood samples were taken immediately before and immediately after administration on the first day.

[0077] Table 2 shows the actual measured blood concentrations (ng / mL) for the first course at 0.5 mg / kg, the second course at 1.0 mg / kg, and the third course at 2.0 mg / kg.

[0078] [Table 2]

[0079] Figure 6 shows a comparison of the actual PK data (Table 2) and the kinetic simulation (the first cycle was 0.5 mg / kg, the second cycle was 1.0 mg / kg, and the third cycle was 2.0 mg / kg, each administered for 5 consecutive days, with a 2-day rest period as one cycle). The horizontal axis represents time (hr), and the vertical axis represents TfR436 blood concentration.

Claims

1. A therapeutic agent comprising a substance that recognizes a target expressed in erythroblasts, wherein the therapeutic agent is administered so that the serum concentration of the substance in a subject to which the therapeutic agent is administered is maintained at 10% or more of the highest value after administration during the administration period.

2. The therapeutic agent according to claim 1, wherein the substance that recognizes a target expressed in erythroblasts has a blood half-life of 24 hours or less when administered in a single dose of 0.25 mg / kg to a human.

3. The therapeutic agent according to claim 1, wherein the therapeutic agent is administered so that the serum concentration of the substance in the subject administered the therapeutic agent is maintained at 100 ng / mL or more during the administration period.

4. The method of claim 1, wherein the method is administered at least once on each of two or more days out of seven consecutive days.

5. The therapeutic agent according to claim 1, wherein the single dose of the substance that recognizes a target expressed in erythroblasts is 0.5 mg / kg or more.

6. The therapeutic agent according to claim 1, wherein the total weekly dose is 2.5 mg / kg or more.

7. The therapeutic agent according to claim 1, wherein the single dose of the substance that recognizes a target expressed in erythroblasts is 0.5 mg / kg and is administered at least once on each of five or more days out of seven consecutive days.

8. The therapeutic agent according to claim 1, wherein the total dose is 40 mg / kg or less.

9. The therapeutic agent according to <1>, which is administered by any one of the following administration methods (1) to (3). (1) An administration method comprising a first course consisting of 5 consecutive days of administration at 0.5 mg / kg and a 2-day drug holiday, a second course consisting of 5 consecutive days of administration at 1.0 mg / kg and a 2-day drug holiday, and a third or subsequent course consisting of 5 consecutive days of administration at 2.0 mg / kg and a 2-day drug holiday; (2) an administration method comprising a first course consisting of 5 consecutive days of administration at 1.0 mg / kg followed by a 2-day drug holiday, and a second or subsequent course consisting of 5 consecutive days of administration at 2.0 mg / kg followed by a 2-day drug holiday; or (3) An administration method including a first or subsequent cycle consisting of 5 consecutive days of administration at 2.0 mg / kg followed by a 2-day washout period;

10. The therapeutic agent according to claim 1, wherein the substance that recognizes a target expressed in erythroblasts is a substance that recognizes a transferrin receptor.

11. The therapeutic agent according to claim 1, which is a therapeutic agent for NK cell tumors.

12. The therapeutic agent according to claim 11, wherein the NK cell tumor is aggressive NK cell leukemia or extranodal NK / T-cell lymphoma, nasal type (ENKL).

13. The therapeutic agent according to claim 10, wherein the substance that recognizes the transferrin receptor is an antibody that recognizes the transferrin receptor.

14. The therapeutic agent according to claim 13, wherein the antibody that recognizes the transferrin receptor is an antibody that recognizes the human transferrin receptor.

15. The therapeutic agent according to claim 13, wherein the antibody that recognizes the human transferrin receptor is an antibody that recognizes the amino acids 629 to 633 of the human transferrin receptor, or an antibody that inhibits the binding of other antibodies to the amino acids 629 to 633 of the human transferrin receptor.

16. The therapeutic agent of claim 13, 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.

17. The therapeutic agent according to claim 13, wherein the antibody has a heavy chain having SEQ ID NO: 7 and a light chain having SEQ ID NO:

8.

18. The therapeutic agent of claim 13, wherein the antibody is a human antibody or a humanized antibody.

19. The antibody is Fab, Fab', F(ab') ２ The therapeutic agent of claim 13, which is an antibody fragment selected from the group consisting of a single-chain antibody (scFv), a multispecific antibody, a disulfide-stabilized V region (dsFv), and a peptide containing CDRs.

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